RESUMO
In the developing nervous system, neural stem cells are polarized and maintain an apical domain facing a central lumen. The presence of apical membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis, cells lose their polarization, and the concomitant loss of the apical domain coincides with a loss of the stem cell identity. Little is known about the molecular signals controlling apical membrane size. Here, we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human embryonic stem cells, to identify a molecular signaling pathway initiated by lysophosphatidic acid that controls apical membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This apical domain size increase occurs independently of effects on proliferation and involves a serum response factor-dependent transcriptional induction of junctional and apical membrane components.
Assuntos
Autorrenovação Celular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Neurogênese , Transdução de Sinais , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular , Membrana Celular/metabolismo , Polaridade Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Imunofluorescência , Expressão Gênica , Humanos , Lisofosfolipídeos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células Neuroepiteliais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas , Transcrição GênicaRESUMO
The salamander is the only tetrapod that functionally regenerates all cell types of the limb and spinal cord (SC) and thus represents an important regeneration model, but the lack of gene-knockout technology has limited molecular analysis. We compared transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) in the knockout of three loci in the axolotl and find that CRISPRs show highly penetrant knockout with less toxic effects compared to TALENs. Deletion of Sox2 in up to 100% of cells yielded viable F0 larvae with normal SC organization and ependymoglial cell marker expression such as GFAP and ZO-1. However, upon tail amputation, neural stem cell proliferation was inhibited, resulting in spinal-cord-specific regeneration failure. In contrast, the mesodermal blastema formed normally. Sox3 expression during development, but not regeneration, most likely allowed embryonic survival and the regeneration-specific phenotype. This analysis represents the first tissue-specific regeneration phenotype from the genomic deletion of a gene in the axolotl.
Assuntos
Ambystoma mexicanum/fisiologia , Proteínas de Anfíbios/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Deleção de Genes , Células-Tronco Neurais/citologia , Regeneração , Fatores de Transcrição SOXB1/genética , Ambystoma mexicanum/embriologia , Ambystoma mexicanum/genética , Animais , Sequência de Bases , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Regeneração da Medula EspinalRESUMO
Salamanders regenerate appendages via a progenitor pool called the blastema. The cellular mechanisms underlying regeneration of muscle have been much debated but have remained unclear. Here we applied Cre-loxP genetic fate mapping to skeletal muscle during limb regeneration in two salamander species, Notophthalmus viridescens (newt) and Ambystoma mexicanum (axolotl). Remarkably, we found that myofiber dedifferentiation is an integral part of limb regeneration in the newt, but not in axolotl. In the newt, myofiber fragmentation results in proliferating, PAX7(-) mononuclear cells in the blastema that give rise to the skeletal muscle in the new limb. In contrast, myofibers in axolotl do not generate proliferating cells, and do not contribute to newly regenerated muscle; instead, resident PAX7(+) cells provide the regeneration activity. Our results therefore show significant diversity in limb muscle regeneration mechanisms among salamanders and suggest that multiple strategies may be feasible for inducing regeneration in other species, including mammals.
Assuntos
Ambystoma mexicanum/fisiologia , Desdiferenciação Celular , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Salamandridae/fisiologia , Células-Tronco/citologia , Animais , Animais Geneticamente Modificados , Proliferação de Células , Extremidades/fisiologia , Genes Reporter , Células Germinativas/citologia , Células Germinativas/metabolismo , Larva/fisiologia , Mesoderma/citologia , Mesoderma/transplante , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Fator de Transcrição PAX7/metabolismoRESUMO
An amputated salamander limb regenerates the correct number of segments. Models explaining limb regeneration were largely distinct from those for limb development, despite the presence of common patterning molecules. Intercalation has been an important concept to explain salamander limb regeneration, but clear evidence supporting or refuting this model was lacking. In the intercalation model, the first blastema cells acquire fingertip identity, creating a gap in positional identity that triggers regeneration of the intervening region from the stump. We used HOXA protein analysis and transplantation assays to show that axolotl limb blastema cells acquire positional identity in a proximal-to-distal sequence. Therefore, intercalation is not the primary mechanism for segment formation during limb regeneration in this animal. Patterning in development and regeneration uses similar mechanisms.
Assuntos
Extremidades/fisiologia , Proteínas de Homeodomínio/metabolismo , Regeneração , Ambystoma mexicanum , Animais , Padronização Corporal , Extremidades/anatomia & histologia , Dados de Sequência MolecularRESUMO
Limb regeneration involves re-establishing a limb development program from cells within adult tissues. Identifying molecular handles that provide insight into the relationship between cell differentiation status and cell lineage is an important step to study limb blastema cell formation. Here, using single cell PCR, focusing on newly isolated Twist1 sequences, we molecularly profile axolotl limb blastema cells using several progenitor cell markers. We link their molecular expression profile to their embryonic lineage via cell tracking experiments. We use in situ hybridization to determine the spatial localization and extent of overlap of different markers and cell types. Finally, we show by single cell PCR that the mature axolotl limb harbors a small but significant population of Twist1(+) cells.
Assuntos
Ambystoma mexicanum/fisiologia , Tecido Conjuntivo/metabolismo , Extremidades/fisiologia , Músculo Esquelético/metabolismo , Regeneração/fisiologia , Células-Tronco/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Linhagem da Célula/fisiologia , Células do Tecido Conjuntivo/metabolismo , Hibridização In Situ , Mesoderma/citologia , Músculo Esquelético/citologia , Reação em Cadeia da Polimerase , Pele/citologia , TranscriptomaRESUMO
We show that after tail amputation in Ambystoma mexicanum (Axolotl) the correct number and spacing of dorsal root ganglia are regenerated. By transplantation of spinal cord tissue and nonclonal neurospheres, we show that the central spinal cord represents a source of peripheral nervous system cells. Interestingly, melanophores migrate from preexisting precursors in the skin. Finally, we demonstrate that implantation of a clonally derived spinal cord neurosphere can result in reconstitution of all examined cell types in the regenerating central spinal cord, suggesting derivation of a cell with spinal cord stem cell properties.