cerebral cortex projection neuron development, diversity, disease and regeneration
Macklis Lab Publications
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Cortical projection neurons with distinct axonal connectivity employ ribosomal complexes with distinct protein compositions. BioRxiv Preprint. December 23, 2024. DOI:https://doi.org/10.1101/2024.12.22.629968 Tran T, Bogdan Budnik, John E. Froberg, and Jeffrey D. Macklis. Cortical projection neurons with distinct axonal connectivity employ ribosomal complexes with distinct protein compositions. BioRxiv Preprint. December 23, 2024. DOI:https://doi.org/10.1101/2024.12.22.629968 Diverse subtypes of cortical projection neurons (PN) form long-range axonal projections that are responsible for distinct sensory, motor, cognitive, and behavioral functions. Translational control has been identified at multiple stages of PN development, but how translational regulation contributes to formation of distinct, subtype-specific long-range circuits is poorly understood. Ribosomal complexes (RCs) exhibit variations of their component proteins, with an increasing set of examples that confer specialized translational control. Here, we directly compare the protein compositions of RCs in vivo from two closely related cortical neuron subtypes–cortical output “subcerebral PN” (SCPN) and interhemispheric “callosal PN” (CPN)– during establishment of their distinct axonal connectivity. Using retrograde labeling of subtype-specific somata, purification by fluorescence-activated cell sorting, ribosome immunoprecipitation, and ultra-low-input mass spectrometry, we identify distinct protein compositions of RCs from these two subtypes. Strikingly, we identify 16 associated proteins reliably and exclusively detected only in RCs of SCPN. 10 of these proteins have known interaction with components of ribosomes; we further validated ribosome interaction with protein kinase C epsilon (PRKCE), a candidate with roles in synaptogenesis. PRKCE and a subset of SCPN-specific candidate ribosome-associated proteins also exhibit enriched gene expression by SCPN. Together, these results indicate that ribosomal complexes exhibit subtypespecific protein composition in distinct subtypes of cortical projection neurons during development, and identify potential candidates for further investigation of function in translational regulation involved in subtype-specific circuit formation.All rights reserved. -
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 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 developing neurons of the cerebral cortex. BioRxiv Preprint. 2024 Jan 23:2023.09.24.559186. PMID: 38328182; PMCID: PMC10849483. 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 developing neurons of the cerebral cortex. BioRxiv Preprint. 2024 Jan 23:2023.09.24.559186. PMID: 38328182; PMCID: PMC10849483. DOI:https://doi.org/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 cytoplasmic adenylation element binding protein 4 (CPEB4), an RNA binding protein regulating localization and translation of mRNAs encoding molecular machinery important for axonal branching and complexity. We also identify RNA binding motif single stranded interacting protein 1 (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.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. -
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.