Relationship Between Tenascin Gene And Zebrafish Embryonic Heart Development

Congenital heart disease (CHD) is a serious congenital malformation which is associated with the abnormal differentiation and migration of myocardial cells and (or) cardiac conduction cells. Extracellular matrix and its receptor play important roles in heart development, and have been one of the hottest research areas of congenital heart disease. Tenascin(TN) is an extracellular matrix oligosaccharide protein which highly conserved in vertebrates. To date, there are four famlily members have been found, including TNC, TNW, TNR, TNX. TNC, a most important family member, plays an important role in embryonic development and pathology. The role of TNC in some heart diseases , such as aqtherosclerotic plaque,myocardial infarction,dilated cardiomyopathies and myocarditis, had been widely studyied. However, the role of TNC in heart development has not yet been clarified .As in vitro fertilization,transparent embryo and easy to be genetically manipulated, zebrafish has became an excellent model for heart research. To explore the causes of congenital heart disease, we studied the temporal and spatial expression pattern of tenascin-c(tnc), tenascin-w(tnw) and tenascin-r(tnr) in zebrafish early development by using in situ hybridization and RT-PCR. We also investigated the formation of heart and the expression of tnc in the abnormal zebrafish heart development by treating with ethanol. Additionally, We investigated the relationship between tnc, retinoic acid signaling and heart development by treating embryos with retinoic acid and retinal dehyfrogenase inhibitor(DEAB), overexpressing the genes of tnc,hand2 by microinjection.Part I: The temporal and spatial expression pattern of tenascin in zebrafish early developmentObject To explore the temporal and spatial expression pattern of tnc, tnw, tnr in zebrafish early development.Methods Zebrafish embryos at different stages (2hpf-72hpf)(hpf: hour post fertilization) were collected for RT-PCR and in situ hybridization.Results tnc, tnw and tnr mRNAs are all expressed in zebrafish from 24hpf to 7dpf, tnc mRNA is expressed at pharyngeal arch, notochord, somite, otic vesicle, pectoral fin and hindbrain, tnw is expressed at pharyngeal arch, notochord, somite, otic vesicle, hindbrain and midbrain, tnr is expressed specifically at central nervous system.Conclusion Zebrafish tnc and tnw genes have special temporal and spatial expression pattern, and share partial overlapping expression pattern, tnr gene can be regarded as a marker of central nervous system.Part II: Expression of tenascin-c in the development of zebrafish embryos induced by ethanolObject To further understand the function of tnc gene in embryo development, we explored the expression pattern of tnc gene in zebrafish abnomal development which was induced by ethanol.Methods Zebrafish embryos at different stages(3hpf-24hpf) were treated with ethanol at 100,200,300,400 and 500mM for different time period. The effects of ethanol on the cardiac development were investigated at 48hpf and 72hpf under the dissecting microscope. Zebrafish embryos which were treated with ethanol from 100mM to 500mM were collected at 24hpf and 48hpf, tnc expression pattern were examined by in situ hybridization and RT-PCR.Results Embryos treated with ethanol at 300mM and above caused mutisystem (including heart) malformations, while ethanol-treated at 200 mM only caused abnormal heart development. The results of RT-PCR and in situ hybridization revealed that the expression of nkx2.5 gene changed when treated with ethanol. Ethanol-treated at 100mM and above causesd up-regulated expression of tnc in zebrafish heart.Conclusion The effect of ethanol on zebrafish heart development is dose and stage dependent. Zebrafish embryos treated with 200 mM ethanol at blastula stage and gastrula stage can be used as an animal model to explore the mechanism of cardiac looping. The effect of ethanol on heart development might be caused by over- expression of nkx2.5 gene. tnc is up-regulated when treated with ethanol , The results indicate that the expression pattern of tnc in pathologic state is highly conserved not only in all vertebrate, but also in adults and embryos.Part III: The roles of tnc gene and retinoic acid signaling in heart development of zebrafish embryosObject To explore the roles of tnc gene and retinoic acid signaling in heart development , and to further provide clues to pathogensis of congenital heart disease, we invesitigated whether the expression of tnc gene was changed with retinoic acid signaling and the association between tnc and the related gene of heart development such as hand2,gata4 and gata.5.Methods The expression of tnc in zebrafish heart which were treated with retinoic acid and retinal dehyfrogenase inhibitor(DEAB) was detected by in situ hybridization. We then studied the expression of hand2, gata5 and gata4 genes by overexpressing tnc gene by microinjection. Moreover,We observed tnc expression by in situ hybridization in the embryos overexpressed hand2 genes.Results The expression of tnc gene was up-regulated in zebrafish heart at 72hpf when treated with DEAB, while there was no expression of tnc in heart when treated with RA. No heart-related transcription factors are changed when overexpress tnc gene. Overexpression of hand.2 gene can up-regulate the expression of tnc in heart.Conclusion Retinoic acid signaling is the up-stream signaling that restricts the expression of tnc gene. hand2 gene can regulate the expression of tnc in zebrafish heart. Therefore, We suggest that retinoic acid signaling might regulate tnc expression via hand2.