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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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EDA shows allele-specific expression differences inseveral tissues, indicating cis-regulatorydivergence.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 of EDA is expressed morehighly than the freshwater allele, suggesting that there are differences inthe cis-regulatory sequences controllingEDA expression. Several other tissues, however, do notshow significant allelic imbalance in EDA 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
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fig1: EDA shows allele-specific expression differences inseveral tissues, indicating cis-regulatorydivergence.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 of EDA is expressed morehighly than the freshwater allele, suggesting that there are differences inthe cis-regulatory sequences controllingEDA expression. Several other tissues, however, do notshow significant allelic imbalance in EDA 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

Mentions: In order to test if EDA is differentially expressed in marine andfreshwater fish due to cis-regulatory differences, we performedallele-specific expression in F1 hybrid fish made by crossing marine and freshwatersticklebacks. The F1 hybrids are heterozygous for both the marine and freshwaterhaplotypes at the EDA locus, and therefore express both alleles inan identical trans-acting environment. We then isolated RNA from 10different developing tissues, and determined whether the freshwater and marineEDA transcripts were expressed at the same or different levelsusing pyrosequencing (Figure 1, see‘Materials and methods’). No significant expression differences betweenmarine and freshwater EDA alleles were observed in the fins or thelower jaw. However, the freshwater EDA allele was expressed almostfourfold lower than the marine allele in the developing anterior and posterior flanks(corresponding to sites where armor plates had already appeared, or were not formingyet; respectively), and in the dorsal and pelvic spines (p < 0.01,Student's t test), as well as twofold lower in the premaxilla(p < 0.05, Student's t test). These data suggest thatthe marine and freshwater haplotypes at the EDA locus havecis-acting regulatory changes that reduce expression of thefreshwater allele in particular tissues, including the flank regions where armorplates normally form.10.7554/eLife.05290.003Figure 1.EDA shows allele-specific expression differences inseveral tissues, indicating cis-regulatorydivergence.


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)

EDA shows allele-specific expression differences inseveral tissues, indicating cis-regulatorydivergence.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 of EDA is expressed morehighly than the freshwater allele, suggesting that there are differences inthe cis-regulatory sequences controllingEDA expression. Several other tissues, however, do notshow significant allelic imbalance in EDA 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
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384742&req=5

fig1: EDA shows allele-specific expression differences inseveral tissues, indicating cis-regulatorydivergence.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 of EDA is expressed morehighly than the freshwater allele, suggesting that there are differences inthe cis-regulatory sequences controllingEDA expression. Several other tissues, however, do notshow significant allelic imbalance in EDA 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
Mentions: In order to test if EDA is differentially expressed in marine andfreshwater fish due to cis-regulatory differences, we performedallele-specific expression in F1 hybrid fish made by crossing marine and freshwatersticklebacks. The F1 hybrids are heterozygous for both the marine and freshwaterhaplotypes at the EDA locus, and therefore express both alleles inan identical trans-acting environment. We then isolated RNA from 10different developing tissues, and determined whether the freshwater and marineEDA transcripts were expressed at the same or different levelsusing pyrosequencing (Figure 1, see‘Materials and methods’). No significant expression differences betweenmarine and freshwater EDA alleles were observed in the fins or thelower jaw. However, the freshwater EDA allele was expressed almostfourfold lower than the marine allele in the developing anterior and posterior flanks(corresponding to sites where armor plates had already appeared, or were not formingyet; respectively), and in the dorsal and pelvic spines (p < 0.01,Student's t test), as well as twofold lower in the premaxilla(p < 0.05, Student's t test). These data suggest thatthe marine and freshwater haplotypes at the EDA locus havecis-acting regulatory changes that reduce expression of thefreshwater allele in particular tissues, including the flank regions where armorplates normally form.10.7554/eLife.05290.003Figure 1.EDA shows allele-specific expression differences inseveral tissues, indicating cis-regulatorydivergence.

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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