Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs

Abstract

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.

The origins, scaling and loss of tetrapod digits

“…Cattle have two toes (digits III and IV) while pigs have two prominent toes (III and IV) and two that are reduced in size (II and V). Expression of the SHH receptor, Ptch1, is highly reduced and limited to the posterior autopod in developing cow [109] and pig [110] forelimb buds compared with mouse ( figure 1). In addition to its role in derepressing the SMOOTHENED receptor in response to SHH binding, PTCH1 is thought to sequester and restrict SHH protein distribution [112,113].…”

“…In addition to these two Shh-dependent and apoptosis-mediated mechanisms of digit loss, Fgf8 expression is lost from the AER overlying truncated and missing digits of cow, pig, camel, horse and three-toed jerboa limb buds at digit-forming stages. This suggests that loss of a mitogenic fibroblast growth factor (FGF) signal might play an additional role reinforcing each of the early limb pattern and late apoptotic mechanisms of digit loss [109,110].…”

“…Given the importance of Shh in determination of digit number, it is perhaps no surprise that the loss of digits in reptiles [107,108] and mammals [109,110] corresponds to changes in the expression of Shh pathway members. Comparison of five different Hemiergis lizard species, with digits ranging from two to five in number, showed that species with the fewest digits were those with the shortest duration of Shh expression in developing limb buds ( figure 1).…”

“…Further, identification of a limb cis-regulatory module (LRM) showed the bovine LRM sequence contains a repeat expansion of variable length in many artiodactyl species, and it fails to promote mesenchymal expansion of a LacZ reporter in transgenic mice [109]. Consistent with these results, limb-specific Ptch1 loss of function causes oligodactyly in mouse forelimbs in a pattern similar to the cow limb [109,114,115]. Together with Shh expression termination in the squamate reptiles, these studies show how modulation of the Shh signal or reception frequently contributes to evolution of digit loss.…”

Perspectives on the history of evo-devo and the contemporary research landscape in the genomics era

“…Sequence differences in a cis-regulatory region have also been suggested to underlie morphological diversification in vertebrate limbs (discussed in the article by Cooper and coworkers [54]). The cis-regulatory region implicated in this case controls expression of a gene encoding a key component of a signalling pathway specifically in the limb bud, and the differences in gene expression found in mice and cows have been suggested to explain the different numbers of digits in the two animals (for details, also see [63]). …”