Novel Targets and Functions for Xenopus pitx3 during Embryonic Development

Pitx3 is a homeodomain transcription factor with an expression pattern conserved across phyla: in the dopaminergic neurons of the midbrain, in the lens during all stages of lens development and in the forming somites. In Xenopus laevis, this gene shows novel expression areas, such as the pituitary gland, the heart and the gut. Morpholino-based knockdown of pitx3 results in phenotypes characterized by small or absent lens, bent dorsal axis and randomized coiling of the heart and gut. Comparing gene expression changes in wild-type versus knockdown embryos by microarray, we generated a vast list of genes possible downstream targets for pitx3. Confirming a number of those genes as affected by the absence of pitx3 allowed for positioning pitx3 in a variety of pathways. Given that a significant number of these genes were known as major players during somitogenesis, corroborated with the bent dorsal axis phenotype initiated the further discovery for the role of pitx3 in this developmental process. To determine direct targets for pitx3 we needed a reporter assay to test the protein-promoter interaction. Since the existing assays were deemed unsatisfactory in terms of accuracy and sensitivity we developed a new technique which permits precise detection of the reporter gene in a homogenous population of cells containing both the transcription factor and the reporter. This also enables the assessment of cooperativity for the tested transcription factors. Lastly, this new technique was employed to examine the promoters of some of the microarray candidate genes and to determine new direct targets for pitx3, thus redesigning existing pathways to incorporate the new interactions.