Studies On The Function Of Zebrafish Tmp3,hprg1b And Cxxc5 Genes In Cardiogenesis

Congenital heart disease is a neonatal birth defect with the highest morbidity and mortality.Most of the congenital heart diseases are found to be caused by abnormal expression of the regulatory genes in cardiogenesis.The investigation of the gene regulatory mechanism of early cardiac development is the key to the treatment of congenital heart disease.As zebrafish has become a vertebrate model for the study of developmental biology and functional genomics,it can provide an effective research strategy for exploring the molecular mechanisms of early heart development and the pathogenesis of congenital heart disease.In this paper,the zebrafish model was used to study the role of tmp3,hprg1 b and cxxc5 genes in the regulation of early embryonic heart development.1.Study on the function of tmp3 gene in cardiogenesistmp3 gene is a cardiac development candidate gene cloned by our lab.In this paper,a tmp3 promoter-driven EGFP expression transgenic zebrafish line Tg(tmp3:EGFP),which briefly called tmp3 EGFP,was constructed using Tol2 transposon system.Tmp3 protein was found to be strongly expressed in the heart and skeletal muscle at 36,48 and 56 hours post fertilization(hpf)by tracking the location of EGFP signal.In situhybridization was used to detect the early expression of EGFP.At 40% epiboly,the expression signal was located at the lateral margin of the blastomere.At 22 somites stage,the signal was located in the cardiac cone and skeletal muscle.At 24 hpf,the signal was located in the heart tube and skeletal muscle.At 48 hpf,the signal is located in the beating heart and skeletal muscle.The tmp3 EGFP transgenic zebrafish were crossed with Tg(myl7: DSRED)or Tg(fli1a:DSRED)transgenic zebrafish which labeled the cardiomyocytes or endocardial cells respectively.The 72 hpf double-transgenic zebrafish Tg(tmp3EGFP;myl7DSRED)and Tg(tmp3EGFP;fli1aDSRED)were scanned by confocal microscopy,and the single slice results showed that Tmp3 protein was localized in the myocardium.A tmp3 gene knockout zebrafish line was constructed using TALEN-mediated gene knockout.Western blot analysis showed that there was no corresponding functional Tmp3 protein synthesis in tmp3 gene knockout zebrafish.The phenotypic observation of tmp3 knockout homozygous found that tmp3 knockout zebrafish showed pericardial edema,decreased cardiac chamber size,abnormal looping and trunk truncation.Observing the phenotype of tmp3 knockout Tg(myl7: DSRED;flk1: EGFP)double transgenic zebrafish revealed that tmp3 gene knockout led to relatively smaller ventricle and atrium and narrowed atrio-ventricular canal.To investigate that whether the smaller heartchambers observed in tmp3 KO larvae were due to reduction in cell number or in cell size.The antibody of cell membrane tight junction protein ZO-1 was used to label the cell membrane,and the size of tmp3 knockout zebrafish cardiac cells was analyzed.The results showed that the size of tmp3 knockout zebrafish heart cells appeared to be unaffected.On the other hand,the 48 hpf zebrafish hearts were co-stained with the nuclear stain DAPI and MHC antibody to specifically identify and count cardiomyocytes.The number of cardiomyocytes in hearts from tmp3 KO larvae was significantly decreased compared to wild-type(WT)larvae,indicating that a reduction in the number of cardiomyocytes results in smaller cardiac chambers.Edu cell proliferation analysis revealed a significant reduction in the number of proliferating cells in hearts from 48 hpf tmp3 KO embryos compared to WT embryos.The results of TUNEL cell apoptosis assay showed that the apoptosis of tmp3 gene knockout zebrafish cardiac cells was unaffected when compared with wildtype control.Therefore,it was demonstrated that the tmp3 gene regulates the development of zebrafish cardiogenesis via affecting the proliferation of zebrafish cardiomyocytes.Previously,Creb2 was selected and proved to be an interacting protein of Tmp3 by our lab via yeast two-hybrid and COIP interaction analysis.Whether Tmp3 protein regulates cardiac development by interacting with Creb2? In this study,the location of endogenous Tmp3and Creb2 proteins in the zebrafish heart was examined by laser confocal microscopy.The results showed that these two proteins were co-localized in the 72 hpf zebrafish heart.Immunofluorescence assay revealed that the endogenous TMP3 protein and CREB2 protein were co-localized in the nucleus of H9c2 rat cardiomyocytes.Next,immunostaining was used to track the location of endogenous TMP3 and CREB2 protein in differentiated P19 cells at 6 and 10 days post-differentiation induction,it found that at day 6 post-differentiation induction TMP3 and CREB2 protein were primarily co-localized in the cytoplasm in majority of the P19 cells examined.At day 10 post-differentiation induction,the expression of TMP3 and CREB2 protein were co-localized in the nucleus of almost all of the cells examined.These results indicated that TMP3 and CREB2 protein translocated to the nucleus in response to cardiomyocyte differentiation stimuli,thus these two proteins are probably associated with the development of cardiomyocytes.As TMP3 is a transmembrane protein without a nuclear location signal(NLS),to uncover that whether TMP3 translocate into nuclear via interaction with CREB2,the fluorescent plasmids of full-length TMP3 and TMP3 without the CREB2 interacting domain(134-240AA)were constructed and revealed that TMP3 full-length plasmids were expressed in the nucleus and cytoplasm,whereas the truncated TMP3 m protein is only located in the cytoplasm.Thus,TMP3 translocated into nucleus through the interaction with theCREB2.The cardiac defects observed in zebrafish embryo that knockdown the expression of gata4,the key regulator of early cardiomyocyte proliferation and differentiation,are similar with tmp3 knockout zebrafish,suggesting that gata4 may be a downstream target of the Tmp3-Creb2 complex.In this study,RT-qPCR was used to detect the expression of ccnd2 a and cdk4,the downstream target of gata4,in tmp3 knockout zebrafish embryos.The results showed that gata4,ccnd2 a,and cdk4 were significantly down-regulated in tmp3 knockout zebrafish,indicating that gata4 may be a downstream target of Tmp3-Creb2 complex.And gata4 mR NA can to some extent rescue the cardiac defects of tmp3 gene knockout zebrafish,further proving that gata4 is the downstream target of Tmp3-Creb2 during early cardiac development.In addition,RT-qPCR was used to detect the expression of TMP3 mR NA in myocardial tissue of patients with TOF(tetralogy of Fallot)and found that the expression level of TMP3 mRNA in myocardial tissue of TOF patients was significantly decreased.Subsequently,a missense mutation of the TMP3 gene was detected in the TOF patient by sequencing(c.820C> T,p.R274C).The mutant TMP3 plasmid(R274C,TMP3m)was further transfected into H9c2 cardiomyocytes.The transcriptional activity of CRE-luc,the upstream response element reporter of GATA4,was found to be significantly reduced in TMP3mtransfected group when compared with control.Moreover,quantification of GATA4 protein expression level in H9c2 rat cardiomyocytes transfected with wildtype TMP3 and TMP3 m by western blot analysis showed that TMP3 m transfected cells was significant reduced in GATA4 protein expression when compared to wildtype TMP3 transfected cells.Taken together,these results suggest that TMP3 mutation affects the expression of GATA4 protein,which may be associated with the pathogenesis of TOF.2.Study on the function of hprg1 b gene in cardiogenesisThe hprg1 b gene is another early cardiac development candidate gene screened by the P-factor mediated mutations in Drosophila by our lab.In this paper,to track the temporal and spatial expression pattern of hprg1 b gene during early cardiogenesis,Tol2 transposon system was used to construct the hprg1 b promoter driven EGFP expression transgenic zebrafish line Tg(hprg1b:EGFP)which briefly called 1bEGFP,and the expression pattern of EGFP signal was captured during embryogenesis.It was found that at 16 hpf hprg1b gene was evidently expressed in the bilateral heart primordia at lateral plate mesoderm.At 20 hpf,the signal was fused at the midline cardiac cone.The signal was then located in the heart tube at 24 hpf and subsequently in the heart chambers at 48 hpf.It can be seen that the temporal and spatial expression profile of hprg1 b gene is consistent with that of early cardiac morphogenesis.In this paper,Tg(1bEGFP)transgenic zebrafish were crossed with Tg(myl7DSRED)or Tg(kdrlMCHERRY)transgenic zebrafish to produce Tg(1bEGFP;myl7DSRED)or Tg(1bEGFP;kdrlMCHERRY)double transgenic zebrafish.By observing the heart of these double transgenic zebrafish at 72 hpf,it was showed that Hprg1 b protein expression was only located in the myocardium.In addition,the 75 hpf Tg(1bEGFP: myl7DSRED)double transgenic zebrafish heart was further investigated by confocal microscopy,and it was found that hprg1 b was strongly expressed in the outflow tract,atrioventricular canal and the inflow tract of the atrium.The 3D stereoscopic images of the heart indicated that hprg1 b was only expressed in a part of the cardiomyocytes.Flow cytometry analysis of the primary cardiac cells of the 2-month old Tg(1bEGFP: myl7DSRED)revealed that only 10.7% of the cardiac cells in heart were hprg1 b positive cells,suggesting that hprg1 b positive cells may be a special type of cardiomyocytes.In this paper,a hprg1 b overexpression plasmid pCV/hf was constructed and transfected into zebrafish stem cell line Z428.After 1 day post transfection,the expression of the early myocardial markers gata4 and nkx2.5,the early endocardial marker etv2,and the cardiomyocyte differentiation marker myh6 were evidently up-regulated as detected by RT-PCR.Furthermore,the expression of these four genes in hpg1 b overexpressing Z428 cells was quantified by ddPCR.It was found thatthe expression of the four genes was significantly up-regulated after 1 day post transfection(dpt).At 2 dpt,the expression of nkx2.5,gata4 and myh6 were increased continually,but the expression of etv2 began to decrease.At 3dpt,the expression of three cardiomyocyte markers continued to increase,but the endocardium marker etv2 continued to decline.Thus,it indicated that hprg1 b regulate heart development via continuous induction of the expression of early myocardial marker genes.3.Study on the function of cxxc5 gene in cardiogenesisCXXC-type zinc-finger protein 5 is reported to have a wide range of physiological and pathologic functions in the tissues it expressed.Previous studies have shown that CXXC5 is highly expressed in the heart and CXXC5 is associated with pathologic and physiological myocardium remodeling,suggesting that CXXC5 is likely to play an important role in cardiogenesis,cardiomyopathy,and pathogenesis of cardiac disease.However,currently there is no direct evidence to illustrate the function of CXXC5 in the heart.Bioinformatics analysis revealed that the functional domain of CXXC5 was evolutionarily highly conserved.Therefore,using zebrafish model to explore the function of CXXC5 in early embryonic heart development will provide clues for the screening of key regulators of early heart development.In this study,the expression of cxxc5 gene in early embryogenesis of zebrafish was detected by RT-PCR.It was found that cxxc5 started toexpress at 40% epiboly and was consistently expressed form embryonic stage to adulthood.The spatial temporal expression pattern of cxxc5 mR NA was detected by in situ hybridization.The results showed that at 40% epiboly,cxxc5 was expressed at lateral margin of blastomere.In 22 somites stage,cxxc5 was expressed in the cardiac cone.And lately expressed in the heart tube at 24 hpf.At 48 hpf,it was found in the heart chambers.The spatial-temporal expression pattern of cxxc5 indicated that cxxc5 may involve in the regulation of early cardiogenesis.Morpholino knockdown the expression of cxxc5 has a significant effect on the heart development.About 70% of the embryos injected with cxxc5 MO showed cardiac looping defect,cardiac dysplasia and pericardial edema.Statistical survival rate analysis showed that most of the cxxc5 morphants died on the 7dpf.Co-injection of cxxc5 capped mR NA with cxxc5 MO could to some extent rescue the phenotype that appeared in cxxc5 morphants,suggesting that the phenotype of cxxc5 morphants is due to decreased expression level of cxxc5 mRNA.The expression of cmlc2 was detected by in situ hybridization.It was found that the expression of cmlc2 in the 24 hpf cxxc5 morphants stayed in the midline,which was different with the wildtype embryo that the cmlc2 has migrated to the left eye.At 48 hpf,the average angle between the longitudinal axis of ventricle atrium of the cxxc5 morphants was 12 °,which was significantly lower than the average angle of 27 ° of the wild type embryos.In addition,physiological function of the embryonic heart was further analyzed using Semi-Automated Optical Heartbeat Analysis system.The results showed that the heart period was significantly prolonged,the heart rate was evidently slower,and fractional area change was decreased in cxxc5 morphants when compared with wildtype control,indicating that the physiological function of the heart was compromised in cxxc5 morphants.Previous studies have demonstrated that human CXXC5 interacts with SMADs protein(SMAD2,SMAD3).Using bioinformatics analysis this paper revealed that human CXXC5,SMAD2 and SMAD3 proteins are evolutionarily highly conserved between human and zebrafish.Our lab previously demonstrated the interaction between zebrafish Cxxc5 and Smads(Smad2,Smad3)by COIP and GST-pulldown.In this study,the transcriptional activity of the TGF-? signal fluorescent reporter plasmids CAGA-LUC,BMP signal fluorescent reporter plasmids BRE-LUC,and the Activin signal fluorescent reporter plasmid 3TP-LUC were detected in the H9c2 cells with overexpression of Cxxc5.The results showed that the transcriptional activity of CAGA-LUC and BRE-LUC was significantly increased,whereas the activity of 3TP-LUC was unchanged,suggesting that cxxc5 may affect the TGF-? signaling and the relevant BMP signal in cardiomyocytes.To uncover the downstream targets of TGF-? signaling regulated by Cxxc5-Smads complex,this paper analyzed the promoter of classical downstream genes of TGF-? signaling,such as nkx2.5,gata4,hand2,and has2,for MH1-binding site SBE(Smad-binding element,CAGAC)and CpG Island.The results showed that the promoter regions of gata4,hand2 and has2 have the Smad-binding element and CpG Island.RT-qPCR revealed that hand2 and has2 were significantly down-regulated in cxxc5 morphants.And the embryos co-injected with hand2 capped mR NA and cxxc5 MO can effectively rescue the cardiac looping defects in cxxc5 morphants.Therefore,these results indicate that cxxc5 may regulate cardiac looping via hand2,the downstream target of TGF-? signaling.The expression of CXXC5 was significantly differentially expressed in the heart of congenital heart disease children before and after cardiac surgery.Here,the expression of CXXC5 in the heart of TOF patients was detected by RT-qPCR.The results showed that CXXC5 was significantly down-regulated in TOF patients when compared with control,suggesting that CXXC5 may be a candidate marker for clinical diagnosis of TOF.The above results indicated that cxxc5 is an endogenous factor expressed in early heart development and may play an essential role in the formation of left-right asymmetry of the heart.And for the first time,this paper provided an in vivo evidence to prove that cxxc5 regulates the cardiac looping and cardiogenesis via TGF-? signaling pathway.