A novel enhancer near the Pitx1 gene influences development and evolution of pelvic appendages in vertebrates

Abstract

Vertebrate pelvic reduction is a classic example of repeated evolution. Recurrent loss of pelvic appendages in sticklebacks has previously been linked to natural mutations in a pelvic enhancer that maps upstream of Pitx1. The sequence of this upstream PelA enhancer is not conserved to mammals, so we have surveyed a large region surrounding the mouse Pitx1 gene for other possible hind limb control sequences. Here we identify a new pelvic enhancer, PelB, that maps downstream rather than upstream of Pitx1. PelB drives expression in the posterior portion of the developing hind limb, and deleting the sequence from mice alters the size of several hind limb structures. PelB sequences are broadly conserved from fish to mammals. A wild stickleback population lacking the pelvis has an insertion/deletion mutation that disrupts the structure and function of PelB, suggesting that changes in this ancient enhancer contribute to evolutionary modification of pelvic appendages in nature.

Recapitulating evolutionary divergence in a singlecis-regulatory element is sufficient to cause expression changes of the lens geneTdrd7

“…Strikingly, replacing the sequence of this single regulatory element is sufficient to cause a more than two-fold increase in expression, which reveals that this repressor element has a major effect on Tdrd7 expression. This result contrasts previous findings that perturbations of individual regulatory elements generally have no measurable or only a small effect on gene expression (47)(48)(49)(50)(51)(52)(53). Previously-observed robustness at the transcriptional level can be attributed to functional redundancy, whereby multiple regulatory elements control the expression of a gene and a substantial change in gene expression can only be achieved by perturbations of multiple elements (50).…”

“…While perturbations or deletions of single regulatory elements can result in drastic (55)(56)(57) or subtle phenotypic changes (47,49,52), often they do not result in detectable phenotypic changes (48,50,51,58). Even in the absence of detectable phenotypes when perturbing a single regulatory element, mutations in several regulatory elements can collectively account for phenotypic differences.…”

Variation in mouse pelvic morphology maps to locations enriched in Sox9 Class II and Pitx1 regulatory features

“…Here, we demonstrated Pitx1 and Sox9 Class II regulation are important mechanisms for the phenotypic integration of the mouse pelvis. That Pitx1 regulation may be an important developmental mechanism for the generation of covariation in this mouse population is particularly exciting given its role in structuring variation allowing adaptation of the pelvic apparatus of populations of stickleback fish to new environments (Chan et al, 2010; Thompson et al, 2018).…”

“…The Pitx1 gene has an important role in specifying the hind limb or pelvic fin structures across vertebrates and has been implicated as a primary contributor to variation in the pelvic apparatus in populations of stickleback fish enabling them to adapt quickly to different environments (Chan et al, 2010; Thompson et al, 2018). Moreover, mutations to Pitx1 in mice result in ilium dysmorphologies among other more subtle pelvic and hind limb alterations (Marcil et al, 2003).…”

“…Our results support this contention as all classes of Pitx1 ChIP‐seq features were overrepresented in the QTL identified here indicating regulation of growth through this set of mechanisms may contribute to individual differences in this population of mammals. In the case of Pitx1 and the evolution of the pelvic apparatus in stickleback fish, the Pitx1 gene itself shows signs of being highly conserved, with adaptive differences among groups being driven by natural selection acting on variation in its associated regulatory features (Chan et al, 2010; Thompson et al, 2018). Our results support the position that Pitx1 cis ‐ and trans ‐regulation is an important regulator of phenotypic integration in the pelvic girdle and may be important for rendering the pelvic girdle evolvable across many species of vertebrates.…”