The role of Tbx6 in the determination of left-right asymmetry in mice

Columbia University Health Sciences, New York NY

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Abstract

The purpose of this research is to understand the role Tbx6 plays in the determination of left/right body axis asymmetry of the mouse embryo. Mutations in genes that regulate body axis asymmetry lead to congenital cardiac malformations as well as other serious health issues related to organ situs. Tbx6 homozygous mutant embryos demonstrate abnormalities in heart looping, embryo turning and vitelline vessel placement at midgestation, all of which are key morphological aspects of left/right asymmetry leads to the hypothesis that Tbx6 plays an important role in the initial events that usher in the breaking of bilateral symmetry. Deviations from bilateral symmetry initially occur in the mouse embryonic node, a bundle of cells located at the anterior most region of the primitive streak. Cilia present in the node all beat in the same direction, creating a unidirectional flow of extra-cellular fluid. This asymmetric flow sets up a cascade of gene expression events, which leads to asymmetric organogenesis. Studies show morphological defects in the nodal cilia of Tbx6 mutant embryos even though Tbx6 is not expressed in the node at the time left/right asymmetry is established. The main hypothesis to be tested is that Tbx6 is involved in left-right asymmetry determination by regulating the formation of the node and nodal cilia and the transfer of asymmetric signals from the embryonic node to the left lateral plate mesoderm (LPM) by affecting different downstream target genes. A Tbx6 lineage tracer will be developed in order to investigate whether the effect of Tbx6 on the embryonic node is direct or indirect. In Situ hybridization will be used to examine the expression pattern of genes involved in the non-canonical Wnt signaling pathway and Nodal signal transduction pathway in Tbx6 mutants to determine whether Tbx6 regulates left-right determination by affecting these genes. Also immunofluorescence will be used to examine whether Tbx6 mutants have altered expression of proteins that are known to phenocopy the Tbx6 mutant cilia defects. Finally, using bacterial artificial chromosomes and molecular cloning a transgenicTbx6ERCre allele will be created in order to conduct lineage trace studies.