Fig. 5: Determining the sufficiency and requirement of FGF and BMP in induction of patterning competency and associated H3K27me3 patterns.

a Treatment scheme of lateral limb wound samples: wounds were treated with beads soaked in different combinations of FGF and BMP growth factors (GF-Bead) and 7-days (7D) later injected with RA. Subsequently, wounds were evaluated for patterning competency as in the CALM. b Histograms of qRT-PCR data of relative Alx4 and Shh expression in treated wounds described in (a) (GF-Beads: PBS control, FGF2 + FGF8 (F2F8), BMP2 + FGF8 (B2F8), and BMP2 + FGF2 + FGF8 (B2FF)). Gene expression is relative to Ef1α (N = 3 biological replicates per sample group; black * indicates significant change in expression relative to PBS treated wounds using the unpaired t-test, *p < 0.05,**p < 0.005, error bares are SEM). c Images of ectopic growth limb phenotypes in the GF-Bead treated lateral wounds (described in a) 9–12 weeks following RA treatment (scale = 2 mm). N = 20–28 biological replicates for each sample group—refer to Supplementary Data 1.2. d FGF and BMP inhibitor treatment scheme for ND-A tissue. e Heat map representing qRT-PCR data of the relative expression of BMP- (Msx1 and Msx2) and FGF- (Prrx1) dependent genes (normalized to Ef1a expression) in the inhibitor treated ND-As (described in d) relative to uninjured limb tissue. Samples are labeled as follows: (+) = DMSO control, BMPi = LDN193189, FGFI = SU5402, BMPi + FGFi = cotreated with LDN193189 and SU5402. N = 3–5 biological replicates per sample group, black * indicates significant difference in expression relative to DMSO (+) treated ND-A using the Mann–Whitney t-test, *p < 0.05). f (right) Heat map of Normalized Enrichment Scores of GO Biological Process Terms from Gene Set Enrichment Analysis (GSEA) of H3K27me3 CUT&RUN data from the treated ND-As relative to DMSO treated ND-As (full gene lists and GOBP lists are provided in Supplementary Data 3.1-6). Scale bar indicates log2FC in H3K27me3 enrichment. K-means clustering identified 6 clusters (black circles 1–6). (right) Plots of the enrichment scores for the top pathway enrichment hits from the ND-A samples treated with both BMP and FGF inhibitors within the three largest clusters. g Log2FC values of 30 most significant “Additive” genomic regions (flipped enrichment trend only with both inhibitors) in the treated ND-As relative to A-lateral wounds. The top 5 most significant gene regions are indicated in bold font. h List of the top 5 GO Biological Processes from the PANTHER Overrepresentation Test (release 20230705) of “Additive” genomic regions. Full gene lists and associated PANTHER analysis are provided in Supplementary Data 3.13-14.