We previously reported that two NF-κB/Rel family members are involved in notochord formation of the ascidian Halocynthia roretzi. Here, we present evidence that the NF-κB/Rel signaling pathway plays important roles in the notochord formation in another ascidian, Ciona intestinalis. We first found that two NF-κB/Rel family members of C. intestinalis, Ci-rel1 and Ci-rel2, are splice variants: Ci-rel1 is a typical member, while Ci-rel2 is a C-terminally truncated short one. Ectopic expression of GFP-fusion proteins in the C. intestinalis notochord revealed that Ci-rel1 transiently moved into the nucleus in the initial tailbud stage, when concomitant expression of Ci-IκB, a C. intestinalis IκB homologue, was observed, indicating that Ci-rel1 is transiently activated in this stage. Ci-rel1, as well as Ci-rel2, is capable of binding to the κB sequence present upstream of Ci-IκB, suggesting that Ci-IκB is a target gene of Ci-rel1. Reporter gene assay suggests that the expression of Ci-IκB in the notochord is controlled by its κB sequence. Gene silencing of Ci-IκB by injection of the corresponding antisense morpholino oligonucleotide resulted in impairment of notochord formation in C. intestinalis, particularly in a defect in intercalation of notochord cells. Taken together, the results suggest that the regulation of Ci-rel1 by Ci-IκB, whose transcription is regulated by Ci-rel1, in the tailbud stage is essential for notochord formation in C. intestinalis.
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