Characterization of NF-κΒ/IκΒ Proteins in Zebra Fish and Their Involvement in Notochord Development

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Abstract
Although largely involved in innate and adaptive immunity, NF-κB plays an important role in vertebrate development. In chicks, the inactivation of the NF-κΒ pathway induces functional alterations of the apical ectodermal ridge, which mediates limb outgrowth. In mice, the complete absence of NF-κB activity leads to prenatal death and neural tube defects. Here, we report the cloning and characterization of NF-κΒ/IκB proteins in zebra fish. Despite being ubiquitously expressed among the embryonic tissues, NF-κΒ/IκB members present distinct patterns of gene expression during the early zebra fish development. Biochemical assays indicate that zebra fish NF-κΒ proteins are able to bind consensus DNA-binding (κB) sites and inhibitory IκBα proteins from mammals. We show that zebra fish IκBαs are degraded in a time-dependent manner after induction of transduced murine embryo fibroblasts (MEFs) and that these proteins are able to rescue NF-κΒ activity in IκBα−/− MEFs. Expression of a dominant-negative form of the murine IκBα (mIκBαM), which is able to block NF-κΒ 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 ntl (Brachyury homologue), which is typically required for the formation of posterior mesoderm and axial development, suggesting that ntl lies downstream of NF-κΒ. We further show that ntl and Brachyury promoter regions contain functional κB sites and NF-κΒ can directly modulate ntl expression. Our study illustrates the conservation and compatibility of NF-κΒ/IκB proteins among vertebrates and the importance of NF-κΒ pathway in mesoderm formation during early embryogenesis.