Fall 2024; SCRB departmental retreat at the New England Aquarium
Whited Lab Publications
Featured Publications
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2018. Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution. Nature communications. 9(1):5153. Pubmed: 30514844 DOI:10.1038/s41467-018-07604-0 Leigh ND, Dunlap GS, Johnson K, Mariano R, Oshiro R, Wong AY, Bryant DM, Miller BM, Ratner A, Chen A, Ye WW, Haas BJ, Whited JL. 2018. Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution. Nature communications. 9(1):5153. Pubmed: 30514844 DOI:10.1038/s41467-018-07604-0 Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, defining the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. Here we report unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs and identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity of fibroblast-like blastema progenitor cells. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. Overall, these data allow for establishment of a putative framework for adult axolotl limb regeneration. -
Bryant DM, Johnson K, DiTommaso T, Tickle T, Couger MB, Payzin-Dogru D, Lee TJ, Leigh ND, Kuo TH, Davis FG, Bateman J, Bryant S, Guzikowski AR, Tsai SL, Coyne S, Ye WW, Freeman RM, Peshkin L, Tabin CJ, Regev A, Haas BJ, Whited JL. 2017. A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors. Cell reports. 18(3):762-776. Pubmed: 28099853 DOI:S2211-1247(16)31770-3 Bryant DM, Johnson K, DiTommaso T, Tickle T, Couger MB, Payzin-Dogru D, Lee TJ, Leigh ND, Kuo TH, Davis FG, Bateman J, Bryant S, Guzikowski AR, Tsai SL, Coyne S, Ye WW, Freeman RM, Peshkin L, Tabin CJ, Regev A, Haas BJ, Whited JL. 2017. A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors. Cell reports. 18(3):762-776. Pubmed: 28099853 DOI:S2211-1247(16)31770-3 Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved. -
Bryant DM, Sousounis K, Farkas JE, Bryant S, Thao N, Guzikowski AR, Monaghan JR, Levin M, Whited JL. 2017. Repeated removal of developing limb buds permanently reduces appendage size in the highly-regenerative axolotl. Developmental biology. 424(1):1-9. Pubmed: 28235582 DOI:S0012-1606(16)30873-9 Bryant DM, Sousounis K, Farkas JE, Bryant S, Thao N, Guzikowski AR, Monaghan JR, Levin M, Whited JL. 2017. Repeated removal of developing limb buds permanently reduces appendage size in the highly-regenerative axolotl. Developmental biology. 424(1):1-9. Pubmed: 28235582 DOI:S0012-1606(16)30873-9 Matching appendage size to body size is fundamental to animal function. Generating an appropriately-sized appendage is a robust process executed during development which is also critical for regeneration. When challenged, larger animals are programmed to regenerate larger limbs than smaller animals within a single species. Understanding this process has important implications for regenerative medicine. To approach this complex question, models with altered appendage size:body size ratios are required. We hypothesized that repeatedly challenging axolotls to regrow limb buds would affect their developmental program resulting in altered target morphology. We discovered that after 10 months following this experimental procedure, limbs that developed were permanently miniaturized. This altered target morphology was preserved upon amputation and regeneration. Future experiments using this platform should provide critical information about how target limb size is encoded within limb progenitors.Copyright © 2017 Elsevier Inc. All rights reserved. -
Farkas JE, Freitas PD, Bryant DM, Whited JL, Monaghan JR. 2016. Neuregulin-1 signaling is essential for nerve-dependent axolotl limb regeneration. Development (Cambridge, England). 143(15):2724-31. Pubmed: 27317805 DOI:10.1242/dev.133363 Farkas JE, Freitas PD, Bryant DM, Whited JL, Monaghan JR. 2016. Neuregulin-1 signaling is essential for nerve-dependent axolotl limb regeneration. Development (Cambridge, England). 143(15):2724-31. Pubmed: 27317805 DOI:10.1242/dev.133363 The Mexican axolotl (Ambystoma mexicanum) is capable of fully regenerating amputated limbs, but denervation of the limb inhibits the formation of the post-injury proliferative mass called the blastema. The molecular basis behind this phenomenon remains poorly understood, but previous studies have suggested that nerves support regeneration via the secretion of essential growth-promoting factors. An essential nerve-derived factor must be found in the blastema, capable of rescuing regeneration in denervated limbs, and its inhibition must prevent regeneration. Here, we show that the neuronally secreted protein Neuregulin-1 (NRG1) fulfills all these criteria in the axolotl. Immunohistochemistry and in situ hybridization of NRG1 and its active receptor ErbB2 revealed that they are expressed in regenerating blastemas but lost upon denervation. NRG1 was localized to the wound epithelium prior to blastema formation and was later strongly expressed in proliferating blastemal cells. Supplementation by implantation of NRG1-soaked beads rescued regeneration to digits in denervated limbs, and pharmacological inhibition of NRG1 signaling reduced cell proliferation, blocked blastema formation and induced aberrant collagen deposition in fully innervated limbs. Taken together, our results show that nerve-dependent NRG1/ErbB2 signaling promotes blastemal proliferation in the regenerating limb and may play an essential role in blastema formation, thus providing insight into the longstanding question of why nerves are required for axolotl limb regeneration.© 2016. Published by The Company of Biologists Ltd. -
Whited JL, Lehoczky JA, Tabin CJ. 2012. Inducible genetic system for the axolotl. Proceedings of the National Academy of Sciences of the United States of America. 109(34):13662-7. Pubmed: 22869739 DOI:10.1073/pnas.1211816109 Whited JL, Lehoczky JA, Tabin CJ. 2012. Inducible genetic system for the axolotl. Proceedings of the National Academy of Sciences of the United States of America. 109(34):13662-7. Pubmed: 22869739 DOI:10.1073/pnas.1211816109 Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology. -
Whited JL, Cassell A, Brouillette M, Garrity PA. 2004. Dynactin is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons. Development (Cambridge, England). 131(19):4677-86. Pubmed: 15329347 Whited JL, Cassell A, Brouillette M, Garrity PA. 2004. Dynactin is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons. Development (Cambridge, England). 131(19):4677-86. Pubmed: 15329347 How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. In this work, we demonstrate that the Dynactin complex (a regulator of the microtubule motor protein Dynein) is required to maintain the position of the nucleus within post-mitotic Drosophila melanogaster photoreceptor neurons. We show that multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and that inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. We find that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function. -
Dunlap GS, Whited JL. 2019. Development: How Tadpoles ROC Tail Regeneration. Current biology : CB. 29(15):R756-R758. Pubmed: 31386855 DOI:S0960-9822(19)30753-5 Dunlap GS, Whited JL. 2019. Development: How Tadpoles ROC Tail Regeneration. Current biology : CB. 29(15):R756-R758. Pubmed: 31386855 DOI:S0960-9822(19)30753-5 Specialized epidermal cells are essential for the complex tissue regeneration required to replace tails and limbs, but their exact identities and molecular roles remain murky. Recent work in Xenopus has identified an epidermal cell population critical for tail regeneration, providing intriguing new directions for the field.Copyright © 2019 Elsevier Ltd. All rights reserved.
All Publications
2018
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Johnson K, Bateman J, DiTommaso T, Wong AY, Whited JL. 2018. Systemic cell cycle activation is induced following complex tissue injury in axolotl. Developmental biology. 433(2):461-472. Pubmed: 29111100 DOI:S0012-1606(17)30195-1 Johnson K, Bateman J, DiTommaso T, Wong AY, Whited JL. 2018. Systemic cell cycle activation is induced following complex tissue injury in axolotl. Developmental biology. 433(2):461-472. Pubmed: 29111100 DOI:S0012-1606(17)30195-1 Activation of progenitor cells is crucial to promote tissue repair following injury in adult animals. In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body. Among two other injury models, limb crush and skin excisional wound, only limb crush injuries were capable of inducing cellular responses in contralateral uninjured limbs but did not achieve activation levels seen following limb loss. We found this systemic activation response to injury is independent of formation of a wound epidermis over the amputation plane, suggesting that injury-induced signals alone can promote cellular activation. In mammals, mTOR signaling has been shown to promote activation of quiescent cells following injury, and we confirmed a subset of activated contralateral cells is positive for mTOR signaling within axolotl limbs. These findings suggest that conservation of an early systemic response to injury exists between mammals and axolotls, and propose that a distinguishing feature in species capable of full regeneration is converting this initial activation into sustained and productive growth at the site of regeneration.Copyright © 2017 Elsevier Inc. All rights reserved. -
Payzin-Dogru D, Whited JL. 2018. An integrative framework for salamander and mouse limb regeneration. The International journal of developmental biology. 62(6-7-8):393-402. Pubmed: 29943379 DOI:10.1387/ijdb.180002jw Payzin-Dogru D, Whited JL. 2018. An integrative framework for salamander and mouse limb regeneration. The International journal of developmental biology. 62(6-7-8):393-402. Pubmed: 29943379 DOI:10.1387/ijdb.180002jw Appendage regeneration is not a simple task. The animal must harness all of its energy and resources to orchestrate perhaps one of the most complicated events since its development. Balancing the immune response, wound healing, proliferation, patterning and differentiation is an elegant job, and how some animals achieve that still leaves researchers enchanted today. In this work, we review some of the molecular aspects of regeneration, with a focus on the axolotl, the champion of tetrapod limb regeneration, and the mouse, an excellent mammalian model for digit tip regeneration. Advances in molecular and genomic tools have enabled the discovery of exciting fundamental features of limb regeneration. Integrating the data from different animal systems will be crucial to understanding the common requirements of successful appendage regeneration and places for flexibility. The combination of these efforts is paving the way to grasping how good regenerators respond to the loss of body parts, how these mechanisms might compare in modest regenerators, and, ultimately, in developing approaches for improving regenerative outcomes in humans. -
Natarajan N, Abbas Y, Bryant DM, Gonzalez-Rosa JM, Sharpe M, Uygur A, Cocco-Delgado LH, Ho NN, Gerard NP, Gerard CJ, MacRae CA, Burns CE, Burns CG, Whited JL, Lee RT. 2018. Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration. Circulation. 137(20):2152-2165. Pubmed: 29348261 DOI:10.1161/CIRCULATIONAHA.117.030801 Natarajan N, Abbas Y, Bryant DM, Gonzalez-Rosa JM, Sharpe M, Uygur A, Cocco-Delgado LH, Ho NN, Gerard NP, Gerard CJ, MacRae CA, Burns CE, Burns CG, Whited JL, Lee RT. 2018. Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration. Circulation. 137(20):2152-2165. Pubmed: 29348261 DOI:10.1161/CIRCULATIONAHA.117.030801 Array© 2018 American Heart Association, Inc. -
Leigh ND, Dunlap GS, Johnson K, Mariano R, Oshiro R, Wong AY, Bryant DM, Miller BM, Ratner A, Chen A, Ye WW, Haas BJ, Whited JL. 2018. Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution. Nature communications. 9(1):5153. Pubmed: 30514844 DOI:10.1038/s41467-018-07604-0 Leigh ND, Dunlap GS, Johnson K, Mariano R, Oshiro R, Wong AY, Bryant DM, Miller BM, Ratner A, Chen A, Ye WW, Haas BJ, Whited JL. 2018. Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution. Nature communications. 9(1):5153. Pubmed: 30514844 DOI:10.1038/s41467-018-07604-0 Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, defining the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. Here we report unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs and identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity of fibroblast-like blastema progenitor cells. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. Overall, these data allow for establishment of a putative framework for adult axolotl limb regeneration. 2017
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Haas BJ, Whited JL. 2017. Advances in Decoding Axolotl Limb Regeneration. Trends in genetics : TIG. 33(8):553-565. Pubmed: 28648452 DOI:S0168-9525(17)30088-4 Haas BJ, Whited JL. 2017. Advances in Decoding Axolotl Limb Regeneration. Trends in genetics : TIG. 33(8):553-565. Pubmed: 28648452 DOI:S0168-9525(17)30088-4 Humans and other mammals are limited in their natural abilities to regenerate lost body parts. By contrast, many salamanders are highly regenerative and can spontaneously replace lost limbs even as adults. Because salamander limbs are anatomically similar to human limbs, knowing how they regenerate should provide important clues for regenerative medicine. Although interest in understanding the mechanics of this process has never wavered, until recently researchers have been vexed by seemingly impenetrable logistics of working with these creatures at a molecular level. Chief among the problems has been the very large size of salamander genomes, and not a single salamander genome has been fully sequenced to date. Recently the enormous gap in sequence information has been bridged by approaches that leverage mRNA as the starting point. Together with functional experimentation, these data are rapidly enabling researchers to finally uncover the molecular mechanisms underpinning the astonishing biological process of limb regeneration.Copyright © 2017 Elsevier Ltd. All rights reserved. -
Bryant DM, Sousounis K, Payzin-Dogru D, Bryant S, Sandoval AGW, Martinez Fernandez J, Mariano R, Oshiro R, Wong AY, Leigh ND, Johnson K, Whited JL. 2017. Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls. NPJ Regenerative medicine. 2:30. Pubmed: 29302364 DOI:10.1038/s41536-017-0034-z Bryant DM, Sousounis K, Payzin-Dogru D, Bryant S, Sandoval AGW, Martinez Fernandez J, Mariano R, Oshiro R, Wong AY, Leigh ND, Johnson K, Whited JL. 2017. Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls. NPJ Regenerative medicine. 2:30. Pubmed: 29302364 DOI:10.1038/s41536-017-0034-z Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl's ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls' ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of , an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine. 2015
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Kuo TH, Whited JL. 2015. Pseudotyped retroviruses for infecting axolotl. Methods in molecular biology (Clifton, N.J.). 1290:127-40. Pubmed: 25740482 DOI:10.1007/978-1-4939-2495-0_10 Kuo TH, Whited JL. 2015. Pseudotyped retroviruses for infecting axolotl. Methods in molecular biology (Clifton, N.J.). 1290:127-40. Pubmed: 25740482 DOI:10.1007/978-1-4939-2495-0_10 The ability to introduce DNA elements into host cells and analyze the effects has revolutionized modern biology. Here we describe a protocol to generate Moloney murine leukemia virus (MMLV)-based, replication-incompetent pseudotyped retrovirus capable of infecting axolotls and incorporating genetic information into their genome. When pseudotyped with vesicular stomatitis virus (VSV)-G glycoprotein, the retroviruses can infect a broad range of proliferative axolotl cell types. However, if the retrovirus is pseudotyped with an avian sarcoma leukosis virus (ASLV)-A envelope protein, only axolotl cells experimentally manipulated to express the cognate tumor virus A (TVA) receptor can be targeted by infections. These strategies enable robust transgene expression over many cell divisions, cell lineage tracing, and cell subtype targeting for gene expression. -
Kuo TH, Kowalko JE, DiTommaso T, Nyambi M, Montoro DT, Essner JJ, Whited JL. 2015. TALEN-mediated gene editing of the thrombospondin-1 locus in axolotl. Regeneration (Oxford, England). 2(1):37-43. Pubmed: 27499866 DOI:10.1002/reg2.29 Kuo TH, Kowalko JE, DiTommaso T, Nyambi M, Montoro DT, Essner JJ, Whited JL. 2015. TALEN-mediated gene editing of the thrombospondin-1 locus in axolotl. Regeneration (Oxford, England). 2(1):37-43. Pubmed: 27499866 DOI:10.1002/reg2.29 Loss-of-function genetics provides strong evidence for a gene's function in a wild-type context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these long-standing questions. Here we demonstrate targeted modification of the thrombospondin-1 (tsp-1) locus using transcription-activator-like effector nucleases (TALENs) and identify a role of tsp-1 in recruitment of myeloid cells during limb regeneration. We find that while tsp-1-edited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALEN-mediated gene edits.