Question being asked
Previous studies showed reduced expression of Pitx1 in sticklebacks that fail to form a pelvis. Is Pitx1 expression lost simply because the pelvis itself is no longer forming in these fish? Or, are there regulatory changes at the Pitx1 locus itself that reduce how much expression comes from a variant Pitx1 gene found in a pelvic-reduced population?
Generate hybrid fish that carry one copy of the Pitx1 gene from pelvic reduced fish (PAXB allele)and one copy of the Pitx1 gene from pelvic complete fish (FRIL allele). Determine if these two forms of the gene are expressed at the same level or different levels in developing pelvic tissue
The F1 hybrid fish generated in this experiment do form a pelvis, since they inherit functional copies of all pelvis-forming genes from one of the parents. If the failutre to express Pitx1 in PAXB pelvic-reduced fish is just a secondary effect of losing the pelvis, then expression of the PAXB form of the gene should be restored in a hybrid fish that now make a pelvis. Conversely, iif thePAXB form of PITX1 still fails to express even in a fish that can form a pelvis, this version of the gene must have a regulatory mutation that causes reduced expression in pelvic tissue.
It is important to consider the possibility that F1 hybrids of any combination may not express the two different alleles at the same level. In this case, any differences observed in PAXB and FRIL expression would not be informative. As a control, the authors generate sticklebacks that have one copy of LITC (pelvic-complete) and one copy of FRIL (pelvic-complete) and measure the expression levels of each allele in the pelvis. Since the two pelvic-complete alleles are not expected to have a regulatory mutation, they should be expressed at similar levels.
Compared to FRIL (the pelvic-complete allele), PAXB (pelvic-reduced allele) expression is significantly reduced in pelvic tissue of F1 hybrid fish. In contrast, in developing heads, PAXB and FRIL are expressed at similar levels.
What this figure shows
The cross on the left is the control experiment: F1 hybrids generated from two pelvic-complete sticklebacks (FRIL x LITC).
On the right is the cross used to generate F1 hybrids from a pelvic-complete and a pelvic-reduced stickleback (FRIL x PAXB).
In the plots, "normalized expression" is a ratio of the expression level of one allele to the expression level of the FRIL allele. PAXB is expressed at similar levels as FRIL in brain tissue and therefore the normalized expression is close to 1. PAXB is expressed at a much lower level compared to FRIL in pelvic tissue and therefore the normalized expression is less than 1 (and would approach 0 if expression in the pelvis was abolished).
The Pitx1 gene of pelvic-reduced fish (PAXB) shows greatly reduced expression in the developing pelvis of F1 hybrid fish. Reduced expression of Pitx1 is thus linked to the PAXB allele, and must be caused by cis-regulatory mutations the Pitx1 gene itself, rather than being an indirect effect of other changes
Down regulation of Pitx1 is NOT observed in the brain, and therefore the regulatory change in pelvic-reduced sticklebacks are is tissue-specific.
Question for 2A
Can we narrow down the regulatory change in Pitx1 to a smaller, specific stretch of DNA? Does this region contain sequences that tend to be conserved among most fish? Are such conserved noncoding regions somehow altered n pelvic-reduced sticklebacks?
Experiment for 2A
Look for the smallest genetic region that is consistently associated with pelvic reduction in genetic crosses and natural populations. Within this candidate region, identify the DNA region that exhibits the most differences in pelvic-reduced versus pelvic-complete sticklebacks.
Rationale for 2A
The specific region responsible for pelvic development is expected to be different in pelvic-reduced sticklebacks compared to pelvic-complete sticklebacks.
Results for 2A
Genetic markers spanning a 23,000 bp region are consistently correlated with pelvic reduction in both lab crosses and interbreeding fish from a wild population. This region corresponds to an intergenic (noncoding) region upstream of Pitx1. co. The coding regions of Pitx1 and Histone 2a (another gene located within the candidate region) did not exhibit significant differences in pelvic-reduced sticklebacks compared with pelvic-complete sticklebacks, and therefore are not likely to cause the differences in pelvic development.
Conclusions for 2A
The genetic changes that regulate pelvic development are located in an intergenic region (as opposed to within the coding region) of Pitx1. This region contains several noncoding sequences that are conserved (that is shows few genetic changes) between other types of fish, but do look different in pelvic-reduced sticklebacks.
Question for 2B
Can we identify the precise DNA sequence that drives Pitx1 expression in pelvic tissue?
The authors break up the candidate region into smaller fragments and clone them into reporter constructs. Different reporter constructs were introduced into fertilized stickleback eggs, to determine which fragment could drive gene expression specifically in pelvic tissue of transgenic fish.
If a transgene contains the fragment of DNA that can drive Pitx1 expression in the developing pelvis, then the transgenic animal will express the Green Fluorescent Protein (GFP) reporter gene in the developing pelvis.
Both a 2.5 kb and a smaller 501 base pair fragment within the candidate region from a pelvis-complete stickleback (SALR) drive enhanced GFP expression (EGFP) in the pelvic tissue of developing sticklebacks. EGFP expression was not observed in other tissues. If the same region is cloned and tested from PAXB, the deleted form of the enhancer found in the pelvic reduced population no longer drives GFP expression.
The Pitx1 gene of pelvic-complete sticklebacks contains a non-coding regulatory sequence that drives expression specifically in the developing pelvis (Pel-501-bpSALR). This 501 bp region is deleted in PAXB sticklebacks, confirming the previously postulated regulatory alteration in pelvic-reduced fish.
Pel can promote Pitx1 expression in the developing pelvis, but is this sufficient to induce the development of a pelvic structures in pelvis-reduced sticklebacks?
Inject a piece of DNA containing a transgene with Pel and Pitx1 into a pelvic-reduced stickleback and check for the development of pelvic structures. As a negative control, the authors use pelvic-reduced sticklebacks not injected with a transgene.
If mutation of Pel is a key factor controlling pelvic loss in sticklebacks, reintroduction of a Pel-Pitx1 transgene generated from a pelvic-complete fish should be able to restore pelvic development in a pelvic-reduced fish.
Pelvic-reduced fish injected with the Pel-Pitx1 transgene develop pelvic structures, while fish that were not injected with the transgene did not develop pelvic tissue.
Reintroduction of a single gene can restore the morphological formation of a pelvis in evolved sticklebacks, providing strong evidence that the correct gene and regulatory sequences governing this trait have been identified.
Question for A and B
Does the Pel region differ among populations of pelvic-reduced sticklebacks? If so, what kind of mutations are observed, and how do they differ? Do different pelvic-reduced populations have similar mutations in Pel?
Experiment for A and B
Analyze the Pel sequence from pelvic-reduced populations and compare it with the Pel sequence in pelvic-complete populations. The Pel sequence from three pelvic-reduced populations was directly sequenced. In 10 additional pelvic-reduced populations, SNP markers were used to identify changes in the Pel region.
Rationale for A and B
Evolution of pelvic reduction could occur either by similar or different mechanisms in different lakes around the world. Having identified the key Pel enhancer for Pitx1 expression in the pelvis, it is now possible to test whether changes in this sequence are a common feature of pelvic evolution in different populations.
Results for A and B
Deletions of the Pel enhancer sequence were found in nine different pelvic-reduced stickleback populations. All the observed deletions removed the Pel region, though the size and end points of the deletions vary from a few hundred to a few thousand base pairs among populations.
Question for C
Is the Pel region particularly susceptible to mutations when compared with other regions of the stickleback genome?
Experiment/Rationale for C
Regions with high DNA flexibility have been previously been found at fragile sites that are more prone to breakage in human chromosomes. A similar high degree of DNA flexibility could lead to the repeated deletions observed in different stickleback populations.
Results for C
Sequences in the Pitx1 gene locus shows some of the highest predicted DNA helical twist flexibility in the entire stickleback genome (twelve times higher than the median flexibility score of the rest of the genome)..
Repeated evolution occurs through repeated deletions of the Pitx1 regulatory region in many different populations, and may be influenced by unusual high DNA flexibility at the Pitx1 locus.
Do deletion mutations in the Pitx1 gene provide a selective advantage in pelvic-reduced populations? Or are pelvic mutations simply the sign of neutral or deleterious mutations that randomly accumulate over time in isolated freshwater populations?
The authors examine DNA sequence variation in multiple fish taken from different types of populations that have retained or lost their pelvis. A Pitx1 mutation that confers a positive fitness advantage will rapidly rise in frequency to become the predominant sequence within a population, a leading to a higher frequency of derived alleles and a reduction in heterozygosity in the corresponding region. These molecular signatures of positive election can be measured by established statistical tests that summarize the patterns of variation seen in different genomic windows across the Pitx1 region.
Sticklebacks from freshwater populations that have lost their pelvis (FW-Reduced) show both decreased heterozygosity, and increased frequency of derived alleles compared to both marine fish (Mar-Complete) and freshwater populations that have retained the pelvis (FW-Complete). These molecular signatures of positive selection are centered on the upstream non-coding region of the Pitx1 gene, the same region that contains the Pel regulatory enhancer.
Large evolutionary differences that map to a particular locus can still be caused by many linked small-effect mutations that have accumulated in that gene (24, 25). However, we find that pelvic-reduction in sticklebacks maps to a type of DNA lesion that may produce a large regulatory change in a single mutational leap: deletions that completely remove a regulatory enhancer. Smaller functional lesions might be found in some pelvic-reduced populations, including four populations without obvious deletions. However, three of these populations show unusual morphological features, suggesting that their pelvic loss may have occurred through non–Pitx1-mediated mechanisms (8, 26).
The Pitx1 locus scores as one of the most flexible regions in the stickleback genome, which may reflect a susceptibility to double-stranded DNA breaks and repair through NHEJ (27–29). We hypothesize that sequence features in the Pitx1 locus may predispose the locus to structural changes, possibly explaining the high prevalence of independent deletion mutations fixed in different pelvic-reduced stickleback populations. A similar spectrum of independent small-deletion mutations has been seen at the vernalization 1 locus of plants (30), suggesting that recurrent deletions in particular genes may also contribute to parallel evolution of other phenotypes in natural populations.
Mutations in developmental control genes are often deleterious in laboratory animals, leading to long-standing doubts about whether mutations in such genes could ever be advantageous in nature (31). Although Pitx1 coding regions are lethal in mice (32), we find clear signatures of positive selection in the Pitx1 gene of pelvic-reduced sticklebacks. Before this work, the primary evidence that pelvic reduction might be adaptive in sticklebacks came from repeated evolution of similar phenotypes in similar ecological environments and the temporal sequence of pelvic reduction in fossil sticklebacks (11, 12, 33). The molecular signatures of selection we have identified in the current study are centered on the tissue-specific Pel enhancer region rather than the Pitx1coding region. Regulatory changes in developmental control genes have often been proposed as a possible basis for morphological evolution (3, 34). However, many proposed examples of regulatory evolution in wild animals have not yet been traced to particular sequences (2) or do not show obvious molecular signatures of selection in natural populations (35). Identification of the Pel enhancer underlying pelvic reduction in sticklebacks connects a major change in vertebrate skeletal structures to specific DNA sequence alterations and provides clear evidence for adaptive evolution surrounding the corresponding region in many different wild populations.
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- We thank M. McLaughlin for fish husbandry, M. Nonet for the gift of the pBH-mcs-YFP vector, Broad Institute for the public gasAcu1 genome assembly, and many individuals for valuable fish samples (table S1). This work was supported by a Stanford Affymetrix Bio-X Graduate Fellowship (Y.F.C.); the Howard Hughes Medical Insititute (HHMI) Exceptional Research Opportunities Program (G.V.); the Burroughs Wellcome Fund (M.D.S.); NSF grants DEB0211391 and DEB0322818 (M.A.B.); a Canada Research Chair and grants from the Natural Sciences and Engineering Research Council of Canada and the Guggenheim Foundation (D.S.); NIH grant P50 HG02568 (R.M.M., D.P., and D.M.K.); and an HHMI investigatorship (D.M.K.). Sequences generated for this study are available in GenBank (accession GU130433-7).