Functional Study On C32 In Zebrafish Tooth Development

Background and PurposeTooth development in vertebrate is a rather complex event.Teeth are specialized structural components of the craniofacial skeleton.Developmental defects occur either alone or in combination with other birth defects.A cascade of genes orchestrate a series of events leading to formation of tooth in an embryo.Both teeth location and shape control,are inseparable from the combined action of dental epithelial and mesenchyma,definition of dental epithelial,differential gene expression in dental mesenchyma and the local signal regulating in morphogenesis of the tooth germ.Many genes play important roles in the development of tooth,such as BMP,FGF,Hedgehog(Hh),Wnt(wingless),Frizzled protein,Shh(sonic hedgehog),Syndecan-I,MSX,DLX,Pax and so on.Being similar to human and other mammals,zebrafish have dental pulp cavity,dentin,enamel,and enamel organ between the teeth.The tooth development of zebrafish,namely the bud stage,cap stage,bell stage,is similar to human,although the period is relatively short.The zebrafish lacks oral teeth,but has teeth implanted on the fifth ceratobranchials,the pharyngeal jaws.Adult zebrafish pharyngeal arch skeleton and tooth are like bone tissue.The sclerous tissues structure of the zebrafish tooth is very similar to the enamel and dentin of human tooth.The zebrafish tooth also have pulp cavity,and there also is pulp tissue inside it.Under SEM,there are numerous dentinal tubules into the dentin wall of zebrafish teeth.Tissue structure and ultramicro structure of zebrafish tooth is similarity to the human tooth.Therefore,zebrafish can be used as a model organism of tooth development research.Recently,C32 was found to be related with dental disease in the study of our lab.The homologous genes of C32 in zebrafish are zgc:153440,zgc:162724,zgc:175175 and zgc:136895.However,their expression profiles and functions in the zebrafish are not clear.Therefor,we researched the expression and function of homologous genes of C32 gene in tooth development in the zebrafish,to realize the complete route of study of tooth development related genes in the zebrafish,and to identify the correlation of human C32 gene and tooth development.Whole mount in situ hybridization technology is widely used to detect gene expression profile.Therefore,we designed and synthesized antisense RNA probe to detect gene expression profiles of homologous genes of C32 gene during embryonic development.Several rmethods have been used in studying the gene function in zebrafish development,such as manual mutation,over-expressing and knocking down.A series of research protocols to intervene the gene expression of zebrafish embryos has been well set up.Morpholino oligos strategy is a representative of these intervention methods.In this report,we focus on C32 function in zebrafish embryos development,using over-expressing and knock-down of C32 and rescue studies.The aim of current study is to identify this gene in zebrafish and to evaluate the role of this gene in the development of zebrafish especially the development of tooth.Samples and Methods1.Identification of homologous gene of human C32 in zebrafishUsing the online sequence-comparing tools,BLAST from NCBI,the homologous gene of human C32 in zebrafish was identified and the similarity between this gene and human C32 was analyzed.We used GeneStar to compare2.We used RT-PCR to determine the expression levels of the homologous gene of human C32 transcripts in zebrafish.Total RNA from zebrafish embryos was extracted with Trizol(Invitrogen,Carlsbad,CA,USA)and purified by chloroform extraction and isopropanol precipitation.Reverse transcription was performed with a reverse-transcription kit.All RT-PCR products were confirmed by direct sequencing.3.We generated the digoxigenin-labeled antisense cRNA probes according to the protocol of the manufacturer by using the cDNA templates.The whole-mount in situ hybridization of albino embryos was performed to to visualize the expression pattern of zgc:153440 and zgc:162724 in zebrafish embryo.4.Design and synthesis of morpholino oligonucleotides.ATG-inhibition,splice-blocking,and standard control morpholino oligonucleotides targeting the homologous gene of human C32 in zebrafish were designed and synthesized by Gene Tools(USA).One was proposed to suppress gene translation and the another aimed to interfere with the normal splicing of the mRNA of this gene to result in abnormal mRNA,followed by the generation of malfunction protein.5.Microinjection into the embryos of zebrafish.Morpholino oligos were injected into the egg yolk of 1-2-cell-stage Oregon AB embryos according to the standard protocol.To verify the evaluation of the effect of zgc:153440-MO2,we detected the splicing alterations for zgc:153440,the homologous gene of human C32 in zebrafish,at different time points after the injection of the morpholino.For rescue studies,MOs were coinjected into 1-2-cell-stage zygotes with either wildtype(WT)or mutant C32 mRNA;these zygotes were expressed in pCS2+ and were purified by a mMSSAGE mMACHINE SP6 Kit(Ambion,USA).6.Observation of the effect of morpholinos on the development of zebrafishThe development of zebrafish embryos after injection of the above morpholinos was observed.Besides,choosing 6dpf as the time point,we observed the dentition by alizarin-staining,and then counted the visually significantly defective embryos and calculated the defective rate and survival rate.ResultsThere were four homologous genes of human C32 in zebrafish genome,the code names of which in Gene Bank are Danio rerio zgc:153440,zgc:162724,zgc:136895 and zgc:175175.These genes are highly homologous with human C32.zgc:153440 and zgc:162724 were transcribed from 1 dpf to 5dpf in zebrafish development,but zgc:175175 gave no RT-PCR product.The transcript for zgc:153440 was expressed in developing ceratobranchials at 48 hpf and later stages.And the probe hybridization of zgc:162724 showed no signal dots.The zgc:153440-MO 1,targeting the 5' UTR through the first 25 bases of the coding sequence of zgc:153440,could block translation initiation of it.The zgc:153440-MO2,targeting splice junctions of zgc:153440.altered normal zgc:153440 splicing by adding intron 2.Injection of either zgc:153440-MO 1 or zgc:153440-MO2 caused an abnormal tooth phenotype in 63%or 68%of the fish,respectively.Coinjection of WT zgc:153440 mRNA with either zgc:153440-MO 1 or zgc:153440-MO2 resulted in a normal phenotype in 30%or 32%of the fish,respectively,whereas coinjection with the same amount of mutant zgc:153440 mRNA caused approximately 5%of the fish to have a normal phenotype.ConclusionsSuccessfully using online sequence-comparing tools,BLAST from NCBI,we searched and determined the homologous gene of human C32 in zebrafish,then found the similarity between them was quite high.RT-PCR confirmed that zgc:153440 and zgc:162724 had been transcribed during a quite early phase of embryonic development of zebrafish.The antisense morpholino targeting Danio rerio zgc:153440 might inhibit the transcription.The embryos treated with either zgc:153440-MO 1 or zgc:153440-MO2 showed developmental delay and tooth defects compared to those treated with the control MOs.The phenotype of altered tooth development induced by either of the zgc:153440-specific morpholinos can be corrected when the embryos are coinjected with WT zgc:153440,but not with the mutant zgc:153440 mRNA.It's indicated that zgc:153440,the homologous gene of human C32 in zebrafish genome,may play a critical role in tooth development of zebrafish.Danio rerio zgc:153440,the homologous gene of human C32 in zebrafish genome,may play a critical role in early tooth development of zebrafish.