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 restricts neural crest induction in both the zebrafish and Xenopus 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 propose that Kctd15 is involved in delineating the neural crest domain by preventing it from expanding beyond its natural limits. Recent experiments 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 micro array mediated analysis of RNA expression patterns. The results of these experiments are being analyzed at present, and are expected to reveal novel information about neural crest induction and the role of Kctd15 in limiting this inductive process. In another project that continues to be pursued we have sought to apply modern genome editing technology to the model systems of interest in this laboratory including Xenopus and the zebrafish. TALENs, referring to chimeric protein molecules that consists of a DNA binding domain derived from plant pathogenic bacteria and a non-specific nuclease domain, have been introduced as specific tools to generate genome breaks and subsequently gene disruptions in various model organisms. Rapid progress in the techniques for constructing DNA sequences that encode TALEN proteins have led to increased applications for these molecules. Further, a different system allowing similar genome editing functions has recently been introduced based on the Cas9 and CRISPR molecules. In a collaborative effort we have tested the efficacy of TALEN mediated gene disruption in Xenopus, using both Xenopus laevis and Xenopus tropicalis as test species. Xenopus has a long history as a premier model system for the study of vertebrate developmental biology, cell cycle control, and for the cloning of animals and reprogramming of somatic cells. A major limitation of the Xenopus system had been the lack of practical methods for gene disruption and genetic manipulation. We have tested the conditions of TALEN expression in Xenopus embryos and have adapted methods for assaying the effect of these nuclease molecules. Through these technical modifications and adaptations we have been able to show that TALENs are effective in introducing mutations in several Xenopus genes, which is likely to encourage the wide use of this technology in this model system. We participated in a collaborative study with Raymond Habas and colleagues at Temple University in studying a previously undescribed protein named Custos, which we show can modulate the Wnt signaling pathway. Custos genes are conserved among vertebrates, but no homologs have been identified in invertebrates. Given the importance of canonical Wnt signaling in development and in human disease, notably cancer, molecules that regulate this pathway have received wide attention. Custos is a protein with little similarity to known protein families. Studying its role in Xenopus embryos and in cultured cells we found that overexpression of Custos inhibits canonical Wnt signaling. In the frog embryo, both overexpression and knock-down of Custos leads to defective development, indicating that the appropriate dosage of this protein is essential for embryogenesis. This was also true in zebrafish embryos, indicating that the role of Custos is conserved in evolution. In epistatic experiments Custos was shown to act at the level of regulation of beta-catenin, the downstream effector of the canonical Wnt signaling pathway. Both in cultured cells and in Xenopus embryos, Custos was able to inhibit nuclear transport of beta-catenin, a key step in its function as an effector of Wnt signaling. Thus we conclude that Custos modulates Wnt signaling in the early embryo by affecting the nuclear import of beta-catenin.
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