cerebral cortex projection neuron development, diversity, disease and regeneration
Macklis Lab Publications
Featured Publications
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2024. Bcl11b orchestrates subcerebral projection neuron axon development via cell-autonomous, non-cell-autonomous, and subcellular mechanisms. bioRxiv. DOI:https://doi.org/10.1101/2024.10.20.619265 Itoh Y, Woodworth MB, Greig LC, Engmann AK, Tillman DE, Hatch JJ, Macklis JD. 2024. Bcl11b orchestrates subcerebral projection neuron axon development via cell-autonomous, non-cell-autonomous, and subcellular mechanisms. bioRxiv. DOI:https://doi.org/10.1101/2024.10.20.619265 Both cell-intrinsic competency and extracellular cues regulate axon projection, but mechanisms that coordinate these elements remain poorly understood. Subcerebral projection neurons (SCPN) extend their primary axons from cortex through subcortical structures, including the striatum, targeting the brainstem and spinal cord. We identify that the transcription factor Bcl11b/Ctip2 functions in multiple independent neuron populations to control SCPN axon development. Bcl11b expressed by SCPN is required cellautonomously for axonal outgrowth and efficient entry into the internal capsule within the striatum, while Bcl11b expressed by medium spiny neurons (MSN) non-cell-autonomously regulates SCPN axon fasciculation within the internal capsule and subsequent pathfinding. Further, integrated investigation of Bcl11b-null SCPN with transcriptomic, immunocytochemical, and in vivo growth cone purification approaches identifies that Cdh13 is localized along axons and on growth cone surfaces of SCPN in vivo, and mediates Bcl11b regulation of SCPN axonal outgrowth. Together, these results demonstrate that Bcl11b controls multiple aspects of SCPN axon development by coordinating intrinsic SCPN cell autonomous subcellular mechanisms and extrinsic MSN non-cell-autonomous mechanisms.Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved. -
Greig LC, Woodworth MB, Poulopoulos A, Lim S, Macklis JD. 2024. BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis. Cell reports. 43(8):114650. Pubmed: 39159043 DOI:S2211-1247(24)01001-5 Greig LC, Woodworth MB, Poulopoulos A, Lim S, Macklis JD. 2024. BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis. Cell reports. 43(8):114650. Pubmed: 39159043 DOI:S2211-1247(24)01001-5 We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved. -
Ozkan A, Padmanabhan HK, Shipman SL, Azim E, Kumar P, Sadegh C, Basak AN, Macklis JD. 2024. Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors. bioRxiv : the preprint server for biology. Pubmed: 38712174 DOI:10.1101/2024.04.21.590488 Ozkan A, Padmanabhan HK, Shipman SL, Azim E, Kumar P, Sadegh C, Basak AN, Macklis JD. 2024. Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors. bioRxiv : the preprint server for biology. Pubmed: 38712174 DOI:10.1101/2024.04.21.590488 Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitry , or for optimally informative disease modeling and/or therapeutic screening , it is important to reproduce the type or subtype of neurons involved. No such appropriate models exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factor is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and that suppresses a latent neurogenic program by repressing inappropriate proneural expression by progenitors. We FACS-purify these genetically accessible progenitors from postnatal mouse cortex and establish a pure culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activating , while antagonizing with ). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors, and find that these neurons differentiate with remarkable fidelity compared with corticospinal neurons . They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employed -driven differentiation. Neurons induced by instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from genetically accessible cortical progenitors sets a precedent and foundation for mechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair. -
Poulopoulos A, Davis P, Brandenburg C, Itoh Y, Galazo MJ, Greig LC, Romanowski AJ, Budnik B, Macklis JD. 2024. Symmetry in levels of axon-axon homophilic adhesion establishes topography in the corpus callosum and development of connectivity between brain hemispheres. bioRxiv : the preprint server for biology. Pubmed: 38585721 DOI:10.1101/2024.03.28.587108 Poulopoulos A, Davis P, Brandenburg C, Itoh Y, Galazo MJ, Greig LC, Romanowski AJ, Budnik B, Macklis JD. 2024. Symmetry in levels of axon-axon homophilic adhesion establishes topography in the corpus callosum and development of connectivity between brain hemispheres. bioRxiv : the preprint server for biology. Pubmed: 38585721 DOI:10.1101/2024.03.28.587108 Specific and highly diverse connectivity between functionally specialized regions of the nervous system is controlled at multiple scales, from anatomically organized connectivity following macroscopic axon tracts to individual axon target-finding and synapse formation. Identifying mechanisms that enable entire subpopulations of related neurons to project their axons with regional specificity within stereotyped tracts to form appropriate long-range connectivity is key to understanding brain development, organization, and function. Here, we investigate how axons of the cerebral cortex form precise connections between the two cortical hemispheres via the corpus callosum. We identify topographic principles of the developing trans-hemispheric callosal tract that emerge through intrinsic guidance executed by growing axons in the corpus callosum within the first postnatal week in mice. Using micro-transplantation of regionally distinct neurons, subtype-specific growth cone purification, subcellular proteomics, and in utero gene manipulation, we investigate guidance mechanisms of transhemispheric axons. We find that adhesion molecule levels instruct tract topography and target field guidance. We propose a model in which transcallosal axons in the developing brain perform a "handshake" that is guided through co-fasciculation with symmetric contralateral axons, resulting in the stereotyped homotopic connectivity between the brain's hemispheres. -
Veeraraghavan P, Engmann AK, Hatch JJ, Itoh Y, Nguyen D, Addison T, Macklis JD. 2024. Dynamic subtype- and context-specific subcellular RNA regulation in growth cones of neocortical projection neurons. bioRxiv. DOI:https://doi.org/10.1101/2023.09.24.559186 Veeraraghavan P, Engmann AK, Hatch JJ, Itoh Y, Nguyen D, Addison T, Macklis JD. 2024. Dynamic subtype- and context-specific subcellular RNA regulation in growth cones of neocortical projection neurons. bioRxiv. DOI:https://doi.org/10.1101/2023.09.24.559186 -
Durak, O*, Kim, JY*, Tillman, DE, Itoh, Y, Wettstein, M, Greig, LC, Macklis, JD. . 2022. ASD gene Bcl11a regulates subcellular RNA localization, associative circuitry, and social behavior. bioRxiv. DOI:10.1101/2022.10.06.511159 Durak, O*, Kim, JY*, Tillman, DE, Itoh, Y, Wettstein, M, Greig, LC, Macklis, JD. . 2022. ASD gene Bcl11a regulates subcellular RNA localization, associative circuitry, and social behavior. bioRxiv. DOI:10.1101/2022.10.06.511159 -
Froberg JE, Durak O, Macklis JD. 2023. Development of nanoRibo-seq enables study of regulated translation by cortical neuron subtypes, showing uORF translation in synaptic-axonal genes. Cell reports. 42(9):112995. Pubmed: 37624698 DOI:S2211-1247(23)01006-9 Froberg JE, Durak O, Macklis JD. 2023. Development of nanoRibo-seq enables study of regulated translation by cortical neuron subtypes, showing uORF translation in synaptic-axonal genes. Cell reports. 42(9):112995. Pubmed: 37624698 DOI:S2211-1247(23)01006-9 Investigation of translation in rare cell types or subcellular contexts is challenging due to large input requirements for standard approaches. Here, we present "nanoRibo-seq" an optimized approach using 10- to 10-fold less input material than bulk approaches. nanoRibo-seq exhibits rigorous quality control features consistent with quantification of ribosome protected fragments with as few as 1,000 cells. We compare translatomes of two closely related cortical neuron subtypes, callosal projection neurons (CPN) and subcerebral projection neurons (SCPN), during their early postnatal development. We find that, while translational efficiency is highly correlated between CPN and SCPN, several dozen mRNAs are differentially translated. We further examine upstream open reading frame (uORF) translation and identify that mRNAs involved in synapse organization and axon development are highly enriched for uORF translation in both subtypes. nanoRibo-seq enables investigation of translational regulation of rare cell types in vivo and offers a flexible approach for globally quantifying translation from limited input material.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Galazo MJ, Sweetser DA, Macklis JD. 2023. Tle4 controls both developmental acquisition and early post-natal maturation of corticothalamic projection neuron identity. Cell reports. 42(8):112957. Pubmed: 37561632 DOI:S2211-1247(23)00968-3 Galazo MJ, Sweetser DA, Macklis JD. 2023. Tle4 controls both developmental acquisition and early post-natal maturation of corticothalamic projection neuron identity. Cell reports. 42(8):112957. Pubmed: 37561632 DOI:S2211-1247(23)00968-3 Identities of distinct neuron subtypes are specified during embryonic development, then maintained during post-natal maturation. In cerebral cortex, mechanisms controlling early acquisition of neuron-subtype identities have become increasingly understood. However, mechanisms controlling neuron-subtype identity stability during post-natal maturation are largely unexplored. We identify that Tle4 is required for both early acquisition and post-natal stability of corticothalamic neuron-subtype identity. Embryonically, Tle4 promotes acquisition of corticothalamic identity and blocks emergence of core characteristics of subcerebral/corticospinal projection neuron identity, including gene expression and connectivity. During the first post-natal week, when corticothalamic innervation is ongoing, Tle4 is required to stabilize corticothalamic neuron identity, limiting interference from differentiation programs of developmentally related neuron classes. We identify a deacetylation-based epigenetic mechanism by which TLE4 controls Fezf2 expression level by corticothalamic neurons. This contributes to distinction of cortical output subtypes and ensures identity stability for appropriate maturation of corticothalamic neurons.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Itoh Y, Sahni V, Shnider SJ, McKee H, Macklis JD. 2023. Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations. Cell reports. 42(3):112182. Pubmed: 36934325 DOI:S2211-1247(23)00193-6 Itoh Y, Sahni V, Shnider SJ, McKee H, Macklis JD. 2023. Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations. Cell reports. 42(3):112182. Pubmed: 36934325 DOI:S2211-1247(23)00193-6 How CNS circuits sculpt their axonal arbors into spatially and functionally organized domains is not well understood. Segmental specificity of corticospinal connectivity is an exemplar for such regional specificity of many axon projections. Corticospinal neurons (CSN) innervate spinal and brainstem targets with segmental precision, controlling voluntary movement. Multiple molecularly distinct CSN subpopulations innervate the cervical cord for evolutionarily enhanced precision of forelimb movement. Evolutionarily newer CSN exclusively innervate bulbar-cervical targets, while CSN are heterogeneous; distinct subpopulations extend axons to either bulbar-cervical or thoraco-lumbar segments. We identify that Lumican controls balance of cervical innervation between CSN and CSN axons during development, which is maintained into maturity. Lumican, an extracellular proteoglycan expressed by CSN, non-cell-autonomously suppresses cervical collateralization by multiple CSN subpopulations. This inter-axonal molecular crosstalk between CSN subpopulations controls murine corticospinal circuitry refinement and forelimb dexterity. Such crosstalk is generalizable beyond the corticospinal system for evolutionary incorporation of new neuron populations into preexisting circuitry.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Song JHT, Ruven C, Patel P, Ding F, Macklis JD*, Sahni V*. 2022. Cbln1 directs axon targeting by corticospinal neurons specifically toward thoraco-lumbar spinal cord. BioRxiv. DOI:https://doi.org/10.1101/2022.04.06.487184 Song JHT, Ruven C, Patel P, Ding F, Macklis JD*, Sahni V*. 2022. Cbln1 directs axon targeting by corticospinal neurons specifically toward thoraco-lumbar spinal cord. BioRxiv. DOI:https://doi.org/10.1101/2022.04.06.487184 Corticospinal neurons (CSN) are centrally required for skilled voluntary movement, which necessitates that they establish precise subcerebral connectivity with the brainstem and spinal cord. However, molecular controls regulating specificity of this projection targeting remain largely unknown. We previously identified that developing CSN subpopulations exhibit striking axon targeting specificity in the spinal white matter. These CSN subpopulations with segmentally distinct spinal projections are also molecularly distinct; a subset of differentially expressed genes between these distinct CSN subpopulations function as molecular controls regulating differential axon projection targeting. Rostrolateral CSN extend axons exclusively to bulbar-cervical segments (CSNBC-lat), while caudomedial CSN (CSNmedial) are more heterogeneous, with distinct, intermingled subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. Here, we report that Cerebellin 1 (Cbln1) is expressed specifically by CSN in medial, but not lateral, sensorimotor cortex. Cbln1 shows highly dynamic temporal expression, with Cbln1 levels in CSN highest during the period of peak axon extension toward thoraco-lumbar segments. Using gain-of-function experiments, we identify that Cbln1 is sufficient to direct thoraco-lumbar axon extension by CSN. Mis-expression of Cbln1 in CSNBC-lat either by in utero electroporation, or in postmitotic CSNBC-lat by AAV-mediated gene delivery, re-directs these axons past their normal bulbar-cervical targets toward thoracic segments. Further, Cbln1 overexpression in postmitotic CSNmedial increases the number of CSNmedial axons that extend past cervical segments into the thoracic cord. Collectively, these results identify that Cbln1 functions as a potent molecular control over thoraco-lumbar CSN axon extension, part of an integrated network of controls over segmentally-specific CSN axon projection targeting. -
Engmann AK, Hatch JJ, Nanda P, Veeraraghavan P, Ozkan A, Poulopoulos A, Murphy AJ, Macklis JD. 2022. Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes. Nature protocols. 17(2):222-251. Pubmed: 35022617 DOI:10.1038/s41596-021-00638-7 Engmann AK, Hatch JJ, Nanda P, Veeraraghavan P, Ozkan A, Poulopoulos A, Murphy AJ, Macklis JD. 2022. Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes. Nature protocols. 17(2):222-251. Pubmed: 35022617 DOI:10.1038/s41596-021-00638-7 During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype- and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate that subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit 'wiring'. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from the rodent central nervous system (CNS) in vivo. By using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is isolated by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting and established FACS of neurons and are suitable for downstream analyses of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes ~3 h, plus sorting time, and ~1-2 h for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, depending on the turnaround time of the core facility involved.© 2022. The Author(s), under exclusive licence to Springer Nature Limited. -
Sahni V, Itoh Y, Shnider SJ, Macklis JD. 2021. Crim1 and Kelch-like 14 exert complementary dual-directional developmental control over segmentally specific corticospinal axon projection targeting. Cell reports. 37(3):109842. Pubmed: 34686337 DOI:S2211-1247(21)01306-1 Sahni V, Itoh Y, Shnider SJ, Macklis JD. 2021. Crim1 and Kelch-like 14 exert complementary dual-directional developmental control over segmentally specific corticospinal axon projection targeting. Cell reports. 37(3):109842. Pubmed: 34686337 DOI:S2211-1247(21)01306-1 The cerebral cortex executes highly skilled movement, necessitating that it connects accurately with specific brainstem and spinal motor circuitry. Corticospinal neurons (CSN) must correctly target specific spinal segments, but the basis for this targeting remains unknown. In the accompanying report, we show that segmentally distinct CSN subpopulations are molecularly distinct from early development, identifying candidate molecular controls over segmentally specific axon targeting. Here, we functionally investigate two of these candidate molecular controls, Crim1 and Kelch-like 14 (Klhl14), identifying their critical roles in directing CSN axons to appropriate spinal segmental levels in the white matter prior to axon collateralization. Crim1 and Klhl14 are specifically expressed by distinct CSN subpopulations and regulate their differental white matter projection targeting-Crim1 directs thoracolumbar axon extension, while Klhl14 limits axon extension to bulbar-cervical segments. These molecular regulators of descending spinal projections constitute the first stages of a dual-directional set of complementary controls over CSN diversity for segmentally and functionally distinct circuitry.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved. -
Sahni V, Shnider SJ, Jabaudon D, Song JHT, Itoh Y, Greig LC, Macklis JD. 2021. Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting. Cell reports. 37(3):109843. Pubmed: 34686320 DOI:S2211-1247(21)01307-3 Sahni V, Shnider SJ, Jabaudon D, Song JHT, Itoh Y, Greig LC, Macklis JD. 2021. Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting. Cell reports. 37(3):109843. Pubmed: 34686320 DOI:S2211-1247(21)01307-3 For precise motor control, distinct subpopulations of corticospinal neurons (CSN) must extend axons to distinct spinal segments, from proximal targets in the brainstem and cervical cord to distal targets in thoracic and lumbar spinal segments. We find that developing CSN subpopulations exhibit striking axon targeting specificity in spinal white matter, which establishes the foundation for durable specificity of adult corticospinal circuitry. Employing developmental retrograde and anterograde labeling, and their distinct neocortical locations, we purified developing CSN subpopulations using fluorescence-activated cell sorting to identify genes differentially expressed between bulbar-cervical and thoracolumbar-projecting CSN subpopulations at critical developmental times. These segmentally distinct CSN subpopulations are molecularly distinct from the earliest stages of axon extension, enabling prospective identification even before eventual axon targeting decisions are evident in the spinal cord. This molecular delineation extends beyond simple spatial separation of these subpopulations in the cortex. Together, these results identify candidate molecular controls over segmentally specific corticospinal axon projection targeting.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved. -
Diaz JL, Siththanandan VB, Lu V, Gonzalez-Nava N, Pasquina L, MacDonald JL, Woodworth MB, Ozkan A, Nair R, He Z, Sahni V, Sarnow P, Palmer TD, Macklis JD, Tharin S. 2020. An evolutionarily acquired microRNA shapes development of mammalian cortical projections. Proceedings of the National Academy of Sciences of the United States of America. 117(46):29113-29122. Pubmed: 33139574 DOI:10.1073/pnas.2006700117 Diaz JL, Siththanandan VB, Lu V, Gonzalez-Nava N, Pasquina L, MacDonald JL, Woodworth MB, Ozkan A, Nair R, He Z, Sahni V, Sarnow P, Palmer TD, Macklis JD, Tharin S. 2020. An evolutionarily acquired microRNA shapes development of mammalian cortical projections. Proceedings of the National Academy of Sciences of the United States of America. 117(46):29113-29122. Pubmed: 33139574 DOI:10.1073/pnas.2006700117 The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are coexpressed across multiple projection neuron subtypes. Here, we discover 17 CSMN-enriched microRNAs (miRNAs), 15 of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is a demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians' increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.Copyright © 2020 the Author(s). Published by PNAS. -
Poulopoulos A, Murphy AJ, Ozkan A, Davis P, Hatch J, Kirchner R, Macklis JD. 2019. Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex. Nature. 565(7739):356-360. Pubmed: 30626971 DOI:10.1038/s41586-018-0847-y Poulopoulos A, Murphy AJ, Ozkan A, Davis P, Hatch J, Kirchner R, Macklis JD. 2019. Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex. Nature. 565(7739):356-360. Pubmed: 30626971 DOI:10.1038/s41586-018-0847-y The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones-subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection. To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting and subcellular RNA-proteome mapping, an approach that identifies and quantifies local transcriptomes and proteomes from labelled growth cones of single projections in vivo. Using this approach on the developing callosal projection of the mouse cerebral cortex, we mapped molecular enrichments in trans-hemispheric growth cones relative to their parent cell bodies, producing paired subcellular proteomes and transcriptomes from single neuron subtypes directly from the brain. These data provide generalizable proof-of-principle for this approach, and reveal molecular specializations of the growth cone, including accumulations of the growth-regulating kinase mTOR, together with mRNAs that contain mTOR-dependent motifs. These findings illuminate the relationships between subcellular distributions of RNA and protein in developing projection neurons, and provide a systems-level approach for the discovery of subtype- and stage-specific molecular substrates of circuit wiring, miswiring, and the potential for regeneration. -
Wuttke TV, Markopoulos F, Padmanabhan H, Wheeler AP, Murthy VN, Macklis JD. 2018. Developmentally primed cortical neurons maintain fidelity of differentiation and establish appropriate functional connectivity after transplantation. Nature neuroscience. 21(4):517-529. Pubmed: 29507412 DOI:10.1038/s41593-018-0098-0 Wuttke TV, Markopoulos F, Padmanabhan H, Wheeler AP, Murthy VN, Macklis JD. 2018. Developmentally primed cortical neurons maintain fidelity of differentiation and establish appropriate functional connectivity after transplantation. Nature neuroscience. 21(4):517-529. Pubmed: 29507412 DOI:10.1038/s41593-018-0098-0 Repair of complex CNS circuitry requires newly incorporated neurons to become appropriately, functionally integrated. One approach is to direct differentiation of endogenous progenitors in situ, or ex vivo followed by transplantation. Prior studies find that newly incorporated neurons can establish long-distance axon projections, form synapses and functionally integrate in evolutionarily old hypothalamic energy-balance circuitry. We now demonstrate that postnatal neocortical connectivity can be reconstituted with point-to-point precision, including cellular integration of specific, molecularly identified projection neuron subtypes into correct positions, combined with development of appropriate long-distance projections and synapses. Using optogenetics-based electrophysiology, experiments demonstrate functional afferent and efferent integration of transplanted neurons into transcallosal projection neuron circuitry. Results further indicate that 'primed' early postmitotic neurons, including already fate-restricted deep-layer projection neurons and/or plastic postmitotic neuroblasts with partially fate-restricted potential, account for the predominant population of neurons capable of achieving this optimal level of integration. -
Galazo MJ, Emsley JG, Macklis JD. 2016. Corticothalamic Projection Neuron Development beyond Subtype Specification: Fog2 and Intersectional Controls Regulate Intraclass Neuronal Diversity. Neuron. 91(1):90-106. Pubmed: 27321927 DOI:S0896-6273(16)30203-3 Galazo MJ, Emsley JG, Macklis JD. 2016. Corticothalamic Projection Neuron Development beyond Subtype Specification: Fog2 and Intersectional Controls Regulate Intraclass Neuronal Diversity. Neuron. 91(1):90-106. Pubmed: 27321927 DOI:S0896-6273(16)30203-3 Corticothalamic projection neurons (CThPN) are a diverse set of neurons, critical for function of the neocortex. CThPN development and diversity need to be precisely regulated, but little is known about molecular controls over their differentiation and functional specialization, critically limiting understanding of cortical development and complexity. We report the identification of a set of genes that both define CThPN and likely control their differentiation, diversity, and function. We selected the CThPN-specific transcriptional coregulator Fog2 for functional analysis. We identify that Fog2 controls CThPN molecular differentiation, axonal targeting, and diversity, in part by regulating the expression level of Ctip2 by CThPN, via combinatorial interactions with other molecular controls. Loss of Fog2 specifically disrupts differentiation of subsets of CThPN specialized in motor function, indicating that Fog2 coordinates subtype and functional-area differentiation. These results confirm that we identified key controls over CThPN development and identify Fog2 as a critical control over CThPN diversity.Copyright © 2016 Elsevier Inc. All rights reserved. -
Greig LC, Woodworth MB, Greppi C, Macklis JD. 2016. Ctip1 Controls Acquisition of Sensory Area Identity and Establishment of Sensory Input Fields in the Developing Neocortex. Neuron. 90(2):261-77. Pubmed: 27100196 DOI:S0896-6273(16)00187-2 Greig LC, Woodworth MB, Greppi C, Macklis JD. 2016. Ctip1 Controls Acquisition of Sensory Area Identity and Establishment of Sensory Input Fields in the Developing Neocortex. Neuron. 90(2):261-77. Pubmed: 27100196 DOI:S0896-6273(16)00187-2 While transcriptional controls over the size and relative position of cortical areas have been identified, less is known about regulators that direct acquisition of area-specific characteristics. Here, we report that the transcription factor Ctip1 functions in primary sensory areas to repress motor and activate sensory programs of gene expression, enabling establishment of sharp molecular boundaries defining functional areas. In Ctip1 mutants, abnormal gene expression leads to aberrantly motorized corticocortical and corticofugal output connectivity. Ctip1 critically regulates differentiation of layer IV neurons, and selective loss of Ctip1 in cortex deprives thalamocortical axons of their receptive "sensory field" in layer IV, which normally provides a tangentially and radially defined compartment of dedicated synaptic territory. Therefore, although thalamocortical axons invade appropriate cortical regions, they are unable to organize into properly configured sensory maps. Together, these data identify Ctip1 as a critical control over sensory area development.Copyright © 2016 Elsevier Inc. All rights reserved. -
Woodworth MB, Greig LC, Liu KX, Ippolito GC, Tucker HO, Macklis JD. 2016. Ctip1 Regulates the Balance between Specification of Distinct Projection Neuron Subtypes in Deep Cortical Layers. Cell reports. 15(5):999-1012. Pubmed: 27117402 DOI:S2211-1247(16)30335-7 Woodworth MB, Greig LC, Liu KX, Ippolito GC, Tucker HO, Macklis JD. 2016. Ctip1 Regulates the Balance between Specification of Distinct Projection Neuron Subtypes in Deep Cortical Layers. Cell reports. 15(5):999-1012. Pubmed: 27117402 DOI:S2211-1247(16)30335-7 The molecular linkage between neocortical projection neuron subtype and area development, which enables the establishment of functional areas by projection neuron populations appropriate for specific sensory and motor functions, is poorly understood. Here, we report that Ctip1 controls precision of neocortical development by regulating subtype identity in deep-layer projection neurons. Ctip1 is expressed by postmitotic callosal and corticothalamic projection neurons but is excluded over embryonic development from corticospinal motor neurons, which instead express its close relative, Ctip2. Loss of Ctip1 function results in a striking bias in favor of subcerebral projection neuron development in sensory cortex at the expense of corticothalamic and deep-layer callosal development, while misexpression of Ctip1 in vivo represses subcerebral gene expression and projections. As we report in a paired paper, Ctip1 also controls acquisition of sensory area identity. Therefore, Ctip1 couples subtype and area specification, enabling specific functional areas to organize precise ratios of appropriate output projections.Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved. -
Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H, Macklis JD. 2013. Molecular logic of neocortical projection neuron specification, development and diversity. Nature reviews. Neuroscience. 14(11):755-69. Pubmed: 24105342 DOI:10.1038/nrn3586 Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H, Macklis JD. 2013. Molecular logic of neocortical projection neuron specification, development and diversity. Nature reviews. Neuroscience. 14(11):755-69. Pubmed: 24105342 DOI:10.1038/nrn3586 The sophisticated circuitry of the neocortex is assembled from a diverse repertoire of neuronal subtypes generated during development under precise molecular regulation. In recent years, several key controls over the specification and differentiation of neocortical projection neurons have been identified. This work provides substantial insight into the 'molecular logic' underlying cortical development and increasingly supports a model in which individual progenitor-stage and postmitotic regulators are embedded within highly interconnected networks that gate sequential developmental decisions. Here, we provide an integrative account of the molecular controls that direct the progressive development and delineation of subtype and area identity of neocortical projection neurons. -
Czupryn A, Zhou YD, Chen X, McNay D, Anderson MP, Flier JS, Macklis JD. 2011. Transplanted hypothalamic neurons restore leptin signaling and ameliorate obesity in db/db mice. Science (New York, N.Y.). 334(6059):1133-7. Pubmed: 22116886 DOI:10.1126/science.1209870 Czupryn A, Zhou YD, Chen X, McNay D, Anderson MP, Flier JS, Macklis JD. 2011. Transplanted hypothalamic neurons restore leptin signaling and ameliorate obesity in db/db mice. Science (New York, N.Y.). 334(6059):1133-7. Pubmed: 22116886 DOI:10.1126/science.1209870 Evolutionarily old and conserved homeostatic systems in the brain, including the hypothalamus, are organized into nuclear structures of heterogeneous and diverse neuron populations. To investigate whether such circuits can be functionally reconstituted by synaptic integration of similarly diverse populations of neurons, we generated physically chimeric hypothalami by microtransplanting small numbers of embryonic enhanced green fluorescent protein-expressing, leptin-responsive hypothalamic cells into hypothalami of postnatal leptin receptor-deficient (db/db) mice that develop morbid obesity. Donor neurons differentiated and integrated as four distinct hypothalamic neuron subtypes, formed functional excitatory and inhibitory synapses, partially restored leptin responsiveness, and ameliorated hyperglycemia and obesity in db/db mice. These experiments serve as a proof of concept that transplanted neurons can functionally reconstitute complex neuronal circuitry in the mammalian brain.
All Publications
2024
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Peter M, Shipman S, Heo J, Macklis JD. 2024. Limitations of fluorescent timer protein maturation kinetics to isolate transcriptionally synchronized human neural progenitor cells. iScience. 27(6):109911. Pubmed: 38784012 DOI:10.1016/j.isci.2024.109911 Peter M, Shipman S, Heo J, Macklis JD. 2024. Limitations of fluorescent timer protein maturation kinetics to isolate transcriptionally synchronized human neural progenitor cells. iScience. 27(6):109911. Pubmed: 38784012 DOI:10.1016/j.isci.2024.109911 Differentiation of human pluripotent stem cells (hPSCs) into subtype-specific neurons holds substantial potential for disease modeling . For successful differentiation, a detailed understanding of the transcriptional networks regulating cell fate decisions is critical. The heterochronic nature of neurodevelopment, during which distinct cells in the brain and during differentiation acquire their fates in an unsynchronized manner, hinders pooled transcriptional comparisons. One approach is to "translate" chronologic time into linear developmental and maturational time. Simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells are unable to achieve this goal, due to asynchronous promotor onset in individual cells. We tested five fluorescent timer (FT) molecules expressed from the endogenous paired box 6 (PAX6) promoter in 293T and human hPSCs. Each of these FT systems faithfully reported chronologic time in 293T cells, but none of the FT constructs followed the same fluorescence kinetics in human neural progenitor cells.© 2024 The Author(s). -
Ozkan A, Padmanabhan HK, Shipman SL, Azim E, Kumar P, Sadegh C, Basak AN, Macklis JD. 2024. Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors. bioRxiv : the preprint server for biology. Pubmed: 38712174 DOI:10.1101/2024.04.21.590488 Ozkan A, Padmanabhan HK, Shipman SL, Azim E, Kumar P, Sadegh C, Basak AN, Macklis JD. 2024. Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors. bioRxiv : the preprint server for biology. Pubmed: 38712174 DOI:10.1101/2024.04.21.590488 Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitry , or for optimally informative disease modeling and/or therapeutic screening , it is important to reproduce the type or subtype of neurons involved. No such appropriate models exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factor is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and that suppresses a latent neurogenic program by repressing inappropriate proneural expression by progenitors. We FACS-purify these genetically accessible progenitors from postnatal mouse cortex and establish a pure culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activating , while antagonizing with ). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors, and find that these neurons differentiate with remarkable fidelity compared with corticospinal neurons . They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employed -driven differentiation. Neurons induced by instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from genetically accessible cortical progenitors sets a precedent and foundation for mechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair. -
Poulopoulos A, Davis P, Brandenburg C, Itoh Y, Galazo MJ, Greig LC, Romanowski AJ, Budnik B, Macklis JD. 2024. Symmetry in levels of axon-axon homophilic adhesion establishes topography in the corpus callosum and development of connectivity between brain hemispheres. bioRxiv : the preprint server for biology. Pubmed: 38585721 DOI:10.1101/2024.03.28.587108 Poulopoulos A, Davis P, Brandenburg C, Itoh Y, Galazo MJ, Greig LC, Romanowski AJ, Budnik B, Macklis JD. 2024. Symmetry in levels of axon-axon homophilic adhesion establishes topography in the corpus callosum and development of connectivity between brain hemispheres. bioRxiv : the preprint server for biology. Pubmed: 38585721 DOI:10.1101/2024.03.28.587108 Specific and highly diverse connectivity between functionally specialized regions of the nervous system is controlled at multiple scales, from anatomically organized connectivity following macroscopic axon tracts to individual axon target-finding and synapse formation. Identifying mechanisms that enable entire subpopulations of related neurons to project their axons with regional specificity within stereotyped tracts to form appropriate long-range connectivity is key to understanding brain development, organization, and function. Here, we investigate how axons of the cerebral cortex form precise connections between the two cortical hemispheres via the corpus callosum. We identify topographic principles of the developing trans-hemispheric callosal tract that emerge through intrinsic guidance executed by growing axons in the corpus callosum within the first postnatal week in mice. Using micro-transplantation of regionally distinct neurons, subtype-specific growth cone purification, subcellular proteomics, and in utero gene manipulation, we investigate guidance mechanisms of transhemispheric axons. We find that adhesion molecule levels instruct tract topography and target field guidance. We propose a model in which transcallosal axons in the developing brain perform a "handshake" that is guided through co-fasciculation with symmetric contralateral axons, resulting in the stereotyped homotopic connectivity between the brain's hemispheres. -
Veeraraghavan P, Engmann AK, Hatch JJ, Itoh Y, Nguyen D, Addison T, Macklis JD. 2024. Dynamic subtype- and context-specific subcellular RNA regulation in growth cones of developing neurons of the cerebral cortex. bioRxiv : the preprint server for biology. Pubmed: 38328182 DOI:10.1101/2023.09.24.559186 Veeraraghavan P, Engmann AK, Hatch JJ, Itoh Y, Nguyen D, Addison T, Macklis JD. 2024. Dynamic subtype- and context-specific subcellular RNA regulation in growth cones of developing neurons of the cerebral cortex. bioRxiv : the preprint server for biology. Pubmed: 38328182 DOI:10.1101/2023.09.24.559186 Molecular mechanisms that cells employ to compartmentalize function via localization of function-specific RNA and translation are only partially elucidated. We investigate long-range projection neurons of the cerebral cortex as highly polarized exemplars to elucidate dynamic regulation of RNA localization, stability, and translation within growth cones (GCs), leading tips of growing axons. Comparison of GC-localized transcriptomes between two distinct subtypes of projection neurons- interhemispheric-callosal and corticothalamic- across developmental stages identifies both distinct and shared subcellular machinery, and intriguingly highlights enrichment of genes associated with neurodevelopmental and neuropsychiatric disorders. Developmental context-specific components of GC-localized transcriptomes identify known and novel potential regulators of distinct phases of circuit formation: long-distance growth, target area innervation, and synapse formation. Further, we investigate mechanisms by which transcripts are enriched and dynamically regulated in GCs, and identify GC-enriched motifs in 3' untranslated regions. As one example, we identify (CPEB4), an RNA binding protein regulating localization and translation of mRNAs encoding molecular machinery important for axonal branching and complexity. We also identify (RBMS1) as a dynamically expressed regulator of RNA stabilization that enables successful callosal circuit formation. Subtly aberrant associative and integrative cortical circuitry can profoundly affect cortical function, often causing neurodevelopmental and neuropsychiatric disorders. Elucidation of context-specific subcellular RNA regulation for GC- and soma-localized molecular controls over precise circuit development, maintenance, and function offers generalizable insights for other polarized cells, and might contribute substantially to understanding neurodevelopmental and behavioral-cognitive disorders and toward targeted therapeutics. -
Greig LC, Woodworth MB, Poulopoulos A, Lim S, Macklis JD. 2024. BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis. Cell reports. 43(8):114650. Pubmed: 39159043 DOI:S2211-1247(24)01001-5 Greig LC, Woodworth MB, Poulopoulos A, Lim S, Macklis JD. 2024. BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis. Cell reports. 43(8):114650. Pubmed: 39159043 DOI:S2211-1247(24)01001-5 We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved. 2023
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Greig LC, Woodworth MB, Poulopoulos A, Lim S, Macklis JD. 2023. BEAM: a combinatorial recombinase toolbox for binary gene expression and mosaic analysis. bioRxiv : the preprint server for biology. Pubmed: 36824714 DOI:10.1101/2023.02.16.528875 Greig LC, Woodworth MB, Poulopoulos A, Lim S, Macklis JD. 2023. BEAM: a combinatorial recombinase toolbox for binary gene expression and mosaic analysis. bioRxiv : the preprint server for biology. Pubmed: 36824714 DOI:10.1101/2023.02.16.528875 Genetic mosaic analysis, in which mutant cells reside intermingled with wild-type cells, is a powerful experimental approach, but has not been widely used in mice because existing genome-based strategies require complicated and protracted breeding schemes. We have developed an alternative approach termed BEAM (for Binary Expression Aleatory Mosaic) that relies on sparse recombinase activation to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Following delivery of DNA constructs by transfection or viral transduction, combinatorial recombinase activity generates two distinct populations of cells labeled with either green or red fluorescent protein. Any gene of interest can be mis-expressed or deleted in one population for comparison with intermingled control cells. We have extensively optimized and characterized this system both and , and demonstrate its power for investigating cell autonomy, identifying temporally or spatially aberrant phenotypes, revealing changes in cell proliferation or death, and controlling for procedural variability. -
Peter M, Shipman S, Macklis JD. 2023. Limitations of fluorescent timer protein maturation kinetics to isolate transcriptionally synchronized cortically differentiating human pluripotent stem cells. bioRxiv : the preprint server for biology. Pubmed: 37609140 DOI:10.1101/2023.08.04.552012 Peter M, Shipman S, Macklis JD. 2023. Limitations of fluorescent timer protein maturation kinetics to isolate transcriptionally synchronized cortically differentiating human pluripotent stem cells. bioRxiv : the preprint server for biology. Pubmed: 37609140 DOI:10.1101/2023.08.04.552012 Differentiation of human pluripotent stem cells (hPSC) into distinct neuronal populations holds substantial potential for disease modeling , toward both elucidation of pathobiological mechanisms and screening of potential therapeutic agents. For successful differentiation of hPSCs into subtype-specific neurons using protocols, detailed understanding of the transcriptional networks and their dynamic programs regulating endogenous cell fate decisions is critical. One major roadblock is the heterochronic nature of neurodevelopment, during which distinct cells and cell types in the brain and during differentiation mature and acquire their fates in an unsynchronized manner, hindering pooled transcriptional comparisons. One potential approach is to "translate" chronologic time into linear developmental and maturational time. Attempts to partially achieve this using simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells have not been able to achieve this goal, due to asynchrony of promotor onset in individual cells. Toward solving this, we generated and tested a range of knock-in hPSC lines that express five distinct dual FP timer systems or single time-resolved fluorescent timer (FT) molecules, either in 293T cells or in human hPSCs driving expression from the endogenous paired box 6 (PAX6) promoter of cerebral cortex progenitors. While each of these dual FP or FT systems faithfully reported chronologic time when expressed from a strong inducible promoter in 293T cells, none of the tested FP/FT constructs followed the same fluorescence kinetics in developing human neural progenitor cells, and were unsuccessful in identification and isolation of distinct, developmentally synchronized cortical progenitor populations based on ratiometric fluorescence. This work highlights unique and often surprising expression kinetics and regulation in specific cell types differentiating from hPSCs. -
Froberg JE, Durak O, Macklis JD. 2023. Development of nanoRibo-seq enables study of regulated translation by cortical neuron subtypes, showing uORF translation in synaptic-axonal genes. Cell reports. 42(9):112995. Pubmed: 37624698 DOI:S2211-1247(23)01006-9 Froberg JE, Durak O, Macklis JD. 2023. Development of nanoRibo-seq enables study of regulated translation by cortical neuron subtypes, showing uORF translation in synaptic-axonal genes. Cell reports. 42(9):112995. Pubmed: 37624698 DOI:S2211-1247(23)01006-9 Investigation of translation in rare cell types or subcellular contexts is challenging due to large input requirements for standard approaches. Here, we present "nanoRibo-seq" an optimized approach using 10- to 10-fold less input material than bulk approaches. nanoRibo-seq exhibits rigorous quality control features consistent with quantification of ribosome protected fragments with as few as 1,000 cells. We compare translatomes of two closely related cortical neuron subtypes, callosal projection neurons (CPN) and subcerebral projection neurons (SCPN), during their early postnatal development. We find that, while translational efficiency is highly correlated between CPN and SCPN, several dozen mRNAs are differentially translated. We further examine upstream open reading frame (uORF) translation and identify that mRNAs involved in synapse organization and axon development are highly enriched for uORF translation in both subtypes. nanoRibo-seq enables investigation of translational regulation of rare cell types in vivo and offers a flexible approach for globally quantifying translation from limited input material.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Galazo MJ, Sweetser DA, Macklis JD. 2023. Tle4 controls both developmental acquisition and early post-natal maturation of corticothalamic projection neuron identity. Cell reports. 42(8):112957. Pubmed: 37561632 DOI:S2211-1247(23)00968-3 Galazo MJ, Sweetser DA, Macklis JD. 2023. Tle4 controls both developmental acquisition and early post-natal maturation of corticothalamic projection neuron identity. Cell reports. 42(8):112957. Pubmed: 37561632 DOI:S2211-1247(23)00968-3 Identities of distinct neuron subtypes are specified during embryonic development, then maintained during post-natal maturation. In cerebral cortex, mechanisms controlling early acquisition of neuron-subtype identities have become increasingly understood. However, mechanisms controlling neuron-subtype identity stability during post-natal maturation are largely unexplored. We identify that Tle4 is required for both early acquisition and post-natal stability of corticothalamic neuron-subtype identity. Embryonically, Tle4 promotes acquisition of corticothalamic identity and blocks emergence of core characteristics of subcerebral/corticospinal projection neuron identity, including gene expression and connectivity. During the first post-natal week, when corticothalamic innervation is ongoing, Tle4 is required to stabilize corticothalamic neuron identity, limiting interference from differentiation programs of developmentally related neuron classes. We identify a deacetylation-based epigenetic mechanism by which TLE4 controls Fezf2 expression level by corticothalamic neurons. This contributes to distinction of cortical output subtypes and ensures identity stability for appropriate maturation of corticothalamic neurons.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Itoh Y, Sahni V, Shnider SJ, McKee H, Macklis JD. 2023. Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations. Cell reports. 42(3):112182. Pubmed: 36934325 DOI:S2211-1247(23)00193-6 Itoh Y, Sahni V, Shnider SJ, McKee H, Macklis JD. 2023. Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations. Cell reports. 42(3):112182. Pubmed: 36934325 DOI:S2211-1247(23)00193-6 How CNS circuits sculpt their axonal arbors into spatially and functionally organized domains is not well understood. Segmental specificity of corticospinal connectivity is an exemplar for such regional specificity of many axon projections. Corticospinal neurons (CSN) innervate spinal and brainstem targets with segmental precision, controlling voluntary movement. Multiple molecularly distinct CSN subpopulations innervate the cervical cord for evolutionarily enhanced precision of forelimb movement. Evolutionarily newer CSN exclusively innervate bulbar-cervical targets, while CSN are heterogeneous; distinct subpopulations extend axons to either bulbar-cervical or thoraco-lumbar segments. We identify that Lumican controls balance of cervical innervation between CSN and CSN axons during development, which is maintained into maturity. Lumican, an extracellular proteoglycan expressed by CSN, non-cell-autonomously suppresses cervical collateralization by multiple CSN subpopulations. This inter-axonal molecular crosstalk between CSN subpopulations controls murine corticospinal circuitry refinement and forelimb dexterity. Such crosstalk is generalizable beyond the corticospinal system for evolutionary incorporation of new neuron populations into preexisting circuitry.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Song JHT, Ruven C, Patel P, Ding F, Macklis JD, Sahni V. 2023. Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord. The Journal of neuroscience : the official journal of the Society for Neuroscience. 43(11):1871-1887. Pubmed: 36823038 DOI:10.1523/JNEUROSCI.0710-22.2023 Song JHT, Ruven C, Patel P, Ding F, Macklis JD, Sahni V. 2023. Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord. The Journal of neuroscience : the official journal of the Society for Neuroscience. 43(11):1871-1887. Pubmed: 36823038 DOI:10.1523/JNEUROSCI.0710-22.2023 Corticospinal neurons (CSN) are centrally required for skilled voluntary movement, which necessitates that they establish precise subcerebral connectivity with the brainstem and spinal cord. However, molecular controls regulating specificity of this projection targeting remain largely unknown. We previously identified that developing CSN subpopulations exhibit striking axon targeting specificity in the spinal white matter. These CSN subpopulations with segmentally distinct spinal projections are also molecularly distinct; a subset of differentially expressed genes between these distinct CSN subpopulations regulate differential axon projection targeting. Rostrolateral CSN extend axons exclusively to bulbar-cervical segments (CSN), while caudomedial CSN (CSN) are more heterogeneous, with distinct, intermingled subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. Here, we report, in male and female mice, that () is expressed specifically by CSN in medial, but not lateral, sensorimotor cortex. shows highly dynamic temporal expression, with levels in CSN highest during the period of peak axon extension toward thoraco-lumbar segments. Using gain-of-function experiments, we identify that Cbln1 is sufficient to direct thoraco-lumbar axon extension by CSN. Misexpression of Cbln1 in CSN either by electroporation, or by postmitotic AAV-mediated gene delivery, redirects these axons past their normal bulbar-cervical targets toward thoracic segments. Further, Cbln1 overexpression in postmitotic CSN increases the number of CSN axons that extend past cervical segments into the thoracic cord. Collectively, these results identify that Cbln1 functions as a potent molecular control over thoraco-lumbar CSN axon extension, part of an integrated network of controls over segmentally-specific CSN axon projection targeting. Corticospinal neurons (CSN) exhibit remarkable diversity and precision of axonal projections to targets in the brainstem and distinct spinal segments; the molecular basis for this targeting diversity is largely unknown. CSN subpopulations projecting to distinct targets are also molecularly distinguishable. Distinct subpopulations degenerate in specific motor neuron diseases, further suggesting that intrinsic molecular differences might underlie differential vulnerability to disease. Here, we identify a novel molecular control, Cbln1, expressed by CSN extending axons to thoraco-lumbar spinal segments. Cbln1 is sufficient, but not required, for CSN axon extension toward distal spinal segments, and expression is controlled by recently identified, CSN-intrinsic regulators of axon extension. Our results identify that Cbln1, together with other regulators, coordinates segmentally precise CSN axon targeting.Copyright © 2023 Song et al. 2022
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Song JHT, Ruven C, Patel P, Ding F, Macklis JD*, Sahni V*. 2022. Cbln1 directs axon targeting by corticospinal neurons specifically toward thoraco-lumbar spinal cord. BioRxiv. DOI:https://doi.org/10.1101/2022.04.06.487184 Song JHT, Ruven C, Patel P, Ding F, Macklis JD*, Sahni V*. 2022. Cbln1 directs axon targeting by corticospinal neurons specifically toward thoraco-lumbar spinal cord. BioRxiv. DOI:https://doi.org/10.1101/2022.04.06.487184 Corticospinal neurons (CSN) are centrally required for skilled voluntary movement, which necessitates that they establish precise subcerebral connectivity with the brainstem and spinal cord. However, molecular controls regulating specificity of this projection targeting remain largely unknown. We previously identified that developing CSN subpopulations exhibit striking axon targeting specificity in the spinal white matter. These CSN subpopulations with segmentally distinct spinal projections are also molecularly distinct; a subset of differentially expressed genes between these distinct CSN subpopulations function as molecular controls regulating differential axon projection targeting. Rostrolateral CSN extend axons exclusively to bulbar-cervical segments (CSNBC-lat), while caudomedial CSN (CSNmedial) are more heterogeneous, with distinct, intermingled subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. Here, we report that Cerebellin 1 (Cbln1) is expressed specifically by CSN in medial, but not lateral, sensorimotor cortex. Cbln1 shows highly dynamic temporal expression, with Cbln1 levels in CSN highest during the period of peak axon extension toward thoraco-lumbar segments. Using gain-of-function experiments, we identify that Cbln1 is sufficient to direct thoraco-lumbar axon extension by CSN. Mis-expression of Cbln1 in CSNBC-lat either by in utero electroporation, or in postmitotic CSNBC-lat by AAV-mediated gene delivery, re-directs these axons past their normal bulbar-cervical targets toward thoracic segments. Further, Cbln1 overexpression in postmitotic CSNmedial increases the number of CSNmedial axons that extend past cervical segments into the thoracic cord. Collectively, these results identify that Cbln1 functions as a potent molecular control over thoraco-lumbar CSN axon extension, part of an integrated network of controls over segmentally-specific CSN axon projection targeting. -
Froberg JE, Durak O, Macklis JD. 2022. Development of ultra-low-input nanoRibo-seq enables quantification of translational control, revealing broad uORF translation by subtype-specific neurons. BioRxiv. DOI:https://doi.org/10.1101/2022.04.05.487068 Froberg JE, Durak O, Macklis JD. 2022. Development of ultra-low-input nanoRibo-seq enables quantification of translational control, revealing broad uORF translation by subtype-specific neurons. BioRxiv. DOI:https://doi.org/10.1101/2022.04.05.487068 While increasingly powerful approaches enable investigation of transcription using small samples of RNA, approaches to investigate translational regulation in small populations of specific cell types, and/or (sub)-cellular contexts are lacking. Comprehensive investigation of mRNAs actively translated into proteins from ultra-low input material would provide important insight into molecular machinery and mechanisms underlying many cellular, developmental, and disease processes in vivo. Such investigations are limited by the large input required for current state-of-the-art Ribo-seq. Here, we present an optimized, ultra-low input “nanoRibo-seq” approach using 102 – 103-fold less input material than standard approaches, demonstrated here in subtype-specific neurons. nanoRibo-seq requires as few as 2.5K neurons, and exhibits rigorous quality control features: 1) strong enrichment for CDS versus UTRs and non-CDS; 2) narrow, distinct length distributions over CDS; 3) ribosome P-sites predominantly in-frame to annotated CDS; and 4) sufficient ribosome-protected fragment (RPF) coverage across thousands of mRNAs. As proof-of-concept, we calculate translation efficiencies from paired Ribo-seq and alkaline fragmented control libraries from “callosal projection neurons” (CPN), revealing divergence between mRNA abundance and RPF abundance for hundreds of genes. Intriguingly, we identify substantial translation of upstream ORFs in the 5’ UTRs of genes involved in axon guidance and synapse assembly. nanoRibo-seq enables previously inaccessible investigation of translational regulation by small, specific cell populations in normal or perturbed contexts. -
Engmann AK, Hatch JJ, Nanda P, Veeraraghavan P, Ozkan A, Poulopoulos A, Murphy AJ, Macklis JD. 2022. Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes. Nature protocols. 17(2):222-251. Pubmed: 35022617 DOI:10.1038/s41596-021-00638-7 Engmann AK, Hatch JJ, Nanda P, Veeraraghavan P, Ozkan A, Poulopoulos A, Murphy AJ, Macklis JD. 2022. Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes. Nature protocols. 17(2):222-251. Pubmed: 35022617 DOI:10.1038/s41596-021-00638-7 During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype- and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate that subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit 'wiring'. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from the rodent central nervous system (CNS) in vivo. By using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is isolated by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting and established FACS of neurons and are suitable for downstream analyses of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes ~3 h, plus sorting time, and ~1-2 h for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, depending on the turnaround time of the core facility involved.© 2022. The Author(s), under exclusive licence to Springer Nature Limited. 2021
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Sadegh C, Ebina W, Arvanites AC, Davidow LS, Rubin LL, Macklis JD. 2021. Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells. PloS one. 16(9):e0254113. Pubmed: 34473715 DOI:10.1371/journal.pone.0254113 Sadegh C, Ebina W, Arvanites AC, Davidow LS, Rubin LL, Macklis JD. 2021. Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells. PloS one. 16(9):e0254113. Pubmed: 34473715 DOI:10.1371/journal.pone.0254113 During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed "cortical" here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro. -
Sahni V, Itoh Y, Shnider SJ, Macklis JD. 2021. Crim1 and Kelch-like 14 exert complementary dual-directional developmental control over segmentally specific corticospinal axon projection targeting. Cell reports. 37(3):109842. Pubmed: 34686337 DOI:S2211-1247(21)01306-1 Sahni V, Itoh Y, Shnider SJ, Macklis JD. 2021. Crim1 and Kelch-like 14 exert complementary dual-directional developmental control over segmentally specific corticospinal axon projection targeting. Cell reports. 37(3):109842. Pubmed: 34686337 DOI:S2211-1247(21)01306-1 The cerebral cortex executes highly skilled movement, necessitating that it connects accurately with specific brainstem and spinal motor circuitry. Corticospinal neurons (CSN) must correctly target specific spinal segments, but the basis for this targeting remains unknown. In the accompanying report, we show that segmentally distinct CSN subpopulations are molecularly distinct from early development, identifying candidate molecular controls over segmentally specific axon targeting. Here, we functionally investigate two of these candidate molecular controls, Crim1 and Kelch-like 14 (Klhl14), identifying their critical roles in directing CSN axons to appropriate spinal segmental levels in the white matter prior to axon collateralization. Crim1 and Klhl14 are specifically expressed by distinct CSN subpopulations and regulate their differental white matter projection targeting-Crim1 directs thoracolumbar axon extension, while Klhl14 limits axon extension to bulbar-cervical segments. These molecular regulators of descending spinal projections constitute the first stages of a dual-directional set of complementary controls over CSN diversity for segmentally and functionally distinct circuitry.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved. -
Itoh Y*, Sahni V*, Shnider SJ, McKee H, Macklis JD. 2021. Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations. BioRxiv. DOI:10.1016/j.celrep.2023.112182 Itoh Y*, Sahni V*, Shnider SJ, McKee H, Macklis JD. 2021. Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations. BioRxiv. DOI:10.1016/j.celrep.2023.112182 Corticospinal neurons (CSN) are the cortical projection neurons that innervate the spinal cord and some brainstem targets with segmental precision to control voluntary movement of specific functional motor groups, limb sections, or individual digits. CSN subpopulations exhibit striking axon targeting specificity from development into maturity: Evolutionarily newer rostrolateral CSN exclusively innervate bulbar-cervical targets (CSNBC-lat), while evolutionarily older caudomedial CSN (CSNmedial) are more heterogeneous, with distinct subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. However, molecular regulation over specificity of CSN segmental target innervation is essentially unknown. The cervical cord, with its evolutionarily enhanced precision of forelimb movement, is innervated by multiple CSN subpopulations, suggesting inter-neuronal interactions in establishing cervical corticospinal circuitry. Here, we identify that Lumican, previously unrecognized in axon development, controls the balance of innervation between CSNBC-lat and CSNmedial within the cervical spinal cord. Remarkably, Lumican, an extracellular matrix protein expressed by CSNBC-lat, non-cell-autonomously suppresses axon collateralization in the cervical cord by CSNmedial. Intersectional viral labeling and mouse genetics further identify that Lumican controls axon collateralization by multiple CSN subpopulations in caudomedial sensorimotor cortex. These results identify inter-axonal molecular crosstalk between CSN subpopulations as a novel mechanism controlling corticospinal circuitry, target density, and competitive specificity. Further, this mechanism has potential implications for evolutionary diversification of corticospinal circuitry with finer scale precision. -
Itoh Y, Sahni V, Shnider S, Macklis JD. 2021. Lumican regulates cervical corticospinal axon collateralization via non-autonomous crosstalk between distinct corticospinal neuron subpopulations. BioRxiv. DOI:10.1101/2021.03.26.437104 Itoh Y, Sahni V, Shnider S, Macklis JD. 2021. Lumican regulates cervical corticospinal axon collateralization via non-autonomous crosstalk between distinct corticospinal neuron subpopulations. BioRxiv. DOI:10.1101/2021.03.26.437104 Corticospinal neurons (CSN) are the cortical projection neurons that innervate the spinal cord and some brainstem targets with segmental precision to control voluntary movement of specific functional motor groups, limb sections, or individual digits. CSN subpopulations exhibit striking axon targeting specificity from development into maturity: Evolutionarily newer rostrolateral CSN exclusively innervate bulbar-cervical targets (CSNBC-lat), while evolutionarily older caudomedial CSN (CSNmedial) are more heterogeneous, with distinct subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. However, molecular regulation over specificity of CSN segmental target innervation is essentially unknown. The cervical cord, with its evolutionarily enhanced precision of forelimb movement, is innervated by multiple CSN subpopulations, suggesting inter-neuronal interactions in establishing cervical corticospinal circuitry. Here, we identify that Lumican, previously unrecognized in axon development, controls the balance of innervation between CSNBC-lat and CSNmedial within the cervical spinal cord. Remarkably, Lumican, an extracellular matrix protein expressed by CSNBC-lat, non-cell-autonomously suppresses axon collateralization in the cervical cord by CSNmedial. Intersectional viral labeling and mouse genetics further identify that Lumican controls axon collateralization by multiple CSN subpopulations in caudomedial sensorimotor cortex. These results identify inter-axonal molecular crosstalk between CSN subpopulations as a novel mechanism controlling corticospinal circuitry, target density, and competitive specificity. Further, this mechanism has potential implications for evolutionary diversification of corticospinal circuitry with finer scale precision.The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. -
Sahni V, Shnider SJ, Jabaudon D, Song JHT, Itoh Y, Greig LC, Macklis JD. 2021. Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting. Cell reports. 37(3):109843. Pubmed: 34686320 DOI:S2211-1247(21)01307-3 Sahni V, Shnider SJ, Jabaudon D, Song JHT, Itoh Y, Greig LC, Macklis JD. 2021. Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting. Cell reports. 37(3):109843. Pubmed: 34686320 DOI:S2211-1247(21)01307-3 For precise motor control, distinct subpopulations of corticospinal neurons (CSN) must extend axons to distinct spinal segments, from proximal targets in the brainstem and cervical cord to distal targets in thoracic and lumbar spinal segments. We find that developing CSN subpopulations exhibit striking axon targeting specificity in spinal white matter, which establishes the foundation for durable specificity of adult corticospinal circuitry. Employing developmental retrograde and anterograde labeling, and their distinct neocortical locations, we purified developing CSN subpopulations using fluorescence-activated cell sorting to identify genes differentially expressed between bulbar-cervical and thoracolumbar-projecting CSN subpopulations at critical developmental times. These segmentally distinct CSN subpopulations are molecularly distinct from the earliest stages of axon extension, enabling prospective identification even before eventual axon targeting decisions are evident in the spinal cord. This molecular delineation extends beyond simple spatial separation of these subpopulations in the cortex. Together, these results identify candidate molecular controls over segmentally specific corticospinal axon projection targeting.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved. 2020
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Diaz JL, Siththanandan VB, Lu V, Gonzalez-Nava N, Pasquina L, MacDonald JL, Woodworth MB, Ozkan A, Nair R, He Z, Sahni V, Sarnow P, Palmer TD, Macklis JD, Tharin S. 2020. An evolutionarily acquired microRNA shapes development of mammalian cortical projections. Proceedings of the National Academy of Sciences of the United States of America. 117(46):29113-29122. Pubmed: 33139574 DOI:10.1073/pnas.2006700117 Diaz JL, Siththanandan VB, Lu V, Gonzalez-Nava N, Pasquina L, MacDonald JL, Woodworth MB, Ozkan A, Nair R, He Z, Sahni V, Sarnow P, Palmer TD, Macklis JD, Tharin S. 2020. An evolutionarily acquired microRNA shapes development of mammalian cortical projections. Proceedings of the National Academy of Sciences of the United States of America. 117(46):29113-29122. Pubmed: 33139574 DOI:10.1073/pnas.2006700117 The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are coexpressed across multiple projection neuron subtypes. Here, we discover 17 CSMN-enriched microRNAs (miRNAs), 15 of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is a demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians' increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.Copyright © 2020 the Author(s). Published by PNAS. -
Ribeiro MC, Moore SM, Kishi N, Macklis JD, MacDonald JL. 2020. Vitamin D Supplementation Rescues Aberrant NF-κB Pathway Activation and Partially Ameliorates Rett Syndrome Phenotypes in Mutant Mice. eNeuro. 7(3). Pubmed: 32393583 DOI:10.1523/ENEURO.0167-20.2020 Ribeiro MC, Moore SM, Kishi N, Macklis JD, MacDonald JL. 2020. Vitamin D Supplementation Rescues Aberrant NF-κB Pathway Activation and Partially Ameliorates Rett Syndrome Phenotypes in Mutant Mice. eNeuro. 7(3). Pubmed: 32393583 DOI:10.1523/ENEURO.0167-20.2020 Rett syndrome (RTT) is a severe, progressive X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator We previously identified aberrant NF-κB pathway upregulation in brains of -null mice and demonstrated that genetically attenuating NF-κB rescues some characteristic neuronal RTT phenotypes. These results raised the intriguing question of whether NF-κB pathway inhibitors might provide a therapeutic avenue in RTT. Here, we investigate whether the known NF-κB pathway inhibitor vitamin D ameliorates neuronal phenotypes in -mutant mice. Vitamin D deficiency is prevalent among RTT patients, and we find that -null mice similarly have significantly reduced 25(OH)D serum levels compared with wild-type littermates. We identify that vitamin D rescues aberrant NF-κB pathway activation and reduced neurite outgrowth of knock-down cortical neurons Further, dietary supplementation with vitamin D in early symptomatic male hemizygous null and female heterozygous mice ameliorates reduced neocortical dendritic morphology and soma size phenotypes and modestly improves reduced lifespan of -nulls. These results elucidate fundamental neurobiology of RTT and provide foundation that NF-κB pathway inhibition might be a therapeutic target for RTT.Copyright © 2020 Ribeiro et al. 2019
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Poulopoulos A, Murphy AJ, Ozkan A, Davis P, Hatch J, Kirchner R, Macklis JD. 2019. Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex. Nature. 565(7739):356-360. Pubmed: 30626971 DOI:10.1038/s41586-018-0847-y Poulopoulos A, Murphy AJ, Ozkan A, Davis P, Hatch J, Kirchner R, Macklis JD. 2019. Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex. Nature. 565(7739):356-360. Pubmed: 30626971 DOI:10.1038/s41586-018-0847-y The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones-subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection. To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting and subcellular RNA-proteome mapping, an approach that identifies and quantifies local transcriptomes and proteomes from labelled growth cones of single projections in vivo. Using this approach on the developing callosal projection of the mouse cerebral cortex, we mapped molecular enrichments in trans-hemispheric growth cones relative to their parent cell bodies, producing paired subcellular proteomes and transcriptomes from single neuron subtypes directly from the brain. These data provide generalizable proof-of-principle for this approach, and reveal molecular specializations of the growth cone, including accumulations of the growth-regulating kinase mTOR, together with mRNAs that contain mTOR-dependent motifs. These findings illuminate the relationships between subcellular distributions of RNA and protein in developing projection neurons, and provide a systems-level approach for the discovery of subtype- and stage-specific molecular substrates of circuit wiring, miswiring, and the potential for regeneration. 2018
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MacDonald JL, Fame RM, Gillis-Buck EM, Macklis JD. 2018. Caveolin1 Identifies a Specific Subpopulation of Cerebral Cortex Callosal Projection Neurons (CPN) Including Dual Projecting Cortical Callosal/Frontal Projection Neurons (CPN/FPN). eNeuro. 5(1). Pubmed: 29379878 DOI:10.1523/ENEURO.0234-17.2017 MacDonald JL, Fame RM, Gillis-Buck EM, Macklis JD. 2018. Caveolin1 Identifies a Specific Subpopulation of Cerebral Cortex Callosal Projection Neurons (CPN) Including Dual Projecting Cortical Callosal/Frontal Projection Neurons (CPN/FPN). eNeuro. 5(1). Pubmed: 29379878 DOI:10.1523/ENEURO.0234-17.2017 The neocortex is composed of many distinct subtypes of neurons that must form precise subtype-specific connections to enable the cortex to perform complex functions. Callosal projection neurons (CPN) are the broad population of commissural neurons that connect the cerebral hemispheres via the corpus callosum (CC). Currently, how the remarkable diversity of CPN subtypes and connectivity is specified, and how they differentiate to form highly precise and specific circuits, are largely unknown. We identify in mouse that the lipid-bound scaffolding domain protein Caveolin 1 (CAV1) is specifically expressed by a unique subpopulation of Layer V CPN that maintain dual ipsilateral frontal projections to premotor cortex. CAV1 is expressed by over 80% of these dual projecting callosal/frontal projection neurons (CPN/FPN), with expression peaking early postnatally as axonal and dendritic targets are being reached and refined. CAV1 is localized to the soma and dendrites of CPN/FPN, a unique population of neurons that shares information both between hemispheres and with premotor cortex, suggesting function during postmitotic development and refinement of these neurons, rather than in their specification. Consistent with this, we find that function is not necessary for the early specification of CPN/FPN, or for projecting to their dual axonal targets. CPN subtype-specific expression of identifies and characterizes a first molecular component that distinguishes this functionally unique projection neuron population, a population that expands in primates, and is prototypical of additional dual and higher-order projection neuron subtypes. -
Wuttke TV, Markopoulos F, Padmanabhan H, Wheeler AP, Murthy VN, Macklis JD. 2018. Developmentally primed cortical neurons maintain fidelity of differentiation and establish appropriate functional connectivity after transplantation. Nature neuroscience. 21(4):517-529. Pubmed: 29507412 DOI:10.1038/s41593-018-0098-0 Wuttke TV, Markopoulos F, Padmanabhan H, Wheeler AP, Murthy VN, Macklis JD. 2018. Developmentally primed cortical neurons maintain fidelity of differentiation and establish appropriate functional connectivity after transplantation. Nature neuroscience. 21(4):517-529. Pubmed: 29507412 DOI:10.1038/s41593-018-0098-0 Repair of complex CNS circuitry requires newly incorporated neurons to become appropriately, functionally integrated. One approach is to direct differentiation of endogenous progenitors in situ, or ex vivo followed by transplantation. Prior studies find that newly incorporated neurons can establish long-distance axon projections, form synapses and functionally integrate in evolutionarily old hypothalamic energy-balance circuitry. We now demonstrate that postnatal neocortical connectivity can be reconstituted with point-to-point precision, including cellular integration of specific, molecularly identified projection neuron subtypes into correct positions, combined with development of appropriate long-distance projections and synapses. Using optogenetics-based electrophysiology, experiments demonstrate functional afferent and efferent integration of transplanted neurons into transcallosal projection neuron circuitry. Results further indicate that 'primed' early postmitotic neurons, including already fate-restricted deep-layer projection neurons and/or plastic postmitotic neuroblasts with partially fate-restricted potential, account for the predominant population of neurons capable of achieving this optimal level of integration. 2017
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Chen Z, Lin K, Macklis JD, Al-Chalabi A. 2017. Proposed association between the hexanucleotide repeat of C9orf72 and opposability index of the thumb. Amyotrophic lateral sclerosis & frontotemporal degeneration. 18(3-4):175-181. Pubmed: 28010125 DOI:10.1080/21678421.2016.1257024 Chen Z, Lin K, Macklis JD, Al-Chalabi A. 2017. Proposed association between the hexanucleotide repeat of C9orf72 and opposability index of the thumb. Amyotrophic lateral sclerosis & frontotemporal degeneration. 18(3-4):175-181. Pubmed: 28010125 DOI:10.1080/21678421.2016.1257024 Array -
Rodriguez-Muela N, Litterman NK, Norabuena EM, Mull JL, Galazo MJ, Sun C, Ng SY, Makhortova NR, White A, Lynes MM, Chung WK, Davidow LS, Macklis JD, Rubin LL. 2017. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease. Cell reports. 18(6):1484-1498. Pubmed: 28178525 DOI:S2211-1247(17)30072-4 Rodriguez-Muela N, Litterman NK, Norabuena EM, Mull JL, Galazo MJ, Sun C, Ng SY, Makhortova NR, White A, Lynes MM, Chung WK, Davidow LS, Macklis JD, Rubin LL. 2017. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease. Cell reports. 18(6):1484-1498. Pubmed: 28178525 DOI:S2211-1247(17)30072-4 The mechanism underlying selective motor neuron (MN) death remains an essential question in the MN disease field. The MN disease spinal muscular atrophy (SMA) is attributable to reduced levels of the ubiquitous protein SMN. Here, we report that SMN levels are widely variable in MNs within a single genetic background and that this heterogeneity is seen not only in SMA MNs but also in MNs derived from controls and amyotrophic lateral sclerosis (ALS) patients. Furthermore, cells with low SMN are more susceptible to cell death. These findings raise the important clinical implication that some SMN-elevating therapeutics might be effective in MN diseases besides SMA. Supporting this, we found that increasing SMN across all MN populations using an Nedd8-activating enzyme inhibitor promotes survival in both SMA and ALS-derived MNs. Altogether, our work demonstrates that examination of human neurons at the single-cell level can reveal alternative strategies to be explored in the treatment of degenerative diseases.Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved. -
Fame RM, Dehay C, Kennedy H, Macklis JD. 2017. Subtype-Specific Genes that Characterize Subpopulations of Callosal Projection Neurons in Mouse Identify Molecularly Homologous Populations in Macaque Cortex. Cerebral cortex (New York, N.Y. : 1991). 27(3):1817-1830. Pubmed: 26874185 DOI:10.1093/cercor/bhw023 Fame RM, Dehay C, Kennedy H, Macklis JD. 2017. Subtype-Specific Genes that Characterize Subpopulations of Callosal Projection Neurons in Mouse Identify Molecularly Homologous Populations in Macaque Cortex. Cerebral cortex (New York, N.Y. : 1991). 27(3):1817-1830. Pubmed: 26874185 DOI:10.1093/cercor/bhw023 Callosal projection neurons (CPN) interconnect the neocortical hemispheres via the corpus callosum and are implicated in associative integration of multimodal information. CPN have undergone differential evolutionary elaboration, leading to increased diversity of cortical neurons-and more extensive and varied connections in neocortical gray and white matter-in primates compared with rodents. In mouse, distinct sets of genes are enriched in discrete subpopulations of CPN, indicating the molecular diversity of rodent CPN. Elements of rodent CPN functional and organizational diversity might thus be present in the further elaborated primate cortex. We address the hypothesis that genes controlling mouse CPN subtype diversity might reflect molecular patterns shared among mammals that arose prior to the divergence of rodents and primates. We find that, while early expression of the examined CPN-enriched genes, and postmigratory expression of these CPN-enriched genes in deep layers are highly conserved (e.g., Ptn, Nnmt, Cited2, Dkk3), in contrast, the examined genes expressed by superficial layer CPN show more variable levels of conservation (e.g., EphA3, Chn2). These results suggest that there has been evolutionarily differential retraction and elaboration of superficial layer CPN subpopulations between mouse and macaque, with independent derivation of novel populations in primates. Together, these data inform future studies regarding CPN subpopulations that are unique to primates and rodents, and indicate putative evolutionary relationships.© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. -
Muralidharan B, Khatri Z, Maheshwari U, Gupta R, Roy B, Pradhan SJ, Karmodiya K, Padmanabhan H, Shetty AS, Balaji C, Kolthur-Seetharam U, Macklis JD, Galande S, Tole S. 2017. LHX2 Interacts with the NuRD Complex and Regulates Cortical Neuron Subtype Determinants Fezf2 and Sox11. The Journal of neuroscience : the official journal of the Society for Neuroscience. 37(1):194-203. Pubmed: 28053041 DOI:10.1523/JNEUROSCI.2836-16.2016 Muralidharan B, Khatri Z, Maheshwari U, Gupta R, Roy B, Pradhan SJ, Karmodiya K, Padmanabhan H, Shetty AS, Balaji C, Kolthur-Seetharam U, Macklis JD, Galande S, Tole S. 2017. LHX2 Interacts with the NuRD Complex and Regulates Cortical Neuron Subtype Determinants Fezf2 and Sox11. The Journal of neuroscience : the official journal of the Society for Neuroscience. 37(1):194-203. Pubmed: 28053041 DOI:10.1523/JNEUROSCI.2836-16.2016 ArrayCopyright © 2017 Muralidharan et al. -
Shipman SL, Nivala J, Macklis JD, Church GM. 2017. CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature. 547(7663):345-349. Pubmed: 28700573 DOI:10.1038/nature23017 Shipman SL, Nivala J, Macklis JD, Church GM. 2017. CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature. 547(7663):345-349. Pubmed: 28700573 DOI:10.1038/nature23017 DNA is an excellent medium for archiving data. Recent efforts have illustrated the potential for information storage in DNA using synthesized oligonucleotides assembled in vitro. A relatively unexplored avenue of information storage in DNA is the ability to write information into the genome of a living cell by the addition of nucleotides over time. Using the Cas1-Cas2 integrase, the CRISPR-Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity. When harnessed, this system has the potential to write arbitrary information into the genome. Here we use the CRISPR-Cas system to encode the pixel values of black and white images and a short movie into the genomes of a population of living bacteria. In doing so, we push the technical limits of this information storage system and optimize strategies to minimize those limitations. We also uncover underlying principles of the CRISPR-Cas adaptation system, including sequence determinants of spacer acquisition that are relevant for understanding both the basic biology of bacterial adaptation and its technological applications. This work demonstrates that this system can capture and stably store practical amounts of real data within the genomes of populations of living cells. -
Itoh Y, Poulopoulos A, Macklis JD. 2017. Unfolding the Folding Problem of the Cerebral Cortex: Movin' and Groovin'. Developmental cell. 41(4):332-334. Pubmed: 28535368 DOI:S1534-5807(17)30393-3 Itoh Y, Poulopoulos A, Macklis JD. 2017. Unfolding the Folding Problem of the Cerebral Cortex: Movin' and Groovin'. Developmental cell. 41(4):332-334. Pubmed: 28535368 DOI:S1534-5807(17)30393-3 The development of reproducible folding in the gyrencephalic cerebral cortex is a topic of great interest to neuroscientists. In a recent paper in Cell, del Toro et al. (2017) show that changing the adhesive properties of neurons in the normally lissencephalic mouse cortex leads to the formation of stereotyped folding.Copyright © 2017 Elsevier Inc. All rights reserved. -
Frangeul L, Kehayas V, Sanchez-Mut JV, Fièvre S, Krishna-K K, Pouchelon G, Telley L, Bellone C, Holtmaat A, Gräff J, Macklis JD, Jabaudon D. 2017. Input-dependent regulation of excitability controls dendritic maturation in somatosensory thalamocortical neurons. Nature communications. 8(1):2015. Pubmed: 29222517 DOI:10.1038/s41467-017-02172-1 Frangeul L, Kehayas V, Sanchez-Mut JV, Fièvre S, Krishna-K K, Pouchelon G, Telley L, Bellone C, Holtmaat A, Gräff J, Macklis JD, Jabaudon D. 2017. Input-dependent regulation of excitability controls dendritic maturation in somatosensory thalamocortical neurons. Nature communications. 8(1):2015. Pubmed: 29222517 DOI:10.1038/s41467-017-02172-1 Input from the sensory organs is required to pattern neurons into topographical maps during development. Dendritic complexity critically determines this patterning process; yet, how signals from the periphery act to control dendritic maturation is unclear. Here, using genetic and surgical manipulations of sensory input in mouse somatosensory thalamocortical neurons, we show that membrane excitability is a critical component of dendritic development. Using a combination of genetic approaches, we find that ablation of N-methyl-D-aspartate (NMDA) receptors during postnatal development leads to epigenetic repression of Kv1.1-type potassium channels, increased excitability, and impaired dendritic maturation. Lesions to whisker input pathways had similar effects. Overexpression of Kv1.1 was sufficient to enable dendritic maturation in the absence of sensory input. Thus, Kv1.1 acts to tune neuronal excitability and maintain it within a physiological range, allowing dendritic maturation to proceed. Together, these results reveal an input-dependent control over neuronal excitability and dendritic complexity in the development and plasticity of sensory pathways. 2016
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Woodworth MB, Greig LC, Liu KX, Ippolito GC, Tucker HO, Macklis JD. 2016. Ctip1 Regulates the Balance between Specification of Distinct Projection Neuron Subtypes in Deep Cortical Layers. Cell reports. 15(5):999-1012. Pubmed: 27117402 DOI:S2211-1247(16)30335-7 Woodworth MB, Greig LC, Liu KX, Ippolito GC, Tucker HO, Macklis JD. 2016. Ctip1 Regulates the Balance between Specification of Distinct Projection Neuron Subtypes in Deep Cortical Layers. Cell reports. 15(5):999-1012. Pubmed: 27117402 DOI:S2211-1247(16)30335-7 The molecular linkage between neocortical projection neuron subtype and area development, which enables the establishment of functional areas by projection neuron populations appropriate for specific sensory and motor functions, is poorly understood. Here, we report that Ctip1 controls precision of neocortical development by regulating subtype identity in deep-layer projection neurons. Ctip1 is expressed by postmitotic callosal and corticothalamic projection neurons but is excluded over embryonic development from corticospinal motor neurons, which instead express its close relative, Ctip2. Loss of Ctip1 function results in a striking bias in favor of subcerebral projection neuron development in sensory cortex at the expense of corticothalamic and deep-layer callosal development, while misexpression of Ctip1 in vivo represses subcerebral gene expression and projections. As we report in a paired paper, Ctip1 also controls acquisition of sensory area identity. Therefore, Ctip1 couples subtype and area specification, enabling specific functional areas to organize precise ratios of appropriate output projections.Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved. -
Sances S, Bruijn LI, Chandran S, Eggan K, Ho R, Klim JR, Livesey MR, Lowry E, Macklis JD, Rushton D, Sadegh C, Sareen D, Wichterle H, Zhang SC, Svendsen CN. 2016. Modeling ALS with motor neurons derived from human induced pluripotent stem cells. Nature neuroscience. 19(4):542-53. Pubmed: 27021939 DOI:10.1038/nn.4273 Sances S, Bruijn LI, Chandran S, Eggan K, Ho R, Klim JR, Livesey MR, Lowry E, Macklis JD, Rushton D, Sadegh C, Sareen D, Wichterle H, Zhang SC, Svendsen CN. 2016. Modeling ALS with motor neurons derived from human induced pluripotent stem cells. Nature neuroscience. 19(4):542-53. Pubmed: 27021939 DOI:10.1038/nn.4273 Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease. -
Kishi N, MacDonald JL, Ye J, Molyneaux BJ, Azim E, Macklis JD. 2016. Reduction of aberrant NF-κB signalling ameliorates Rett syndrome phenotypes in Mecp2-null mice. Nature communications. 7:10520. Pubmed: 26821816 DOI:10.1038/ncomms10520 Kishi N, MacDonald JL, Ye J, Molyneaux BJ, Azim E, Macklis JD. 2016. Reduction of aberrant NF-κB signalling ameliorates Rett syndrome phenotypes in Mecp2-null mice. Nature communications. 7:10520. Pubmed: 26821816 DOI:10.1038/ncomms10520 Mutations in the transcriptional regulator Mecp2 cause the severe X-linked neurodevelopmental disorder Rett syndrome (RTT). In this study, we investigate genes that function downstream of MeCP2 in cerebral cortex circuitry, and identify upregulation of Irak1, a central component of the NF-κB pathway. We show that overexpression of Irak1 mimics the reduced dendritic complexity of Mecp2-null cortical callosal projection neurons (CPN), and that NF-κB signalling is upregulated in the cortex with Mecp2 loss-of-function. Strikingly, we find that genetically reducing NF-κB signalling in Mecp2-null mice not only ameliorates CPN dendritic complexity but also substantially extends their normally shortened lifespan, indicating broader roles for NF-κB signalling in RTT pathogenesis. These results provide new insight into both the fundamental neurobiology of RTT, and potential therapeutic strategies via NF-κB pathway modulation. -
Greig LC, Woodworth MB, Greppi C, Macklis JD. 2016. Ctip1 Controls Acquisition of Sensory Area Identity and Establishment of Sensory Input Fields in the Developing Neocortex. Neuron. 90(2):261-77. Pubmed: 27100196 DOI:S0896-6273(16)00187-2 Greig LC, Woodworth MB, Greppi C, Macklis JD. 2016. Ctip1 Controls Acquisition of Sensory Area Identity and Establishment of Sensory Input Fields in the Developing Neocortex. Neuron. 90(2):261-77. Pubmed: 27100196 DOI:S0896-6273(16)00187-2 While transcriptional controls over the size and relative position of cortical areas have been identified, less is known about regulators that direct acquisition of area-specific characteristics. Here, we report that the transcription factor Ctip1 functions in primary sensory areas to repress motor and activate sensory programs of gene expression, enabling establishment of sharp molecular boundaries defining functional areas. In Ctip1 mutants, abnormal gene expression leads to aberrantly motorized corticocortical and corticofugal output connectivity. Ctip1 critically regulates differentiation of layer IV neurons, and selective loss of Ctip1 in cortex deprives thalamocortical axons of their receptive "sensory field" in layer IV, which normally provides a tangentially and radially defined compartment of dedicated synaptic territory. Therefore, although thalamocortical axons invade appropriate cortical regions, they are unable to organize into properly configured sensory maps. Together, these data identify Ctip1 as a critical control over sensory area development.Copyright © 2016 Elsevier Inc. All rights reserved. -
Smith EC, Luc S, Croney DM, Woodworth MB, Greig LC, Fujiwara Y, Nguyen M, Sher F, Macklis JD, Bauer DE, Orkin SH. 2016. Strict in vivo specificity of the erythroid enhancer. Blood. 128(19):2338-2342. Pubmed: 27707736 DOI:10.1182/blood-2016-08-736249 Smith EC, Luc S, Croney DM, Woodworth MB, Greig LC, Fujiwara Y, Nguyen M, Sher F, Macklis JD, Bauer DE, Orkin SH. 2016. Strict in vivo specificity of the erythroid enhancer. Blood. 128(19):2338-2342. Pubmed: 27707736 DOI:10.1182/blood-2016-08-736249 BCL11A, a repressor of human fetal (γ-)globin expression, is required for immune and hematopoietic stem cell functions and brain development. Regulatory sequences within the gene, which are subject to genetic variation affecting fetal globin expression, display hallmarks of an erythroid enhancer in cell lines and transgenic mice. As such, this enhancer is a novel, attractive target for therapeutic gene editing. To explore the roles of such sequences in vivo, we generated mice in which the orthologous 10-kb intronic sequences were removed. enhancer-deleted mice, (Δenh), phenocopy the BCL11A-null state with respect to alterations of globin expression, yet are viable and exhibit no observable blood, brain, or other abnormalities. These preclinical findings provide strong in vivo support for genetic modification of the enhancer for therapy of hemoglobin disorders.© 2016 by The American Society of Hematology. -
Galazo MJ, Emsley JG, Macklis JD. 2016. Corticothalamic Projection Neuron Development beyond Subtype Specification: Fog2 and Intersectional Controls Regulate Intraclass Neuronal Diversity. Neuron. 91(1):90-106. Pubmed: 27321927 DOI:S0896-6273(16)30203-3 Galazo MJ, Emsley JG, Macklis JD. 2016. Corticothalamic Projection Neuron Development beyond Subtype Specification: Fog2 and Intersectional Controls Regulate Intraclass Neuronal Diversity. Neuron. 91(1):90-106. Pubmed: 27321927 DOI:S0896-6273(16)30203-3 Corticothalamic projection neurons (CThPN) are a diverse set of neurons, critical for function of the neocortex. CThPN development and diversity need to be precisely regulated, but little is known about molecular controls over their differentiation and functional specialization, critically limiting understanding of cortical development and complexity. We report the identification of a set of genes that both define CThPN and likely control their differentiation, diversity, and function. We selected the CThPN-specific transcriptional coregulator Fog2 for functional analysis. We identify that Fog2 controls CThPN molecular differentiation, axonal targeting, and diversity, in part by regulating the expression level of Ctip2 by CThPN, via combinatorial interactions with other molecular controls. Loss of Fog2 specifically disrupts differentiation of subsets of CThPN specialized in motor function, indicating that Fog2 coordinates subtype and functional-area differentiation. These results confirm that we identified key controls over CThPN development and identify Fog2 as a critical control over CThPN diversity.Copyright © 2016 Elsevier Inc. All rights reserved. -
Fame RM, MacDonald JL, Dunwoodie SL, Takahashi E, Macklis JD. 2016. Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity. The Journal of neuroscience : the official journal of the Society for Neuroscience. 36(24):6403-19. Pubmed: 27307230 DOI:10.1523/JNEUROSCI.4067-15.2016 Fame RM, MacDonald JL, Dunwoodie SL, Takahashi E, Macklis JD. 2016. Cited2 Regulates Neocortical Layer II/III Generation and Somatosensory Callosal Projection Neuron Development and Connectivity. The Journal of neuroscience : the official journal of the Society for Neuroscience. 36(24):6403-19. Pubmed: 27307230 DOI:10.1523/JNEUROSCI.4067-15.2016 ArrayCopyright © 2016 the authors 0270-6474/16/366404-17$15.00/0. -
Shipman SL, Nivala J, Macklis JD, Church GM. 2016. Molecular recordings by directed CRISPR spacer acquisition. Science (New York, N.Y.). 353(6298):aaf1175. Pubmed: 27284167 DOI:10.1126/science.aaf1175 Shipman SL, Nivala J, Macklis JD, Church GM. 2016. Molecular recordings by directed CRISPR spacer acquisition. Science (New York, N.Y.). 353(6298):aaf1175. Pubmed: 27284167 DOI:10.1126/science.aaf1175 The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution so as to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.Copyright © 2016, American Association for the Advancement of Science. 2015
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Jara JH, Genç B, Cox GA, Bohn MC, Roos RP, Macklis JD, Ulupınar E, Özdinler PH. 2015. Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function. Cerebral cortex (New York, N.Y. : 1991). 25(11):4259-72. Pubmed: 25596590 DOI:10.1093/cercor/bhu318 Jara JH, Genç B, Cox GA, Bohn MC, Roos RP, Macklis JD, Ulupınar E, Özdinler PH. 2015. Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function. Cerebral cortex (New York, N.Y. : 1991). 25(11):4259-72. Pubmed: 25596590 DOI:10.1093/cercor/bhu318 Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1(nm3419) (UCHL1(-/-)) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1(-/-) mice as a tool to study CSMN pathology.© The Author 2015. Published by Oxford University Press. -
Jones AR, Troakes C, King A, Sahni V, De Jong S, Bossers K, Papouli E, Mirza M, Al-Sarraj S, Shaw CE, Shaw PJ, Kirby J, Veldink JH, Macklis JD, Powell JF, Al-Chalabi A. 2015. Stratified gene expression analysis identifies major amyotrophic lateral sclerosis genes. Neurobiology of aging. 36(5):2006.e1-9. Pubmed: 25801576 DOI:S0197-4580(15)00117-7 Jones AR, Troakes C, King A, Sahni V, De Jong S, Bossers K, Papouli E, Mirza M, Al-Sarraj S, Shaw CE, Shaw PJ, Kirby J, Veldink JH, Macklis JD, Powell JF, Al-Chalabi A. 2015. Stratified gene expression analysis identifies major amyotrophic lateral sclerosis genes. Neurobiology of aging. 36(5):2006.e1-9. Pubmed: 25801576 DOI:S0197-4580(15)00117-7 Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons resulting in progressive paralysis. Gene expression studies of ALS only rarely identify the same gene pathways as gene association studies. We hypothesized that analyzing tissues by matching on degree of disease severity would identify different patterns of gene expression from a traditional case-control comparison. We analyzed gene expression changes in 4 postmortem central nervous system regions, stratified by severity of motor neuron loss. An overall comparison of cases (n = 6) and controls (n = 3) identified known ALS gene, SOX5, as showing differential expression (log2 fold change = 0.09, p = 5.5 × 10(-5)). Analyses stratified by disease severity identified expression changes in C9orf72 (p = 2.77 × 10(-3)), MATR3 (p = 3.46 × 10(-3)), and VEGFA (p = 8.21 × 10(-4)), all implicated in ALS through genetic studies, and changes in other genes in pathways involving RNA processing and immune response. These findings suggest that analysis of gene expression stratified by disease severity can identify major ALS genes and may be more efficient than traditional case-control comparison.Copyright © 2015 Elsevier Inc. All rights reserved. 2014
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Sadegh C, Macklis JD. 2014. Established monolayer differentiation of mouse embryonic stem cells generates heterogeneous neocortical-like neurons stalled at a stage equivalent to midcorticogenesis. The Journal of comparative neurology. 522(12):2691-706. Pubmed: 24610556 DOI:10.1002/cne.23576 Sadegh C, Macklis JD. 2014. Established monolayer differentiation of mouse embryonic stem cells generates heterogeneous neocortical-like neurons stalled at a stage equivalent to midcorticogenesis. The Journal of comparative neurology. 522(12):2691-706. Pubmed: 24610556 DOI:10.1002/cne.23576 Two existing and widely applied protocols of embryonic stem (ES) cell differentiation have been developed to enable in vitro generation of neurons resembling neocortical projection neurons in monolayer culture and from embryoid bodies. The monolayer approach offers advantages for detailed in vitro characterizations and potential mechanistic and therapeutic screening. We investigated whether mouse ES cells undergoing largely undirected neocortical differentiation in monolayer culture recapitulate progressive developmental programs of in vivo progenitor and postmitotic differentiation and whether they develop into specific neocortical subtypes. We find that ES-derived mitotic cells that have been dorsalized by the sonic hedgehog antagonist cyclopamine, and that express, as a total population, cardinal markers of telencephalic progenitors, are, in fact, molecularly heterogeneous. We next show that these progenitors subsequently generate small numbers of heterogeneous neocortical-like neurons that are "stalled" at an immature stage of differentiation, based on multiple developmental criteria. Although some aspects of neocortical development are recapitulated by existing protocols of ES cell differentiation, these data indicate that mouse ES-derived neocortical progenitors both are more heterogeneous than their in vivo counterparts and seemingly include many incorrectly specified progenitors. Furthermore, these ES-derived progenitors spontaneously differentiate into sparse, and incompletely and largely imprecisely differentiated, neocortical-like neurons that fail to adopt specific neuronal identities in vitro. These results provide both foundation and motivation for refining and enhancing directed differentiation of clinically important neocortical projection neuron subtypes.© 2014 Wiley Periodicals, Inc. -
Sohur US, Padmanabhan HK, Kotchetkov IS, Menezes JR, Macklis JD. 2014. Anatomic and molecular development of corticostriatal projection neurons in mice. Cerebral cortex (New York, N.Y. : 1991). 24(2):293-303. Pubmed: 23118198 DOI:10.1093/cercor/bhs342 Sohur US, Padmanabhan HK, Kotchetkov IS, Menezes JR, Macklis JD. 2014. Anatomic and molecular development of corticostriatal projection neurons in mice. Cerebral cortex (New York, N.Y. : 1991). 24(2):293-303. Pubmed: 23118198 DOI:10.1093/cercor/bhs342 Corticostriatal projection neurons (CStrPN) project from the neocortex to ipsilateral and contralateral striata to control and coordinate motor programs and movement. They are clinically important as the predominant cortical population that degenerates in Huntington's disease and corticobasal ganglionic degeneration, and their injury contributes to multiple forms of cerebral palsy. Together with their well-studied functions in motor control, these clinical connections make them a functionally, behaviorally, and clinically important population of neocortical neurons. Little is known about their development. "Intratelencephalic" CStrPN (CStrPNi), projecting to the contralateral striatum, with their axons fully within the telencephalon (intratelencephalic), are a major population of CStrPN. CStrPNi are of particular interest developmentally because they share hodological and axon guidance characteristics of both callosal projection neurons (CPN) and corticofugal projection neurons (CFuPN); CStrPNi send axons contralaterally before descending into the contralateral striatum. The relationship of CStrPNi development to that of broader CPN and CFuPN populations remains unclear; evidence suggests that CStrPNi might be evolutionary "hybrids" between CFuPN and deep layer CPN-in a sense "chimeric" with both callosal and corticofugal features. Here, we investigated the development of CStrPNi in mice-their birth, maturation, projections, and expression of molecular developmental controls over projection neuron subtype identity. 2013
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Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H, Macklis JD. 2013. Molecular logic of neocortical projection neuron specification, development and diversity. Nature reviews. Neuroscience. 14(11):755-69. Pubmed: 24105342 DOI:10.1038/nrn3586 Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H, Macklis JD. 2013. Molecular logic of neocortical projection neuron specification, development and diversity. Nature reviews. Neuroscience. 14(11):755-69. Pubmed: 24105342 DOI:10.1038/nrn3586 The sophisticated circuitry of the neocortex is assembled from a diverse repertoire of neuronal subtypes generated during development under precise molecular regulation. In recent years, several key controls over the specification and differentiation of neocortical projection neurons have been identified. This work provides substantial insight into the 'molecular logic' underlying cortical development and increasingly supports a model in which individual progenitor-stage and postmitotic regulators are embedded within highly interconnected networks that gate sequential developmental decisions. Here, we provide an integrative account of the molecular controls that direct the progressive development and delineation of subtype and area identity of neocortical projection neurons. -
Ravits J, Appel S, Baloh RH, Barohn R, Brooks BR, Elman L, Floeter MK, Henderson C, Lomen-Hoerth C, Macklis JD, McCluskey L, Mitsumoto H, Przedborski S, Rothstein J, Trojanowski JQ, van den Berg LH, Ringel S. 2013. Deciphering amyotrophic lateral sclerosis: what phenotype, neuropathology and genetics are telling us about pathogenesis. Amyotrophic lateral sclerosis & frontotemporal degeneration. 14 Suppl 1(0 1):5-18. Pubmed: 23678876 DOI:10.3109/21678421.2013.778548 Ravits J, Appel S, Baloh RH, Barohn R, Brooks BR, Elman L, Floeter MK, Henderson C, Lomen-Hoerth C, Macklis JD, McCluskey L, Mitsumoto H, Przedborski S, Rothstein J, Trojanowski JQ, van den Berg LH, Ringel S. 2013. Deciphering amyotrophic lateral sclerosis: what phenotype, neuropathology and genetics are telling us about pathogenesis. Amyotrophic lateral sclerosis & frontotemporal degeneration. 14 Suppl 1(0 1):5-18. Pubmed: 23678876 DOI:10.3109/21678421.2013.778548 Amyotrophic lateral sclerosis (ALS) is characterized phenotypically by progressive weakness and neuropathologically by loss of motor neurons. Phenotypically, there is marked heterogeneity. Typical ALS has mixed upper motor neuron (UMN) and lower motor neuron (LMN) involvement. Primary lateral sclerosis has predominant UMN involvement. Progressive muscular atrophy has predominant LMN involvement. Bulbar and limb ALS have predominant regional involvement. Frontotemporal dementia has significant cognitive and behavioral involvement. These phenotypes can be so distinctive that they would seem to have differing biology. However, they cannot be distinguished, at least neuropathologically or genetically. In sporadic ALS (SALS), they are mostly characterized by ubiquitinated cytoplasmic inclusions of TDP-43. In familial ALS (FALS), where phenotypes are indistinguishable from SALS and similarly heterogeneous, each mutated gene has its own genetic and molecular signature. Overall, since the same phenotypes can have multiple causes including different gene mutations, there must be multiple molecular mechanisms causing ALS - and ALS is a syndrome. Since, however, multiple phenotypes can be caused by one single gene mutation, a single molecular mechanism can cause heterogeneity. What the mechanisms are remain unknown, but active propagation of the pathology neuroanatomically seems to be a principal component. Leading candidate mechanisms include RNA processing, cell-cell interactions between neurons and non-neuronal neighbors, focal seeding from a misfolded protein that has prion-like propagation, and fatal errors introduced during neurodevelopment of the motor system. If fundamental mechanisms could be identified and understood, ALS therapy could rationally target progression and stop the disease - a goal that seems increasingly achievable. -
Cederquist GY, Azim E, Shnider SJ, Padmanabhan H, Macklis JD. 2013. Lmo4 establishes rostral motor cortex projection neuron subtype diversity. The Journal of neuroscience : the official journal of the Society for Neuroscience. 33(15):6321-32. Pubmed: 23575831 DOI:10.1523/JNEUROSCI.5140-12.2013 Cederquist GY, Azim E, Shnider SJ, Padmanabhan H, Macklis JD. 2013. Lmo4 establishes rostral motor cortex projection neuron subtype diversity. The Journal of neuroscience : the official journal of the Society for Neuroscience. 33(15):6321-32. Pubmed: 23575831 DOI:10.1523/JNEUROSCI.5140-12.2013 The mammalian neocortex is parcellated into anatomically and functionally distinct areas. The establishment of area-specific neuronal diversity and circuit connectivity enables distinct neocortical regions to control diverse and specialized functional outputs, yet underlying molecular controls remain largely unknown. Here, we identify a central role for the transcriptional regulator Lim-only 4 (Lmo4) in establishing the diversity of neuronal subtypes within rostral mouse motor cortex, where projection neurons have particularly diverse and multi-projection connectivity compared with caudal motor cortex. In rostral motor cortex, we report that both subcerebral projection neurons (SCPN), which send projections away from the cerebrum, and callosal projection neurons (CPN), which send projections to contralateral cortex, express Lmo4, whereas more caudal SCPN and CPN do not. Lmo4-expressing SCPN and CPN populations are comprised of multiple hodologically distinct subtypes. SCPN in rostral layer Va project largely to brainstem, whereas SCPN in layer Vb project largely to spinal cord, and a subset of both rostral SCPN and CPN sends second ipsilateral caudal (backward) projections in addition to primary projections. Without Lmo4 function, the molecular identity of neurons in rostral motor cortex is disrupted and more homogenous, rostral layer Va SCPN aberrantly project to the spinal cord, and many dual-projection SCPN and CPN fail to send a second backward projection. These molecular and hodological disruptions result in greater overall homogeneity of motor cortex output. Together, these results identify Lmo4 as a central developmental control over the diversity of motor cortex projection neuron subpopulations, establishing their area-specific identity and specialized connectivity. 2012
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Sohur US, Arlotta P, Macklis JD. 2012. Developmental Controls are Re-Expressed during Induction of Neurogenesis in the Neocortex of Young Adult Mice. Frontiers in neuroscience. 6:12. Pubmed: 22347158 DOI:10.3389/fnins.2012.00012 Sohur US, Arlotta P, Macklis JD. 2012. Developmental Controls are Re-Expressed during Induction of Neurogenesis in the Neocortex of Young Adult Mice. Frontiers in neuroscience. 6:12. Pubmed: 22347158 DOI:10.3389/fnins.2012.00012 Whether induction of low-level neurogenesis in normally non-neurogenic regions of the adult brain mimics aspects of developmental neurogenesis is currently unknown. Previously, we and others identified that biophysically induced, neuron subtype-specific apoptosis in mouse neocortex results in induction of neurogenesis of limited numbers of subtype-appropriate projection neurons with axonal projections to either thalamus or spinal cord, depending on the neuron subtype activated to undergo targeted apoptosis. Here, we test the hypothesis that developmental genes from embryonic corticogenesis are re-activated, and that some of these genes might underlie induction of low-level adult neocortical neurogenesis. We directly investigated this hypothesis via microarray analysis of microdissected regions of young adult mouse neocortex undergoing biophysically activated targeted apoptosis of neocortical callosal projection neurons. We compared the microarray results identifying differentially expressed genes with public databases of embryonic developmental genes. We find that, following activation of subtype-specific neuronal apoptosis, three distinct sets of normal developmental genes are selectively re-expressed in neocortical regions of induced neurogenesis in young adult mice: (1) genes expressed by subsets of progenitors and immature neurons in the developing ventricular and/or subventricular zones; (2) genes normally expressed by developmental radial glial progenitors; and (3) genes involved in synaptogenesis. Together with previous results, the data indicate that at least some developmental molecular controls over embryonic neurogenesis can be re-activated in the setting of induction of neurogenesis in the young adult neocortex, and suggest that some of these activate and initiate adult neuronal differentiation from endogenous progenitor populations. Understanding molecular mechanisms contributing to induced adult neurogenesis might enable directed CNS repair. -
Tharin S, Kothapalli CR, Ozdinler PH, Pasquina L, Chung S, Varner J, DeValence S, Kamm R, Macklis JD. 2012. A microfluidic device to investigate axon targeting by limited numbers of purified cortical projection neuron subtypes. Integrative biology : quantitative biosciences from nano to macro. 4(11):1398-405. Pubmed: 23034677 DOI:10.1039/c2ib20019h Tharin S, Kothapalli CR, Ozdinler PH, Pasquina L, Chung S, Varner J, DeValence S, Kamm R, Macklis JD. 2012. A microfluidic device to investigate axon targeting by limited numbers of purified cortical projection neuron subtypes. Integrative biology : quantitative biosciences from nano to macro. 4(11):1398-405. Pubmed: 23034677 DOI:10.1039/c2ib20019h While much is known about general controls over axon guidance of broad classes of projection neurons (those with long-distance axonal connections), molecular controls over specific axon targeting by distinct neuron subtypes are poorly understood. Corticospinal motor neurons (CSMN) are prototypical and clinically important cerebral cortex projection neurons; they are the brain neurons that degenerate in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases, and their injury is central to the loss of motor function in spinal cord injury. Primary culture of purified immature murine CSMN has been recently established, using either fluorescence-activated cell sorting (FACS) or immunopanning, enabling a previously unattainable level of subtype-specific investigation, but the resulting number of CSMN is quite limiting for standard approaches to study axon guidance. We developed a microfluidic system specifically designed to investigate axon targeting of limited numbers of purified CSMN and other projection neurons in culture. The system contains two chambers for culturing target tissue explants, allowing for biologically revealing axonal growth "choice" experiments. This device will be uniquely enabling for investigation of controls over axon growth and neuronal survival of many types of neurons, particularly those available only in limited numbers. -
Macklis JD. 2012. Human adult olfactory bulb neurogenesis? Novelty is the best policy. Neuron. 74(4):595-6. Pubmed: 22632715 DOI:10.1016/j.neuron.2012.05.005 Macklis JD. 2012. Human adult olfactory bulb neurogenesis? Novelty is the best policy. Neuron. 74(4):595-6. Pubmed: 22632715 DOI:10.1016/j.neuron.2012.05.005 There is ongoing controversy as to whether the understanding of adult mammalian neurogenesis gained from rodent studies is applicable to humans. In this issue of Neuron, Bergmann et al. (2012) propose that adult human olfactory bulb neurogenesis with long-term neuronal survival is extremely limited.Copyright © 2012 Elsevier Inc. All rights reserved. -
Emsley JG, Menezes JR, Madeiro Da Costa RF, Martinez AM, Macklis JD. 2012. Identification of radial glia-like cells in the adult mouse olfactory bulb. Experimental neurology. 236(2):283-97. Pubmed: 22634209 DOI:10.1016/j.expneurol.2012.05.012 Emsley JG, Menezes JR, Madeiro Da Costa RF, Martinez AM, Macklis JD. 2012. Identification of radial glia-like cells in the adult mouse olfactory bulb. Experimental neurology. 236(2):283-97. Pubmed: 22634209 DOI:10.1016/j.expneurol.2012.05.012 Immature neurons migrate tangentially within the rostral migratory stream (RMS) to the adult olfactory bulb (OB), then radially to their final positions as granule and periglomerular neurons; the controls over this transition are not well understood. Using adult transgenic mice with the human GFAP promoter driving expression of enhanced GFP, we identified a population of radial glia-like cells that we term adult olfactory radial glia-like cells (AORGs). AORGs have large, round somas and simple, radially oriented processes. Confocal reconstructions indicate that AORGs variably express typical radial glial markers, only rarely express mouse GFAP, and do not express astroglial, oligodendroglial, neuronal, or tanycyte markers. Electron microscopy provides further supporting evidence that AORGs are not immature neurons. Developmental analyses indicate that AORGs are present as early as P1, and are generated through adulthood. Tracing studies show that AORGs are not born in the SVZa, suggesting that they are born either in the RMS or the OB. Migrating immature neurons from the adult SVZa are closely apposed to AORGs during radial migration in vivo and in vitro. Taken together, these data indicate a newly-identified population of radial glia-like cells in the adult OB that might function uniquely in neuronal radial migration during adult OB neurogenesis.Copyright © 2012 Elsevier Inc. All rights reserved. -
Woodworth MB, Greig LC, Kriegstein AR, Macklis JD. 2012. SnapShot: cortical development. Cell. 151(4):918-918.e1. Pubmed: 23141546 DOI:S0092-8674(12)01186-5 Woodworth MB, Greig LC, Kriegstein AR, Macklis JD. 2012. SnapShot: cortical development. Cell. 151(4):918-918.e1. Pubmed: 23141546 DOI:S0092-8674(12)01186-5 -
Jabaudon D, Shnider SJ, Tischfield DJ, Galazo MJ, Macklis JD. 2012. RORβ induces barrel-like neuronal clusters in the developing neocortex. Cerebral cortex (New York, N.Y. : 1991). 22(5):996-1006. Pubmed: 21799210 DOI:10.1093/cercor/bhr182 Jabaudon D, Shnider SJ, Tischfield DJ, Galazo MJ, Macklis JD. 2012. RORβ induces barrel-like neuronal clusters in the developing neocortex. Cerebral cortex (New York, N.Y. : 1991). 22(5):996-1006. Pubmed: 21799210 DOI:10.1093/cercor/bhr182 Neurons in layer IV of the rodent whisker somatosensory cortex are tangentially organized in periodic clusters called barrels, each of which is innervated by thalamocortical axons transmitting sensory information from a single principal whisker, together forming a somatotopic map of the whisker pad. Proper thalamocortical innervation is critical for barrel formation during development, but the molecular mechanisms controlling layer IV neuron clustering are unknown. Here, we investigate the role in this mapping of the nuclear orphan receptor RORβ, which is expressed in neurons in layer IV during corticogenesis. We find that RORβ protein expression specifically increases in the whisker barrel cortex during barrel formation and that in vivo overexpression of RORβ is sufficient to induce periodic barrel-like clustering of cortical neurons. Remarkably, this clustering can be induced as early as E18, prior to innervation by thalamocortical afferents and whisker derived-input. At later developmental stages, these ectopic neuronal clusters are specifically innervated by thalamocortical axons, demonstrated by anterograde labeling from the thalamus and by expression of thalamocortical-specific synaptic markers. Together, these data indicate that RORβ expression levels control cytoarchitectural patterning of neocortical neurons during development, a critical process for the topographical mapping of whisker input onto the cortical surface. 2011
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Ozdinler PH, Benn S, Yamamoto TH, Güzel M, Brown RH, Macklis JD. 2011. Corticospinal motor neurons and related subcerebral projection neurons undergo early and specific neurodegeneration in hSOD1G⁹³A transgenic ALS mice. The Journal of neuroscience : the official journal of the Society for Neuroscience. 31(11):4166-77. Pubmed: 21411657 DOI:10.1523/JNEUROSCI.4184-10.2011 Ozdinler PH, Benn S, Yamamoto TH, Güzel M, Brown RH, Macklis JD. 2011. Corticospinal motor neurons and related subcerebral projection neurons undergo early and specific neurodegeneration in hSOD1G⁹³A transgenic ALS mice. The Journal of neuroscience : the official journal of the Society for Neuroscience. 31(11):4166-77. Pubmed: 21411657 DOI:10.1523/JNEUROSCI.4184-10.2011 Amyotrophic lateral sclerosis (ALS) is characterized by predominant vulnerability and central degeneration of both corticospinal/corticobulbar motor neurons (CSMN; "upper motor neurons") in cerebral cortex, and spinal/bulbar motor neurons (SMN; "lower motor neurons") in spinal cord and brainstem. Increasing evidence indicates broader cerebral cortex pathology in cognitive, sensory, and association systems in select cases. It remains unclear whether widely accepted transgenic ALS models, in particular hSOD1(G93A) mice, undergo degeneration of CSMN and molecularly/developmentally closely related populations of nonmotor projection neurons [e.g., other subcerebral projection neurons (SCPN)], and whether potential CSMN/SCPN degeneration is specific and early. This relative lack of knowledge regarding upper motor neuron pathology in these ALS model mice has hindered both molecular-pathophysiologic understanding of ALS and their use toward potential CSMN therapeutic approaches. Here, using a combination of anatomic, cellular, transgenic labeling, and newly available neuronal subtype-specific molecular analyses, we identify that CSMN and related nonmotor SCPN specifically and progressively degenerate in hSOD1(G93A) mice. Degeneration starts quite early and presymptomatically, by postnatal day 30. Other neocortical layers, cortical interneurons, and other projection neuron populations, even within layer V, are not similarly affected. Nonneuronal pathology in neocortex (activated astroglia and microglia) is consistent with findings in human ALS cortex and in affected mouse and human spinal cord. These results indicate previously unknown neuron type-specific vulnerability of CSMN/sensory and association SCPN, and identify that characteristic dual CSMN and SMN degeneration is conserved in hSOD1(G93A) mice. These results provide a foundation for detailed investigation of CSMN/SCPN vulnerability and toward potential CSMN therapeutics in ALS. -
Czupryn A, Zhou YD, Chen X, McNay D, Anderson MP, Flier JS, Macklis JD. 2011. Transplanted hypothalamic neurons restore leptin signaling and ameliorate obesity in db/db mice. Science (New York, N.Y.). 334(6059):1133-7. Pubmed: 22116886 DOI:10.1126/science.1209870 Czupryn A, Zhou YD, Chen X, McNay D, Anderson MP, Flier JS, Macklis JD. 2011. Transplanted hypothalamic neurons restore leptin signaling and ameliorate obesity in db/db mice. Science (New York, N.Y.). 334(6059):1133-7. Pubmed: 22116886 DOI:10.1126/science.1209870 Evolutionarily old and conserved homeostatic systems in the brain, including the hypothalamus, are organized into nuclear structures of heterogeneous and diverse neuron populations. To investigate whether such circuits can be functionally reconstituted by synaptic integration of similarly diverse populations of neurons, we generated physically chimeric hypothalami by microtransplanting small numbers of embryonic enhanced green fluorescent protein-expressing, leptin-responsive hypothalamic cells into hypothalami of postnatal leptin receptor-deficient (db/db) mice that develop morbid obesity. Donor neurons differentiated and integrated as four distinct hypothalamic neuron subtypes, formed functional excitatory and inhibitory synapses, partially restored leptin responsiveness, and ameliorated hyperglycemia and obesity in db/db mice. These experiments serve as a proof of concept that transplanted neurons can functionally reconstitute complex neuronal circuitry in the mammalian brain. -
Fame RM, MacDonald JL, Macklis JD. 2011. Development, specification, and diversity of callosal projection neurons. Trends in neurosciences. 34(1):41-50. Pubmed: 21129791 DOI:10.1016/j.tins.2010.10.002 Fame RM, MacDonald JL, Macklis JD. 2011. Development, specification, and diversity of callosal projection neurons. Trends in neurosciences. 34(1):41-50. Pubmed: 21129791 DOI:10.1016/j.tins.2010.10.002 Callosal projection neurons (CPN) are a diverse population of neocortical projection neurons that connect the two hemispheres of the cerebral cortex via the corpus callosum. They play key roles in high-level associative connectivity, and have been implicated in cognitive syndromes of high-level associative dysfunction, such as autism spectrum disorders. CPN evolved relatively recently compared to other cortical neuron populations, and have undergone disproportionately large expansion from mouse to human. While much is known about the anatomical trajectory of developing CPN axons, and progress has been made in identifying cellular and molecular controls over midline crossing, only recently have molecular-genetic controls been identified that specify CPN populations, and help define CPN subpopulations. In this review, we discuss the development, diversity and evolution of CPN.Copyright © 2010 Elsevier Ltd. All rights reserved. 2010
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Tomassy GS, De Leonibus E, Jabaudon D, Lodato S, Alfano C, Mele A, Macklis JD, Studer M. 2010. Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI. Proceedings of the National Academy of Sciences of the United States of America. 107(8):3576-81. Pubmed: 20133588 DOI:10.1073/pnas.0911792107 Tomassy GS, De Leonibus E, Jabaudon D, Lodato S, Alfano C, Mele A, Macklis JD, Studer M. 2010. Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI. Proceedings of the National Academy of Sciences of the United States of America. 107(8):3576-81. Pubmed: 20133588 DOI:10.1073/pnas.0911792107 Transcription factors with gradients of expression in neocortical progenitors give rise to distinct motor and sensory cortical areas by controlling the area-specific differentiation of distinct neuronal subtypes. However, the molecular mechanisms underlying this area-restricted control are still unclear. Here, we show that COUP-TFI controls the timing of birth and specification of corticospinal motor neurons (CSMN) in somatosensory cortex via repression of a CSMN differentiation program. Loss of COUP-TFI function causes an area-specific premature generation of neurons with cardinal features of CSMN, which project to subcerebral structures, including the spinal cord. Concurrently, genuine CSMN differentiate imprecisely and do not project beyond the pons, together resulting in impaired skilled motor function in adult mice with cortical COUP-TFI loss-of-function. Our findings indicate that COUP-TFI exerts critical areal and temporal control over the precise differentiation of CSMN during corticogenesis, thereby enabling the area-specific functional features of motor and sensory areas to arise. -
Kishi N, Macklis JD. 2010. MeCP2 functions largely cell-autonomously, but also non-cell-autonomously, in neuronal maturation and dendritic arborization of cortical pyramidal neurons. Experimental neurology. 222(1):51-8. Pubmed: 20025874 DOI:10.1016/j.expneurol.2009.12.007 Kishi N, Macklis JD. 2010. MeCP2 functions largely cell-autonomously, but also non-cell-autonomously, in neuronal maturation and dendritic arborization of cortical pyramidal neurons. Experimental neurology. 222(1):51-8. Pubmed: 20025874 DOI:10.1016/j.expneurol.2009.12.007 Rett syndrome is a human neurodevelopmental disorder presenting almost exclusively in female infants; it is the second most common cause of mental retardation in girls, after Down's syndrome. The identification in 1999 that mutation of the methyl-CpG-binding protein 2 (MECP2) gene on the X chromosome causes Rett syndrome has led to a rapid increase in understanding of the neurobiological basis of the disorder. However, much about the functional role of MeCP2, and the cellular phenotype of both patients with Rett syndrome and mutant Mecp2 mouse models, remains unclear. Building on prior work in which we demonstrated that cortical layer 2/3 pyramidal neurons (primarily interhemispheric "callosal projection neurons" (CPN)) have reduced dendritic complexity and smaller somata in Mecp2-null mice, here we investigate whether Mecp2 loss-of-function affects neuronal maturation cell-autonomously and/or non-cell-autonomously by creating physical chimeras. We transplanted Mecp2-null or wild-type (wt) E17-18 cortical neuroblasts and immature neurons from mice constitutively expressing enhanced green fluorescent protein (eGFP) into wt P2-3 mouse cortices to generate chimeric cortices. Mecp2-null layer 2/3 pyramidal neurons in both Mecp2-null and wt neonatal cortices exhibit equivalent reduction in dendritic complexity, and are smaller than transplanted wt neurons, independent of recipient environment. These results indicate that the phenotype of Mecp2-null pyramidal neurons results largely from cell-autonomous mechanisms, with additional non-cell-autonomous effects. Dysregulation of MeCP2 target genes in individual neuronal populations such as CPN is likely centrally involved in Rett syndrome pathogenesis. Our results indicating MeCP2 function in the centrally affected projection neuron population of CPN themselves provide a foundation and motivation for identification of transcriptionally regulated MeCP2 target genes in developing CPN.Copyright 2009 Elsevier Inc. All rights reserved. 2009
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Azim E, Jabaudon D, Fame RM, Macklis JD. 2009. SOX6 controls dorsal progenitor identity and interneuron diversity during neocortical development. Nature neuroscience. 12(10):1238-47. Pubmed: 19657336 DOI:10.1038/nn.2387 Azim E, Jabaudon D, Fame RM, Macklis JD. 2009. SOX6 controls dorsal progenitor identity and interneuron diversity during neocortical development. Nature neuroscience. 12(10):1238-47. Pubmed: 19657336 DOI:10.1038/nn.2387 The neuronal diversity of the CNS emerges largely from controlled spatial and temporal segregation of cell type-specific molecular regulators. We found that the transcription factor SOX6 controls the molecular segregation of dorsal (pallial) from ventral (subpallial) telencephalic progenitors and the differentiation of cortical interneurons, regulating forebrain progenitor and interneuron heterogeneity. During corticogenesis in mice, SOX6 and SOX5 were largely mutually exclusively expressed in pallial and subpallial progenitors, respectively, and remained mutually exclusive in a reverse pattern in postmitotic neuronal progeny. Loss of SOX6 from pallial progenitors caused their inappropriate expression of normally subpallium-restricted developmental controls, conferring mixed dorsal-ventral identity. In postmitotic cortical interneurons, loss of SOX6 disrupted the differentiation and diversity of cortical interneuron subtypes, analogous to SOX5 control over cortical projection neuron development. These data indicate that SOX6 is a central regulator of both progenitor and cortical interneuron diversity during neocortical development. -
Molyneaux BJ, Arlotta P, Fame RM, MacDonald JL, MacQuarrie KL, Macklis JD. 2009. Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 29(39):12343-54. Pubmed: 19793993 DOI:10.1523/JNEUROSCI.6108-08.2009 Molyneaux BJ, Arlotta P, Fame RM, MacDonald JL, MacQuarrie KL, Macklis JD. 2009. Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 29(39):12343-54. Pubmed: 19793993 DOI:10.1523/JNEUROSCI.6108-08.2009 Little is known about the molecular development and heterogeneity of callosal projection neurons (CPN), cortical commissural neurons that connect homotopic regions of the two cerebral hemispheres via the corpus callosum and that are critical for bilateral integration of cortical information. Here we report on the identification of a series of genes that individually and in combination define CPN and novel CPN subpopulations during embryonic and postnatal development. We used in situ hybridization analysis, immunocytochemistry, and retrograde labeling to define the layer-specific and neuron-type-specific distribution of these newly identified CPN genes across different stages of maturation. We demonstrate that a subset of these genes (e.g., Hspb3 and Lpl) appear specific to all CPN (in layers II/III and V-VI), whereas others (e.g., Nectin-3, Plexin-D1, and Dkk3) discriminate between CPN of the deep layers and those of the upper layers. Furthermore, the data show that several genes finely subdivide CPN within individual layers and appear to label CPN subpopulations that have not been described previously using anatomical or morphological criteria. The genes identified here likely reflect the existence of distinct programs of gene expression governing the development, maturation, and function of the newly identified subpopulations of CPN. Together, these data define the first set of genes that identify and molecularly subcategorize distinct populations of callosal projection neurons, often located in distinct subdivisions of the canonical cortical laminae. -
Azim E, Shnider SJ, Cederquist GY, Sohur US, Macklis JD. 2009. Lmo4 and Clim1 progressively delineate cortical projection neuron subtypes during development. Cerebral cortex (New York, N.Y. : 1991). 19 Suppl 1(Suppl 1):i62-9. Pubmed: 19366868 DOI:10.1093/cercor/bhp030 Azim E, Shnider SJ, Cederquist GY, Sohur US, Macklis JD. 2009. Lmo4 and Clim1 progressively delineate cortical projection neuron subtypes during development. Cerebral cortex (New York, N.Y. : 1991). 19 Suppl 1(Suppl 1):i62-9. Pubmed: 19366868 DOI:10.1093/cercor/bhp030 Molecular controls over the development of the exceptional neuronal subtype diversity of the cerebral cortex are now beginning to be identified. The initial subtype fate decision early in the life of a neuron, and the malleability of this fate when the balance of key postmitotic signals is modified, reveals not only that a neuron is deterministically set on a general developmental path at its birth, but also that this program must be precisely executed during postmitotic differentiation. Here, we show that callosal projection neurons (CPN) and subcerebral projection neurons (subcerebral PN) in layer V of the neocortex share aspects of molecular identity after their birth that are progressively resolved during differentiation. The LIM-homeodomain-related genes Lmo4 and Clim1 are initially expressed by both CPN and subcerebral PN in layer V, and only during mid to late differentiation does expression of Lmo4 and Clim1 become largely segregated into distinct neuronal subtypes. This progressive postmitotic resolution of molecular identity reveals similarities and possibly shared evolutionary origin between layer V CPN and subcerebral PN, and provides insight into how and when these neuronal subtypes achieve their distinct identities during cortical development. 2008
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Joshi PS, Molyneaux BJ, Feng L, Xie X, Macklis JD, Gan L. 2008. Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron. 60(2):258-72. Pubmed: 18957218 DOI:10.1016/j.neuron.2008.08.006 Joshi PS, Molyneaux BJ, Feng L, Xie X, Macklis JD, Gan L. 2008. Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron. 60(2):258-72. Pubmed: 18957218 DOI:10.1016/j.neuron.2008.08.006 While progenitor-restricted factors broadly specify area identities in developing neocortex, the downstream regulatory elements involved in acquisition of those identities in postmitotic neurons are largely unknown. Here, we identify Bhlhb5, a transcription factor expressed in layers II-V, as a postmitotic regulator of area identity. Bhlhb5 is initially expressed in a high caudomedial to low rostrolateral gradient that transforms into a sharp border between sensory and rostral motor cortices. Bhlhb5 null mice exhibit aberrant expression of area-specific genes and structural organization in the somatosensory and caudal motor cortices. In somatosensory cortex, Bhlhb5 null mice display postsynaptic disorganization of vibrissal barrels. In caudal motor cortex, Bhlhb5 null mice exhibit anomalous differentiation of corticospinal motor neurons, accompanied by failure of corticospinal tract formation. Together, these results demonstrate Bhlhb5's function as an area-specific transcription factor that regulates the postmitotic acquisition of area identities and elucidate the genetic hierarchy between progenitors and postmitotic neurons driving neocortical arealization. -
Magavi SS, Macklis JD. 2008. Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo. Methods in molecular biology (Clifton, N.J.). 438:335-43. Pubmed: 18369768 DOI:10.1007/978-1-59745-133-8_25 Magavi SS, Macklis JD. 2008. Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo. Methods in molecular biology (Clifton, N.J.). 438:335-43. Pubmed: 18369768 DOI:10.1007/978-1-59745-133-8_25 Bromodeoxyuridine, variously abbreviated as BrdU, BudR, and BrdUrd, is a halogenated thymidine analog that is permanently integrated into the DNA of dividing cells during DNA synthesis in S phase. BrdU can be immunocytochemically detected in vitro and in vivo, allowing the identification of cells that were dividing the period of BrdU exposure. In vivo, it has been used to identify the "birthdate" of cells during development, to examine the fate of postnatally generated cells, and to label cells before transplantation, for subsequent identification. -
Catapano LA, Magavi SS, Macklis JD. 2008. Neuroanatomical tracing of neuronal projections with Fluoro-Gold. Methods in molecular biology (Clifton, N.J.). 438:353-9. Pubmed: 18369770 DOI:10.1007/978-1-59745-133-8_27 Catapano LA, Magavi SS, Macklis JD. 2008. Neuroanatomical tracing of neuronal projections with Fluoro-Gold. Methods in molecular biology (Clifton, N.J.). 438:353-9. Pubmed: 18369770 DOI:10.1007/978-1-59745-133-8_27 The study of neuronal connectivity requires the ability to trace axons from the neuronal cell body to its axon terminal (anterograde tracing) and from the terminal back to the soma (retrograde tracing). Such neuroanatomical tracing is frequently used to identify neurons on the basis of their pre- or postsynaptic connections. Neuroanatomical tracing has become particularly important in nervous system regeneration and repair, allowing investigators to follow the axon projections of newly born, transplanted, or axotomized neurons in lesioned or neurodegenerative environments. To allow further study of neurons identified and labeled in this way, it is particularly important that tracers are compatible with other tissue processing such as immunocytochemistry. Fluoro-Gold (Fluorochrome Inc., Denver CO) is one such highly flexible fluorescent retrograde marker commonly used for neuronal labeling and neuroanatomical tracing. -
Magavi SS, Macklis JD. 2008. Immunocytochemical analysis of neuronal differentiation. Methods in molecular biology (Clifton, N.J.). 438:345-52. Pubmed: 18369769 DOI:10.1007/978-1-59745-133-8_26 Magavi SS, Macklis JD. 2008. Immunocytochemical analysis of neuronal differentiation. Methods in molecular biology (Clifton, N.J.). 438:345-52. Pubmed: 18369769 DOI:10.1007/978-1-59745-133-8_26 Fully understanding the phenotype of neurons in vivo involves examining their morphology, immunocytochemically analyzing their protein expression, examining their afferent and efferent integration into neuronal microcircuitry, and functionally examining their activity. This task is significantly more difficult when you are attempting to determine whether multipotent precursor cells, often referred to as stem cells, differentiate into neurons in vivo. Transplanted or endogenous precursor cells often produce relatively small numbers of new neurons in the adult brain, making electron microscopy or electrophysiological analysis extremely challenging, and functional analysis difficult. Studying such cells usually depends heavily on immunocytochemical approaches. We review a range of immunocytochemical techniques for identifying whether transplanted or endogenous neural precursors have differentiated into mature neurons. We provide immunocytochemical protocols for the migratory neuronal marker Doublecortin (Dcx), the early expressed marker Hu, and mature neuronal marker NeuN. In Chapters 25 and 27 of Part IV, we provide protocols for identifying newborn cells by using the mitotic label bromodeoxyuridine and for examining axonal projections by using the retrograde label FluoroGold. -
Arlotta P, Molyneaux BJ, Jabaudon D, Yoshida Y, Macklis JD. 2008. Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum. The Journal of neuroscience : the official journal of the Society for Neuroscience. 28(3):622-32. Pubmed: 18199763 DOI:10.1523/JNEUROSCI.2986-07.2008 Arlotta P, Molyneaux BJ, Jabaudon D, Yoshida Y, Macklis JD. 2008. Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum. The Journal of neuroscience : the official journal of the Society for Neuroscience. 28(3):622-32. Pubmed: 18199763 DOI:10.1523/JNEUROSCI.2986-07.2008 Striatal medium spiny neurons (MSN) are critically involved in motor control, and their degeneration is a principal component of Huntington's disease. We find that the transcription factor Ctip2 (also known as Bcl11b) is central to MSN differentiation and striatal development. Within the striatum, it is expressed by all MSN, although it is excluded from essentially all striatal interneurons. In the absence of Ctip2, MSN do not fully differentiate, as demonstrated by dramatically reduced expression of a large number of MSN markers, including DARPP-32, FOXP1, Chrm4, Reelin, MOR1 (mu-opioid receptor 1), glutamate receptor 1, and Plexin-D1. Furthermore, MSN fail to aggregate into patches, resulting in severely disrupted patch-matrix organization within the striatum. Finally, heterotopic cellular aggregates invade the Ctip2-/- striatum, suggesting a failure by MSN to repel these cells in the absence of Ctip2. This is associated with abnormal dopaminergic innervation of the mutant striatum and dramatic changes in gene expression, including dysregulation of molecules involved in cellular repulsion. Together, these data indicate that Ctip2 is a critical regulator of MSN differentiation, striatal patch development, and the establishment of the cellular architecture of the striatum. -
Lai T, Jabaudon D, Molyneaux BJ, Azim E, Arlotta P, Menezes JR, Macklis JD. 2008. SOX5 controls the sequential generation of distinct corticofugal neuron subtypes. Neuron. 57(2):232-47. Pubmed: 18215621 DOI:10.1016/j.neuron.2007.12.023 Lai T, Jabaudon D, Molyneaux BJ, Azim E, Arlotta P, Menezes JR, Macklis JD. 2008. SOX5 controls the sequential generation of distinct corticofugal neuron subtypes. Neuron. 57(2):232-47. Pubmed: 18215621 DOI:10.1016/j.neuron.2007.12.023 The molecular mechanisms controlling the development of distinct subtypes of neocortical projection neurons, and CNS neuronal diversity more broadly, are only now emerging. We report that the transcription factor SOX5 controls the sequential generation of distinct corticofugal neuron subtypes by preventing premature emergence of normally later-born corticofugal neurons. SOX5 loss-of-function causes striking overlap of the identities of the three principal sequentially born corticofugal neuron subtypes: subplate neurons, corticothalamic neurons, and subcerebral projection neurons. In Sox5(-/-) cortex, subplate neurons aberrantly develop molecular hallmarks and connectivity of subcerebral projection neurons; corticothalamic neurons are imprecisely differentiated, while differentiation of subcerebral projection neurons is accelerated. Gain-of-function analysis reinforces the critical role of SOX5 in controlling the sequential generation of corticofugal neurons--SOX5 overexpression at late stages of corticogenesis causes re-emergence of neurons with corticofugal features. These data indicate that SOX5 controls the timing of critical fate decisions during corticofugal neuron production and thus subtype-specific differentiation and neocortical neuron diversity. 2007
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Breunig JJ, Arellano JI, Macklis JD, Rakic P. 2007. Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses. Cell stem cell. 1(6):612-27. Pubmed: 18371403 DOI:10.1016/j.stem.2007.11.008 Breunig JJ, Arellano JI, Macklis JD, Rakic P. 2007. Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses. Cell stem cell. 1(6):612-27. Pubmed: 18371403 DOI:10.1016/j.stem.2007.11.008 Adult neurogenesis research has made enormous strides in the last decade but has been complicated by several failures to replicate promising findings. Prevalent use of highly sensitive methods with inherent sources of error has led to extraordinary conclusions without adequate crossvalidation. Perhaps the biggest culprit is the reliance on molecules involved in DNA synthesis and genetic markers to indicate neuronal neogenesis. In this Protocol Review, we present an overview of common methodological issues in the field and suggest alternative approaches, including viral vectors, siRNA, and inducible transgenic/knockout mice. A multipronged approach will enhance the overall rigor of research on stem cell biology and related fields by allowing increased replication of findings between groups and across systems. -
Gao X, Arlotta P, Macklis JD, Chen J. 2007. Conditional knock-out of beta-catenin in postnatal-born dentate gyrus granule neurons results in dendritic malformation. The Journal of neuroscience : the official journal of the Society for Neuroscience. 27(52):14317-25. Pubmed: 18160639 Gao X, Arlotta P, Macklis JD, Chen J. 2007. Conditional knock-out of beta-catenin in postnatal-born dentate gyrus granule neurons results in dendritic malformation. The Journal of neuroscience : the official journal of the Society for Neuroscience. 27(52):14317-25. Pubmed: 18160639 Neurons are continuously added to the brain throughout life, and these neurons must develop dendritic arbors and functional connections with existing neurons to be integrated into neuronal circuitry. The molecular mechanisms that regulate dendritic development of newborn neurons in the hippocampal dentate gyrus are still unclear. Here, we show that beta-catenin is expressed in newborn granule neurons and in neural progenitor cells in the hippocampal dentate gyrus. Specific knock-out of beta-catenin in newborn neurons, without affecting beta-catenin expression in neural progenitor cells, led to defects in dendritic morphology of these newborn neurons in vivo. Majority of newborn neurons that cannot extend dendrites survive <1 month after they were born. Our results indicate that beta-catenin plays an important role in dendritic development of postnatal-born neurons in vivo, and is therefore essential for the neurogenesis in the postnatal brain. -
Molyneaux BJ, Arlotta P, Menezes JR, Macklis JD. 2007. Neuronal subtype specification in the cerebral cortex. Nature reviews. Neuroscience. 8(6):427-37. Pubmed: 17514196 Molyneaux BJ, Arlotta P, Menezes JR, Macklis JD. 2007. Neuronal subtype specification in the cerebral cortex. Nature reviews. Neuroscience. 8(6):427-37. Pubmed: 17514196 In recent years, tremendous progress has been made in understanding the mechanisms underlying the specification of projection neurons within the mammalian neocortex. New experimental approaches have made it possible to identify progenitors and study the lineage relationships of different neocortical projection neurons. An expanding set of genes with layer and neuronal subtype specificity have been identified within the neocortex, and their function during projection neuron development is starting to be elucidated. Here, we assess recent data regarding the nature of neocortical progenitors, review the roles of individual genes in projection neuron specification and discuss the implications for progenitor plasticity. -
Molyneaux BJ, Arlotta P, Macklis JD. 2007. Molecular development of corticospinal motor neuron circuitry. Novartis Foundation symposium. 288:3-15; discussion 15-20, 96-8. Pubmed: 18494249 Molyneaux BJ, Arlotta P, Macklis JD. 2007. Molecular development of corticospinal motor neuron circuitry. Novartis Foundation symposium. 288:3-15; discussion 15-20, 96-8. Pubmed: 18494249 The organization, function and evolution of the brain depends centrally on the precise development of a wide diversity of distinct neuronal subtypes. Furthermore, given the heterogeneity of neuronal subtypes within the CNS and the complexity of their connections, attempts to functionally repair circuitry will require a detailed understanding of the molecular controls over differentiation, connectivity and survival of specific lineages. Toward these goals, we recently identified developmentally regulated transcriptional programmes for specific lineages of long-distance neocortical projection neurons as they develop in vivo (in particular, for corticospinal motor neurons; CSMN). We purified CSMN, a clinically important population of neocortical projection neurons, at distinct stages of development in vivo, and compared their gene expression to that of two other pure populations of neocortical projection neurons. We identified novel and largely uncharacterized genes that are instructive for CSMN development and implicated in key developmental processes. These include Fezf2 (also known as Fezl), a regulator of subcerebral projection neuron identity, and Ctip2 (also known as Bcl1b), a regulator of the fasciculation, outgrowth and pathfinding of CSMN axonal projections to the spinal cord. Loss-of-function and gain-of-function analysis for multiple identified genes reveal programmes of combinatorial molecular controls over the precise development of key neocortical and other forebrain projection neuron populations that elucidate organization and function of the forebrain, and that might be manipulated toward functional cellular repair of complex brain circuitry. 2006
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Sohur US, Emsley JG, Mitchell BD, Macklis JD. 2006. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 361(1473):1477-97. Pubmed: 16939970 Sohur US, Emsley JG, Mitchell BD, Macklis JD. 2006. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 361(1473):1477-97. Pubmed: 16939970 Recent work in neuroscience has shown that the adult central nervous system (CNS) contains neural progenitors, precursors and stem cells that are capable of generating new neurons, astrocytes and oligodendrocytes. While challenging the previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them the future possibilities for development of novel neural repair strategies. The purpose of this review is to present the current knowledge about constitutively occurring adult mammalian neurogenesis, highlight the critical differences between 'neurogenic' and 'non-neurogenic' regions in the adult brain, and describe the cardinal features of two well-described neurogenic regions-the subventricular zone/olfactory bulb system and the dentate gyrus of the hippocampus. We also provide an overview of presently used models for studying neural precursors in vitro, mention some precursor transplantation models and emphasize that, in this rapidly growing field of neuroscience, one must be cautious with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims towards molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what might be the function of newly generated neurons in the adult brain, and provide a summary of present thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease. -
Ozdinler PH, Macklis JD. 2006. IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nature neuroscience. 9(11):1371-81. Pubmed: 17057708 Ozdinler PH, Macklis JD. 2006. IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nature neuroscience. 9(11):1371-81. Pubmed: 17057708 Corticospinal motor neurons (CSMN) are among the most complex CNS neurons; they control voluntary motor function and are prototypical projection neurons. In amyotrophic lateral sclerosis (ALS), both spinal motor neurons and CSMN degenerate; their damage contributes centrally to the loss of motor function in spinal cord injury. Direct investigation of CSMN is severely limited by inaccessibility in the heterogeneous cortex. Here, using new CSMN purification and culture approaches, and in vivo analyses, we report that insulin-like growth factor-1 (IGF-I) specifically enhances the extent and rate of murine CSMN axon outgrowth, mediated via the IGF-I receptor and downstream signaling pathways; this is distinct from IGF-I support of neuronal survival. In contrast, brain-derived neurotrophic factor (BDNF) enhances branching and arborization, but not axon outgrowth. These experiments define specific controls over directed differentiation of CSMN, indicate a distinct role of IGF-I in CSMN axon outgrowth during development, and might enable control over CSMN derived from neural precursors. -
Steele AD, Emsley JG, Ozdinler PH, Lindquist S, Macklis JD. 2006. Prion protein (PrPc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis. Proceedings of the National Academy of Sciences of the United States of America. 103(9):3416-21. Pubmed: 16492732 Steele AD, Emsley JG, Ozdinler PH, Lindquist S, Macklis JD. 2006. Prion protein (PrPc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis. Proceedings of the National Academy of Sciences of the United States of America. 103(9):3416-21. Pubmed: 16492732 The misfolding of the prion protein (PrP(c)) is a central event in prion diseases, yet the normal function of PrP(c) remains unknown. PrP(c) has putative roles in many cellular processes including signaling, survival, adhesion, and differentiation. Given the abundance of PrP(c) in the developing and mature mammalian CNS, we investigated the role of PrP(c) in neural development and in adult neurogenesis, which occurs constitutively in the dentate gyrus (DG) of the hippocampus and in the olfactory bulb from precursors in the subventricular zone (SVZ)/rostral migratory stream. In vivo, we find that PrP(c) is expressed immediately adjacent to the proliferative region of the SVZ but not in mitotic cells. In vivo and in vitro studies further find that PrP(c) is expressed in multipotent neural precursors and mature neurons but is not detectable in glia. Loss- and gain-of-function experiments demonstrate that PrP(c) levels correlate with differentiation of multipotent neural precursors into mature neurons in vitro and that PrP(c) levels positively influence neuronal differentiation in a dose-dependent manner. PrP(c) also increases cellular proliferation in vivo; in the SVZ, PrP(c) overexpresser (OE) mice have more proliferating cells compared with wild-type (WT) or knockout (KO) mice; in the DG, PrP(c) OE and WT mice have more proliferating cells compared with KO mice. Our results demonstrate that PrP(c) plays an important role in neurogenesis and differentiation. Because the final number of neurons produced in the DG is unchanged by PrP(c) expression, other factors must control the ultimate fate of new neurons. -
Emsley JG, Macklis JD. 2006. Astroglial heterogeneity closely reflects the neuronal-defined anatomy of the adult murine CNS. Neuron glia biology. 2(3):175-86. Pubmed: 17356684 Emsley JG, Macklis JD. 2006. Astroglial heterogeneity closely reflects the neuronal-defined anatomy of the adult murine CNS. Neuron glia biology. 2(3):175-86. Pubmed: 17356684 Astroglia comprise an extremely morphologically diverse cell type that have crucial roles in neural development and function. Nonetheless, distinct regions of the CNS have traditionally been defined by the phenotypic characteristics and connectivity of neuros. In a complementary fashion, we present evidence that discrete regions of the adult CNS can be delineated based solely on the morphology, density and proliferation rates of astroglia. We used transgenic hGFAP-GFP mice in which robust expression of GFP in adult astroglia enables detailed morphological characterization of this diversely heterogeneous cell population with 3D confocal microscopy. By using three complementary methods for labeling adult astroglia (hGFAP-GFP expression, and GFAP and S100beta immunostaining), we find that there is a remarkably diverse, regionally stereotypical array of astroglial morphology throughout the CNS, and that discrete anatomical regions can be defined solely on the morphology of astroglia within that region. Second, we find that the density of astroglia varies dramatically across the CNS, and that astroglial density effectively delineates even the sub-regions of complex structures, such as the thalamus. We also find that regional astroglial density varies depending on how astroglia are labeled. To quantify and illustrate these broad differences in astroglial density, we generated an anatomical density atlas of the CNS. Third, the proliferation rate, or mitotic index, of astroglia in the adult CNS also effectively defines anatomical regions. These differences are present regardless of the astroglial-labeling method used. To supplement our atlas of astroglial density we generated an atlas of proliferation density for the adult CNS. Together, these studies demonstrate that the morphology, density and proliferation rate of astroglia can independently define the discrete cytoarchitecture of the adult mammalian CNS, and support the concept that regional astroglial heterogeneity reflects important molecular and functional differences between distinct classes of astroglia, much like the long-accepted heterogeneity of neuronal populations. 2005
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Molyneaux BJ, Arlotta P, Hirata T, Hibi M, Macklis JD. 2005. Fezl is required for the birth and specification of corticospinal motor neurons. Neuron. 47(6):817-31. Pubmed: 16157277 Molyneaux BJ, Arlotta P, Hirata T, Hibi M, Macklis JD. 2005. Fezl is required for the birth and specification of corticospinal motor neurons. Neuron. 47(6):817-31. Pubmed: 16157277 The molecular mechanisms controlling the differentiation of neural progenitors into distinct subtypes of neurons during neocortical development are unknown. Here, we report that Fezl is required for the specification of corticospinal motor neurons and other subcerebral projection neurons, which are absent from Fezl null mutant neocortex. There is neither an increase in cell death in Fezl(-/-) cortex nor abnormalities in migration, indicating that the absence of subcerebral projection neurons is due to a failure in fate specification. In striking contrast, other neuronal populations in the same and other cortical layers are born normally. Overexpression of Fezl results in excess production of subcerebral projection neurons and arrested migration of these neurons in the germinal zone. These data indicate that Fezl plays a central role in the specification of corticospinal motor neurons and other subcerebral projection neurons, controlling early decisions regarding lineage-specific differentiation from neural progenitors. -
Arlotta P, Macklis JD. 2005. Archeo-cell biology: carbon dating is not just for pots and dinosaurs. Cell. 122(1):4-6. Pubmed: 16009125 Arlotta P, Macklis JD. 2005. Archeo-cell biology: carbon dating is not just for pots and dinosaurs. Cell. 122(1):4-6. Pubmed: 16009125 Defining the life span of specific human cell populations is limited by our inability to mark the exact time when cells are born in a way that can be detected over many years. In this issue of Cell, Spalding et al. (2005) describe a clever strategy for retrospectively birth dating human cells in vivo, based on their incorporation of 14C during a peak in atmospheric levels of this isotope resulting from above-ground nuclear arms testing in the 1950s. -
Arlotta P, Molyneaux BJ, Chen J, Inoue J, Kominami R, Macklis JD. 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron. 45(2):207-21. Pubmed: 15664173 Arlotta P, Molyneaux BJ, Chen J, Inoue J, Kominami R, Macklis JD. 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron. 45(2):207-21. Pubmed: 15664173 Within the vertebrate nervous system, the presence of many different lineages of neurons and glia complicates the molecular characterization of single neuronal populations. In order to elucidate molecular mechanisms underlying the specification and development of corticospinal motor neurons (CSMN), we purified CSMN at distinct stages of development in vivo and compared their gene expression to two other pure populations of cortical projection neurons: callosal projection neurons and corticotectal projection neurons. We found genes that are potentially instructive for CSMN development, as well as genes that are excluded from CSMN and are restricted to other populations of neurons, even within the same cortical layer. Loss-of-function experiments in null mutant mice for Ctip2 (also known as Bcl11b), one of the newly characterized genes, demonstrate that it plays a critical role in the development of CSMN axonal projections to the spinal cord in vivo, confirming that we identified central genetic determinants of the CSMN population. -
Emsley JG, Mitchell BD, Kempermann G, Macklis JD. 2005. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Progress in neurobiology. 75(5):321-41. Pubmed: 15913880 Emsley JG, Mitchell BD, Kempermann G, Macklis JD. 2005. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Progress in neurobiology. 75(5):321-41. Pubmed: 15913880 Recent work in neuroscience has shown that the adult central nervous system contains neural progenitors, precursors, and stem cells that are capable of generating new neurons, astrocytes, and oligodendrocytes. While challenging previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them future possibilities for the development of novel neural repair strategies. The purpose of this review is to present current knowledge about constitutively occurring adult mammalian neurogenesis, to highlight the critical differences between "neurogenic" and "non-neurogenic" regions in the adult brain, and to describe the cardinal features of two well-described neurogenic regions-the subventricular zone/olfactory bulb system, and the dentate gyrus of the hippocampus. We also provide an overview of currently used models for studying neural precursors in vitro, mention some precursor transplantation models, and emphasize that, in this rapidly growing field of neuroscience, one must take caution with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims toward molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what the function might be of newly generated neurons in the adult brain and provide a summary of current thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease. -
Magavi SS, Mitchell BD, Szentirmai O, Carter BS, Macklis JD. 2005. Adult-born and preexisting olfactory granule neurons undergo distinct experience-dependent modifications of their olfactory responses in vivo. The Journal of neuroscience : the official journal of the Society for Neuroscience. 25(46):10729-39. Pubmed: 16291946 Magavi SS, Mitchell BD, Szentirmai O, Carter BS, Macklis JD. 2005. Adult-born and preexisting olfactory granule neurons undergo distinct experience-dependent modifications of their olfactory responses in vivo. The Journal of neuroscience : the official journal of the Society for Neuroscience. 25(46):10729-39. Pubmed: 16291946 Neurogenesis continues throughout adulthood in the mammalian olfactory bulb and hippocampal dentate gyrus, suggesting the hypothesis that recently generated, adult-born neurons contribute to neural plasticity and learning. To explore this hypothesis, we examined whether olfactory experience modifies the responses of adult-born neurons to odorants, using immediate early genes (IEGs) to assay the response of olfactory granule neurons. We find that, shortly after they differentiate and synaptically integrate, the population of adult-born olfactory granule neurons has a greater population IEG response to novel odors than mature, preexisting neurons. Familiarizing mice with test odors increases the response of the recently incorporated adult-born neuron population to the test odors, and this increased responsiveness is long lasting, demonstrating that the response of the adult-born neuron population is altered by experience. In contrast, familiarizing mice with test odors decreases the IEG response of developmentally generated neurons, suggesting that recently generated adult-born neurons play a distinct role in olfactory processing. The increased IEG response is stimulus specific; familiarizing mice with a set of different, "distractor" odors does not increase the adult-born neuron population response to the test odors. Odor familiarization does not influence the survival of adult-born neurons, indicating that the changes in the population response of adult-born neurons are not attributable to increased survival of odor-stimulated neurons. These results demonstrate that recently generated adult-born olfactory granule neurons and older, preexisting granule neurons undergo contrasting experience-dependent modifications in vivo and support the hypothesis that adult-born neurons are involved in olfactory learning. -
Mitchell BD, Macklis JD. 2005. Large-scale maintenance of dual projections by callosal and frontal cortical projection neurons in adult mice. The Journal of comparative neurology. 482(1):17-32. Pubmed: 15612019 Mitchell BD, Macklis JD. 2005. Large-scale maintenance of dual projections by callosal and frontal cortical projection neurons in adult mice. The Journal of comparative neurology. 482(1):17-32. Pubmed: 15612019 Integration of sensory-motor information in premotor cortex of rodents occurs largely through callosal and frontal cortical association projections directed in a hierarchically organized manner. Although most anatomical studies in rodents have been performed in rats, mammalian genetic models have focused on mice, because of their successful manipulation on the genetic and cell biological levels. It is therefore important to establish the normal patterns of anatomical connectivity in mice, which potentially differ from those in rats. The goal of this study is to investigate the anatomical development of callosal and frontal premotor projection neurons (CPN and FPN, respectively) in mouse sensory-motor and premotor cortex and to investigate quantitatively the potential laminar differences between these neurons with simultaneous callosal and frontal projections during development. The retrograde tracers Fluoro-Gold and DiI were injected into sensory-motor and premotor cortices, respectively, C57Bl/6 mice at different developmental times (P2, P8, P21, adult). We found that, in contrast to the case in primate and cat, there is widespread overlap in populations of long-distance projection neurons in mice; many projection neurons have simultaneous projections to both contralateral somatosensory cortex and ipsilateral frontal cortex, and a considerable number of these dual projections persist into adulthood. In addition, there are significant laminar differences in the percentage of neurons with simultaneous callosal and frontal projections, and an isolated population of layer V FPN has bilateral projections to both premotor cortical hemispheres. Taken together, our results indicate that a large proportion of individual projection neurons maintains simultaneous callosal and frontal projections in adult mice, suggesting that these dual projections might serve the critical function of integrating motor coordination information with multimodal association areas.2004 Wiley-Liss, Inc. -
Kishi N, Macklis JD. 2005. Dissecting MECP2 function in the central nervous system. Journal of child neurology. 20(9):753-9. Pubmed: 16225831 Kishi N, Macklis JD. 2005. Dissecting MECP2 function in the central nervous system. Journal of child neurology. 20(9):753-9. Pubmed: 16225831 Rett syndrome is a neurodevelopmental disorder and an important cause of mental retardation and autistic behavior in girls and in a small group of boys. In 1999, mutation of the methyl-CpG binding protein 2 (MECP2) gene encoding a transcriptional repressor on the X chromosome was found to cause Rett syndrome. Since this discovery, significant research has focused on the elucidation of its specific role in the central nervous system. Recent studies revealed that MECP2 is expressed in more differentiated neurons rather than in less differentiated neuroblasts and that MECP2 is involved in the maturation and maintenance of neurons, including dendritic arborization and axonal projections, rather than in early cell fate decisions in the mammalian brain. In this review, we summarize recent findings regarding regional, temporal, and cell type-specific MECP2 expression in the central nervous system; neurobiologic abnormalities in MECP2 -mutant mice; and MECP2 target genes in the central nervous system. 2004
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Emsley JG, Mitchell BD, Magavi SS, Arlotta P, Macklis JD. 2004. The repair of complex neuronal circuitry by transplanted and endogenous precursors. NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics. 1(4):452-71. Pubmed: 15717047 Emsley JG, Mitchell BD, Magavi SS, Arlotta P, Macklis JD. 2004. The repair of complex neuronal circuitry by transplanted and endogenous precursors. NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics. 1(4):452-71. Pubmed: 15717047 During the past three decades, research exploring potential neuronal replacement therapies has focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain. However, in the last decade, the development of novel approaches has resulted in an explosion of new research showing that neurogenesis, the birth of new neurons, normally occurs in two limited and specific regions of the adult mammalian brain, and that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain. Recent advances in our understanding of related events of neural development and plasticity, including the role of radial glia in developmental neurogenesis, and the ability of endogenous precursors present in the adult brain to be induced to produce neurons and partially repopulate brain regions affected by neurodegenerative processes, have led to fundamental changes in the views about how the brain develops, as well as to approaches by which transplanted or endogenous precursors might be used to repair the adult brain. For example, recruitment of new neurons can be induced in a region-specific, layer-specific, and neuronal type-specific manner, and, in some cases, newly recruited neurons can form long-distance connections to appropriate targets. Elucidation of the relevant molecular controls may both allow control over transplanted precursor cells and potentially allow for the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that might not require transplantation of exogenous cells. -
Catapano LA, Arlotta P, Cage TA, Macklis JD. 2004. Stage-specific and opposing roles of BDNF, NT-3 and bFGF in differentiation of purified callosal projection neurons toward cellular repair of complex circuitry. The European journal of neuroscience. 19(9):2421-34. Pubmed: 15128396 Catapano LA, Arlotta P, Cage TA, Macklis JD. 2004. Stage-specific and opposing roles of BDNF, NT-3 and bFGF in differentiation of purified callosal projection neurons toward cellular repair of complex circuitry. The European journal of neuroscience. 19(9):2421-34. Pubmed: 15128396 Cellular repair of neuronal circuitry affected by neurodegenerative disease or injury may be approached in the adult neocortex via transplantation of neural precursors ("neural stem cells") or via molecular manipulation and recruitment of new neurons from endogenous precursors in situ. A major challenge for potential future approaches to neuronal replacement will be to specifically direct and control progressive differentiation, axonal projection and connectivity of neural precursors along a specific neuronal lineage. This goal will require a progressively more detailed understanding of the molecular controls over morphologic differentiation of specific neuronal lineages, including neurite outgrowth and elongation, in order to accurately permit and direct proper neuronal integration and connectivity. Here, we investigate controls over the morphologic differentiation of a specific prototypical lineage of cortical neurons: callosal projection neurons (CPN). We highly enriched CPN to an essentially pure population, and cultured them at three distinct stages of development from embryonic and postnatal mouse cortex by retrograde fluorescence labelling, followed by fluorescence-activated cell sorting. We find that specific peptide growth factors exert direct stage-specific positive and negative effects over the morphologic differentiation and process outgrowth of CPN. These effects are distinct from the effects of these growth factors on CPN survival [Catapano et al. (2001)J. Neurosci., 21, 8863-8872]. These data may be critical for the future goal of directing lineage-specific neuronal differentiation of transplanted or endogenous precursors/"stem cells" toward cellular repair of complex cortical circuitry. -
Mellough CB, Cui Q, Spalding KL, Symons NA, Pollett MA, Snyder EY, Macklis JD, Harvey AR. 2004. Fate of multipotent neural precursor cells transplanted into mouse retina selectively depleted of retinal ganglion cells. Experimental neurology. 186(1):6-19. Pubmed: 14980806 Mellough CB, Cui Q, Spalding KL, Symons NA, Pollett MA, Snyder EY, Macklis JD, Harvey AR. 2004. Fate of multipotent neural precursor cells transplanted into mouse retina selectively depleted of retinal ganglion cells. Experimental neurology. 186(1):6-19. Pubmed: 14980806 In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype. -
Kishi N, Macklis JD. 2004. MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Molecular and cellular neurosciences. 27(3):306-21. Pubmed: 15519245 Kishi N, Macklis JD. 2004. MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Molecular and cellular neurosciences. 27(3):306-21. Pubmed: 15519245 Rett syndrome is a neurodevelopmental disorder and one of the causes of mental retardation and autistic behavior in girls, as well as in a small group of boys. It was recently discovered that mutation of the methyl-CpG-binding protein 2 (MECP2) gene encoding a transcriptional repressor on the X chromosome causes Rett syndrome. Although it is evident that phenotypes of MECP2 mutant mice that resemble those of Rett syndrome are attributable to lack of the MECP2 gene in the central nervous system (CNS), there is little understanding of the neuropathological abnormalities in the CNS of MECP2-null mice. Here, we investigated the developmental regulation and specific cellular expression of MECP2 during neural development both in vitro and in vivo. MECP2 is expressed in mature neurons, but not in astroglia or oligodendroglia, and is increasingly expressed during development of the mouse neocortex. In addition, in vitro culture studies suggest that MECP2 is expressed in more differentiated neurons rather than in less differentiated neuroblasts. Under in vitro conditions using neural precursor cultures, we find that MECP2 mutant neural precursors differentiate into morphologically mature neurons and glia, and no significant differences in differentiation are detected between cells from wild-type and MECP2 mutant mice, suggesting that MECP2 may play a different role in mice than it does in Xenopus embryos. In agreement with this hypothesis, neocortical projection layers in MECP2 -/y mice are thinner than those in wild-type mice, and pyramidal neurons in layer II/III in MECP2 -/y mice are smaller and less complex than those in wild-type mice. Taken together, our results indicate that MECP2 is involved in the maturation and maintenance of neurons, including dendritic arborization, rather than in cell fate decisions. -
Chen J, Magavi SS, Macklis JD. 2004. Neurogenesis of corticospinal motor neurons extending spinal projections in adult mice. Proceedings of the National Academy of Sciences of the United States of America. 101(46):16357-62. Pubmed: 15534207 Chen J, Magavi SS, Macklis JD. 2004. Neurogenesis of corticospinal motor neurons extending spinal projections in adult mice. Proceedings of the National Academy of Sciences of the United States of America. 101(46):16357-62. Pubmed: 15534207 The adult mammalian CNS shows a very limited capacity to regenerate after injury. However, endogenous precursors, or stem cells, provide a potential source of new neurons in the adult brain. Here, we induce the birth of new corticospinal motor neurons (CSMN), the CNS neurons that die in motor neuron degenerative diseases, including amyotrophic lateral sclerosis, and that cause loss of motor function in spinal cord injury. We induced synchronous apoptotic degeneration of CSMN and examined the fates of newborn cells arising from endogenous precursors, using markers for DNA replication, neuroblast migration, and progressive neuronal differentiation, combined with retrograde labeling from the spinal cord. We observed neuroblasts entering the neocortex and progressively differentiating into mature pyramidal neurons in cortical layer V. We found 20-30 new neurons per mm(3) in experimental mice vs. 0 in controls. A subset of these newborn neurons projected axons into the spinal cord and survived >56 weeks. These results demonstrate that endogenous precursors can differentiate into even highly complex long-projection CSMN in the adult mammalian brain and send new projections to spinal cord targets, suggesting that molecular manipulation of endogenous neural precursors in situ may offer future therapeutic possibilities for motor neuron degenerative disease and spinal cord injury. -
Emsley JG, Arlotta P, Macklis JD. 2004. Star-cross'd neurons: astroglial effects on neural repair in the adult mammalian CNS. Trends in neurosciences. 27(5):238-40. Pubmed: 15111002 Emsley JG, Arlotta P, Macklis JD. 2004. Star-cross'd neurons: astroglial effects on neural repair in the adult mammalian CNS. Trends in neurosciences. 27(5):238-40. Pubmed: 15111002 Astroglia have long been thought to play merely a supporting role in the life of the neuron. However, these star-shaped cells have recently been the focus of intense study that has begun to emphasize remarkable and novel roles for these amazing cells. While astroglia play positive roles in the life of the neuron, they can simultaneously exert negative influences. Kinouchi et al. convincingly demonstrate and characterize an inhibitory role played by astroglia after neuronal transplantation. These findings remind us that astroglia exert positive and negative influences on neuronal survival, migration, neurite outgrowth and functional integration. Here, we review the complementary and often contradictory roles of astroglia during neuronal integration. -
Mitchell BD, Emsley JG, Magavi SS, Arlotta P, Macklis JD. 2004. Constitutive and induced neurogenesis in the adult mammalian brain: manipulation of endogenous precursors toward CNS repair. Developmental neuroscience. 26(2-4):101-17. Pubmed: 15711054 Mitchell BD, Emsley JG, Magavi SS, Arlotta P, Macklis JD. 2004. Constitutive and induced neurogenesis in the adult mammalian brain: manipulation of endogenous precursors toward CNS repair. Developmental neuroscience. 26(2-4):101-17. Pubmed: 15711054 Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. During the past 3 decades, research exploring potential neuronal replacement therapies has focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain. However, in the last decade, the development of new techniques has resulted in an explosion of new research showing that neurogenesis, the birth of new neurons, normally occurs in two limited and specific regions of the adult mammalian brain and that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain. Recent advances in our understanding of related events of neural development and plasticity, including the role of radial glia in developmental neurogenesis and the ability of endogenous precursors present in the adult brain to be induced to produce neurons and partially repopulate brain regions affected by neurodegenerative processes, have led to fundamental changes in the views about how the brain develops as well as to approaches by which endogenous precursors might be recruited to repair the adult brain. Recruitment of new neurons can be induced in a region-specific, layer-specific and neuronal-type-specific manner, and, in some cases, newly recruited neurons can form long-distance connections to appropriate targets. Elucidation of the relevant molecular controls may both allow control over transplanted precursor cells and potentially allow the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that do not require transplantation of exogenous cells.Copyright 2004 S. Karger AG, Basel. 2003
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Eyding D, Macklis JD, Neubacher U, Funke K, Wörgötter F. 2003. Selective elimination of corticogeniculate feedback abolishes the electroencephalogram dependence of primary visual cortical receptive fields and reduces their spatial specificity. The Journal of neuroscience : the official journal of the Society for Neuroscience. 23(18):7021-33. Pubmed: 12904463 Eyding D, Macklis JD, Neubacher U, Funke K, Wörgötter F. 2003. Selective elimination of corticogeniculate feedback abolishes the electroencephalogram dependence of primary visual cortical receptive fields and reduces their spatial specificity. The Journal of neuroscience : the official journal of the Society for Neuroscience. 23(18):7021-33. Pubmed: 12904463 The role of corticogeniculate feedback in the organization, function, and state dependence of visual responses and receptive fields (RFs) is not well understood. We investigated the contribution of the corticogeniculate loop to state-dependent changes of characteristics of the primary visual cortex response by using a novel approach of eliminating corticogeniculate projection neurons with targeted neuronal apoptosis. Experiments were performed in anesthetized cats (N2O plus halothane) with parallel recordings of single units from experimental (right) and control (left) hemispheres approximately 2 weeks after induction of apoptosis. Within the experimental hemispheres, neurons of area 17 and of the dorsal lateral geniculate nucleus (dLGN) showed an unusually enhanced and prolonged tonic visual response during episodes of synchronized (syn) EEG activity, whereas response levels during less synchronized states were almost normal. In addition, dLGN cells showed a reduced tendency for burst firing and a less regular spike interval distribution compared with those of controls. These changes are likely attributable to a tonic depolarization of dLGN relay neurons or, more likely, to a decreased responsiveness of thalamic inhibitory processes to cortical feedback. Cortical neurons also displayed an activity-dependent increase in RF size, in contrast to an almost activity-invariant RF size of controls, a phenomenon likely related to the elimination of collateral, intracortical projections of layer 6 neurons. Together, these results demonstrate that selective chronic elimination of corticogeniculate feedback results in the loss of EEG-correlated differences of visual processing in the remaining thalamocortical network, accompanied by a significant increase in excitability during syn EEG, at the expense of noticeably reduced spatial receptive-field specificity in the remaining cortical neurons. -
Arlotta P, Magavi SS, Macklis JD. 2003. Molecular manipulation of neural precursors in situ: induction of adult cortical neurogenesis. Experimental gerontology. 38(1-2):173-82. Pubmed: 12543275 Arlotta P, Magavi SS, Macklis JD. 2003. Molecular manipulation of neural precursors in situ: induction of adult cortical neurogenesis. Experimental gerontology. 38(1-2):173-82. Pubmed: 12543275 Over the past three decades, research exploring potential neuronal replacement therapies have focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain. Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. However, in the last decade, the development of new techniques has resulted in an explosion of new research showing that neurogenesis, the birth of new neurons, normally occurs in two limited and specific regions of the adult mammalian brain, and that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain. Recent findings from our lab demonstrate that it is possible to induce neurogenesis de novo in the adult mammalian brain, particularly in the neocortex where it does not normally occur, and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons. Recruitment of new neurons can be induced in a region-specific, layer-specific, and neuronal type-specific manner, and newly recruited neurons can form long-distance connections to appropriate targets. Elucidation of the relevant molecular controls may both allow control over transplanted precursor cells and potentially allow the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that do not require transplantation of exogenous cells. -
Arlotta P, Magavi SS, Macklis JD. 2003. Induction of adult neurogenesis: molecular manipulation of neural precursors in situ. Annals of the New York Academy of Sciences. 991:229-36. Pubmed: 12846990 Arlotta P, Magavi SS, Macklis JD. 2003. Induction of adult neurogenesis: molecular manipulation of neural precursors in situ. Annals of the New York Academy of Sciences. 991:229-36. Pubmed: 12846990 Over most of the past century, it was thought that the adult brain was completely incapable of generating new neurons. However, in the last decade, the development of new techniques has resulted in an explosion of new research showing that (i) neurogenesis, the birth of new neurons, is not restricted to embryonic development, but normally also occurs in two limited regions of the adult mammalian brain (the olfactory bulb and the dentate gyrus of the hippocampus); (ii) that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain; and (iii) that it is possible to induce neurogenesis even in regions of the adult mammalian brain, like the neocortex, where it does not normally occur, via manipulation of endogenous multipotent precursors in situ. In the neocortex, recruitment of small numbers of new neurons can be induced in a region-specific, layer-specific, and neuronal type-specific manner, and newly recruited neurons can form long-distance connections to appropriate targets. This suggests that elucidation of the relevant molecular controls over adult neurogenesis from endogenous neural precursors/stem cells may allow the development of neuronal replacement therapies for neurodegenerative disease and other central nervous system injuries that may not require transplantation of exogenous cells. 2002
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Magavi SS, Macklis JD. 2002. Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo. Methods in molecular biology (Clifton, N.J.). 198:283-90. Pubmed: 11951629 Magavi SS, Macklis JD. 2002. Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo. Methods in molecular biology (Clifton, N.J.). 198:283-90. Pubmed: 11951629 -
Magavi SS, Macklis JD. 2002. Immunocytochemical analysis of neuronal differentiation. Methods in molecular biology (Clifton, N.J.). 198:291-7. Pubmed: 11951631 Magavi SS, Macklis JD. 2002. Immunocytochemical analysis of neuronal differentiation. Methods in molecular biology (Clifton, N.J.). 198:291-7. Pubmed: 11951631 -
Wörgötter F, Eyding D, Macklis JD, Funke K. 2002. The influence of the corticothalamic projection on responses in thalamus and cortex. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 357(1428):1823-34. Pubmed: 12626015 Wörgötter F, Eyding D, Macklis JD, Funke K. 2002. The influence of the corticothalamic projection on responses in thalamus and cortex. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 357(1428):1823-34. Pubmed: 12626015 We review results on the in vivo properties of neurons in the dorsal lateral geniculate nucleus (dLGN) that receives its afferent input from the retina and projects to the visual cortex. In addition, the dLGN receives input from the brain stem and from a rather strong corticothalamic back-projection, which originates in layer 6 of the visual cortex. We compare the behaviour of dLGN cells during spontaneous changes of the frequency contents of the electroencephalograph (EEG) (which are mainly related to a changing brain stem influence), with those that are obtained when experimentally silencing the corticothalamic feedback. The spatial and temporal response properties of dLGN cells are compared during these two conditions, and we report that the neurons behave similarly during a synchronized EEG state and during inactive corticothalamic feedback. In both situations, dLGN cells are rather phasic and their remaining tonic activity is temporally dispersed, indicating a hyperpolarizing effect. By means of a novel method, we were able to chronically eliminate a large proportion of the corticothalamic projection neurons from the otherwise intact cortex. In this condition, we found that cortical cells also lose their EEG specific response differences but, in this instance, probably due to a facilitatory (depolarizing) plasticity reaction of the remaining network. -
Magavi SS, Macklis JD. 2002. Induction of neuronal type-specific neurogenesis in the cerebral cortex of adult mice: manipulation of neural precursors in situ. Brain research. Developmental brain research. 134(1-2):57-76. Pubmed: 11947937 Magavi SS, Macklis JD. 2002. Induction of neuronal type-specific neurogenesis in the cerebral cortex of adult mice: manipulation of neural precursors in situ. Brain research. Developmental brain research. 134(1-2):57-76. Pubmed: 11947937 Over the past 3 decades, research exploring potential neuronal replacement therapies have focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain [Nat. Neurosci. 3 (2000) 67-78]. Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. However, in the last decade, the development of new techniques has resulted in an explosion of new research showing that neurogenesis, the birth of new neurons, normally occurs in two limited and specific regions of the adult mammalian brain, and that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain [Mol. Cell. Neurosci. 19 (1999) 474-486]. Recent findings from our laboratory demonstrate that it is possible to induce neurogenesis de novo in the adult mammalian brain, particularly in the neocortex where it does not normally occur, and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons [Nature 405 (2000) 951-955; Neuron 25 (2000) 481-492]. Recruitment of new neurons can be induced in a region-specific, layer-specific, and neuronal type-specific manner, and newly recruited neurons can form long-distance connections to appropriate targets. Elucidation of the relevant molecular controls may both allow control over transplanted precursor cells and potentially allow the development of neuronal replacement therapies for neurodegenerative disease and other central nervous system injuries that do not require transplantation of exogenous cells. -
Catapano LA, Magavi SS, Macklis JD. 2002. Neuroanatomical tracing of neuronal projections with Fluoro-Gold. Methods in molecular biology (Clifton, N.J.). 198:299-304. Pubmed: 11951632 Catapano LA, Magavi SS, Macklis JD. 2002. Neuroanatomical tracing of neuronal projections with Fluoro-Gold. Methods in molecular biology (Clifton, N.J.). 198:299-304. Pubmed: 11951632 -
Fricker-Gates RA, Shin JJ, Tai CC, Catapano LA, Macklis JD. 2002. Late-stage immature neocortical neurons reconstruct interhemispheric connections and form synaptic contacts with increased efficiency in adult mouse cortex undergoing targeted neurodegeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 22(10):4045-56. Pubmed: 12019324 Fricker-Gates RA, Shin JJ, Tai CC, Catapano LA, Macklis JD. 2002. Late-stage immature neocortical neurons reconstruct interhemispheric connections and form synaptic contacts with increased efficiency in adult mouse cortex undergoing targeted neurodegeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 22(10):4045-56. Pubmed: 12019324 In the neocortex, the effectiveness of potential cellular repopulation therapies for diseases involving neuronal loss may depend critically on whether newly incorporated cells can differentiate appropriately into precisely the right kind of neuron, re-establish precise long-distance connections, and reconstruct complex functional circuitry. Here, we test the hypothesis that increased efficiency of connectivity could be achieved if precursors could be more fully differentiated toward desired phenotypes. We compared embryonic neuroblasts and immature murine neurons subregionally dissected from either embryonic day 17 (E17) (Shin et al., 2000) or E19 primary somatosensory (S1) cortex and postnatal day 3 (P3) purified callosal projection neurons (CPNs) with regard to neurotransmitter and receptor phenotype and afferent synapse formation after transplantation into adult mouse S1 cortex undergoing targeted apoptotic degeneration of layer II/III and V CPNs. Two weeks after transplantation, neurons from all developmental stages were found dispersed within layers II/III and V, many with morphological features typical of large pyramidal neurons. Retrograde labeling with FluoroGold revealed that 42 +/- 2% of transplanted E19 immature S1 neurons formed connections with the contralateral S1 cortex by 12 weeks after transplantation, compared with 23 +/- 7% of E17 neurons. A greater percentage of E19-derived neurons received synapses (77 +/- 1%) compared with E17-derived neurons (67 +/- 2%). Similar percentages of both E17 and E19 donor-derived neurons expressed neurotransmitters and receptors [glutamate, aspartate, GABA, GABA receptor (GABA-R), NMDA-R, AMPA-R, and kainate-R] appropriate for endogenous adult CPNs progressively over a period of 2-12 weeks after transplantation. Although P3 fluorescence-activated cell sorting-purified neurons also expressed these mature phenotypic markers after transplantation, their survival in vivo was poor. We conclude that later-stage and region-specific immature neurons develop a mature CPN phenotype and make appropriate connections with recipient circuitry with increased efficiency. However, at postnatal stages of development, limitations in survival outweigh this increased efficiency. These results suggest that efforts to direct the differentiation of earlier precursors precisely along specific desired neuronal lineages could potentially make possible the highly efficient reconstruction of complex neocortical and other CNS circuitry. 2001
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Magavi SS, Macklis JD. 2001. Manipulation of neural precursors in situ: induction of neurogenesis in the neocortex of adult mice. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 25(6):816-35. Pubmed: 11750176 Magavi SS, Macklis JD. 2001. Manipulation of neural precursors in situ: induction of neurogenesis in the neocortex of adult mice. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 25(6):816-35. Pubmed: 11750176 Over the past three decades, research exploring potential neuronal replacement therapies have focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain. Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. However, in the last decade, the development of new techniques has resulted in an explosion of new research showing that neurogenesis, the birth of new neurons, normally occurs in two limited and specific regions of the adult mammalian brain, and that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain. Recent findings from our lab demonstrate that it is possible to induce neurogenesis de novo in the adult mammalian brain, particularly in the neocortex where it does not normally occur, and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons. Elucidation of the relevant molecular controls may allow the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that do not require transplantation of exogenous cells. -
Catapano LA, Arnold MW, Perez FA, Macklis JD. 2001. Specific neurotrophic factors support the survival of cortical projection neurons at distinct stages of development. The Journal of neuroscience : the official journal of the Society for Neuroscience. 21(22):8863-72. Pubmed: 11698598 Catapano LA, Arnold MW, Perez FA, Macklis JD. 2001. Specific neurotrophic factors support the survival of cortical projection neurons at distinct stages of development. The Journal of neuroscience : the official journal of the Society for Neuroscience. 21(22):8863-72. Pubmed: 11698598 Repair of specific neuronal circuitry in the neocortex may be possible via neural precursor transplantation or manipulation of endogenous precursors in situ. These approaches will almost certainly require a detailed understanding of the mechanisms that control survival and differentiation of specific neuronal lineages. Such analysis has been hampered by the overwhelming diversity of neuronal types intermixed in neocortex and the inability to isolate individual lineages. To elucidate stage-specific controls over the survival of individual lineages of cortical neurons, we purified immature callosal projection neurons (CPN) at distinct stages of development from embryonic and postnatal mouse cortex by retrograde fluorescence labeling, followed by fluorescence-activated cell sorting. Purified CPN survive well in culture, acquire stage-specific projection neuron morphologies, and express appropriate neurotransmitters and growth factor receptors. Purified CPN are dependent on exogenous trophic support for survival in a stage-specific manner. Survival of postnatal day 2 (P2) to P3 and P6-P7 CPN is promoted by overlapping but distinct sets of neurotrophic factors, whereas embryonic day 19 CPN show less specificity of dependence on peptide factors. These studies demonstrate for the first time the stage-specific control by peptide growth factors over the survival of a specific cortical neuronal lineage. Such information may be critical for the future goal of directed differentiation of transplanted or endogenous precursors toward cellular repair of complex cortical circuitry. -
Macklis JD. 2001. Neurobiology: New memories from new neurons. Nature. 410(6826):314-5, 317. Pubmed: 11268187 Macklis JD. 2001. Neurobiology: New memories from new neurons. Nature. 410(6826):314-5, 317. Pubmed: 11268187 2000
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Magavi SS, Leavitt BR, Macklis JD. 2000. Induction of neurogenesis in the neocortex of adult mice. Nature. 405(6789):951-5. Pubmed: 10879536 Magavi SS, Leavitt BR, Macklis JD. 2000. Induction of neurogenesis in the neocortex of adult mice. Nature. 405(6789):951-5. Pubmed: 10879536 Neurogenesis normally only occurs in limited areas of the adult mammalian brain--the hippocampus, olfactory bulb and epithelium, and at low levels in some regions of macaque cortex. Here we show that endogenous neural precursors can be induced in situ to differentiate into mature neurons, in regions of adult mammalian neocortex that do not normally undergo any neurogenesis. This differentiation occurs in a layer- and region-specific manner, and the neurons can re-form appropriate corticothalamic connections. We induced synchronous apoptotic degeneration of corticothalamic neurons in layer VI of anterior cortex of adult mice and examined the fates of dividing cells within cortex, using markers for DNA replication (5-bromodeoxyuridine; BrdU) and progressive neuronal differentiation. Newly made, BrdU-positive cells expressed NeuN, a mature neuronal marker, in regions of cortex undergoing targeted neuronal death and survived for at least 28 weeks. Subsets of BrdU+ precursors expressed Doublecortin, a protein found exclusively in migrating neurons, and Hu, an early neuronal marker. Retrograde labelling from thalamus demonstrated that BrdU+ neurons can form long-distance corticothalamic connections. Our results indicate that neuronal replacement therapies for neurodegenerative disease and CNS injury may be possible through manipulation of endogenous neural precursors in situ. -
Scharff C, Kirn JR, Grossman M, Macklis JD, Nottebohm F. 2000. Targeted neuronal death affects neuronal replacement and vocal behavior in adult songbirds. Neuron. 25(2):481-92. Pubmed: 10719901 Scharff C, Kirn JR, Grossman M, Macklis JD, Nottebohm F. 2000. Targeted neuronal death affects neuronal replacement and vocal behavior in adult songbirds. Neuron. 25(2):481-92. Pubmed: 10719901 In the high vocal center (HVC) of adult songbirds, increases in spontaneous neuronal replacement correlate with song changes and with cell death. We experimentally induced death of specific HVC neuron types in adult male zebra finches using targeted photolysis. Induced death of a projection neuron type that normally turns over resulted in compensatory replacement of the same type. Induced death of the normally nonreplaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. We infer that neuronal death regulates the recruitment of replaceable neurons. Song deteriorated in some birds only after elimination of replaceable neurons. Behavioral deficits were transient and followed by variable degrees of recovery. This raises the possibility that induced neuronal replacement can restore a learned behavior. -
Shin JJ, Fricker-Gates RA, Perez FA, Leavitt BR, Zurakowski D, Macklis JD. 2000. Transplanted neuroblasts differentiate appropriately into projection neurons with correct neurotransmitter and receptor phenotype in neocortex undergoing targeted projection neuron degeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 20(19):7404-16. Pubmed: 11007899 Shin JJ, Fricker-Gates RA, Perez FA, Leavitt BR, Zurakowski D, Macklis JD. 2000. Transplanted neuroblasts differentiate appropriately into projection neurons with correct neurotransmitter and receptor phenotype in neocortex undergoing targeted projection neuron degeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 20(19):7404-16. Pubmed: 11007899 Reconstruction of complex neocortical and other CNS circuitry may be possible via transplantation of appropriate neural precursors, guided by cellular and molecular controls. Although cellular repopulation and complex circuitry repair may make possible new avenues of treatment for degenerative, developmental, or acquired CNS diseases, functional integration may depend critically on specificity of neuronal synaptic integration and appropriate neurotransmitter/receptor phenotype. The current study investigated neurotransmitter and receptor phenotypes of newly incorporated neurons after transplantation in regions of targeted neuronal degeneration of cortical callosal projection neurons (CPNs). Donor neuroblasts were compared to the population of normal endogenous CPNs in their expression of appropriate neurotransmitters (glutamate, aspartate, and GABA) and receptors (kainate-R, AMPA-R, NMDA-R. and GABA-R), and the time course over which this phenotype developed after transplantation. Transplanted immature neuroblasts from embryonic day 17 (E17) primary somatosensory (S1) cortex migrated to cortical layers undergoing degeneration, differentiated to a mature CPN phenotype, and received synaptic input from other neurons. In addition, 23.1 +/- 13.6% of the donor-derived neurons extended appropriate long-distance callosal projections to the contralateral S1 cortex. The percentage of donor-derived neurons expressing appropriate neurotransmitters and receptors showed a steady increase with time, reaching numbers equivalent to adult endogenous CPNs by 4-16 weeks after transplantation. These results suggest that previously demonstrated changes in gene expression induced by synchronous apoptotic degeneration of adult CPNs create a cellular and molecular environment that is both permissive and instructive for the specific and appropriate maturation of transplanted neuroblasts. These experiments demonstrate, for the first time, that newly repopulating neurons can undergo directed differentiation with high fidelity of their neurotransmitter and receptor phenotype, toward reconstruction of complex CNS circuitry. -
Gates MA, Fricker-Gates RA, Macklis JD. 2000. Reconstruction of cortical circuitry. Progress in brain research. 127:115-56. Pubmed: 11142025 Gates MA, Fricker-Gates RA, Macklis JD. 2000. Reconstruction of cortical circuitry. Progress in brain research. 127:115-56. Pubmed: 11142025 -
Gates MA, Tai CC, Macklis JD. 2000. Neocortical neurons lacking the protein-tyrosine kinase B receptor display abnormal differentiation and process elongation in vitro and in vivo. Neuroscience. 98(3):437-47. Pubmed: 10869838 Gates MA, Tai CC, Macklis JD. 2000. Neocortical neurons lacking the protein-tyrosine kinase B receptor display abnormal differentiation and process elongation in vitro and in vivo. Neuroscience. 98(3):437-47. Pubmed: 10869838 The spatial and temporal expression of the protein-tyrosine kinase B (TrkB) receptor and its ligands has been correlated with the development of the neocortex. Activation of the receptor has been associated with neocortical neuronal survival, differentiation, connectivity and neurotransmitter release. Although such findings suggest an important role for TrkB signaling in corticogenesis, conclusive evidence from targeted gene deletion ("knockout"; TrkB -/-) mice has been limited, due in part to the neonatal lethality of most of these mutant mice and the confounding variables associated with the poor health of those few surviving slightly longer postnatally. In the present study, the effects of TrkB signaling on the survival, differentiation and integration of neocortical neurons was directly investigated in vitro and in vivo. First, we conducted a neuron-specific immunocytochemical analysis of TrkB -/- mice to determine whether early cortical structure and patterns of histogenesis were normal or perturbed. We then employed in vitro and in vivo approaches to extend the life of TrkB -/- neocortical neurons beyond the period possible in TrkB -/- mutant mice themselves: (i) dissociated cell culture to directly compare the developmental potential of TrkB -/-, +/- and +/+ neurons; and (ii) neural transplantation into homochronic wild-type recipients to investigate the cell-autonomous effects of the receptor knockout on the differentiation, growth and integration of neocortical neurons. These latter experiments allowed, for the first time, study of the survival and differentiation potential of TrkB -/- neocortical neurons beyond the initial stages of corticogenesis. Direct comparison of brains of TrkB -/-, +/- and +/+ littermates immunocytochemically labeled with antibodies to microtubule-associated protein-2, neurofilament and beta-tubulin III revealed subtle anatomical anomalies in the mutant mice. These anomalies include abnormally diffuse microtubule-associated protein-2 positive neurons just dorsal to the corpus callosum, and heterotopic aggregations of postmitotic neurons in the subventricular zones of the ganglionic eminences, both suggesting delayed neuronal migration and differentiation. Cell culture experiments revealed substantially reduced survival by TrkB -/- neocortical neurons, and a significant reduction in neurite outgrowth by surviving TrkB -/- neurons. In experiments where prelabeled embryonic or neonatal TrkB -/- neocortical neurons were transplanted into the cerebral cortices of neonatal wild-type recipients, a similar quantitatively significant defect in the formation of dendrites, as well as reduced integration of TrkB -/- neocortical neurons, was also evident. These findings demonstrate cell-autonomous abnormalities in the development of neocortical neurons from TrkB -/- mice, and the subtle, but potentially critical, role of protein-tyrosine kinase B signaling in neocortical neuronal survival, differentiation and connectivity. 1999
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Lanier LM, Gates MA, Witke W, Menzies AS, Wehman AM, Macklis JD, Kwiatkowski D, Soriano P, Gertler FB. 1999. Mena is required for neurulation and commissure formation. Neuron. 22(2):313-25. Pubmed: 10069337 Lanier LM, Gates MA, Witke W, Menzies AS, Wehman AM, Macklis JD, Kwiatkowski D, Soriano P, Gertler FB. 1999. Mena is required for neurulation and commissure formation. Neuron. 22(2):313-25. Pubmed: 10069337 Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton. -
Leavitt BR, Hernit-Grant CS, Macklis JD. 1999. Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons. Experimental neurology. 157(1):43-57. Pubmed: 10222107 Leavitt BR, Hernit-Grant CS, Macklis JD. 1999. Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons. Experimental neurology. 157(1):43-57. Pubmed: 10222107 Neuronal migration is an essential step in normal mammalian neocortical development, and the expression of defined cellular and molecular signals within the developing cortical microenvironment is likely crucial to this process. Therapy via transplanted or manipulated endogenous precursors for diseases which involve neuronal loss may depend critically on whether newly incorporated cells can actively migrate to repopulate areas of neuronal loss within the adult brain. Previous studies demonstrated that embryonic neurons and multipotent precursors transplanted into the neocortex of adult mice undergoing targeted apoptosis of pyramidal neurons migrate long distances into neuron-deficient regions, undergo directed differentiation, accept afferent synaptic input, and make appropriate long-distance projections. The experiments presented here: (1) use time-lapse digital confocal imaging of neuronal migration in living slice cultures to assess cellular mechanisms utilized by immature neurons during such long distance migration, and (2) identify changes within the host cortical astroglial population that may contribute to this migration. Prelabeled embryonic day 17 mouse neocortical neurons were transplanted into adult mouse primary somatosensory cortex undergoing targeted apoptotic degeneration of callosal projection neurons. Four to 7 days following transplantation, living slice cultures containing the region of transplanted cells were prepared and observed. Sequential time-lapse images were recorded using a video-based digital confocal microscope. Transplanted cells displayed bipolar morphologies characteristic of migrating neuroblasts and moved in a saltatory manner with mean rates of up to 14 microm/h. To investigate whether a permissive glial phenotype may provide a potential substrate for this directed form of neuronal migration, slice cultures were immunostained with the RC2 monoclonal antibody, which identifies radial glia that act as a substrate for neuronal migration during corticogenesis. RC2 does not label mature stellate astrocytes, which express glial fibrillary acidic protein (GFAP). RC2 expression was observed in glial cells closely apposed to migrating donor neurons within the slice cultures. The timing and specificity of RC2 expression was examined immunocytochemically at various times following transplantation. RC2 immunostaining within regions of neuronal degeneration was transient, with peak staining between 3 and 7 days following transplantation. Strongly RC2-immunoreactive cells that did not express GFAP were found within these regions, but not in distant cortical regions or within control brains. RC2-positive cells were identified in recipient transgenic mice which express beta-galactosidase under a glial specific promoter. Coexpression of RC2 and beta-galactosidase identified these cells as host astroglia. These results demonstrate that adult cortical astrocytes retain the capacity to reexpress an earlier developmental phenotype that may partially underlie the observed active migration of transplanted neurons and neural precursors. Further understanding of these processes could allow directed migration of transplanted or endogenous precursors toward therapeutic cellular repopulation and complex circuit reconstruction in neocortex and other CNS regions.Copyright 1999 Academic Press. -
Sheen VL, Arnold MW, Wang Y, Macklis JD. 1999. Neural precursor differentiation following transplantation into neocortex is dependent on intrinsic developmental state and receptor competence. Experimental neurology. 158(1):47-62. Pubmed: 10448417 Sheen VL, Arnold MW, Wang Y, Macklis JD. 1999. Neural precursor differentiation following transplantation into neocortex is dependent on intrinsic developmental state and receptor competence. Experimental neurology. 158(1):47-62. Pubmed: 10448417 Reconstruction of neocortical circuitry by transplantation of neural precursors, or by manipulation of endogenous precursors, may depend critically upon both local microenvironmental control signals and the intrinsic competence of populations of precursors to appropriately respond to external molecular controls. Dependence on the developmental state of donor or endogenous precursor cells in achieving appropriate differentiation, integration, and connectivity is not clearly understood. Recent studies have demonstrated the ability to generate expandable, often clonal neural precursors at various stages of development. Transplantation of a variety of these precursors suggests that precursor differentiation and integration within the central nervous system (CNS) may depend directly on the level of cellular maturation, with less differentiated, earlier stage precursors offering more flexible but less efficient integration and more differentiated, later stage precursors offering more efficient differentiation to specific phenotypes. To further investigate this hypothesis within neocortex, we used the relatively immature HiB5 multipotent neural precursor cell line derived from embryonic day 16 hippocampus, which is less mature than precursor types that have demonstrated neuronal differentiation in adult neocortex. HiB5 cells labeled fluorescently, radioactively, and genetically were transplanted into murine neocortex under three different conditions expected to offer varying levels of instructive and permissive microenvironmental signals: (1) the developing cortex in utero; (2) regions of adult neocortex undergoing targeted pyramidal neuronal degeneration in which developmental signals are upregulated and in which later stage precursors and immature neurons undergo directed pyramidal neuron differentiation; or (3) the intact adult neocortex. Differentiation and integration of transplanted cells were examined histologically and immunocytochemically by morphology and using neuronal- and glial-specific markers. We found that these precursors underwent differentiation toward cortical neuron phenotypes with characteristic morphologies when transplanted in utero, but failed to do so under either of the adult conditions. HiB5 precursors demonstrated highly immature characteristics in vitro, consistently expressing neuroepithelial but not glial or neuronal markers. Under all conditions, donor cells survived and migrated 1-2 mm from the injection track 2 to 4 weeks after transplantation. HiB5 neural precursors transplanted into the developing cortex of embryonic mice in utero migrated within the cortex, integrated well into the host parenchyma, and differentiated toward morphologically diverse, neuronal phenotypes. HiB5 cells transplanted into the intact cortex of adult mice survived, but did not show neuronal differentiation. In contrast to slightly later stage neural precursors and embryonic neurons used in previous transplantation studies, the HiB5 cells also failed to undergo neuronal differentiation after transplantation into regions undergoing induced apoptotic neuronal degeneration in adult cortex. These results suggested that these early hippocampal-derived precursors might not be fully competent to respond to later stage differentiation and/or survival signals important in neocortex and known to be upregulated in regions undergoing targeted neuronal apoptosis, including the TrkB neurotrophin receptor ligands BDNF and NT-4/5. We investigated this hypothesis and found that undifferentiated HiB5 cells lack catalytic trkB neurotrophin receptors at the mRNA and protein levels, while confirming that they express trkC receptors under the same conditions. Taken together, these findings support a progressive sequence of neural precursor differentiation and a spectrum of competence by precursors to respond to instructive microenvironmental signals. (ABSTRACT TRUNCATED) -
Catapano LA, Sheen VL, Leavitt BR, Macklis JD. 1999. Differentiation of transplanted neural precursors varies regionally in adults striatum. Neuroreport. 10(18):3971-7. Pubmed: 10716243 Catapano LA, Sheen VL, Leavitt BR, Macklis JD. 1999. Differentiation of transplanted neural precursors varies regionally in adults striatum. Neuroreport. 10(18):3971-7. Pubmed: 10716243 In the current experiments, we address the emerging hypothesis that transplanted neural precursor cells can respond to local microenvironmental signals in the post-developmental brain and exhibit patterns of differentiation that depend critically on specific location within the brain. HiB5 precursor cells were transplanted into adult mouse cortex, corpus callosum, and multiple positions in striatum, and assessed for differentiation by morphology and immunocytochemistry. Our results indicate that the likelihood of both neuronal and glial differentiation of transplanted precursors depends on proximity to the medial striatum or subventricular zone of the adult host, supporting the concept that microenvironmental signals can critically affect the differentiation fate of neural precursors, and suggesting the potential to manipulate such signals in the adult brain. 1998
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Wang Y, Sheen VL, Macklis JD. 1998. Cortical interneurons upregulate neurotrophins in vivo in response to targeted apoptotic degeneration of neighboring pyramidal neurons. Experimental neurology. 154(2):389-402. Pubmed: 9878177 Wang Y, Sheen VL, Macklis JD. 1998. Cortical interneurons upregulate neurotrophins in vivo in response to targeted apoptotic degeneration of neighboring pyramidal neurons. Experimental neurology. 154(2):389-402. Pubmed: 9878177 Intercellular signals provided by growth and neurotrophic factors play a critical role during neurogenesis and as part of cellular repopulation strategies directed toward reconstruction of complex CNS circuitry. Local signals influence the differentiation of transplanted and endogenous neurons and neural precursors, but the cellular sources and control over expression of these molecules remain unclear. We have previously examined microenvironmental control in neocortex over neuron and neural precursor migration and differentiation following transplantation, using an approach of targeted apoptotic neuronal degeneration to specific neuronal populations in vivo. Prior results suggested the hypothesis that upregulated or reexpressed developmental signal molecules, produced by degenerating pyramidal neurons and/or by neighboring neurons or nonneuronal cells, may be responsible for observed events of directed migration, differentiation, and connectivity by transplanted immature neurons and precursors. To directly investigate this hypothesis, we analyzed the gene expression of candidate and control neurotrophins, growth factors, and receptors within regions of targeted neuronal cell death, first by quantitative Northern blot analysis and then by in situ hybridization combined with immunocytochemical analysis. The genes for BDNF, NT-4/5, trkB receptors, and to a lesser extent NT-3 were upregulated specifically within the regions of neocortex undergoing targeted neuronal degeneration and specifically during the period of ongoing pyramidal neuron apoptosis. Upregulation occurred during the same 3-week period as the previously investigated cellular events of directed migration, differentiation, and integration. No upregulation was seen in panels of control neurotrophins, growth factors, and receptors that are not as developmentally regulated in cortex or that are thought to have primary actions in other CNS regions. In situ hybridization and immunocytochemistry revealed that BDNF mRNA expression was upregulated specifically by local interneurons adjacent to degenerating pyramidal neurons. These findings suggest specific effects of targeted apoptosis on neurotrophin and other gene expression via mechanisms, including intercellular signaling between degenerating pyramidal neurons and surrounding interneurons. Further understanding of these and other controls over neocortical projection neuron differentiation may provide insight regarding normal neocortical development, intercellular signaling induced by apoptosis, and toward reconstruction and cellular repopulation of complex neocortical and other CNS circuitry.Copyright 1998 Academic Press. 1997
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Snyder EY, Yoon C, Flax JD, Macklis JD. 1997. Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proceedings of the National Academy of Sciences of the United States of America. 94(21):11663-8. Pubmed: 9326667 Snyder EY, Yoon C, Flax JD, Macklis JD. 1997. Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proceedings of the National Academy of Sciences of the United States of America. 94(21):11663-8. Pubmed: 9326667 Neurons undergoing targeted photolytic cell death degenerate by apoptosis. Clonal, multipotent neural precursor cells were transplanted into regions of adult mouse neocortex undergoing selective degeneration of layer II/III pyramidal neurons via targeted photolysis. These precursors integrated into the regions of selective neuronal death; 15 +/- 7% differentiated into neurons with many characteristics of the degenerated pyramidal neurons. They extended axons and dendrites and established afferent synaptic contacts. In intact and kainic acid-lesioned control adult neocortex, transplanted precursors differentiated exclusively into glia. These results suggest that the microenvironmental alterations produced by this synchronous apoptotic neuronal degeneration in adult neocortex induced multipotent neural precursors to undergo neuronal differentiation which ordinarily occurs only during embryonic corticogenesis. Studying the effects of this defined microenvironmental perturbation on the differentiation of clonal neural precursors may facilitate identification of factors involved in commitment and differentiation during normal development. Because photolytic degeneration simulates some mechanisms underlying apoptotic neurodegenerative diseases, these results also suggest the possibility of neural precursor transplantation as a potential cell replacement or molecular support therapy for some diseases of neocortex, even in the adult. 1996
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Hernit-Grant CS, Macklis JD. 1996. Embryonic neurons transplanted to regions of targeted photolytic cell death in adult mouse somatosensory cortex re-form specific callosal projections. Experimental neurology. 139(1):131-42. Pubmed: 8635560 Hernit-Grant CS, Macklis JD. 1996. Embryonic neurons transplanted to regions of targeted photolytic cell death in adult mouse somatosensory cortex re-form specific callosal projections. Experimental neurology. 139(1):131-42. Pubmed: 8635560 In the neocortex, the effectiveness of potential transplantation therapy for diseases involving neuronal loss may depend upon whether donor neurons can reestablish the precise long-distance projections that form the basis of sensory, motor, and cognitive function. During corticogenesis, the formation of these connections is affected by tropic factors, extracellular matrix, structural pathways, and developmental cell death. Previous studies demonstrated that embryonic neurons and multipotent neural precursors transplanted into neocortex or mice undergoing photolytically induced, synchronous, apoptotic neuronal degeneration selectively migrate into these regions, where they differentiate into pyramidal neurons and accept afferent synaptic input. The experiments presented here assess whether embryonic neurons transplanted into regions of somatosensory cortex undergoing targeted cell death differentiate further and develop long-distance axons and whether this outgrowth is target specific. Neocortical neurons from Gestational Day 17 mouse embryos were dissociated, prelabeled with fluorescent nanospheres and a lipophilic dye (DiI or PKH), and transplanted into adult mouse primary somatosensory cortex (S1) undergoing apoptotic degeneration of callosal projection neurons. Donor neurons selectively migrated into and differentiated within regions of targeted neuronal death in lamina II/III over a 2-week period, in agreement with our prior studies. To detect possible projections made by donor neurons 2, 4, 6, 8, or 10 weeks following transplantation, the retrogradely transported dye fluorogold (FG) was stereotaxically injected into contralateral S1, ipsilateral secondary somatosensory cortex (S2), or ipsilateral thalamus. Ten weeks following transplantation, 21 +/- 5% of the labeled donor neurons were labeled by FG injections into contralateral S1, demonstrating that donor neurons sent projections to the distant area, the original target of host neurons undergoing photolytically induced cell death. No donor neurons were labeled with FG injections into ipsilateral S2 or thalamus, nearby targets of other subpopulations of neurons in S1. These data indicate that in the adult neocortex: (1) transplanted immature neurons are capable of extending long-distance projections between hemispheres through the mature white matter of the corpus callosum and (2) these projections are formed with specificity to replace projections by neurons undergoing synchronous degeneration. These experiments provide an experimental system with which to test factors affecting such outgrowth and connectivity. Taken together, these results suggest that the reconstruction and repair of cortical circuitry responsible for sensory, motor, or cognitive function may be possible in the mature neocortex, if donor neurons or precursor cells are provided with the correct combination of local and distant signals within an appropriately permissive host environment. 1995
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Sheen VL, Macklis JD. 1995. Targeted neocortical cell death in adult mice guides migration and differentiation of transplanted embryonic neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 15(12):8378-92. Pubmed: 8613770 Sheen VL, Macklis JD. 1995. Targeted neocortical cell death in adult mice guides migration and differentiation of transplanted embryonic neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 15(12):8378-92. Pubmed: 8613770 Local expression of cellular and molecular signals is required for normal neuronal migration and differentiation during neocortical development and during periods of plasticity in the adult brain. We have previously shown that neonatal and juvenile mice that induction of apoptotic degeneration in neocortical pyramidal neurons by targeted photolysis provides an altered environment that directs migration and differentiation of transplanted embryonic neurons. Here we employ the same paradigm in adult mice to test whether targeted photolysis induces the reexpression in the mature brain of developmental signals that control migration, differentiation and integration of embryonic neurons. We examined both the time course of migration and the morphologic and immunocytochemical differentiation of embryonic neurons transplanted into regions of targeted photolytic cell death. Pyramidal neurons in neocortical lamina II/III underwent photolytically induced apoptosis after retrograde incorporation of the photoactive chromophore chlorine e6 and transdural exposure to 674 nm near-infrared laser energy. Embryonic day 17 neocortical neurons were prelabeled with fluorescent nanospheres and the lipophilic dye PKH26, transplanted into regions of ongoing neuronal degeneration in adult mice, and examined histologically and immunocytochemically. Transplanted neurons began migration into regions of neuronal death within 3 d and differentiated into large pyramidal neurons similar to those degenerating. In contrast, neurons transplanted into intact cortex did not migrate, and they differentiate into small presumptive interneurons. Migration up to 430 microM in experimental mice was complete by 2 weeks; approximately 45% of the donor neurons migrated greater than 3 SDs beyond the mean for neurons transplanted into intact neocortex of age-matched adult hosts. Following migration, dendrites and axons of many donor neurons were properly oriented toward the pial surface and corpus callosum, indicating integration into the host parenchyma. Neurofilament and neuron-specific enolase staining further support appropriate differentiation and integration. These results indicate that signals guiding neuronal migration and differentiation in neocortex are reexpressed in adult mice well beyond the period of corticogenesis within regions of targeted photolytic cell death. Elucidating the molecular mechanisms underlying these events by comparison with adjacent unperturbed regions will contribute to efforts toward future therapeutic transplantation and control over endogenous plasticity. 1994
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Sheen VL, Macklis JD. 1994. Apoptotic mechanisms in targeted neuronal cell death by chromophore-activated photolysis. Experimental neurology. 130(1):67-81. Pubmed: 7821398 Sheen VL, Macklis JD. 1994. Apoptotic mechanisms in targeted neuronal cell death by chromophore-activated photolysis. Experimental neurology. 130(1):67-81. Pubmed: 7821398 Apoptosis influences early development and later refinement in adult tissues. Experiments in which embryonic neurons or multipotent neural precursor cells are transplanted into regions of neuronal degeneration following targeted photolytic cell death show similar regulation of neuronal migration and differentiation. In those experiments, transplanted cells sought to restore normal cytoarchitecture by preferential migration into neuron deficient regions, assumption of pyramidal morphology, and early process elongation. Control transplants into intact and kainic acid lesioned cortex failed to elicit similar responses. We investigated the possibility that mechanisms of neuronal death common to apoptosis and targeted photolysis could explain the similar developmental influences. We assessed the pathways of cellular injury and eventual cell death in neuroblastoma and PC12 cell cultures labeled with nanospheres carrying the chromophore NH4-chlorin e6 and subjected to photoactivation (1) pharmacologically by scavengers of singlet oxygen and inhibitors of lysosomal proteases, (2) histologically by electron, fluorescence, and light microscopy, and (3) biochemically with binding of cellular DNA by propridium iodide, 3'-OH DNA end terminal labeling, and gel electrophoresis. We found that nanospheres were incorporated into lysosomes, and exposure to light energy led to singlet oxygen (1O2) production and cell death within both neuroblastoma and PC-12 cell lines. Scavengers of 1O2 prevented cell toxicity, while inactivation of lysosomal proteases reduced cell death. Morphologically, degenerating cells revealed release of proteases from lysosomes and disruption of cytoskeletal proteins. Apoptotic characteristics including early loss of cell adhesion, plasma membrane blebbing, and nuclear condensation and convolution were observed. Biochemically, DNA fragmentation was present in cells stained with propridium iodide and observed by 3'-OH end terminal labeling and gel electrophoresis. Thus, cells targeted by photolytically generated 1O2 undergo a form of cell autolysis whose final common pathway is apoptotic. The slow, nonnecrotic process of targeted neuronal cell death in vivo may activate many of the same physiological cues activated by programmed cell death during normal development and during organizational refinement in the adult vertebrate nervous system. This may potentially explain the migration and differentiation of neocortical neurons and neural precursors transplanted into these regions of neuronal degeneration. 1993
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Macklis JD. 1993. Transplanted neocortical neurons migrate selectively into regions of neuronal degeneration produced by chromophore-targeted laser photolysis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 13(9):3848-63. Pubmed: 8366349 Macklis JD. 1993. Transplanted neocortical neurons migrate selectively into regions of neuronal degeneration produced by chromophore-targeted laser photolysis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 13(9):3848-63. Pubmed: 8366349 Selective degeneration of neocortical callosal pyramidal neurons by noninvasive laser illumination was used for directed studies of neocortical transplantation, to test the hypothesis that transplanted embryonic neurons may seek to restore normal cytoarchitecture within an appropriately permissive local environment. At long wavelengths that penetrate through tissue without major absorption, photolysis can cause extremely selective degeneration to desired subpopulations of targeted neurons in vivo (Macklis and Madison, 1991; Madison and Macklis, 1993). Cell death is geographically defined and slowly progressive, allowing control over the anatomical substrate for transplantation. Targeting occurs by retrograde incorporation of cytolytic chromophores that are activated by specific-wavelength light. Intermixed neurons, glia, axons, blood vessels, and connective tissue remain intact. Degeneration was effected within neocortical lamina II/III of neonatal mouse pups following targeting in utero or early postnatally with photoactive nanospheres. Total neuron density was reduced typically by 25-30% within defined areas, with approximately 60% loss of large projection neurons and no change in the number of small, presumptive interneurons. Embryonic day 17 neocortical cell suspensions, which included recently postmitotic neurons destined to form lamina II/III, were transplanted lateral to these regions of ongoing neuron degeneration in juvenile mice. Cellular injections spanned laminae II-V, to provide donor neurons with both lateral and laminar choice for possible migration and integration. Donor cells were labeled in vitro with unique fluorescent and electron-dense nanospheres that allowed distinct identification of donor cells at both light and electron microscopic levels. Control experiments included neocortical transplants into intact age-matched hosts, into hosts with kainic acid lesions to neocortex, or distant to the region of photolytic neuronal degeneration; embryonic cerebellar transplants to the regions of selective photolytic degeneration; and grafts of hypoosmotically lysed neocortical cells to lesioned regions. After survival times of 1 hr to 12 weeks, labeled neurons were identified morphologically and positions were digitized for qualitative and quantitative analysis of position and specificity of migration and cellular integration; electron microscopy was used to confirm further the donor identities of migrated neurons. Neurons placed near host zones of photolytic neuron degeneration migrated up to 780 microns specifically within these zones; approximately 44% of donor neurons migrated significantly beyond the injection site to enter these regions. Migration and integration did not occur in normal, unaffected deeper layers IV-VI of these experimental mice, or in the normal lamina II/III bordering the transplantation site on the side opposite the neuron-deficient region. Control grafts of all five types revealed only minimal local spread without laminar preference.(ABSTRACT TRUNCATED AT 400 WORDS) -
Madison RD, Macklis JD. 1993. Noninvasively induced degeneration of neocortical pyramidal neurons in vivo: selective targeting by laser activation of retrogradely transported photolytic chromophore. Experimental neurology. 121(2):153-9. Pubmed: 8339767 Madison RD, Macklis JD. 1993. Noninvasively induced degeneration of neocortical pyramidal neurons in vivo: selective targeting by laser activation of retrogradely transported photolytic chromophore. Experimental neurology. 121(2):153-9. Pubmed: 8339767 Interactions among neuronal subpopulations determine brain development and function. The present study illustrates the ability to noninvasively and selectively lesion targeted subpopulations of neurons in a highly specific, temporally defined, and geographically localized manner. This method provides a fundamental advance toward rigorous investigation of the importance of identified neuronal subtypes. Projecting pyramidal neurons of rats and mice were targeted by retrograde transport of latex nanospheres from contralateral motor cortex containing a nontoxic chromophore (chlorin e6) that produces cytotoxic singlet oxygen during photoactivation by deeply penetrating 670-nm light. Geographically defined regions of cortex were exposed to laser illumination at 670 nm to activate singlet oxygen production by the intracellular chromophore, and animals were sacrificed from 4 h to 7 days following laser exposure. Brains were processed to display degenerating neurons using sensitive silver-staining procedures. Selective damage occurred to pyramidal neurons with known callosal projections, solely within layers II/III and V of the illuminated region. Such subpopulation specificity provides models for neural transplantation and analysis of anatomically distributed neuronal networks. 1992
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Macklis RM, Macklis JD. 1992. Historical and phrenologic reflections on the nonmotor functions of the cerebellum: love under the tent?. Neurology. 42(4):928-32. Pubmed: 1565255 Macklis RM, Macklis JD. 1992. Historical and phrenologic reflections on the nonmotor functions of the cerebellum: love under the tent?. Neurology. 42(4):928-32. Pubmed: 1565255 -
Sheen VL, Dreyer EB, Macklis JD. 1992. Calcium-mediated neuronal degeneration following singlet oxygen production. Neuroreport. 3(8):705-8. Pubmed: 1520860 Sheen VL, Dreyer EB, Macklis JD. 1992. Calcium-mediated neuronal degeneration following singlet oxygen production. Neuroreport. 3(8):705-8. Pubmed: 1520860 NONINVASIVE photolytic injury to targeted neuronal subpopulations in vivo causes unique, slowly progressive neuronal degeneration. To examine the mechanisms of degeneration toward development and transplantation studies, cytosolic calcium levels were measured in vitro from neocortical neurons after incorporation of photoactive nanospheres and laser-activated singlet oxygen production within lysosomes. Cytosolic calcium increased irreversibly, predominantly from extracellular sources through channel-mediated mechanisms and increased membrane porosity. Propidium iodide studies demonstrated gradual loss of membrane integrity over hours to days. The calcium channel blocker nimodipine, or calcium-free medium, partially protected neurons from calcium flux and cell death. Results suggest calcium-dependent and independent mechanisms of neuronal degeneration following singlet oxygen production. 1991
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Macklis JD, Quattrochi JJ. 1991. Restricted diffusion and stability of carbachol-fluorescent nanospheres in-vivo. Neuroreport. 2(5):247-50. Pubmed: 1912455 Macklis JD, Quattrochi JJ. 1991. Restricted diffusion and stability of carbachol-fluorescent nanospheres in-vivo. Neuroreport. 2(5):247-50. Pubmed: 1912455 Mapping neuronal populations that induce behavioral state changes after pharmacological activation requires discrete localization of drug injection sites, and is limited by widespread diffusion of molecular drugs. Nanospheres with diameters of 50-100 nm can reduce diffusion significantly because of their relatively large sizes. The cholinergic agonist carbachol was radiolabeled with methyl14C and incorporated within a latex nanosphere delivery system (LNDS). We quantitatively compared diffusion of 14C-carbachol within these nanospheres with that of free 14C-carbachol, demonstrating approximately ten-fold reduced radial diffusion by nanospheres 10 min to 24 h post-injection; approximately 90% of injected radioactivity was restricted to regions within approximately 100-150 microns and 1400-1500 microns respectively. Thus, incorporation of active agents such as drugs within nanospheres dramatically increases the precision of their delivery in-vivo (here about 1,000-fold by volume). -
Macklis JD, Madison RD. 1991. Neuroblastoma grafts are noninvasively removed within mouse neocortex by selective laser activation of intracellular photolytic chromophore. The Journal of neuroscience : the official journal of the Society for Neuroscience. 11(7):2055-62. Pubmed: 2066774 Macklis JD, Madison RD. 1991. Neuroblastoma grafts are noninvasively removed within mouse neocortex by selective laser activation of intracellular photolytic chromophore. The Journal of neuroscience : the official journal of the Society for Neuroscience. 11(7):2055-62. Pubmed: 2066774 Studies of neural cell transplantation would be aided by the ability to damage or destroy, noninvasively and extremely selectively, grafted cells at defined times following their initial implantation. Mechanisms of graft integration and performance could be investigated, also providing insight into natural injury and repair mechanisms. At long wavelengths between 650 and 850 nm, laser energy can penetrate several millimeters of brain tissue without absorption or damage to the unpigmented tissue. However, targeted cells are selectively damaged by illumination at these long wavelengths if they contain latex nanospheres with incorporated cytolytic chromophores (e.g., chlorin e6). Light penetration allows many thousands of cells to be lesioned simultaneously, noninvasively, and deep within a surrounding matrix of other tissue. Such laser-activated damage has been termed laser photolysis (PL). We studied damage to C1300 neuroblastoma (NB) cells grafted into mouse neocortex in vivo by this process of PL. NB cells provided a simple and reproducible model of neural grafting, allowing direct histologic assessment of cellular growth and viability by distinct morphologic and mitotic criteria. Cells were cultured by standard methods, labeled in vitro by brief exposure to nanospheres containing chlorin e6, and grafted to sites within deep layers of mouse neocortex. Mice were exposed to transcranial, fractionated, unfocused pulses of 670-nm-wavelength energy totaling 90-120 J/cm2. We histologically assessed graft growth and cellular viability over a period from 2 d to 4 weeks, measured graft volumes quantitatively during the period of early rapid growth in controls (2 and 7 d), and generated 3-D reconstructions from serial sections to assist in visual analysis.(ABSTRACT TRUNCATED AT 250 WORDS) -
Liu GT, Carrazana EJ, Macklis JD, Mikati MA. 1991. Delayed oculogyric crises associated with striatocapsular infarction. Journal of clinical neuro-ophthalmology. 11(3):198-201. Pubmed: 1836805 Liu GT, Carrazana EJ, Macklis JD, Mikati MA. 1991. Delayed oculogyric crises associated with striatocapsular infarction. Journal of clinical neuro-ophthalmology. 11(3):198-201. Pubmed: 1836805 Oculogyric crises are dystonic, usually upward, conjugate eye deviations. We describe an 11-year-old girl who developed oculogyric crises 3 1/2 years after infarction of the right caudate, putamen, and internal capsule. Her abnormal eye movements responded to anticholinergic agents. This is the first reported association between oculogyric crises and striatocapsular infarction. 1990
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Macklis JD, Madison RD. 1990. Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity. Journal of neuroscience methods. 31(1):43-6. Pubmed: 2308380 Macklis JD, Madison RD. 1990. Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity. Journal of neuroscience methods. 31(1):43-6. Pubmed: 2308380 We describe a visual assay of neuronal electrophysiologic status for use with cultured neurons, based on the exclusion of propidium iodide (PI) by intact cellular membranes. We use this fluorescent dye, which binds to nucleic acids, at concentrations suitable for long-term exposure to neurons without toxicity. We correlate the progressive loss of resting membrane potential and the progressive inability to generate stimulated action potentials by cultured mouse dorsal root ganglion neurons with increasing incorporation of PI. The scoring system used to gauge incorporation of PI is rapid and highly reproducible using a standard fluorescence microscope. Applications exist for studies of neuronal toxicity, survival, and electrophysiology in vitro. -
Madison R, Macklis JD, Thies C. 1990. Latex nanosphere delivery system (LNDS): novel nanometer-sized carriers of fluorescent dyes and active agents selectively target neuronal subpopulations via uptake and retrograde transport. Brain research. 522(1):90-8. Pubmed: 2224519 Madison R, Macklis JD, Thies C. 1990. Latex nanosphere delivery system (LNDS): novel nanometer-sized carriers of fluorescent dyes and active agents selectively target neuronal subpopulations via uptake and retrograde transport. Brain research. 522(1):90-8. Pubmed: 2224519 A wide range of latex particles are described which are capable of carrying high concentrations of fluorescent dyes, drugs, and photoactive agents selectively to subpopulations of neurons in vitro and in vivo. Particle size, charge, and concentration were all found to influence uptake into cultured neurons or retrograde transport in vivo. Chromophore loadings of greater than 14% (w/w) were obtained. Incorporation of a photoactivated dye (chlorin e6) into the polymer did not compromise the ability of the dye to produce singlet oxygen following light exposure. We refer to this unique family of latex particles as the latex nanosphere delivery system (LNDS). The LNDS will be usefull for studies of neuroanatomy and nervous system development, as well as more general areas of biomedical research where it is desirable to selectively label subpopulations of cells. The LNDS also offers a means of providing targeted delivery of drugs or photoactive agents to selected subpopulations of cells; this will allow experimentation not currently possible using any existent methodology. 1989
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Quattrochi JJ, Mamelak AN, Madison RD, Macklis JD, Hobson JA. 1989. Mapping neuronal inputs to REM sleep induction sites with carbachol-fluorescent microspheres. Science (New York, N.Y.). 245(4921):984-6. Pubmed: 2475910 Quattrochi JJ, Mamelak AN, Madison RD, Macklis JD, Hobson JA. 1989. Mapping neuronal inputs to REM sleep induction sites with carbachol-fluorescent microspheres. Science (New York, N.Y.). 245(4921):984-6. Pubmed: 2475910 The cholinergic agonist carbachol was conjugated to latex microspheres that were fluorescently labeled with rhodamine and used as neuroanatomical probes that show little diffusion from their injection site and retrogradely label neurons projecting to the injection site. Microinjection of this pharmacologically active probe into the gigantocellular field of the cat pontine brain stem caused the awake cats to fall into rapid movement (REM) sleep indistinguishable from that produced by free carbachol. Three-dimensional computer reconstruction of the retrogradely labeled neurons revealed a widely distributed neuronal network in the pontine tegmentum. These pharmacologically active microspheres permit a new precision in the characterization and mapping of neurons associated with the control of behavioral state and of other cholinergic networks. -
Schick RM, Jolesz F, Barnes PD, Macklis JD. 1989. MR diagnosis of dural venous sinus thrombosis complicating L-asparaginase therapy. Computerized medical imaging and graphics : the official journal of the Computerized Medical Imaging Society. 13(4):319-27. Pubmed: 2743289 Schick RM, Jolesz F, Barnes PD, Macklis JD. 1989. MR diagnosis of dural venous sinus thrombosis complicating L-asparaginase therapy. Computerized medical imaging and graphics : the official journal of the Computerized Medical Imaging Society. 13(4):319-27. Pubmed: 2743289 Dural venous sinus thrombosis is an important complication of L-asparaginase chemotherapy. The diagnosis and followup of this condition, using spin echo and fast imaging techniques, is described in three patients. Magnetic Resonance (MR) imaging is a rapid, noninvasive technique for diagnosis and follow-up of this condition. Fast imaging techniques can improve the assessment of these patients. 1988
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Madison RD, Macklis JD, Frosch MP. 1988. Non-invasive laser microsurgery selectively damages populations of labeled mouse neurons: dependence on incident laser dose and absorption. Brain research. 445(1):101-10. Pubmed: 3365549 Madison RD, Macklis JD, Frosch MP. 1988. Non-invasive laser microsurgery selectively damages populations of labeled mouse neurons: dependence on incident laser dose and absorption. Brain research. 445(1):101-10. Pubmed: 3365549 Selective photothermolysis (SP) is a novel technique by which brief, unfocused laser pulses are selectively absorbed by, and cause selective thermal damage to, endogenously pigmented structures. This report describes the use of an exogenous non-fluorescent chromophore (Procion blue) to effect cellular damage by SP. Cultured dorsal root ganglia neurons were selectively labeled with Procion blue and subjected to varying doses of laser illumination. Progressive cellular damage was assessed by leakage of propidium iodide through damaged membranes. The neurons targeted via an exogenous chromophore sustained damage which was proportionate to laser dose and chromophore concentration. The results of these experiments demonstrated that the rate and extent of neuronal damage can be controlled by adjusting either the incident dose of laser energy or the amount of target chromophore within cells. Selective photothermolysis will provide an experimental tool for neurobiologists in particular and will find general use within the biomedical field. 1987
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Brasier AR, Macklis JD, Vaughan D, Warner L, Kirshenbaum JM. 1987. Myopericarditis as an initial presentation of meningococcemia. Unusual manifestation of infection with serotype W135. The American journal of medicine. 82(3 Spec No):641-4. Pubmed: 3103444 Brasier AR, Macklis JD, Vaughan D, Warner L, Kirshenbaum JM. 1987. Myopericarditis as an initial presentation of meningococcemia. Unusual manifestation of infection with serotype W135. The American journal of medicine. 82(3 Spec No):641-4. Pubmed: 3103444 Acute meningococcemia is a dramatic clinical syndrome from infection with the gram-negative diplococcus, Neisseria meningitidis. Although pericarditis may complicate the course of meningococcemia, it is distinctly unusual as a presenting sign. A case of disseminated meningococcemia presenting as acute myopericarditis is reported. The serotype isolated, type W135, was a sporadic cause of N. meningitidis in the Boston area. Although the patient had meningitis, bacteremia, and myopericarditis, his course was uncomplicated with early institution of antibiotic therapy. 1985
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Anderson P, Macklis J, Brown M, Ory D. 1985. Eosinophilic cerebrospinal fluid pleocytosis and cryptococcal meningitis. Annals of internal medicine. 103(2):306-7. Pubmed: 4014918 Anderson P, Macklis J, Brown M, Ory D. 1985. Eosinophilic cerebrospinal fluid pleocytosis and cryptococcal meningitis. Annals of internal medicine. 103(2):306-7. Pubmed: 4014918 -
Macklis JD, Sidman RL, Shine HD. 1985. Cross-linked collagen surface for cell culture that is stable, uniform, and optically superior to conventional surfaces. In vitro cellular & developmental biology : journal of the Tissue Culture Association. 21(3 Pt 1):189-94. Pubmed: 3859483 Macklis JD, Sidman RL, Shine HD. 1985. Cross-linked collagen surface for cell culture that is stable, uniform, and optically superior to conventional surfaces. In vitro cellular & developmental biology : journal of the Tissue Culture Association. 21(3 Pt 1):189-94. Pubmed: 3859483 A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. -
Macklis JD, Madison R. 1985. Unfocused laser illumination kills dye-targeted mouse neurons by selective photothermolysis. Brain research. 359(1-2):158-65. Pubmed: 3841019 Macklis JD, Madison R. 1985. Unfocused laser illumination kills dye-targeted mouse neurons by selective photothermolysis. Brain research. 359(1-2):158-65. Pubmed: 3841019 Selective photothermolysis (SP) is a novel technique by which brief, unfocused laser pulses are selectively absorbed by, and cause selective thermal damage to, endogenously pigmented structures. The present experiments demonstrate the feasibility of using an exogenous non-fluorescent chromophore (procion blue) to effect cellular damage by SP. Dorsal root ganglia neurons in vitro were selectively labeled with procion blue and subsequently damaged by unfocused laser illumination. Progressive cellular damage was assessed by propidium iodide (PI), a fluorescent dye that leaks through damaged membranes and binds to nucleic acids. Graded scores of intracellular PI fluorescence demonstrated a highly significant difference in amount of damage between groups of experimental and control cells. Selective photothermolysis is discussed as an experimental tool for neurobiologists in particular and for general use within the biomedical field. 1979
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Macklis JD, Ketterer FD, Cravalho EG. 1979. Temperature dependence of the microwave properties of aqueous solutions of ethylene glycol between +15 degrees C and -70 degrees C. Cryobiology. 16(3):272-86. Pubmed: 477370 Macklis JD, Ketterer FD, Cravalho EG. 1979. Temperature dependence of the microwave properties of aqueous solutions of ethylene glycol between +15 degrees C and -70 degrees C. Cryobiology. 16(3):272-86. Pubmed: 477370 1978
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Macklis JD, Ketterer FD. 1978. Microwave properties of cryoprotectants. Cryobiology. 15(6):627-35. Pubmed: 743887 Macklis JD, Ketterer FD. 1978. Microwave properties of cryoprotectants. Cryobiology. 15(6):627-35. Pubmed: 743887 -
Snyder EY, Macklis JD. Multipotent neural progenitor or stem-like cells may be uniquely suited for therapy for some neurodegenerative conditions. Clinical neuroscience (New York, N.Y.). 3(5):310-6. Pubmed: 8914798 Snyder EY, Macklis JD. Multipotent neural progenitor or stem-like cells may be uniquely suited for therapy for some neurodegenerative conditions. Clinical neuroscience (New York, N.Y.). 3(5):310-6. Pubmed: 8914798 Multipotent neural progenitors or stem cells (or cells which mimic their behavior) are capable of differentiating along multiple central nervous system (CNS) cell-type lineages, neuronal and glial. They can engraft as integral members of normal structures throughout the host CNS without disturbing other neurobiological processes. By exploiting their basic biologic properties, these cells may be able to disseminate therapeutic gene products in a sustained, direct fashion throughout the CNS. In addition, they may replace dysfunctional neurons and glia in both a site-specific and global manner. They may play a therapeutic role in neurodegenerative conditions that occur both during development and in the mature brain. The ability of neural stem cells to respond to neurogenic cues not only when they occur during their normal developmental expression but even when induced or "reactivated" at later stages following injury, may entrance their utility in reconstituting damaged CNS regions. Thus, these vehicles may overcome many of the limitations of viral and non-neural cellular vectors, as well as pharmacologic and genetic interventions. The feasibility of this broadly applicable neural stem cell-based strategy has been demonstrated in a number of murine models of neurodegenerative disease. The focus of this review will be our recent observation of a possible tropism of such cells for neurodegenerative environments.