Summary The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatio-temporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then, we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior and posterior polarity of the nascent limb bud mesenchyme by impacting on Gli3 and Tbx3 expression. TBX3 is required downstream of HAND2 to refine the posterior Gli3 expression boundary. Our analysis uncovers the transcriptional circuits that function in establishing distinct mesenchymal compartments downstream of HAND2 and upstream of SHH signaling.

SUMMARY Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G1–S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation.

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.