ALM experiments at the four positions.

(A) Schematic image of anatomy at the stylopod level of axolotl limb. HE staining (bright field) and acetylated alpha tubulin, visualized by immunofluorescence (green, dark field), are shown in the right panels. Black and white arrows, respectively, indicate major blood vessels and nerves. H: humerus, AHL: Anconaeus humeralis lateralis, HAB: Humeroantebrachialis, CBL: coracobrachialis longus. (B‒I) Blastemas induced at the anterior, posterior, dorsal, or ventral region by skin wounding plus nerve deviation without (B‒E) or with (F‒I) skin grafting from the opposite side of the limb. Images were captured at 10 and 60 dps. Scale bar = 3 mm.

The induction rate of bump/limb formation in ALM.

Gene expression patterns of the ALM-induced blastemas.

Sections of anteriorly (A‒D), posteriorly (F‒I), dorsally (K‒N), or ventrally (P‒S) induced blastemas at 10 dps. Acetylated alpha tubulin (A‒P) was visualized by immunofluorescence. Expression of Lmx1b (B‒Q), Fgf8 (C‒R), and Shh (D‒S) in the regions indicated by white boxes in (A‒P) was visualized by in situ hybridization. Black and white arrowheads indicate the signals of Fgf8 and Shh expression, respectively. The dotted line indicates the epithelial-mesenchyme border. (E‒T) Schematic images of gene expression patterns. Scale bar = 700 μm.

Co-existence of dorsal and ventral cells induces Shh expression.

(A) Experimental scheme. Posterior half of dorsal (PDgfp) or ventral (PVgfp) GFP-expressing skin was grafted on VentBL (VentBL+PDgfp; C, D / VentBL+PVgfp; G, H), or DorBL (DorBL+PDgfp; E, F / DorBL+PVgfp; I, J) region. (B) Induced blastemas at 10 dps; images of bright and dark fields are merged. (C‒J) Dark and bright fields of the same sections of induced blastemas at 10 dps. Red boxes in (C‒I) indicate the corresponding regions of lower images. Expressions of Shh and Lmx1b were visualized by in situ hybridization. GFP signals were visualized by immunofluorescence. Arrowheads indicate the cells expressing Shh. The dotted line indicates the epithelial-mesenchyme border. Scale bar = 3 mm (B) and 700 μm (C).

Limb formation without the co-existence of dorsal and ventral cells by SHH supplementation.

(A) Experimental scheme. Shh-p2A-GFP- or GFP-containing pCS2 vector was electroporated (EP) into AntBL, DorBL, and VentBL. (B) Induced blastemas at 14 dps; images of bright and dark fields are merged. (C‒G) Phenotypes at 90 dps. (H‒K) Histological analysis of intact and induced limbs. Standard Masson’s trichrome staining was performed on the transverse sections. The dotted boxes indicate the regions shown in (L). (L) Upper panels: analyzed regions for calculating symmetry scores. Lower panels: images after pixel classification by machine learning. (C‒I) Symmetry scores of each class. Scores obtained from the same limb are plotted at the same x-coordinates. n.s.; no significant difference, *p<0.05, **p<0.005. Scale bar = 2 mm (B), 4 mm (C‒ G), and 1 mm (H‒K).

Identification of candidate molecules of the dorsal and ventral positional cues.

RNA-Seq was performed on DorBL and VentBL at 10 dps. (A) MA plot of the result. (B) List of DEGs annotated as “intercellular signaling molecules.” (C) Bright and dark fields of sections of DorBL and VentBL at 10 dps with candidate genes introduced. Expression of Shh and Lmx1b was visualized by in situ hybridization, and GFP signals were visualized by immunofluorescence. Arrowheads indicate the cells expressing Shh. The white boxes indicate the regions of the lower panels. Images of dark and bright fields of Shh are obtained from the same section. (D, E) Quantitative analysis of Shh expression in DorBL with FGF2-soaked bead and with DDW-soaked bead (D) and in VentBL treated with 1μM BIO and with DMSO (E). RNA was prepared from 8 identical DorBL for both (D) and from 4 identical VentBL for both (E). Technological replicates are plotted in the same color. (F, G) The limbs formed from VentBL with Wnt10b and DorBL with Fgf2 at 90 dps. Histological analysis and pixel classification was performed in the same way as in Fig. 4. The dotted boxes indicate the regions shown in the right panels. (H) Symmetry scores of each class. Scores obtained from the same limb are plotted at the same x-coordinates. The plots of “int” are the same plots as in Fig. 4. n.s.; no significant difference, *p<0.05, **p<0.005. Scale bar = 700 μm (C), 4 mm (F, G, upper panels), 1 mm (F, G, lower panels).

Limb formation at the dorsal region by BMP2+FGF2+FGF8 supplementation.

(A, B) 14 and 60 dps phenotypes of BMP2+FGF2+FGF8 supplementation by bead grafting at the dorsal (A) or ventral (B) region. Neither nerve deviation nor skin grafting was performed. (C‒H) Expression patterns of Fgf8, Shh and Lmx1b of induced blastemas at 10 dps. Expression was visualized by in situ hybridization. The dotted line indicates the external shape of the blastema. (I‒ K) Phenotype obtained by BMP2+FGF2+FGF8 supplementation to two dorsal regions of an identical limb. (L) Phenotype obtained by BMP2+FGF2+FGF8 supplementation to five dorsal regions of an identical limb. Scale bar = 3 mm (B), 700 μm (H), 2 cm (J), 1 cm (K, L).

The integration model of the four positional cues.

Schematic images of a normal blastema (A), AntBL (B), PostBL (C), DorBL (D), and VentBL (E). Green, red, blue, and yellow boxes within the blastema represent cells derived from anterior, posterior, dorsal, and ventral regions, respectively. Colored arrows indicate the presence of the corresponding positional cue molecules. In this model, limb patterning requires both FGF8 and SHH, and Shh expression in posteriorly derived cells is induced by the co-existence of WNT10B and FGF2.

Limb patterning from BIO-treated VentBL.

(A, B) VentBL treated with 1μM BIO (A) or DMSO (B) at 90 dps. (C‒D) Histological analysis and pixel classification was performed in the same way as in Fig. 4. The dotted boxes indicate the regions shown in (D). (E) Symmetry scores of each class. Scores obtained from the same limb are plotted at the same x-coordinates. The plots of “int” are the same plots as in Fig. 4. n.s.; no significant difference, *p<0.05, **p<0.005. Scale bar = 4 mm (B), 1 cm (C).