The molecular basis of embryogenesis in Xenopus laevis is the subject of this project. We have used DNA microarray technology and other methods for gene discovery in the early embryo, with the aim to obtain information on gene expression patterns and gene function in development, and through this to lead to improved understanding of the molecular basis of normal embryogenesis and abnormalities that can arise by loss of function or malfunction of various genes. Several genes with a role in vertebrate embryogenesis have been studied in the recent past. Our longstanding interest in neural crest development has been continued in the study of the novel factor Kctd15 that inhibits neural crest induction in both the zebrafish and Xenopus when overexpressed in the embryo. Kctd15 is a BTB-domain containing protein that is first expressed in the embryo at the neural plate border, and subsequently in pharyngeal arches and other regions. Overexpression of Kctd15 strongly inhibits neural crest specification in whole embryos and in animal explants, as studied in so-called animal caps from Xenopus embryos. Many transcription factor encoding genes that are characteristically induced during neural crest formation were inhibited by overexpression of Kctd15. We have shown that Kctd15 interacts with and inhibits the function of transcription factor AP-2. AP-2 is known as a major regulatory factor required for the formation of the neural crest in Xenopus and in other animals where it was tested. We conclude that inhibition of AP-2 function is a major basis for the effect of Kctd15 on neural crest formation. To further explore the biological effects of Kctd15 and its interaction with transcription factor AP-2 in the environment of the embryo we have embarked in a collaborative project with Hui Zhao at the Chinese University of Hong-Kong to analyze changes in gene expression patterns after overexpression of Kctd15. For this purpose we have used the well-established explant system from Xenopus embryos, named the animal cap method. In this approach, the animal region of early frog embryos is injected with the desired synthetic mRNAs, the embryos raised to the blastula stage, and then the animal region, or cap, is excised and cultured for an appropriate time period. In our case, various combinations of mRNA were injected to induce neural crest differentiation and inhibit this differentiation by Kctd15, and the resulting samples were subjected to microarray mediated analysis of RNA expression patterns. The results of these experiments show that known neural crest markers are inhibited by Kctd15. In addition, many genes not previously known to be affected have been shown to be inhibited by Kctd15 in the context of neural crest induction. Several of these genes were studied further by analysis of their expression patterns. Expression was detected both in the neural crest and in other tissues of the Xenopus embryos. The work in this laboratory in studying the development of Xenopus laevis is terminating with the completion of the study described above.
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