CgBrachyury regulates haemocyte proliferation as a target gene of CgWnt signal in oyster Crassostrea gigas.

Abstract

The transcription factor Brachyury plays a key role in mesoderm development and notochord formation in most animals. In the present study, a homologue of Brachyury with a conserved Brachyury domain was identified from the Pacific oyster Crassostrea gigas. During the embryogenesis of oyster, CgBrachyury showed high mRNA expression level from the morula to the D-shaped larval stage, with highest level in gastrula stage. In situ hybridization assay yielded consistent results, in which CgBrachyury featured high expression in the blastopore region of gastrula. In adult oysters, CgBrachyury exhibited high expression level in haemocytes, especially in semi-granulocytes. Immunofluorescence assay indicated that CgBrachyury was localized in both the cytoplasm and nucleus of haemocytes. Moreover, the CgBrachyury transcripts in haemocytes were significantly increased at 6 h and 12 h after Vibrio splendidus stimulation. After the expression of CgBrachyury was inhibited by RNAi, the percentage of EDU⁺ haemocytes, the mRNA expression level of haematopoietic transcription factor CgSCL and CgRunx were significantly decreased after V. splendidus stimulation, while the expression level of cell cycle regulatory protein CgCDC6 and CgCDK2 was significantly increased after V. splendidus stimulation. There were two Wnt signal sites (TCF/LEF) identified in CgBrachyury promoter. The dual-luciferase reporter assay showed that CgWnt was able to activate the CgBrachyury promoter in HEK293T cell, and the knockdown of CgWnt by RNAi down-regulated the mRNA expression of CgBrachyury in haemocytes after V. splendidus stimulation. These results collectively indicated that CgBrachyury involved in embryogenesis and haemocyte proliferation as the target gene of CgWnt signal in the oyster.