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

O'Brown NM, Summers BR, Jones FC, Brady SD, Kingsley DM - Elife (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States.

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.

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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 in D is 2 mm long.DOI:http://dx.doi.org/10.7554/eLife.05290.007
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fig4s1: 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 in D is 2 mm long.DOI:http://dx.doi.org/10.7554/eLife.05290.007

Mentions: We next performed site-directed mutagenesis to change the T found in high-plated fishto the G found in all sequenced low-plated fish, while maintaining the sequence ofthe high-plated marine haplotype throughout the rest of the enhancer construct. Thep3.2mar(T → G)-GFP plasmid still drove detectable expression in the anteriorplates, cranial ganglia, jaws, and pectoral fin base, but showed greatly reduced GFPexpression in the posterior armor plates and pelvic girdle junction (Figure 4C,D, Figure 4—figure supplement 1). Thus, the single base pair changeshared by all low-plated sticklebacks produces striking but localized differences ingene expression, with prominent reduction occurring in the flank region where platesnormally develop in marine fish.


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

O'Brown NM, Summers BR, Jones FC, Brady SD, Kingsley DM - Elife (2015)

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 in D is 2 mm long.DOI:http://dx.doi.org/10.7554/eLife.05290.007
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fig4s1: 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 in D is 2 mm long.DOI:http://dx.doi.org/10.7554/eLife.05290.007
Mentions: We next performed site-directed mutagenesis to change the T found in high-plated fishto the G found in all sequenced low-plated fish, while maintaining the sequence ofthe high-plated marine haplotype throughout the rest of the enhancer construct. Thep3.2mar(T → G)-GFP plasmid still drove detectable expression in the anteriorplates, cranial ganglia, jaws, and pectoral fin base, but showed greatly reduced GFPexpression in the posterior armor plates and pelvic girdle junction (Figure 4C,D, Figure 4—figure supplement 1). Thus, the single base pair changeshared by all low-plated sticklebacks produces striking but localized differences ingene expression, with prominent reduction occurring in the flank region where platesnormally develop in marine fish.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States.

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.

Show MeSH
Related in: MedlinePlus