Digit loss/reductions are evolutionary adaptations in cursorial mammals such as pigs. To gain mechanistic insight into these processes, we performed a comparative molecular analysis of limb development in mouse and pig embryos, which revealed a loss of anterior-posterior polarity during distal progression of pig limb bud development. These alterations in pig limb buds are paralleled by changes in the mesenchymal response to Sonic hedgehog (SHH) signaling, which is altered upstream of the reduction and loss of Fgf8 expression in the ectoderm that overlaps the reduced and vestigial digit rudiments of the pig handplate, respectively. Furthermore, genome-wide open chromatin profiling using equivalent developmental stages of mouse and pig limb buds reveals the functional divergence of about one-third of the regulatory genome. This study uncovers widespread alterations in the regulatory landscapes of genes essential for limb development that likely contributed to the morphological diversion of artiodactyl limbs from the pentadactyl archetype of tetrapods.

Mouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Major aspects of limb bud development are controlled by feedback loops that define a self-regulatory signalling system. The SHH/GREM1/AER-FGF feedback loop forms the core of this signalling system that operates between the posterior mesenchymal organiser and the ectodermal signalling centre. The BMP antagonist Gremlin1 (GREM1) is a critical node in this system, whose dynamic expression is controlled by BMP, SHH, and FGF signalling and key to normal progression of limb bud development. Previous analysis identified a distant cis-regulatory landscape within the neighbouring Formin1 (Fmn1) locus that is required for Grem1 expression, reminiscent of the genomic landscapes controlling HoxD and Shh expression in limb buds.

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 spatiotemporal 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 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.