Expression And Function Of Tbx2 In Zebrafish Heart Development

PartⅠSpatiotemporal expression pattern of tbx2 gene in zebrafish heartdevelopmentObjective tbx2, a member of the T-box transcription factor gene family, is expressed in a variety of tissues and organs during embryogenesis. In the developing heart of the mouse, tbx2 is expressed in the outflow tract, inner curvature, atrioventricular canal and inflow tract, corresponding to a non-chamber myocardial zone. The role of tbx2 in cardiac development is poorly understood and little is known in zebrafish. Zebrafish is a powerful ideal organism for the analysis of cardiac development. We show here, for the first time, the spatiotemporal expression pattern of tbx2 in the developing heart of zebrafish.Methods The plasmid DNA of tbx2 was linearized and transcribed in vitro by T7 RNA polymerases for Digoxin labelled antisense RNA probes. Embryos of various stages were fixed and processed for whole-mount in situ hybridization to study the expression pattern of tbx2 gene during cardiogenesis.Results tbx2 is first detected within bilateral regions of anterior lateral plate mesoderm at the 5-somite stage. And by 24hpf (hours post fertilization) in the primitive heart tube. It is gradually extinguished from most of the tube except for the borders between presumptive chambers and after 36hpf, tbx2 predominantly restricted to the atrioventricular junctions and disappeared by 96hpf.Conclusion Whole-mount in situ hybridization is effective for the study of spatiotemporal expression pattern of gene. The expression pattern of tbx2 indicates its function of chamber differentiation and formation of atrioventricular canal. The presence of zebrafish tbx2 in heart indicates extensive evolutionary conservation of expression with mouse tbx2 gene. In our test, tbx2 is first detected within bilateral regions of anterior lateral plate mesoderm at the 5-somite stage in zebrafish, which is a little earlier than the while in mouse embryonic heart until E8．75, when a linear heart tube has formed. It suggests that the regulation of tbx2 is earlier in zebrafish than in the mouse.Part IIMorpholino-mediated translational inhibition and overexpression oftbx2 in zebrafish cardiogenesisObjective To elucidate the role of tbx2 in zebrafish cardiogenesis via morpholino-mediated translational inhibition and overexpression on the base of expression pattern of tbx2．Methods Gene-specific antisense morpholino oligonucleotides were designed to against ATG region of zebrafish tbx2 to knockdown endogenous tbx2 gene. At the one-cell stage, each embryo was injected with gradient doses of 2-8ng MO. The control morpholino (c-MO), was purchased from Gene-Tools.To test the knockdown effectiveness of the morpholino, the tbx2-EGFP (enhanced green fluorescent protein) construct was generated. And coinjection of morpholino with mRNA tests its specificity of the morpholino. Overexpression of tbx2 via mRNA microinjection helps us understand gaining function of tbx2．Results The tbx2-EGFP construct tests the knockdown effectiveness of the morpholino. Zebrafish embryos lacking tbx2 function have defects in cardiac contractility, rhythm and morphology in a dose-dependent manner. The embryos with c-MO are normal in development. Coinjection of morpholino with mRNA rescue the phenotype of tbx2 knocking down. The phenotypes include pericardial edema, hypogenetic ventricle, dilation of atria, arrhythmia, bradycardia, asystole, abnormal atrioventricular canal, aberrant valve and blood regurgitation. The phenotypes could be categorized into three major classes according to the degree of abnormality. Conversely, overexpression of tbx2 fails to form chambers.Conclusion Zebrafish is a powerful ideal organism for the analysis of cardiac development. Morpholino is a good way to inhibit the translation of the gene. Our results demonstrate that tbx2 is essential for chamber differentiation and formation of atrioventricular canal. PartⅢExpression patterns of cardiac specific markers in zebrafish withtbx2 gene knock-downObjective amhc and vmhc represent the atrial and ventricular precursors and fates. nppa, a general marker of myocardial stress, is one of the earliest differentiation markers of working atrial and ventricular myocardium. bmp4 and versican are genes involved in cardiac valve formation, and bmp4 still represents the formation of sinus venosus-atrial, atrio-ventricular and ventricular-arterial junctions. We analyse the expression patterns of cardiac specific markers in zebrafish with tbx2 knock-down.Methods The plasmid DNA of markers were linearized and transcribed in vitro by RNA polymerases for Digoxin labelled antisense RNA probes. Embryos with tbx2 knock-down of various stages were fixed and processed for whole-mount in situ hybridization to study the expression patterns of genes during cardiogenesis.Results Expression pattern of amhc becomes dilated and the ventricle smaller. In comparison with wild-type embryos, tbx2-MOs exhibit striking upregulation of nppa in both chambers. Expression pattern of nkx2．5 is normal. The expression of versican and bmp4 in MO embryos are broadly upregulated and expanded aberrantly throughout the heart.Conclusion The data of RNA whole mount in situ hybridization indicate that chamber differentiation, valve formation at the atrioventricular boundary, sinus venosus-atrial and ventricular-arterial junctions in the MO embryos were significantly impaired. tbx2 is essential for chamber differentiation and formation of junctions in heart.