The Role Of Goosecoid In The Early Development Of Zebrafish

Wnt/? catenin pathway play an important role in the early embryo development,it also regulate embryo dorsal-ventral patterning.goosecoid(gsc)is one of the targets of Wnt pathway,conversely,it play an role of negative feed-back of Wnt pathway.In previous reports,it suggests that gsc mutants display skeleton dysmorphia in mouse and human.However,currently,it is also unclear about gsc function in zebrafish.In previous research,gsc knock-down by morpholino(MO)in zebrafish,no obvious defects were found.As the short function time of MO,we can not figure out the gsc function in later period in zebrafish development.Recently,TALENS gene edit become a new tool in gene function research,as it has the advantage of high target effect and easy operation.In research of gsc function in zebrafish,we bulit gsc mutant model by TALENs.The obtained results as follows:(1)By TALENs technology,we knocked out zebrash gsc,screened and got frame shift mutant(-7bp).(2)In gsc heterozygote self-crossover siblings,no obvious dorsal-ventral defects were found in early time,while swimbladder defect at 5dpf(day after fertilization).The results of sequencing show that siblings with swimbladder are homozugous,statistical data show that the proportion of swimbladder defects embryos is 21%.It sugget that gsc is related to swimbladder defect.(3)Then we perform paraffin section and HE staining in gsc mutants,it show that swimbladder exist.(4)In situ hybridization in use of swmbladder marker hb9,swimbladder budding was normal.The results indicate swimbladder exist,but it can not inflate.(5)Swimbladder inflation involves coordination of mouth,pharynx and pneumatic duct,while expression of gsc mainly focus in pharynx and forebrain.Then we did alcian blue staining and paraffin section of pharynx in gsc mutants,we found that pharynx are normal.Thus,the reason of swimbladder non-inflation in gsc mutants may not relate to pharynx.(6)In addition,swimbladder inflation also need Innervation,gsc expressed in telencephalon and diencephalon,it may relate to swimbladder inflation.we treated wild embryos by LiCl,telencephalon and diencephalon disappeared,and swimbladder can not inflate.lt show that nerve in forehead is related to swimbladder inflation.Further woks needed to prove the abnormity of nerve in forehead in gsc mutants.Thus,we can build the relationship of gsc mutation and swimbladder non-inflation.