Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer.
Chan YF, Marks ME, Jones FC, Villarreal G Jr, Shapiro MD, Brady SD, Southwick AM, Absher DM, Grimwood J, Schmutz J, Myers RM, Petrov D, Jónsson B, Schluter D, Bell MA, Kingsley DM.
Science. 2010 Jan 15;327(5963):302-5.
This paper is most recent in a series of papers over the past decade in which the Kingsley Lab has used stickleback fish as a model to investigated the genetic bases of adaptive natural variation. Marine populations of stickleback have a pelvic apparatus that consists of articulating spines along the fishes lateral sides. Interestingly, several independently derived freshwater populations have lost this structure. Previous work had determined that a chromosome region containing the Pitx1 gene was responsible for pelvic structure loss in multiple populations, and that Pitx1 expression is lost in pelvic reduced stickleback. These and other data suggested that cis-regulatory mutations at the Pitx1 locus were responsible for pelvic reduction. However, regulatory mutations are difficult to identify and the exact sequence changes controlling pelvic reduction had not been identified. In this paper, the authors identify the exact genetic changes responsible for this loss in multiple populations.
The authors start off by confirming that cis-regulatory changes in Pitx1 are responsible for the previously described loss in Pitx1 expression. To do this, the authors generated F1 hybrids between pelvic-complete and pelvic-reduced sticklebacks and measured expression levels of the pelvic-complete and reduced-pelvic alleles. They found that the pelvic-reduced allele was expressed at lower levels than the pelvic-complete allele. The authors conclude that this allele-specific down-regulation must be due to cis-regulatory changes at Pitx1 itself because the two alleles are present in an identical trans-acting environment.
To further localize the position of the cis-acting changes, the authors took advantage of recombination in natural populations. They tested whether markers in the Pitx1 region were associated with the presence or absence of pelvic structures in lakes with dimorphic stickleback forms. They found that microsatellite markers in an intergenic region upstream of Pitx1 showed highly significant allele frequency differences in fish with contrasting pelvic phenoytpes. These results suggested that the regulatory changes controlling pelvic reduction were located within this upstream region.
Next, the authors test this region for regulatory function by cloning different subfragments in front of a reporter and examining there expression transgenic stickleback. Using this method, the authors identified a small fragment from pelvic-complete fish that drove expression in the developing pelvic region. If regulatory changes in Pitx1 are responsible for pelvic reduction in sticklebacks, restoring pelvic expression of Pitx1 should rescue pelvic structure development. Therefore, the authors injected a construct containing the tissue-specific enhancer upstream of the Pitx1 coding region into pelvic-reduced stickleback to see if this would restore pelvic structure development. They found that transgenic stickleback showed enhanced pelvic structure development. These data provided functional evidence that the enhancer is a major determinant of pelvic formation in sticklebacks.
To identify the sequence changes that disrupt this enhancer in pelvic-reduced populations, the authors examined this region in 13 different pelvic-reduced populations from disparate geographic locations. They found that 9 of the 13 pelvic-reduced stickleback populations had deletions removing the enhancer region. Interestingly, individuals within the same population had identical deletions, but individuals from different populations had independently formed deletions. Next, the authors examine if this enhancer is located within a genomic region with high DNA flexibility, as highly flexible regions are know to be fragile and this could help explain the abundance of independently formed deletions. The DNA flexibility analysis revealed that the Pitx1 region was exceptionally flexible compared to other regions of the genome. Furthermore, the tissue-specific enhancer was one of the most flexible regions within the entire Pitx1 locus. These results suggest that the Pitx1 locus is exceptionally fragile and that this fragility may explain the abundance of independently formed deletions.
This paper is exceptional for a number of reasons. Identifying the exact changes that contribute to natural phenotypic variation is difficult, especially when regulatory changes are responsible. This paper is exceptional just for the fact that the authors were able to achieve this. Furthermore, previous work from the Kingsley lab has shown that the parallel evolution commonly occurs by repeated use of identical alleles. Remarkably, not only do the authors find that this is not the case for pelvic structure evolution, but they provide convincing evidence for why pelvic structure evolution occurs through independently formed mutations.