Positive and negative regulation of pattern formation during Xenopus embryogenesis

Dynamic spatiotemporal expression of the nodal gene and its orthologs is involved in the dose-dependent induction and patterning of mesendoderm during early vertebrate embryogenesis. In loss of function studies, a strong knockdown of Xenopus antivin/lefty ( Xatv/Xlefty) function was achieved by coinjecting translation- and splicing-blocking morpholino oligonucleotides that target both the XatvA and XatvB alloalleles. A greater expansion of the Organizer and mesendoderm tissues in my studies than noted in previous Xlefty/Xatv knockdown experiments in Xenopus, with long-term maintenance of expanded axial tissues strongly suggests that the regulation of Xenopus nodal-related (Xnr) signaling by Xlefty/Xatv is essential for proper cell layer specification during early embryogenesis, and tissue patterning at late stages. Inhibitors specific to Xnr signaling were used to provide evidence that Xnr-mediated induction was inherently long-range in the Xlefty/Xatv-deficient embryo, essentially being capable of spreading over the entire animal hemisphere. While the expansion of Xnr1 and Xnr2 expression is limited to the marginal zone by Xlefty/Xatv deficiency, inhibition of Xbra function using Xbra-EnR in Xlefty/Xatv-deficient embryos caused a much larger increase in the level and spatial extent of Xnr expression. However, Xnr2 expression was constrained to the superficial cell layer in any experimental conditions, suggesting a fundamental tissue-specific competence to express Xnrs. These studies reveal a two-level suppressive mechanism for restricting the strength, range and duration of Xnr signaling via both Xlefty/Xatv-mediated extracellular inhibition and Xbra-mediated indirect transcriptional repression. Therefore, the tight regulation of Xnr signaling and expression by multiple influences is essential for precisely refining cell layer specification and patterning during gastrulation.;Conserved Nodal/Lefty/Pitx2 cassette in left LPM has been implicated in left-right (L-R) axis patterning during post-gastrulation and later asymmetric morphogenesis. Detailed genetic pathways for these processes, however, are still elusive because of lack of focused studies on L-R development. Under this rationale, I tried to identify new genes involved in L-R pathways using several screening methods such as PCR-based subtractive cDNA screening and microarrays. Though any candidates were not isolated in these studies, results suggest that an unbiased and rapid screening with Affymetrix GeneChips RTM seems to be an appropriate method for isolating new molecules involved in L-R patterning because of its reliable detection of controls, such as Xnr1 and Xlefty/Xatv..