| The notochord is the defining feature of the chordate clade, and provides a source of patterning signals and structural support to the developing embryo. The knowledge of cis-regulatory sequences directing gene expression in notochord cells and of the transcription factors which bind them in vertebrates is still fragmentary. Because of the genome duplications undergone by vertebrates and the relative difficulty of working with vertebrate embryos, it is difficult to gain a deeper understanding of the inner workings of notochord development. Ciona intestinalis is an invertebrate chordate which possesses a primitive yet fully functional notochord, but has not undergone the genome duplications that vertebrates have. Due to these qualities, we chose to use Ciona intestinalis as a model organism to study notochord development. As a first step toward elucidating the similarities and differences between the ascidian and vertebrate notochords, I used literature searches, BLAST searches, and whole mount in situ hybridizations to identify 20 genes expressed in the notochord in both vertebrates and ascidians. I also determined whether these genes were responsive to changes in the level of Brachyury, a key notochord transcription factor.;I next more closely examined the role of one of these conserved genes in notochord development. Ci-XBP1, an orthologue of a transcription factor activated during the unfolded protein response, appears to affect notochord intercalation when I introduce it into embryos in a dominant-negative form. Possible targets of Ci-XBP1 include genes in the secretory pathway, leading me to hypothesize that Ci-XBP1 is involved in promoting secretion of the extracellular matrix proteins necessary for notochordal integrity. Analysis of Ci-XBP1 sequences from UPR-induced Ciona embryos suggests that Ci-XBP1 does not undergo the cytoplasmic splicing characteristic of its orthologues in other species.;Finally, I examined the regulation of the transcription factor expressed soonest after Ci-Brachyury in the Ciona notochord, Ci-bHLH1, and also investigated some of its target genes. I found that Ci-Brachyury affects Ci-bHLH1 transcription. I participated in a microarmy screen that identified ten Ci-bHLH1 targets expressed in tissues in which Ci-bHLH1 is active, four of which were expressed in the notochord.;Based on the above findings, I propose that the Ciona notochord transcriptional network is simple but not shallow, and may employ both Brachyury-dependent and independent activation mechanisms. |
