Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution

Abstract

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.

Remembering where we are: Positional information in salamander limb regeneration

“…Currently, there is little evidence that maintained rather than reactivated expression of Hox and other homeobox genes is required for adult cells to contribute to the blastema in a position‐appropriate way in salamander limb regeneration. The resolution at which individual cells can be transcriptionally profiled in mature and regenerating salamander limbs continues to increase . To date, no studies have used high resolution approaches to directly investigate whether small populations of cells reside within the adult salamander limb that maintain known positional‐identity markers, but future studies may identify previously overlooked populations of cells that preserve positional identity at a molecular level.…”

“…The immense genomes and long generation times of these animals have made them recede as model organisms in the modern era of molecular genetics; yet, in recent years, the number of laboratories using salamanders as their primary model organism has rapidly grown. In a matter of a few years, the ongoing sequencing and assembly of salamander genomes, advances in targeted mutagenesis and transgenesis, and the advent of high‐throughput single‐cell transcriptional approaches, among many other advances, have turned salamanders into powerful genetically tractable model organisms. These tools have converged to open up new avenues for the investigation of positional information.…”

“…The resolution at which individual cells can be transcriptionally profiled in mature and regenerating salamander limbs continues to increase. 100,101 To date, no studies have used high resolution approaches to directly investigate whether small populations of cells reside within the adult salamander limb that maintain known positional-identity markers, but future studies may identify previously overlooked populations of cells that preserve positional identity at a molecular level.…”

Single-Cell RNA-Seq Reveals Novel Mitochondria-related Musculoskeletal Cell Populations during Adult Axolotl Limb Regeneration Process

“…ScRNA-seq reveals novel cell types and reconstructs lineage hierarchies of tissue types such as human pluripotent stem cells (Han et al, 2018a), blood dendritic cells (Villani et al, 2017), neurons , and uterus epithelium , whereafter it can even maps an atlas (Han et al, 2018b). Recently, the scRNA-seq was performed on axolotl to uncover the connective tissue subpopulations with their characters and mesenchymal cellular diversity during regeneration (Gerber et al, 2018;Leigh et al, 2018). However, the alterations of global limb cell populations during regeneration process were not clarified.…”

Comparison of Gene Expression During Early Phases of Limb Regeneration Between Regeneration-permissive Neotenic and Regeneration-deficient Metamorphic Axolotl

“…But still, much of the established knowledge on axolotl limb regeneration has relied on and is mostly still contingent upon, microarray such as (Knapp et al 2013;Voss et al 2015) and high-throughput technologies including transcriptomics (e.g. (Bryant et al 2017;Leigh et al 2018)) and proteomics (Rao et al 2009;Demircan et al 2017) . In spite of the valuable insights which RNA based gene expression analysis methods have to offer, an inherent limitation of them lies within their failure to accurately reflect the gene expression activity up to the protein level.…”