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.

Fig. 1

Single-cell RNA-seq of the adult axolotl limb. a Hematoxylin and eosin staining of medium-bud blastema with overlying wound epidermis. Cell types labeled in a, b based on morphology/location. Scale bars are 100 μm, n = 4 animals. c Strategy for identifying the cell populations that compose the regenerating axolotl limb. d t-SNE plot of cells collected from homeostasis (n = 2 animals), wound healing (n = 3 animals), early-bud blastema (n = 2 animals), and medium-bud blastema (n = 6 animals). Each dot is a cell and colors represent individual populations and do not reflect the same population between time points

Fig. 2

Wound epidermis gene expression dynamics during regeneration. a Violin plots depicting log₂ values of normalized expression of krt12, otog, and frem2 at homeostasis. b–d Representative RNA in situ hybridizations in limbs at homeostasis probing for krt12 (b) (n = 5 animals), otog (c) (n = 3 animals), and frem2 (d) (n = 3 animals). e Violin plots depicting log₂ values of normalized expression of krt12, otog, and frem2 in the medium-bud blastema. f–h Representative RNA in situ hybridizations in medium-bud blastemas probing for krt12 (f) (n = 5 animals), otog (g) (n = 3 animals), and frem2 (h) (n = 5 animals). In violin plots (a, e), each dot represents an individual cell; epidermal populations of interest are depicted in color, all others are in gray. Yellow line in f, g denotes boundary between the wound epidermis and blastema. Epi epidermis, WE wound epidermis, FLB fibroblast-like blastema cell. Magnification, ×10, scale bars are 100 μm

Fig. 3

The epidermis follows an outward differentiating trajectory. a–d Pseudotime analysis of epidermis populations during homeostasis (a, b) and in the medium-bud blastema (c, d), shown split by overall pseudotime plots (a, c) and colored by Seurat population determined in Fig. 1 (b, d). Abbreviations used are small secretory cells (SSCs), intermediate epidermis (IE), basal epidermis (BE), basal wound epidermis #1 (BW1), intermediate wound epidermis (IW), and basal wound epidermis #2 (BW2). e Expression plot of krt17 in epidermal cells captured at the medium-bud blastema stage over pseudotime, colored using Seurat populations as in d. f–o Representative RNA in situ hybridization probing for krt17 across a time course of limb regeneration. Magnification ×10 in f, h, j, l, n, ×20 in g, i, k, m, o. All scale bars are 100μm. n = 4 animals per time point

Fig. 4

The blastema contains T cells. a Violin plots depicting log₂ values of normalized expression of the T cell-specific gene, trac, at homeostasis, wound healing, early-bud blastema and medium-bud blastema stages. Each dot represents an individual cell. b, c Representative RNA in situ hybridization probing for trac in a medium-bud-stage regenerate. b Magnification, ×20 scale bar is 100 μm; c area outlined in b at ×40 magnification, scale bar is 50 μm. n = 4 animals

Fig. 5

Myeloid cells are present throughout the course of regeneration. a Violin plots depicting log₂ values of normalized expression of myeloid cell enriched apoeb. Each dot represents an individual cell; myeloid cell populations are in color, while all other populations are in gray. b–g Representative RNA in situ hybridizations probing for apoeb across a time course of regeneration. b–g Magnification ×10 during wound healing, specifically at b 3 h post amputation (hpa), c 12hpa, and d 24hpa. e–g Magnification ×10 of a e early-bud blastema, f medium-bud blastema, and a g palette stage-regenerating limb; yellow line denotes border of blastema and WE. All scale bars are 100 μm. n = 4 animals per time point

Fig. 6

Fibroblasts-like cells are found during homeostasis and regeneration. a Violin plots depicting log₂ values of normalized expression of fibroblast and fibroblast-like blastema (FLB) cell-enriched genes lum and dpt at homeostasis, wound healing, early-bud blastema, and medium-bud blastema stages. Each dot represents an individual cell; fibroblast/FLB populations of interest are depicted in color, while all other populations are in gray. b Representative RNA in situ hybridization probing for lum in a medium-bud blastema. c Representative RNA in situ hybridization probing for dpt in a medium-bud blastema. Magnification ×4, scale bar is 500 μm. n = 4 animals per probe

Fig. 7

Putative differentiation trajectories of blastema cells. a t-SNE plot of blastema cells colored by respective time point sampled. b Distribution of pseudotime of blastema cells colored by time point sampled. c t-SNE plot of blastema cell populations in the medium-bud-stage blastema. d Blastema differentiation trajectories colored by blastema cell populations identified in c. Syn. fibros synovial fibroblasts, Endo. endothelial cells, FAPs fibro-adipogenic progenitors, WH wound healing, EB early-bud blastema