Dissection of Ptch1 cis-regulatory robustness in the context of artiodactyl limb evolution

How genes are regulated during development and how this regulation has changed during evolution are two of the most appealing questions of modern biology. Indeed, variation of cis-regulatory elements (CRE) is considered to be one of the main mechanisms underlying morphological evolution. The limb is an excellent model to study both of these processes: its development is genetically well understood and it has adopted a wide range of different morphologies during evolution to adapt to different forms of locomotion (Zuniga, 2015; Petit et al., 2017). Most particularly, I am interested in the evolutionary loss and reduction of digits that happened in artiodactyls, as extant members of this order only have 2 or 4 digits and therefore represent a deviation from the ancestral pentadactyl state of tetrapods (Polly, 2007).
Ptch1, the receptor of the SHH pathway has been shown to play a central role in these evolutionary processes in bovines (Bos taurus). Indeed, failure to up-regulate this gene contributes to the evolutionary loss of anterior-posterior polarity in the limbs of these animals. In this thesis, I first dissected the mouse Ptch1 cis-regulatory landscape and identified three new limb enhancers of Ptch1, increasing the total number of candidate elements regulating this gene in the limb to six. In particular, I identified a distant CRE located 385 kb upstream of the Ptch1 gene body that drives reporter expression in the posterior-distal limb bud mesenchyme similar to the endogenous Ptch1 gene and the previously identified LRM enhancer. Individual and combined knockout of cis-regulatory elements in the mouse using the CRISPR/Cas9 technology led to little variation in Ptch1 expression in the limb, suggesting that Ptch1 regulation is exceptionally robust. To this robustness also likely contributes the upregulation of Ptch1 itself in response to a dose diminution.
Additionally, I analyzed the limb development of the pig (Sus scrofa), another artiodactyl and showed that the evolutionary mechanism underlying pig and bovine limb development were similar. The expression of known genes involved in limb development displayed a similar loss of molecular anterior-posterior polarity in pig and in bovine. This loss of asymmetry is paralleled by changes in the SHH pathway: Ptch1 is more restricted posteriorly in pig than in mouse and Gli1, anteriorized. Furthermore, the evolutionary reduction of digits 2 and 5 in pig was linked with a premature downregulation of AER-FGFs signaling over the primordia of these digits compared with the central ones, providing an explanation to the reduction of lateral digits in pig versus their complete loss in bovines. Next, I also used functional genomics approaches to obtain a comprehensive and unbiased picture of the regulatory variation underlying morphological evolution of the pig limb in comparison to the my reference model, the mouse. In particular, I resorted to ATAC-seq to catalog all regions from the pig and mouse genomes harboring regulatory activity during limb development. This analysis showed that that ∼35% of accessible regions in one of the species are evolutionary conserved at the sequence level, but closed in the other species, suggesting a high level of regulatory re-wiring. Furthermore, many of these alterations are in close proximity to genes that encode components of the major morphological pathways governing limb development such as those of FGF, SHH, WNT or BMP. This study provides a baseline for gene-centric studies that address how regulatory variation impacts on morphological evolution of the limb in artiodactyls.
Finally, I used lacZ reporter assays in transgenic mice to address the activity of the main limb regulatory elements identified in the Ptch1 cis-regulatory landscape in artiodactyls, in comparison to the corresponding mouse constructs. This analysis revealed that that some regulatory elements had functionally degenerated in the artiodactyl lineage (e.g. the LRM enhancer), while other CREs had retained a similar activity in mouse and artiodactyls (egg. E385 enhancer) despite >95 Myr of evolutionary distance. Combined with the thorough molecular analysis of pig limb bud development, these results establish a link between the loss of anterior-posterior polarity and the partial degeneration of the Ptch1 cis-regulatory landscape, which likely originated in the common ancestor of the pig and bovine lineages.