Identification Of The Interacting Proteins Of CFL And Analysis Of Its Function In Zebrafish Heart Development

Heart is an important organ of human. The normal function of heart developmental genes is critical for the development of heart.To study the function of heart developmental genes will provide a base for understanding the pathogenesis of congenital heart disease and interventional treatment.CFL gene is a heart developmental gene which is cloned from zebrafish embryonic cDNA library, it may play an important role in the process of zebrafish heart development.In order to study the function of CFL, bioinformatics analysis of CFL gene was carried out, rabbit polyclonal antibody of CFL protein was generated,the expression profile of CFL during zebrafish embroynic development,the effect of RA signal on CFL expression and the interaction between CFL and DVL2were also identified.Bioinformatics analysis indicated that CFL gene encodes a polypeptide of278amino acids which contains a ZF-CXXC domain. The cDNA of CFL gene was obtained through RT-PCR. CFL protein was expressed and purified. Specific rabbit polyclonal antibody of CFL was genereated. Mount in situ hybridization showed that, CFL mRNA was expressed in every stage of zebrafish development.Western blot results showed that during embryonic development,the expression of CFL was increased from embryonic stage to36hpf, with the peak at24hpf; after36hpf,the expression of CFL decrease somehow. CFL is expressed in adult zebrafish heart, muscle, egg, eye, fin, cheek and notochord.Embryonic antibody staining showed that the expression of CFL protein was in a wide range in zebrafish embryo. The expression of CFL is high in notochord. In heart, CFL is expressed mainly in ventricular area.Preliminary data of our lab showed that CFL may be regulated by RA signal. In order to study the regulation of CFL on zebrafish heart development,we treated zebrafish embryo with RA inhibitor DEAB, and carried out mout in situ hybrization at24hpr, to analysize the effect of DEAB on CFL expression. The result showed that DEAB promoted the expression of CFL. Co-immunoprecipitation analysis of RA treated zebrafish embryos showed that expression of CFL protein increased Thus, RA signal promoted the expression of CFL. CO-IP experiments verified the interaction between CFL and DVL2, which is an important member of WNT pathway. This result suggests that CFL may be the crosstalk molecule between RA signal and WNT pathway.In conclusion, CFL may link RA signal and WNT pathway through its interaction with DVL2be interaction with DVL2, thus paly an important role in zebrafish heart development.