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Theses Doctoral

The roles of T and Tbx6 during gastrulation and determination of left/right asymmetry

Concepcion, Daniel

T and Tbx6 belong to the T-box family of transcription factors. T homozygous null mutants lack posterior somites after the seventh pair, have no distinguishable notochord, have a convoluted neural tube and die at E.10.5. In this study we investigate the phenotype of a dominant negative allele of T, TWis. Like homozygous embryos carrying the null allele of T, TWis homozygous mutants have left/right asymmetry defects. We demonstrate that left/right specific genes Cer2, Nodal, and Gdf1 are not expressed in TWis mutants and that the Notch signaling pathway, a pathway necessary for expression of left/right specific genes, is severely perturbed. We find, through the use of confocal and scanning electron microscopy, that TWis homozygous mutants have severe node morphological defects. Molecular analysis shows that TWis mutants have altered expression of the genes Shh, Foxa2 and Gsc, that not only mark structures of the midline and node, but whose expression is essential for the formation and patterning of these structures.
Tbx6 homozygous null mutants have enlarged tail buds and ectopic neural tubes in place of posterior somites. Tbx6 homozygous null mutants also have irregular development of anterior somites and left/right asymmetry defects that lead to randomization of heart looping.
In this study we made use of a microarray to find transcriptional regulators that are potentially directly regulated by Tbx6 in order to find genes that govern the establishment of presomitic mesoderm identity. Tbx6 homozygous mutant embryos were used to investigate how Tbx6 affects multiple processes in the establishment of left-right asymmetry, which include node and node cilia development and the expression of genes necessary for nodal signal transduction from the perinodal region to the left lateral plate mesoderm (LPM). We saw that the expression of transcription factors involved in node and node cilia development and genes involved in the non-canonical Wnt signaling pathway are not affected in Tbx6 homozygous mutants. We also examined whether the left-right asymmetry defects in Tbx6 homozygous mutants are due to a role for Tbx6 to directly regulate the perinodal expression of Gdf1. We show that perinodal expression of Gdf1 is lost in Tbx6 homozygous mutants at the stage in which Gdf1 is hypothesized to help shuttle Nodal from the perinodal region to the left lateral plate mesoderm. We saw that Tbx6 does not regulate expression of Gdf1 through putative binding sites located in its promoter region and did not detect a genetic interaction between the two genes in determining left/right asymmetry. Finally we saw that addition of a transgene that expresses Gdf1 in Tbx6 homozygous mutants leads to rescue of asymmetric expression of Pitx2 in the left LPM.

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More About This Work

Academic Units
Genetics and Development
Thesis Advisors
Papaioannou, Virginia E.
Degree
Ph.D., Columbia University
Published Here
June 5, 2013
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