The Regulation Of Wnt Signaling On Expression Of Neuromast Specific Gene Six2b In The Zebrafish

The Six gene family encodes a class of important transcription factors.The Six protein has a homologous domain similar to the Hox protein.The Six protein is extensively involved in the development of various organs,especially in the development of fish lateral lines.From the promotion of the formation and differentiation of Cranial placodes,to the activation of Atoh1 a with Eya1 to promote the differentiation of hair cells,are inseparable from the participation of Six proteins.In addition,the Six family members are clustered on chromosomes and are extremely conserved during the evolution of vertebrates.The expression patterns of Six subfamily members are tissue-specific,and their expression in embryos is correlated with their chromosomal arrangement(collinearity),such as Six1/2 is mainly expressed in the lateral line system,Six3/6 is mainly expressed in the eye.So far there are lots of the investigation of Six1 gene in cranial placode and lateral line development.Six1 and Six2 are both so subfamily,whether Six2 gene is involved in lateral line development just like Six1 is unclear.The precise regulation of the expression pattern of the Six gene is important for its function.Wnt signaling pathways play an important role in embryonic development,apoptosis and neurological development,and are involved in the development of the cranial placode,as well as the development of the inner ear and lateral line systems.At present,the expression of the Six2 b gene is also regulated by Wnt signaling pathway is also our concern.189b transgenic fish in our laboratory was identified via a large scale enhancer trap screen mediated by Tol2 transposon in zebrafish.We found that at 16 hpf green fluorescent protein(EGFP)of the transgenic fish was expressed in anterior placode,at 19 hpf was expressed in posterior placode,at 30 hpf and 48 hpf was expressed in otic vesicle,olfactory vesicle,gill arches and neuromasts,and also expressed in the migrating lateral line primordium.We further investigated by immunofluorescence assay that EGFP in 189 b transgenic fish was expressed in neuromast,which was coincided with the immunofluorescence signal of Claudinb,and further demonstrated that EGFP of 189 b transgenic zebrafish was indeed expressed in the neuromast.We used the chromosome walking technique to identify the EGFP insertion site of the 189 b transgenic fish at the upstream of the Six2 b gene.Whole-mount in situ hybridization(WISH)analysis showed that:expression patterns of Six2 b gene and EGFP expression in 189 b transgenic fish have space-time consistency,which indicated that the green fluorescent signal of 189 b transgenic zebrafish could reflect the expression of Six2 b gene.Therefore,we can use 189 b transgenic zebrafish to study the regulation of Six1/2 gene expression.In order to study the regulatory signal upstream of the Six1/2,we used the heat-induced expression system of the wnt signal,hsp: wnt8a-egfp and hsp: dkk1-egfp.Wnt8 a is a positive signal protein of zebrafish classical wnt signaling pathway,and Dkk1(Dickkopf1)is one of the important antagonist molecules in Wnt signaling pathway,which can specifically inhibit classical Wnt signaling pathway.We heat-treated hsp: wnt8a-egfp and hsp: dkk1-egfp transgenic zebrafish embryos,then performed with alkaline phosphatase staining,YO-PRO-1 staining and hair cell counting techniques,we found that neuromasts of the lateral line can not be formed normally after heat shock induced Wnt8 a and Dkk1 expression,which proves that Wnt signaling pathway plays a regulatory role in the development of zebrafish lateral line system.We crossed the 189 b transgenic zebrafish with and hsp: dkk1-egfp transgenic zebrafish,and then heat-treated their embryos at 18-19 h.The expression of EGFP in neuromasts was significantly decreased at 40 and 48 hpf.This indicates that the expression of EGPF in 189 b transgenic zebrafish is controlled by the Wnt signaling pathway.Through in situ hybridization,we further confirmed change of the expression of Six2 b and related genes in the zebrafish neuromasts after expression of Wnt and Dkk1 induced by heat shock.In order to further study the effect of Six2 b gene on the early development of zebrafish lateral line system,the Six2 b gene was knocked out by using the technique of microinjection of Cas9 mRNA and Six2 b gRNA to the zebrafish first cell stage fertilized egg.Sequencing and comparison,T7 EI enzyme digestion method successfully detected the deletion of Six2 b gene.And then through the fluorescent PCR and capillary electrophoresis technology successfully selected Six2 b missing F1 adult fish.The deletion of Six2 b F1 zebrafish can be used for subsequent studies on lateral line phylogeny.In conclusion,we established a transgenic zebrafish system that detected the expression of Six2 gene.It was also found that Wnt signaling could regulate the expression of Six2 b gene in zebrafish.This laid the foundation for the further study of the development of lateral line system in fish and the regulation of the Six family expression in vertebrates.