Characterization of NF-κB/IκB proteins in zebra fish and their involvement in notochord development
Characterization of NF-κB/IκB proteins in zebra fish and their involvement in notochord development
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DOI：
10.1128/mcb.24.12.5257-5268.2004
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发表时间：
2004-06-01
影响因子：
5.3
通讯作者：
Verma, IM
中科院分区：
文献类型：
--
作者：
Correa, RG;Tergaonkar, V;Verma, IM
尽管核因子-kappaB主要参与先天免疫和获得性免疫，但它在脊椎动物的发育过程中发挥着重要作用。在雏鸡中，核因子-kappaB途径的失活导致顶端外胚层脊的功能改变，而顶端外胚层脊介导了肢体的生长。在小鼠中，完全缺乏核因子-kappaB活性会导致产前死亡和神经管缺陷。在此，我们报道了斑马鱼中核因子-kappaB/IkappaB蛋白的克隆和鉴定。尽管在胚胎组织中普遍表达，但在斑马鱼的早期发育过程中，NF-kappaB/IkappaB成员呈现出不同的基因表达模式。生化分析表明，斑马鱼NF-kappaB蛋白能够结合哺乳动物的一致DNA结合位点和抑制性IkappaBalpha蛋白。我们发现斑马鱼IkappaBalpas在转导的小鼠胚胎成纤维细胞(MEF)诱导后以时间依赖的方式降解，这些蛋白能够恢复IkappaBalpha(-/-)MEF中的NF-kappaB活性。在斑马鱼细胞中表达显性-阴性形式的小鼠IkappaBalpha(MIkappaBalphaM)能够阻断NF-kappaB，干扰脊索分化，不产生类似尾巴(NTL)的胚胎。这种表型可以通过共注射T-box基因ND(短臂同源基因)来挽救，这通常是后中胚层形成和轴向发育所必需的，表明ND位于核因子-kappaB的下游。我们进一步证明了nd和brachyury启动子区域含有功能性kappaB位点，并且核因子-kappaB可以直接调控nD的表达。我们的研究阐明了脊椎动物中核因子-kappaB/IkappaB蛋白的保守性和亲和性，以及核因子-kappaB通路在胚胎发育早期中胚层形成中的重要性。
Although largely involved in innate and adaptive immunity, NF-kappaB plays an important role in vertebrate development. In chicks, the inactivation of the NF-kappaB pathway induces functional alterations of the apical ectodermal ridge, which mediates limb outgrowth. In mice, the complete absence of NF-kappaB activity leads to prenatal death and neural tube defects. Here, we report the cloning and characterization of NF-kappaB/IkappaB proteins in zebra fish. Despite being ubiquitously expressed among the embryonic tissues, NF-kappaB/IkappaB members present distinct patterns of gene expression during the early zebra fish development. Biochemical assays indicate that zebra fish NF-kappaB proteins are able to bind consensus DNA-binding (kappaB) sites and inhibitory IkappaBalpha proteins from mammals. We show that zebra fish IkappaBalphas are degraded in a time-dependent manner after induction of transduced murine embryo fibroblasts (MEFs) and that these proteins are able to rescue NF-kappaB activity in IkappaBalpha(-/-) MEFs. Expression of a dominant-negative form of the murine IkappaBalpha (mIkappaBalphaM), which is able to block NF-kappaB in zebra fish cells, interferes with the notochord differentiation, generating no tail (ntl)-like embryos. This phenotype can be rescued by coinjection of the T-box gene nd (Brachyury homologue), which is typically required for the formation of posterior mesoderm and axial development, suggesting that nd lies downstream of NF-kappaB. We further show that nd and Brachyury promoter regions contain functional kappaB sites and NF-kappaB can directly modulate nd expression. Our study illustrates the conservation and compatibility of NF-kappaB/IkappaB proteins among vertebrates and the importance of NF-kappaB pathway in mesoderm formation during early embryogenesis.