Extended Data Figure 2: Apoptosis in bovine forelimb buds during digit formation and gene expression during the onset of limb bud development.

a, The SOX9 transcriptional regulator (red immunofluorescence) marks all chondrogenic progenitors of the forming digit primordia in the bovine forelimb autopod. Apoptotic cells (TUNEL; green fluorescence) are only detected in the interdigit region that is being eliminated at these developmental stages (arrowhead in top panel). No apoptosis is detected in the digit-forming territories, except in the forming joints (arrowhead in bottom panel). Sections were counterstained with Hoechst-33258 to label cell nuclei. b, During initiation of limb bud outgrowth, the complementary expression of Gli3 and Hand2 marks the establishment of anterior–posterior asymmetry and is required to activate Shh expression. Mesenchymal cells responding to SHH signalling upregulate the expression of the SHH receptor Ptch1. Msx2 is a transcriptional sensor of BMP activity and its distribution marks territories of active BMP signalling. The expression of these genes is comparable in early mouse and bovine forelimb buds. In all Extended Data figures, limb buds are oriented with anterior to the top. Per marker and species, the distribution was reproduced in at least n = 3 independent limb bud samples in independent experiments. Scale bar, 0.25 mm.