| The developmental toxicity associated with ethanol was first reported in human fetuses in 1968. Ethanol exposure during pregnancy can cause fetal alcohol syndrome (FAS). The typical characteristics of FAS are delay in development, cardiac abnormalities, central nervous system abnormalities, abnormal craniofacial features, skeletal defects and so on. By estimate, the incidence of FAS is approximately 10-20 per 10,000 live births in the general population. It has been shown that the central nervous system (CNS) is particularly susceptible to ethanol. Embryonic ethanol exposure can cause neural tube defects (NTDs), which are among the most common human birth defects. The incidence of NTDs is approximately 0.5-12 per 10,000 live births, but its underlying molecular mechanism is still unknown. Zebrafish has been proved to be an ideal animal model system for human diseases and for the evaluation of chemical and drug toxicity. In this study, we use zebrafish as an animal model to study the effects of ethanol exposure on early embryonic development, and try to uncover the teratogenic mechanism. We hope to provide theoretic basis for the prevention of human inborn NTDs and provide scientific directions for pregnant women to avoid drinking alcohol.In this study, zebrafish embryos at different developmental stages have been treated with different doses of ethanol. The results show that ethanol exposure appears stage-dependent and dose-dependent, with 3% ethanol exposure can most easily induce split axis. Morphological and histological observation and in situ hybridization results show that double notochords, double neural tubes and two sets of somites appear in the posterior trunk, with the phenotype similar to spina bifida in human. It can be inferred that double neural tubes and two sets of somites are probably induced by split notochord. In addition, ethanol exposure can obviously delay the embryonic development. When the ethanol exposure is ended, the treated embryos are at about 50%-epiboly stage, while the untreated embryos are at about 70% epiboly stage. At about 11 hours post fertilization, the treated embryos are still at gastrulation stage, while convergence and extension movement is the key event during this stage. In order to uncover the mechanism of ethanol induced split axis, whole-mount in situ hybridization has been performed to investigate the expression of several important genes in ethanol exposure embryos. The results indicate that ethanol exposure obviously alters the expression pattern of genes including ntl, MyoD, neurogenin1, tbx6 and gsc, which are related to axis development. At about 11 hours post fertilization, the expression domain of ntl splits into two parts at dorsal region of the trunk, and the expression domain of MyoD becomes wider in the ethanol exposure embryos. The expression of neurogenin1 in ventral proneural clusters appears split, and that of tbx6 becomes wider and irregular in presumptive segmented plate. In addition, the expression of gsc is split into two parts at dorsal region. All of these suggest that ethanol disturbs convergence and extension movement of neuroectoderm and mesoderm cells, and the cells expressing ntl can not converge to the midline. In zebrafish, ntl has been proved to have close relationship with convergence and extension movement, and to be a key regulator for the convergence of notochord domain. It can cooperate with nocanonical Wnt signalling pathway to regulate the posterior mophogenesis. So it can be inferred that disturbing convergence and extention movement is the mechanism of ethanol induced split axis.Amphioxus, a cephalochordate, has been widely considered as the closest living invertebrate relatives of the vertebrates. As many vertebrate homologous genes have been found in amphioxus, it becomes a typical material for the study on the origin and evolution of vertebrates. Zebrafish has been become an ideal animal model for the study of vertebrate developmental mechanism. Now great progress has been made in developmental mechanism of both amphioxus and zebrafish at the molecular level, and many important genes have been found. However, as the animal embryonic development is dynamicly controlled by network genes, further studies should be carried out. In this study, we have cloned the full length cDNAs of AmphiMef2, Amphi-mMRLC and hadh2 in amphioxus for the first time. In order to uncover the changes of genes during evolution, we cloned Z-MRLC and hadh2 in zebrafish and performed comparative study between amphioxus and zebrafish. This study will be certainly helpful to give insight into the developmental mechanism of amphioxus and zebrafish, and to elucidate the function changes of genes during evolution.A novel full-length cDNA has been isolated from amphioxus by large scale sequencing of the cDNA library and RT-PCR, which contains a 1287 bp open reading frame and encodes a putative protein of 428 amino acids. The predicted protein has MADS and MEF domains, similar to other MEF2 members of MADS family. The MADS and MEF domains show more than 90% identity with the corresponding regions in other species. So we named this cDNA clone as AmphiMef2. Phylogenetic analysis indicates that it appears more closely related to its vertebrate homologues. Myocyte enhancer factor 2 proteins are members of family of transcription factors, which can control the expression of muscle-specific genes in vertebrates. However, not all genes are essential for muscle development in invertebrates. So we performed a study on the developmental expression of AmphiMef2 in amphioxus. Results from whole-mount in situ hybridization show that the expression of AmphiMef2 initially appears in the presomitic mesoderm at early neurula stage (10h), then the transcripts are detected in both the somites and the unsegmented presomitic mesoderm. From early larval stage (24h), the expression level in the anterior somites is downregulated. At 36h larval stage, the expression is only detected in the posterior somites. By 48h larval stage, the expression is shifted to the preoral pit (a homologous organ to the vertebrate adenohypophysis) and persists through 72h larval stage. The results suggest that AmphiMef2 may be involved in not only the myogenesis but also the development or function of the preoral pit in amphioxus. Whether AmphiMef2 functions in the reproductive endocrine system of amphioxus is worth to examine further.Another full-length cDNA encoding MRLC protein has been obtained from amphioxus by large screening and RT-PCR, which we named Amphi-mMRLC. Amphi-mMRLC encoding 167 amino acids exhibits high identity with its homologue in zebrafish (similar to 19.9 KD myosin light chain isoform 1, Z-MRLC). Z-MRLC cDNA has been cloned by RT-PCR based on its sequence in GenBank, which encodes 172 amino acids. By comparative analysis of their deduced protein characteristics, some similarities have been found, such as containing EF-hand motif, nearly equal theoretical pI, similar percentage of random coil, and so on. However, some differences are also existed, such as sequence length, components of amino acids, numbers of phosphorylation sites, and so on. The predicted protein of Amphi-mMRLC shares 46% overall identity with zebrafish Z-MRLC and 46%-47% with some MRLC homologues from frog, chicken, human, rat and sponge. Phylogenetic analysis indicates that Amphi-mMRLC and zebrafish Z-MRLC fall in the invertebrate clade and vertebrate clade, respectively, in accordance with animal taxology. Whole-mount in situ hybridization results show that distinct expression pattern of MRLC occurs in amphioxus and zebrafish. The expression of Amphi-mMRLC initially appears in the presomitic mesoderm at early neurula stage, and then is restricted to the somites and myotomes until at least 72 larval stages, while no transcripts can be detected in nonmyotomal muscles. However, the expression of zebrafish Z-MRLC is undetectable in the somites. It is strongly expressed in the heart primordium, head mesoderm and weakly in the differentiating endoderm at segmentation stage. At 72 hpf, the expression is restricted to the heart, dorsal aorta, the epithelium of gut and otic vesicle. As amphioxus Amphi-mMRLC shows similar expression in muscle tissues with other homologous gene in other invertebrates, it can be inferred that MRLC may be involved in myogenesis in invertebrates. However, vertebrate MRLC family genes have more members and wider functions, which may be implicated in the development of skeletal, cardiac, smooth muscle and nonmuscle tissues. In conclusion, members and functions of the MRLC family genes may change during evolution.Hydroxyacyl-coenzyme A dehydrogenase type II (hadh2) is a multifunctional mitochondrial enzyme. It has multiple physiological functions and close relationship with some human diseases, especially some inborn errors. Mutations in the hadh2 gene can lead to 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency which can cause some inborn errors of isoleucine metabolism. A full-length hadh2 cDNA has been isolated from amphioxus by large scale screening and RT-PCR. In order to investigate the function of hadh2 during amphioxus and zebrafish embryogenesis and its change during evolution, zebrafish hadh2 fragment was obtained by RT-PCR based on the GenBank sequence. Comparative analysis of their deduced protein characteristics indicates that both of them encode 260 amino acids, have nearly equal theoretical pI and contain adh-short domain. Sequence alignment and phylogenetic analysis show that amphioxus hadh2 protein shares 70% overall identity with zebrafish hadh2, and branches off at the base of the vertebrates in the phylogenetic tree and has close relationship with its vertebrate homologoues. The developmental expression of hadh2 has been detected in amphioxus and zebrafish embryos and larvae by whole-mount in situ hybridization. The results show that amphioxus hadh2 initially appears in the differentiating endoderm and mesoderm at mid-neurula stage (12h), and then it is expressed in the endoderm, visceral mesoderm and posterior neural tube. By 72 hpf larval stage, the expression is restricted to the gut epithelium. Compared with amphioxus hadh2, zebrafish hadh2 is expressed maternally, and downregulated during gastrulation. By late segmentation stage, it is strongly expressed in the head mesoderm, endoderm and eyes, and weakly in the CNS. During segmentation stage, no transcripts are detected in the posterior neural tube. The results suggest that hadh2 may play important role during amphioxus and zebrafish embryogenesis, but some functions may change during gene evolution. |
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