Dataset Information

Zebrafish Dkk3a protein regulates the activity of myf5 promoter through interaction with membrane receptor integrin ?6b.

ABSTRACT: 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 repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin ?6b (Itg?6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itg?6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itg?6b for Dkk3a. We further proved that the ?-propeller repeat domains of Itg?6b are key segments bound by Dkk3a. Moreover, when dkk3a and itg?6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itg?6b mRNA and itg?6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itg?6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the 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 Itg?6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.

SUBMITTER: Fu CY

PROVIDER: S-EPMC3501020 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Zebrafish Dkk3a protein regulates the activity of myf5 promoter through interaction with membrane receptor integrin α6b.

Fu Chuan-Yang CY Su Ying-Fang YF Lee Ming-Hsuan MH Chang Geen-Dong GD Tsai Huai-Jen HJ

The Journal of biological chemistry 20120928 47

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 repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane recep ...[more]

PMID: 23024366

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Project description:BackgroundMyf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood.ResultsWe isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP) reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1) the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2) the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3) the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4) the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5) the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm.ConclusionWe suggest that the cell lineage-specific expression of myf5 is delicately orchestrated by multiple modules within the distal upstream region. This study provides an insight to understand the molecular control of myf5 and myogenesis in the zebrafish.

Dataset Information

Zebrafish Dkk3a protein regulates the activity of myf5 promoter through interaction with membrane receptor integrin ?6b.

Publications

Zebrafish Dkk3a protein regulates the activity of myf5 promoter through interaction with membrane receptor integrin α6b.

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