The Role Of RMND5B In Zebrafish Heart Development

The heart is one of the earliest organs during embryonic development. The heart disease is the biggest killer of human health life. Therefore, studies on the mechanism of heart development and its disease is of great significance.The preliminary research obtained in our lab indicated that CG3295, the Drosophila homologues gene of RMND5B, could be involved in Drosophila heart development. In this thesis, we studied the role of RMND5B in vertebrate heart development used the zebrafish as a animal model.Whole amount embryo in situ hybridization analysis with zebrafish RMND5B probe showed that RMND5B was expressed at each stages of zebrafish development. In order to study the expression of the protein, the polyclonal antibodies of zebrafish RMND5B was prepared. Western-blotting results showed that the RMND5B was expressed in each stages of zebrafish embryos. In the adult zebrafish, it was expressed in heart, muscle, eyes, skin, eggs and fins with a stronger expression in the heart.Whole amount embryonic immunohistochemisty showed that RMND5B was strongly expressed in a single-cell at 128-cell stage in the zebrafish embryo. At 256-cell stage, the single-cell was divided into two, and at 512-cell stage, the single-cell was divided into four. At 72-hour stage of development RMND5B was expressed stronger in the heart. Sub-cell Localization analysis showed that the protein was located in the cytoplasm.The role of RMND5B in zebrafish heart development was studied by using the cardiac zebrafish nppa:GFP model. Injection of zebrafish embryos with a morpholino that is against RMND5B to knockdown its protein expression, caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and lower heart rate. These results suggested that RMND5B may be involved in early heart morphogenesis and play an important role during the development of the heart.To explore the molecular mechanism underlying these cardiac defects, we analysed the interaction of RMND5B with Smads by co-immunoprecipitation, and four interaction proteins:SMAD4, SMAD3, SMAD7 and SMAD8 were found to be interacted with RMND5B, suggesting that it could be involved in heart development though TGFβsignaling pathway.To sum up, RMND5B may play an important role in zebrafish heart development, possiblely interacting with Smad4, Smad3, Smad7and Smad8 to affect TGF-β(BMP) signaling pathway. The thesis gives a foundation for further study of the role of this gene in the heart development.