Studies On The Function Of The Gene FoxP4 In Zebrafish Heart Development

In this paper, we have screened three proteins CatD, Tcap and FBXL5, which interacted with FoxP4 from heart cDNA library by yeast two-hybrid screening. In order to further study their interactions, we chose wild-type and nppa:GFP transgenic zebrafish as model to explore the expression and function of FoxP4 protein and its interacted proteins CatD, Tcap and FBXL5 in heart development at early stages by use of RNA interference and morpholino technology.FoxP4 gene is on chromosome VI and contains a total of 17 exons by analysis based on bioinformatics prediction. The genomic gene is up to 54kb.The cDNA has 5965 bp nucleotides which encode a 680 amino acids long protein. FoxP4 protein contains an N-terminal C2H2-type zinc finger domain and a C-terminal Fork Head domain to perform bindng DNA functions. FoxP4 is a transcription factor and belongs to the subfamily of Foxp which has a helix structure flanking. FoxP4 gene has a very high degree of homology with the sub-class of FoxPl, FoxP2. RT-PCR and Northern Blot showed that human FoxP4 gene had a higher expression in lung, intestine, brain, liver, heart and nerve tissue during the embryonic development. Human FoxP4 expresses widely in many tissues at the early embryonic development. In situ hybridization showed that the zebrafish FoxP4 homologous gene expressed persistently,suggesting that transcription factor FoxP4 is necessary for the whole embryo early development. Using yeast two-hybrid system, we screened three proteins CatD, Tcap and FBXL5, which interacted with FoxP4. The interactions between FoxP4 and these three proteins were verified by CO-IP and subcellular localization experiments.FoxP4 and its interaction proteins CatD, Tcap and FBXL5 were studied by using the model of zebrafish in this paper. RNAi and Morpholino interference experiments showed that knock-down FoxP4, CatD and Tcap or overexpressing FBXL5 could strengthen the expression of Nkx2.5, which induced severe heart malformations in zebrafish embryos, including increasing atrial size, arrhythmia, unlooping heart, suggesting that FoxP4 gene and their interactions proteins CatD, Tcap and FBXL5 were necessary for heart development through Nkx2.5 signal transduction pathway. RT-PCR experiment also showed that knock-down FoxP4 and their interactions Tcap and CatD genes may block Nkx2.5 signaling pathway at the heart development and affect the downstream gene expression, such as cardiac-actin and tnc. All these results indicated that FoxP4 and its interaction proteins CatD and Tcap, are located downstream of Nkx2.5 signal pathway and regulated by Nkx2.5. FoxP4 and CatD may locate between Nkx2.5 and cardiac-act in. RT-PCR, luciferase reporter assay analysis and Western blot further indicated overexpression of FBXL5in HEK293 cells would enhance the ehancer activity of Nkx2.5 and increase the expression of Nkx2.5. Therefore, we speculate FBXL5 also is involved in the expression of Nkx2.5 regulation process. In a word, our experiments showed FoxP4 gene and its interactions proteins CatD, Tcap and FBXL5 play important roles during heart development and these genes are involved in Nkx2.5 signal pathway.We have generated a homozygous heart-specific GFP-positive zebrafish line, nppa:GFP line, which we have used to analyze heart morphology and physiological function. This zebrafish line was produced by a large-scale insertional mutagenesis project that generated a transgenic zebrafish with heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiment showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expression of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFPline developed normally compared with wild type fishes. There was no difference between the nappa:GFP transgenic and wildtype lines with respect to heart rate, overall size, ejection volume, and fractional shortening, although there was a minor but significant difference in the relaxed ventricle length. Thus, the nppa:GFP line should provide a convenient model for studying in vivo cardiac development and function and could be used to establish biological indices for bioactive molecules such as environmental pollutants, and therapeutic drugs.