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doi: 10.7554/eLife.05290.

A recurrent regulatory change underlying altered expression and Wnt response of the stickleback armor plates gene EDA

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A recurrent regulatory change underlying altered expression and Wnt response of the stickleback armor plates gene EDA

Natasha M O'Brown et al. Elife..

Abstract

Armor plate changes in sticklebacks are a classic example of repeated adaptive evolution. Previous studies identified ectodysplasin (EDA) gene as the major locus controlling recurrent plate loss in freshwater fish, though the causative DNA alterations were not known. Here we show that freshwater EDA alleles have cis-acting regulatory changes that reduce expression in developing plates and spines. An identical T → G base pair change is found in EDA enhancers of divergent low-plated fish. Recreation of the T → G change in a marine enhancer strongly reduces expression in posterior armor plates. Bead implantation and cell culture experiments show that Wnt signaling strongly activates the marine EDA enhancer, and the freshwater T → G change reduces Wnt responsiveness. Thus parallel evolution of low-plated sticklebacks has occurred through a shared DNA regulatory change, which reduces the sensitivity of an EDA enhancer to Wnt signaling, and alters expression in developing armor plates while preserving expression in other tissues.

Keywords: Wnt signaling; chromosomes; ectodysplasin gene; enhancer; evolutionary biology; genes; genomics; recurrent mutation; skeletal development; sticklebacks.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Figure 1.
Figure 1..EDA shows allele-specific expression differences in several tissues, indicatingcis-regulatory divergence.
Allele-specific expression in F1 freshwater-marine heterozygous larvaereveals significant differential expression of the marine and freshwateralleles in dorsal spines 1 and 2, the pelvic spine, the premaxilla, and thepresumptive armor plates (anterior and posterior flanks). In all of thesebony tissues the marine allele ofEDA is expressed morehighly than the freshwater allele, suggesting that there are differences inthecis-regulatory sequences controllingEDA expression. Several other tissues, however, do notshow significant allelic imbalance inEDA expression; theirallelic ratios are close to 1 (dashed line). The control shows results froma 1:1 mixture of plasmids containing the freshwater and marine alleles.Red-shaded structures and bars indicate tissues with significantallelic-imbalance compared to control (***p <0.001, **p < 0.01, *p < 0.05 bytwo-tailed t-test).DOI:http://dx.doi.org/10.7554/eLife.05290.003
Figure 2.
Figure 2.. All low-plated populations share a single base pair change in the genetic region controlling armor plates.
Genome-wide comparisons of low- and high-plated fish reveal a T → Gbase pair change (black box) that is shared between all low-platedpopulations tested, including the low-plated Japanese NAKA fish thatotherwise shows a primarily marine-like haplotype in theEDA region. Geographic population codes and DNAsequences from marine high-plated populations and freshwater low-platedpopulations are shown in red and blue, respectively, along withrepresentative Alizarin Red stained fish showing typical armor platepatterns in different fish. The 16 kb candidate interval controlling armorplate number (blue bar, Colosimo et al.,2005) is shown beneath predicated genes in the region. Also shownare the numbered positions (4–16) of previously identified SNPs thatdifferentiate most low- and high-plated sticklebacks other than NAKA (Colosimo et al., 2005). These numberedSNPs correspond to positions chrIV: 12800508, 12808303, 12808630, 12811933,12813328, 12813394, 12815024, 12815027, 12816201, 12816202, 12816360,12816402, and 12816464 in the stickleback genome assembly (Jones et al., 2012). Blank positionsrepresent occasional gaps in sequence coverage for individual fish fromlarge population surveys (Colosimo et al.,2005; Jones et al.,2012). The position of the shared T → G change(chrIV:12811481) is indicated with a short black vertical line in theoverall genomic interval, and in a 3.2 kb region that was used to test forpossible regulatory enhancers in theEDA region (orangebar, chrIV:12808949–12812120).DOI:http://dx.doi.org/10.7554/eLife.05290.004
Figure 3.
Figure 3.. Reporter expression driven by anEDA enhancer matches several regions of endogenousEDA expression.
(A,D,G,J) Negativecontrol DapB RNAscope in situ staining shows no positive brown signalappearing around the face (A andD), the plates(G), or the pelvic junction (J). The slightbrown color in the pelvic spine is due to natural pigmentation at this site.(B,E,H,K)EndogenousEDA expression is localized to the premaxilla,lips, lower jaw, cranial ganglia, gill and pectoral fin base (BandE); armor plates (H); and the junction betweenthe pelvic spine and the pelvic girdle (K). (C,F,I,L) The p3.2mar-GFP constructdrives reporter expression at several corresponding sites, including thelips, premaxilla, lower jaw and cranial ganglia surrounding the eyes(C andF); in the armor plates;(I) and at the pelvic junction (L). Anatomicalabbreviations as in other figures, including: lips (L), premaxilla (PM),lower jaw (J), cranial ganglia (CG), gills (G), pectoral fin base (PF),anterior plates (AP), and pelvic spine junction (PSJ). Scale bars are 1 mmlong.DOI:http://dx.doi.org/10.7554/eLife.05290.005
Figure 4.
Figure 4.. Enhancer expression in plates and other structures is reduced by a single base pair change.
(A,B) A 3.2 kb enhancer region fromhigh-plated fish drives GFP expression in all armor plates (AP) of2-month-old (20 mm long) marine stickleback larvae, with expressionpreceding plate ossification, and stronger expression in the first 7armor plates. The p3.2mar-GFP construct also drives expression in thelips (L), premaxilla (PM), lower jaw (J), cranial ganglia (CG), the baseof the pectoral fins (PF), and the pelvic spine-girdle junction (PSJ).PanelB is a higher magnification view of the area boxed inpanelA. (C,D) The single basepair change in the p3.2mar(T → G)-GFP construct results in greatlyreduced enhancer activity in the posterior plates, and reduced butdetectable expression in plates 4–7 (D). This stableline also retains expression in the cranial ganglia and lips, reducedexpression in the pelvic junction and the pectoral fin base, and novelstrong expression in the spinal cord. PanelD is a highermagnification view of the area boxed in panelB. The hsp70promoter in the GFP vector drives strong expression in the lens (LN) ofall transgenic fish, helping to identify carriers followingmicroinjection experiments (Chan etal., 2010). Scale bars are 1 mm long.DOI:http://dx.doi.org/10.7554/eLife.05290.006
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Plate enhancer activity is altered by a single base pair change (additional examples from independent transgenic fish).
(A,B) Examples of transient transgenics withmosaic GFP expression under the control of the marine high-plated marineEDA enhancer (p3.2mar-GFP). Multiple transgenicfounders share expression in the cranial ganglia (CG) surrounding theeyes and the lips (L), the premaxilla (PM), under the jaw (J), and inarmor plates (AP). (C,D) Site-directedmutagenesis of the p3.2mar-GFP construct generating p3.2mar(T →G)-GFP results in loss of armor plate expression in transienttransgenics. However, expression in the cranial ganglia (CG) around theeyes and lips (L), as well as some expression surrounding the base of thepelvic spine-girdle junction (PSJ) remains in several fish. Copy number,integration sites, and mosaicism can vary in injected sticklebacks,giving rise to a range of expression levels. Despite this variability,consistent expression patterns can still be detected by comparing resultsfrom multiple injected fish. Overall, posterior plate expression was seenin 9 of 20 transgenic larvae with green eyes following injection ofp3.2mar-GFP, vs of 0 of 27 transgenic larvae following injection ofp3.2mar(T → G)-GFP. Scale bar inD is 2 mm long.DOI:http://dx.doi.org/10.7554/eLife.05290.007
Figure 5.
Figure 5.. Wnt signaling regulates armor plate development.
Live Calcein staining of 6-month-old fish marks newly ossified bones ingreen. (A) Armor plates in an untreated high-plated adultmarine fish. The normal morphologies of two individual plates are outlinedwith dashed lines. (B) Control beads soaked in PBS wereimplanted between the two outlined plates at two months of age. After beadimplantation, fish continued to develop a full set of armor plates, withminimal changes in plate morphology (n = 8). (C)Implantation of Wnt-3a beads results in hypermorphic growth and armor platefusion in the regions surrounding the exogenous Wnt-3a signal (n =11). (D) Conversely, beads soaked in the Wnt inhibitor Dkk-1inhibit plate formation surrounding the site of bead implantation (n= 10). Scale bar inD is 2 mm long.DOI:http://dx.doi.org/10.7554/eLife.05290.008
Figure 6.
Figure 6.. Single point mutation alters Wnt responsiveness of theEDA plate enhancer.
Beads soaked in either PBS or Wnt-3a protein were implanted in the flanks of2-month-old (24 mm long) marine fish. All images were taken at 48 hr postbead implantation. (A,B) RNAscope in situhybridization forEDA expression induced by control beadplacement (A) or Wnt-3a protein (B). The additionof Wnt-3a beads induces a ring ofEDA expression (browncolor inB) directly surrounding the implantation site.(C,D) Bead implantation into the stablep3.2mar-GFP transgenic fish line. Control beads fail to induce GFP activity(C), whereas Wnt-3a beads induce a strong GFP response, seenin a ring surrounding the bead implantation site (D).(E,F) Bead implantation into the stablep3.2mar(T → G)-GFP line of transgenic fish. A ring of GFP expressionis only seen at a distance from the implantation site of either control(E) or Wnt-3a (F) beads, corresponding to thelocation where cyanoacrylate glue was placed following implantation. Strongexpression immediately surrounding the bead is not seen with Wnt-3a beads,in contrast to the result seen with p3.2mar-GFP transgenic fish (comparepanelsF andD). Scale bar inF is 1mm long. (G) In vitro analysis of enhancer response to Wntsignaling via β-catenin co-transfection shows a strong induction ofp3.2mar-Luc (green squares) with 50 ng or more ofβ-catenin in human HaCaT keratinocyte cells. Theβ-catenin-responsiveness of the p3.2mar(T →G)-Luc is significantly lower (black triangles).Combined p-values were calculated using Meta-P (***p< 0.001, **p < 0.01).DOI:http://dx.doi.org/10.7554/eLife.05290.009
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