The Role Of SUMOylation In Zebrafish Cardiac Development

The thesis comprises of two parts: part one,the role of SUMOylation in zebrafish cardiac development.part two,the biological function of vgll4 in zebrafish cardiac development.Chapter ?: The role of SUMOylation in zebrafish cardiac developmentThe SUMOylationof protein is a biological process that SUMO protein is convalently attached to lysine residue of substrate protein by isopeptide through a series of enzyme reactions,thereby modulating protein function.The SUMOylation is one of the key protein regulatory modifications,and involved in a variety of cellular events including cytokinesis,ion channel activity,signal transduction,DNA replication and transcription,mitosis and meiosis and telomere maintenance etc.The role of SUMOylation in embryos development is emerging,and our research focus on the role of SUMOylation in cardiac development.The cardiac development is dependent on the finely balanced SUMOylation,and both hyper-SUMOylation and hypo-SUMOylation will lead to cardiac defects.At present,the mechanisms underlying the cardiac defects caused by the dysregulation of SUMOylation remain largely unknown.To explorethe function and mechanism of SUMOylation in cardiac development,we combined TALEN and morpholino technique to abrogate the function of ubc9 in zebrafish model.The heart of ubc9 deficiency zebrafish manifested abnormal cardiac chamber morphology,failure of looping and atrioventricular canal(AVC)defect.The analysis of cardiac morphogenesis revealed,the multiple stages of cardiac morphogenesis including midline migration,cardiac cone assemble,primary heart tube elongation and looping were perturbed.In addition,the differentiation of myocardium and endocardium was aberrant.The cardiac phenotype of ubc9 deficiency zebrafish is reminiscence of that of gata5 mutants,so we investigated the possible role of gata5 SUMOylation in cardiac development.First,in vitro experiments showed GATA5 was modified by both SUMO1 and SUMO2,and K324 and K360 were two major modification sites.The SUMOylation of GATA5 did not influence its subcelluar localization,but affected its transcription activity.In vivo experiments showed,while gata5 is able to increase the number of cardiac progenitors and result in enlarged hearts in WT zebrafish embryos,this potency was lost in ubc9 deficiency zebrafish embryos.To further evaluate the impact of SUMOylation on GATA5 in vivo,we tested whether K324 R or K360 R m RNA of gata5 can rescue the cardiac defects caused by gata5 morpholino.We found that both K324 and K360 had significant impact on the potency of GATA5 in regulating the number and differentiation of cardiac progenitors during cardiac development,and K360 was the major functional site.More importantly,compared with WT GATA5,the SUMO2-GATA5 or SUMO1-GATA5 fusion protein can partially restore its potency of increasing cardiac progenitors number in ubc9 deficiency zebrafish embryo,furthermore the heart morphology tended to be normal in partial ubc9 deficiency zebrafish embryo.Therefore,the essential role of SUMOylation in zebrafish cardiac development is at least partially mediated by gata5.Our research contribute to the understating of cellular and molecular mechanism underlying SUMOylation in cardiac development,and unravel the functional role of posttranslational modification of GATA5.Chapter ?: The biological function of vgll4 in zebrafish cardiac developmentCongenital heart diseases(CHDs)is one of the most common birth defects with high mortality,posing grave threats to infant health.Chromosome 3pter-p25 deletion syndrome(3p-syndrome)is a hereditary disease caused by short arm deficiency of number 3 chromosome.More than one third of 3p-syndrome patients manifest CHDs,and atrioventricular septal defect(AVSD)is common among CHDs.A number of studied had shown the deletion of 3p25.3 band is the key contributing factor to the major clinicalsymptomsof3p-syndrome.To investigate the mechanisms of 3p-syndrome,we focus on a transcription cofactor--vestigial-like protein 4(VGLL4),which is located in the 3p25.3 band,as a candidate gene involved in cardiac development.To dissect the role of vgll4 in cardiac development,we combined morpholinos with CRISPR/Cas9 or TALEN,and other techniques to study the biological function of VGLL4.There are three homologs of human VGLL4 in zebrafish----vgll4 b,vgll4a and vgll4 l.We first analyzed the spacio-temporal expression pattern of vgll4 b,vgll4a and vgll4 l,and found all these three genes had similar expression in pharynx,pectoral fin and heart,with vgll4 l exhibited unique expression in nose,pronephric duct,cloaca,epidermis etc.The vgll4 b homozygote,vgll4 a and vgll4 l morphants manifested abnormal cardiac morphology,looping and AVC forming defects.In addition,bmp4 and notch1 b which are involved in AVC development were ectopically expressed in ventricle.Due to the highest percentage and severity of abnormality of vgll4 l morphants,we started investigating the possible molecular mechanism of cardiac defects caused by vgll4 lknockdown.Cardiac development transcription factor MEF2 C interact with VGLL4,and its activity is inhibited by VGLL4.Overexpressed zebrafish MEF2 CB dominant negative mutant can partially rescue cardiac defects of vgll4 l morphant,which strongly suggested vgll4 l probably affects zebrafish cardiac development through MEF2 C pathway.