Disruption of the folate pathway in zebrafish causes developmental defects.
Disruption of the folate pathway in zebrafish causes developmental defects.
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DOI：
10.1186/1471-213x-12-12
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发表时间：
2012-04-05
影响因子：
--
通讯作者：
Brody LC
中科院分区：
文献类型：
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作者：
Lee MS;Bonner JR;Bernard DJ;Sanchez EL;Sause ET;Prentice RR;Burgess SM;Brody LC
补充叶酸可以降低神经管缺陷和先天性心脏缺陷的风险。叶酸预防出生缺陷的生物学机制尚不清楚。我们探索使用斑马鱼作为模型系统来研究叶酸代谢在发育过程中的作用。我们首先在斑马鱼中发现了12个人类叶酸代谢基因的同源物。RT-PCR和原位分析表明，母体转录本为胚胎提供mRNA，使胚胎具有完整的叶酸通路。为了扰乱叶酸代谢，我们将斑马鱼胚胎暴露在甲氨蝶呤（MTX）中，甲氨蝶呤是叶酸代谢途径中必需的二氢叶酸还原酶（Dhfr）的有效抑制剂。暴露于高剂量MTX的胚胎在早期分化之前表现出发育停滞。较低剂量的MTX导致胚胎前后轴缩短和心脏缺陷：线形心管或心环不完整。反义morpholino寡核苷酸抑制dhfr mRNA可导致胚胎致死。叶酸途径的一个功能是为dTMP合成提供必要的单碳单位，dTMP是DNA合成的限速步骤。暴露于高水平MTX 24小时后，突变胚胎继续纳入胸腺嘧啶类似物BrdU。然而，额外的实验表明，这些胚胎有丝分裂细胞较少，用磷酸组蛋白H3抗体检测，并且处理过的胚胎细胞周期受到干扰。我们的研究表明，人类和斑马鱼利用类似的单碳途径。我们的数据表明，叶酸代谢对斑马鱼的早期发育至关重要。斑马鱼对叶酸通路及其缺陷的研究可以为人类出生缺陷的潜在病因和叶酸在发育中的自然作用提供见解。
Folic acid supplementation reduces the risk of neural tube defects and congenital heart defects. The biological mechanisms through which folate prevents birth defects are not well understood. We explore the use of zebrafish as a model system to investigate the role of folate metabolism during development. We first identified zebrafish orthologs of 12 human folate metabolic genes. RT-PCR and in situ analysis indicated maternal transcripts supply the embryo with mRNA so that the embryo has an intact folate pathway. To perturb folate metabolism we exposed zebrafish embryos to methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (Dhfr) an essential enzyme in the folate metabolic pathway. Embryos exposed to high doses of MTX exhibited developmental arrest prior to early segmentation. Lower doses of MTX resulted in embryos with a shortened anterior-posterior axis and cardiac defects: linear heart tubes or incomplete cardiac looping. Inhibition of dhfr mRNA with antisense morpholino oligonucleotides resulted in embryonic lethality. One function of the folate pathway is to provide essential one-carbon units for dTMP synthesis, a rate-limiting step of DNA synthesis. After 24 hours of exposure to high levels of MTX, mutant embryos continue to incorporate the thymidine analog BrdU. However, additional experiments indicate that these embryos have fewer mitotic cells, as assayed with phospho-histone H3 antibodies, and that treated embryos have perturbed cell cycles. Our studies demonstrate that human and zebrafish utilize similar one-carbon pathways. Our data indicate that folate metabolism is essential for early zebrafish development. Zebrafish studies of the folate pathway and its deficiencies could provide insight into the underlying etiology of human birth defects and the natural role of folate in development.