The Molecular Mechanism Of VSX1 Repressing Flh Gene Expression

Gastrulation is a key step of forming embryo body plan, its movement pattern and regulation mechanism is one of the most important basic issues in life science exploration for hundred of years. Paraxial protocadherin(papc) is a transmembrane protein that plays an important role in gastrulation morphogenetic movement. Recent research finds that papc takes part in Wnt/PCP signaling pathway, and regulate the convergent-extension movements. On one hand, papc has adhesion properties in its extracellular part, and on the other hand, it can switch the received signals to the actual cell movement. papc expresses in the lateral and ventral mesoderm of germ ring in 50% epiboly of zebrafish embryos. Recently, it was found that in gastrula stage of zebrafish embryos homeobox gene flh expressed in chorda-mesoderm where spt transcript was directly inhibited by flh; Thus spt expressed in paraxial mesoderm where spt activated papc expression. So flh and spt played an important part in gastrulation by mediating papc expression in space. Former research in our lab showed that inhibition of vsxl, another homeobox gene in zebrafish, lead to gastrulation movement block, flh ectopic expression in lateral and ventral mesoderm and inhibition of spt and papc in lateral and ventral mesoderm; When vsxl was overexpressed, flh expression was inhibited in chorda-mesoderm. These results indicated that vsxl could inhibit flh ectopic expression in early development of embryos. In order to further investigate the function of vsxl in controlling gastrulation movement, we deeply analysis the mechanism of vsxl in mediating flh. Main results and conclusions are as following:1. By coinjection of vsxl mRNA and GFP reporter gene controlled by length-different flh promoter sequence separately, we could detect the effect of vsxl mRNA on the expression of GFP reporter gene, it was verified that vsxl could effectively inhibit the expression of reporter gene controlled by flh promoter, and confirmed that from -204bp to -156bp in flh promoter there may be a key site binded by vsxl or its downstream gene. 2. Protein containing homeodomain could bind DNA at the potent binding site TAAT(NN). According to the rule, we found a TAATTG site when analysis the flh promoter sequence of 49bp which was cut from 220bp flh promoter into 172bp. Thus we thought out to mutate TAATTG into TCGATG and constructed a MPZF-220GFP site-mutation reporter gene. When coinjected it with vsxl mRNA into embryos, the reporter gene show no inhibition. The result suggested that the protein encoded by vsxl may bind to TAATTG sequence to directly inhibit flh transcription, which was not indirectly inhibited by the protein encoded by vsxl downstream gene.3. In order to further confirm the site with TAATTG sequence in flh promoter is the site binded directly by VSX1, we expressed a recombinant protein GST+ VSX1 HD containing VSX1 homeodomain in E. coli by taking advantage of pGEX prokaryotic expression system, and special for TAATTG site among the 49bp in flh promoter, we designed and synthesized a biotin-labeled probe containing the TAATTG site, a probe without biotin and another probe with the TAATTG site mutant to TCCCCG, then adopted gel shift assay(EMSA) to detect the in vitro binding of VSX1 protein and TAATTG sequence in flh promoter. EMSA results showed after reaction of binding probe with GST+VSX1 HD, in non-denatured gel there is a retarded band after the band formed by free probe; But there is no retarded band in the reaction with previous adding the competitor 500 times of binding probe. While with the mutant probe previous adding to the reaction, there is a retarded band again. All this in vitro results indicated that VSX1 can direct bind to flh promoter at the TAATTG site.4. In order to test whether VSX1 could bind flh promoter directly in the embryonic cells with normal development, and to discover the number and the position of the site in flh promoter, we prepared the polyclonal antibody and adopted it for Chromotin immunoprecipitation(ChIP) analysis. In analysis with 13 TAATTN sites in the 1.9kb flh promoter, we only got the positive signal with the TAATTG site between -204bp and-156bp, while detect no band for other sites. In vivo ChIP analysis results sufficiently demonstrated in normal development process VSXl protein indeed bind to TAATTG sequence in flh promoter to mediate the flh gene expression. These results indicate that vsxl can regulate the special expression patterns of flh, spt and papc in a cell-autonomous manner by direct inhibiting flh ectopic expression and play a crucial role in global control of gastrulation model.