Major phenotypic changes evolve in parallel in nature by molecular mechanisms that are largely unknown. Here, we use positional cloning methods to identify the major chromosome locus controlling armor plate patterning in wild threespine sticklebacks. Mapping, sequencing, and transgenic studies show that the Ectodysplasin (EDA) signaling pathway plays a key role in evolutionary change in natural populations and that parallel evolution of stickleback low-plated phenotypes at most freshwater locations around the world has occurred by repeated selection of Eda alleles derived from an ancestral low-plated haplotype that first appeared more than two million years ago. Members of this clade of low-plated alleles are present at low frequencies in marine fish, which suggests that standing genetic variation can provide a molecular basis for rapid, parallel evolution of dramatic phenotypic change in nature.

The molecular mechanisms underlying major phenotypic changes that have evolved repeatedly in nature are generally unknown. Pelvic loss in different natural populations of threespine stickleback fish has occurred by regulatory mutations deleting a tissue-specific enhancer of the Pituitary homeobox transcription factor 1 (Pitx1) gene. The high prevalence of deletion mutations at Pitx1 may be influenced by inherent structural features of the locus. Although Pitx1 null mutations are lethal in laboratory animals, Pitx1 regulatory mutations show molecular signatures of positive selection in pelvic-reduced populations. These studies illustrate how major expression and morphological changes can arise by single mutational leaps in natural populations, producing new adaptive alleles via recurrent regulatory alterations in a key developmental control gene.

Background Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2. Methods and Results Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells (HUVECs), suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3’ untranslated region (3’UTR) of KLF2 mRNA. Overexpression of miR-92a precursor (pre-92a) decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase (eNOS) and thrombomodulin (TM) at mRNA and protein levels. A complementary finding is that miR-92a inhibitor (anti-92a) increased the mRNA and protein expression of KLF2, eNOS, and TM. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a to induce KLF2 and the level of this flow-induced KLF2 was reduced by pre-92a. Furthermore, miR-92a level was lower in HUVECs exposed to the atheroprotective pulsatile shear flow (PS) than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunopreciptation coupled with RT-PCR revealed that PS decreased the functional targeting of miR-92a/KLF2 mRNA in HUVECs. Consistent with these findings, mouse carotid arteries receiving pre-92a exhibited impaired vasodilatory response to flow. Conclusions Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.