Fig. 5: Tgfbr1 regulates accessibility of limb regulatory regions.

A, A’ Chromatin regions following pattern 2 and pattern 3. A Venn diagram showing interception between the regions more accessible in mutant extra limb than in mutant GT [FDR < 0.001, logFC > 1.5] and more accessible in control hindlimb than in control GT [FDR < 0.001, logFC > 1.5], but at the same time not differentially accessible between mutant extra hindlimb and control hindlimb [FDR > 0.01, logFC < 0.5]. A’ Heatmap showing log2 normalized counts from genomic regions following pattern 2 across the samples. B, B’ Chromatin regions following pattern 3. B Venn diagram showing interception between regions tested more accessible in control hindlimb than in other tissues by pairwise comparisons [FDR < 0.001, logFC > 1.5]. B’ Heatmap showing log2 normalized counts for the chromatin regions following pattern 3 across samples. C, D ATAC-Seq profiles showing Fgf10 putative regulatory element (C) and Grem1 enhancer (D) that gains accessibility in the mutant extra hindlimb (highlighted with the blue shadow). Also shown are ChiP-Seq tracks of ChIP seq data from wild type forelimbs for Gli3 (yellow) and Hoxa13 (magenta) (obtained from GSE81356 and GSE133710, respectively), indicating their binding to those elements in the forelimb bud. The lower track shows conservation in placental mammals. Grem1 expression in E10.5 wild type (E) or Tgfbr1-cKO (E’) embryos showing the absence of pericloacal expression in wild type embryos and ectopic activation in the caudal-most region of the pericloacal mesoderm of the mutant embryo (white arrow). Grem1 is expressed in the developing limb bud of the control embryo, but it is almost not detectable in the developing limb buds of the mutant embryo (white arrowheads) (n = 3 embryos are analyzed per probe). F ATAC-seq data through the ZRS (highlighted with the blue shadow), showing that it is accessible in the control limb but not in any of the other samples. Two independent biological replicates were analyzed for all ATAC-seq experiments. CR conserved region, cHL control hindlimb, mExHL mutant extra hindlimb, cGT control GT, mGT mutant GT.