Zebrafish DKK3a regulates the promoter activity of myf5 through interaction with membrane receptor Integrinα6b

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repressing myf5 promoter activity. However, the membrane receptor which interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS mass spectrometry to screen putative membrane receptors of Dkk3a, and Integrinα6b (Itgα6b) was finally identified. To further confirm this, we employed cell-surface binding assays which showed that Dkk3a and Itgα6b co-expressed at the cell membrane of HEK-293T cells. Crosslinking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the beta-propeller repeated domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was upregulated four-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a-knockdown embryos co-injected with itgα6b mRNA and itgα6b-knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker, or absent, myf5 expression, and reduced protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a-knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.