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. One of the projects conducted in the laboratory using DNA microarray technology concerns the formation of the notochord in Xenopus;the notochord is the defining structure of chordates, the phylum that includes both frogs and humans. The notochord has been known to contain vacuoles and to be surrounded by a sheath;both of these structures are required to give it the mechanical strength that is an important characteristic of this tissue. Both vacuole and sheath formation involves the secretion of proteins, and previous studies in several laboratories have shown that secreted proteins from different classes are required for notochord formation. In our DNA microarray studies we found that activation of the genes encoding secretory pathway components is a hallmark of notochord differentiation. The great majority of genes that are differentially expressed in the notochord as compared to the rest of the embryo belong to this functional class. We have shown that the coordinate activation of secretory pathway genes in the developing notochord involves the function of two transcription factors named XBP1 and Creb3L2. These factors themselves are preferentially expressed in the early notochord, and their activation in precursor cells is an important step in the specification of the notochord. These studies contribute to an understanding of the molecular basis of differentiation of one of the earliest-forming tissues characteristic of the vertebrate embryo. One of the factors that emerged from our screening efforts is vvp1. In a collaborative effort we found that the vvp1 gene is an excellent marker for a population of pancreatic cells that arise early in development and mark the ventral pancreatic precursors. Using this novel marker gene we have studied pancreas development in Xenopus embryos. The results show that the homeobox gene hhex has a major regulatory function in pancreas development. In tail bud embryos, vpp1 expression can be used to specifically label two ventral pancreatic buds,whereas hhex expression is primarily seen in the liver diverticulum. Ectopic over-expression of a carefully titrated level of hhex mRNA led to a substantial increase in the domain of vpp1-positive cells, which later led to increased development of the ventral pancreas. These giant ventral pancreata may arise by a re-specification of intestinal precursor cells to ventral pancreatic precursor cells. In using antisense morpholino oligonucleotides to study loss-of-function ohenotypes, the knockdown of hhex led to a reduction of the expression of the vpp1 gene and the specific interference with the development of the ventral pancreas. By using a regulated construct to express hhex at different stages of development we obtained evidence to suggest that hhex activity is required during gastrulation to mediate the specification of ventral pancreatic prcursor cells in teh Xenopus embryo. On the basis of these observations we suggest that hhex has an essential role in the formation of a vpp1-positive cell population in the endoderm of the gastrula embryo that is essential for the subsequent development of the ventral pancreas. 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 proteinn 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. All 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 separate placode and neural crest domains by preventing the neural crest 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. 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. 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, of cell cycle control, and for the cloning of animals and the reprogramming of somatic cells. A major limitation of the Xenopus system has been the lack of practical methods for gene disruption and genetic manipulation. While some techniques have been introduced in this area for some time, there remains a great need for improved robust and effective methodologies. 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 adaptation 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.
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