RESUMO
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.
Assuntos
Extremidades/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regeneração , Transcriptoma , Ambystoma mexicanum/genética , Ambystoma mexicanum/fisiologia , Experimentação Animal , Animais , Diferenciação Celular , Linhagem da Célula , Células Epidérmicas , Epiderme/patologia , Epiderme/fisiologia , Extremidades/embriologia , Extremidades/patologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Sistema Imunitário/fisiologia , Hibridização In Situ , Macrófagos , Células-Tronco Mesenquimais , Células Mieloides/fisiologia , Regeneração Nervosa/fisiologia , Neurônios/fisiologia , Regeneração/genética , Análise de Sequência de RNA , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
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.