The Regulatory Mechamisms Of Zebrafih Tbx1 In Cardiac Development

The regulatory mechamisms of zebrafih Tbx1 in Cardiac DevelopmentTbx1, one of the genes mapped within the de122q11 locus in human, appears to be one of the earliest genes involved in pharyngeal arch development. Combined studies of heterozygous deletions within the DGCR and knockout experiments in mice have identified Tbx1 as a major genetic determinant of aortic arch malformations in DGS. In addition, homozygous for Tbx1 mutation in mice displayed a wide range of developmental anomalies encompassing almost all of the common features, including hypoplasia of the thymus and parathyroid glands, cardiac outflow tract abnormalities, abnormal facial structures, abnormal vertebrae and cleft palate. As a result of all these studies, Tbx1 currently represents the most promising candidate gene for DGS.DGS is the most common genetic deletion syndrome in humans, with an incidence of 1 in 4000 live births. DGS is characterized by congenital heart defects, thymus and parathyroid aplasia/hypoplasia, and craniofacial anomalies. These structures are derived from neural crest cells during embryogenesis. In common with mouse, chick, lamprey and zebrafish embryos, Tbx1 is expressed in the pharyngeal endoderm and mesoderm, not in neural crest cells. Thus, the expression pattern of Tbx1 raises a question which roles of Tbx1 played during the development of neural crest cells.The formation of pharyngeal arches is critically dependent on retinoic acid (RA). Defects caused by excessive or insufficient RA in mouse recapitulate the typical phenotypes seen in human DGS/VCFS syndrome. Likewise, RALDH2 knockout and experimental manipulation of retinoid receptor in zebrafish also produce phenotypes of DGS. Over expression and haploinsufficieny of Tbx1 also produce similar DGS-like defects to those caused by RA treated models. The strong phenotypic reminiscence between RA treated DGS-like syndrome and Tbx1-/- mutants prompted us to investigate the effects of RA on the expression pattern of Tbx1.The zebrafish, Danio rerio, offers several distinct advantages as a genetic and embryological model system, including the external fertilization, rapid development and optical clarity of its embryos. Being a vertebrate, the zebrafish has a notochord, blood, heart and vasculature, kidney and optical systems that share many features with corresponding human systems. The zebrafish system bridges the gap between fruit fly/worm and mouse/human genetics, making it feasible to address issues of early development, organ formation, integrative physiology, pharmacology and complex disease.The zebrafish also have the outstanding feature for studying on cardiovascular development. 1. They are completely transparent during the first 3 days of development, which facilitates the experiment on cardiac development. 2. It is easy to trace the cardiac movement and circulation in living embryos. 3. Because of their small size, zebrafish embryos are not completely dependent on a functional cardiovascular system. Even in the total absence of blood circulation, they receive enough oxygen by passive diffusion to survive and continue to develop in a relatively normal fashion for several days, thereby allowing a detailed analysis of animals with severe cardiovascular defects. By contrast, avian and mammalian embryos die rapidly in the absence of a functional cardiovascular system. Forward genetics in zebrafish has led to the identification of several mutations affecting cardiac contractility.Tbx1 shows that both patterns of expression and gene function are generally conserved across vertebrate species. Morpholino (MO) antisense technology is a reliable gene knockdown method in zebrafish and can prevent translation of the target gene. Therefore, to investigate the developmental role of Tbxl, Tbxl specific morpholino antisense oligonucleotides were used in our study. However, knowledge remains limited concerning the upstream regulation of Tbx1 during pharyngeal arch development.Section 1 the expression patterns of Tbx1 and phenotypie analysis of Tbx1 knock-down zebrafish embryosIn this study, we describe that Tbx1 is highly expressed in heart as well as pharyngeal arches and otic vesicle during zebrafish embryogenesis. To determine the function of Tbx1 in zebrafish development, two different morpholino antisense oligonucleotides targeting the zebrafish Tbx1 were used. The cartilages in the mandibular and hyoid arches are drastically reduced in size. The cartilages in pharyngeal arches 3-7 are either seriously reduced in their number or completely absent in Tbx1 knock-down embryos. Tbx1 functionally knock-down produce smaller size in otic vesicle. We observed that the thymus are absent in nearly 60% Tbx1 morphant embryos. Counting the numbers of aortic arches reveals a significant reduction in their number after morpholinos injection when compared to control embryos. Conclusively, Tbx1 knock-down embryos are characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus.Section 2 Altered gene expression profiles in tbx1 morphant embryosT-box genes play important roles during embryogenesis in the vertebrate. Tbx2 is another member of T-box family. In this section, we found that Tbx1 knock-down can lead to altered expression of Tbx2 in the pharyngeal arches; this result raised a possibility that Tbx1 can regulate Tbx2 expression in the pharyngeal arches directly or indirectly. In addition, the expression of Tbx20 is also reduced in Tbx1 knock-down embryos.A large number of genes are expressed in the pharyngeal arches and implicated in pharyngeal arch development, such as Bmp2b, fgf8 and hand2. We showed that the expression of Hand2 and Bmp2b are reduced in the pharyngeal arches. We observed that there is no difference in the expression of Fgf8 between Tbx1 knock-down ad control embryo. The expression of Nppa is down-regulated in the ventricle in Tbx1 knock-down embryos. Taken together, these data indicate Tbx1 knock-down can lead to an altered gene expression profiles in zebrafish.Section 3 Tbx1 knock-down can cause reduced pharyngeal neural crest cells and cardiac dysfunctionWe have found that the structures derived from neural crest cells are seriously interrupted in Tbx1 knock-down embryos.Therefore these results raise a question whether Tbx1 plays an important roles in the control of neural crest cells fate. To understand the function of Tbx1 on neural crest development, we examined the expressions of some neural crest markers, such as foxd3, tfap-2, dlx2αand msx in Tbx1 knock-down embryos. We found that the expression ofdlx2αis seriously reduced by knockdown of Tbx1. The expressions of msx and trap-2 in pharyngeal neural crest cells are also decreased in Tbx1 knock-down embryos. In addition, Tbx1 Knock-down leads to an increase of cell apoptosis in the midbrain, hindbrain and pharyngeal arches. Therefore, we conclude that Tbx1 knock-down can produce the reduced amount of pharyngeal neural crest cells; in addition, at least in part, due to elevated level of apoptotic cells in this region.We also found defects in cardiac function in Tbx1 knock-down embryos. Depressed heart rate and reduced ventricular shortening fraction were observed in the Tbx1 knock-down embryos compared with control embryos. These results demonstrated that knock-down of Tbx1 can cause cardiac dysfunction due to reduced cardiac neural crest cells.Section 4 Exogenous RA down-regulates Tbx1 expression in zebrafishIn this part, we found that zebrafish embryo treated with 10^-7-10^-8mol/L RA at the end of gastrulation or the early segmentation, can be used as an experimental model to explore the mechanisms of cardiac development. Phenotypes of these embryos were analyzed, no detectable effect on the overall body plan was found. However these normal looking embryos, as the animals develop, exhibited selective defects of the heart tube.Time course analysis of zebrafish tbx1 expression by real-time quantitative PCR reveals that the expression level of Tbx1 was depressed due to RA treatment. The analysis of whole-mount in situ hybridization showed that exogenous RA can down-regulate Tbx1 expression in pharyngeal arches and out flow tract when compared to control embryos.