Figure 1.

Connective tissue cells are source cells of the adult frog blastema

(A) Schematic comparison of limb regeneration after amputation in axolotls versus post-metamorphic frogs. Axolotls regenerate a complete limb while post-metamorphic frogs form a blastema but generate an abortive cartilage spike.

(B) Schematic illustration of the Prrx1:CreER line (left) and the CAGGs:lp-Cherry reporter line (right). The CreER cassette was codon optimized and each genotype was generated individually with the REMI protocol (see STAR methods for details). Germline transmitted Prrx1:CreER founders were mated with a CAGGs:lp-Cherry reporter line. The F1 carrying both CreER and reporter cassettes were screened and used for the experiments in this study.

(C) 4-hydroxytamoxifen (4-OHT) treatment paradigm used to convert the reporter cassette in F1.

(D) Whole-mount image of mCherry fluorescence of a hindlimb bud from a converted Prrx1:CreER;CAGGs:lp-Cherry transgenic stage 51 tadpole. Proximal: left; Distal: right. Scale bar represents 20 μm.

(E–G) Magnified fluorescence images of a longitudinal section of (D) immunostained for Prrx1 (mesenchymal CT cells, green) (E), Cherry (converted cells, magenta) (F), and the merged image with DAPI (nuclei, blue) (G). White dashed line denotes the border between Prrx1-positive mesenchyme (mes) and Prrx1-negative epidermis (epi). mCherry-expressing cells are confined to mesenchyme. Scale bars represent 20 μm.

(H) Transverse section of uninjured upper hindlimb skin from a converted Prrx1:CreER;CAGGs:lp-Cherry transgenic froglet, immunostained for Cherry (converted cells, magenta), Prrx1 (dermal CT cells, green), and DAPI (nuclei, blue). White arrowheads indicate examples of converted dermal fibroblasts identified as Cherry- and Prrx1-double positive cells at the mesenchyme (mes)-epidermis (epi) border (white dashed line). Asterisks indicate non-specific tissue autofluorescence. Scale bars represent 50 μm.

(I) Transverse section of cartilage region from a converted Prrx1:CreER;CAGGs:lp-Cherry transgenic froglet, immunostained for Cherry (converted cells, magenta) and DAPI (nuclei, blue). White dashed line denotes the border of cartilage (ca). White arrows indicate examples of converted Cherry-positive chondrocytes and white arrowheads the converted perichondria. Scale bars represent 50 μm.

(J) Transverse section of muscle bundle region from a converted Prrx1:CreER;CAGGs:lp-Cherry transgenic froglet, immunostained for Cherry (converted cells, magenta), Prrx1 (interstitial CT cells, green), and DAPI (nuclei, blue). White arrowheads indicate examples of converted interstitial fibroblasts identified as Cherry- and Prrx1-double positive cells. Scale bars represent 50 μm.

(K) Magnified fluorescence image of a longitudinal section of a 28 dpa blastema from a converted Prrx1:CreER;CAGGs:lp-Cherry transgenic froglet, immunostained for Cherry (converted cells, magenta), Prrx1 (blastema CT cells, green), and DAPI (nuclei, blue). Arrowheads show examples of the Prrx1-converted Cherry-positive cells contributing to the blastema. Scale bars represent 50 μm.

(L) Percentage of Prrx1-expressing mesenchymal cells in limb buds (Lb, 84.4% ± 17.7%, n = 3), uninjured leg (Uninj, 39.9% ± 5.6%, n = 5), and blastema (BL, 68.2% ± 3.0%, n = 3), determined by immunohistochemistry. Statistical significance is calculated using unpaired Student’s t test. ∗∗∗p < 0.001. Data are represented as mean ± SD.

(M) Percentage of converted Cherry+ CT precursors (Cherry+/Prrx1+) in limb buds (Lb, 13.2% ± 1.2%, n = 3), uninjured leg (Uninj, 11.7% ± 3.6%, n = 5), and blastema (BL, 22.0% ± 6.5%, n = 3), determined by immunohistochemistry. Statistical significance is calculated using unpaired Student’s t test. n.s.: p > 0.05; ∗p < 0.01. Data are represented as mean ± SD.

(N) Uniform manifold approximation and projection (UMAP) embedding of scRNA-seq data of 3,817 cells from a 14-dpa frog blastema. Cells are colored by cluster and cell type annotations are noted. Insets show the expression of blastema cells with a CT signature (prrx1), pericyte (myh11), and cartilage (col9a2) markers projected onto the UMAP embedding.

(O) Violin plots show the expression of canonical marker genes defining cluster cell type annotations in the 14 dpa blastema as shown in (N).