The goals of this project are to determine how the anterior of a vertebrate embryo becomes correctly positioned. The extreme anterior ectoderm in the frog Xenopus forms the cement gland, a mucus-secreting organ that is an excellent positional indicator. The focus of this project is to define ectodermal genes that regulate cement gland determination. One transcription factor expressed in the cement gland that is key for cement gland formation is the homeobox gene otx2. Ectopically expressed otx2 activates ectopic cement gland development, while ablation of otx2 activity using a dominant negative protein prevents cement gland formation. A hormone-inducible otx2 protein, otx2GR, activates the cement gland marker XAG1, however only with ongoing protein synthesis, suggesting that otx2 activates expression of downstream genes which in turn activate XAG1 expression. In this proposal, genes activated by otx2GR that may directly activate XAG1 expression will be isolated using a subtractive cloning strategy. In order to further analyze cement gland positioning, dissection of the XAG1 promoter has been carried out. A 200bp XAG1 fragment directs expression of a reporter to the cement gland. This fragment is also responsive to otx2GR, although it contains no otx2 binding sites. Further analysis of the XAG1 promoter will be performed to define promoter elements necessary for cement gland formation. Factors that interact with XAG1 regulatory sequences will be identified and characterized. Since the cement gland is an indicator of normal head development, and since genes and developmental strategies are evolutionarily conserved, what we learn in Xenopus is likely to have relevance for understanding normal and abnormal development of human embryos.
