A Study Of Screening Of The Proteins Interacting With Zebrafish Angiomotin-Like2 Via Yeast Two-hybrid System

Zebrafish ( Brachydanio rerio) , as a new model animal wildly used on embryodevelopment study of vertebrate ,became popular and useful. In recent ten years, thestudy of vertebrate embryo development using this model has developed rapidly.There are several advantages of zebrafish to be a model animal. Firstly, they are smalland easy to be raised. The female can lay more than 200 eggs once a week and theembryos need only 3 months to be hatched. Secondly, the zygotes of zebrafish are bigand limpid, so it is quite convenient to observe the whole developmental processes ofzebrafish embryo and analyse the feasible endpoints in embryotoxicological testing.Finally, the development of zebrafish embryo is quite rapid, only need 3 days fromimpregnated to incubated. Recently a new method which microinject Morpholino intothe embryo of zebrafish to inhibit special genes activity has effectively to be used. Asa result, we decided to use zebrafish as a model to study an important protein namedzebrafish Angiomotin Like 2(zAmotL2).A novel protein, named angiomotin, have been recently identified by its abilityto bind the angiogenesis inhibitor angiostatin in the yeast two-hybrid system.Angiomotin belongs to a family with two other members, AmotL-1 and AmotL--2characterized by coiled-coil and C-terminal PDZ binding domains. Here we show thatthe putative PDZ binding motif of angiomotin serves as a protein recognition site andthat deletion of three amino acids in this site results in inhibition of chemotaxis.Furthermore, endothelial cells expressing mutant angiomotin failed to migrate andform tubes in an in vitro tube formation assay. These results suggest that the putativePDZ binding motif of angiomotin plays a critical role in regulating the responsivenessof endothelial cells to chemotactic cues. Angiostatin, a circulating inhibitor ofangiogenesis, was identified by its ability to maintain dormancy of establishedmetastases in vivo. In vitro, angiostatin inhibits endothelial cell migration,proliferation, and tube formation, and induces apoptosis in a cell type–specificmanner.These findings indicate that angiostatin inhibits cell migration by interferingwith angiomotin activity in endothelial cells.We have screened AmotL2 gene using gene chip method from zebrafish earlydevelopmental embryos cDNA library. It can respond to the FGF signaling pathway.There is no reports of amotl2 in zebrafish field now. The full sequence length ofamotl2 is 2166 bp, and the protein has 721 amino acids. We have identified amotl2homolog in mice from sequence analysis, the alignment of the amino acid sequencesencode in this two proteins are highly homologic. Via whole mount in situhybridization we have discovered the development expression pattern of AmotL2 inzebrafish embryo, and found the expression of AmotL2 may be connected with thecell migration and was regulated by FGF signaling pathway.AmotL2 is highly homologic to the members of motin family, the function of itwhich may be related to cell migration and can be regulated by FGF signal open anew study of the molecular mechanism of zebrafish embryo cell migration andangiogenesis. So we hope to screen the proteins which can interact with AmotL2 andcan find a new way to cure cancer with a new medication angiostation.We used the mixture of mRNA of zebrafish embryo in different developmentalstages such as 1cell,40% epiboly, 5 somite and 24h as the template to construct thecDNA library, and screened about 30 kinds of library proteins interacting with amotl2through yeast two-hybrid system. From the results we selected Dapper homolog2(Dpr2) and Glycogen synthase kinase 3(Gsk3)as the next study objects because theyare both important proteins in many key signaling pathway in vertebrate development.It is sure these results are useful to discover the molecular mechanism of zebrafishembryo cell migration and angiogenesis.