Regulation of the homeobox gene Cdx2 and its downstream target Hoxc8 in embryonic patterning

This dissertation represents studies on homeobox gene regulation during mouse embryonic development. Homeobox genes are key determinants of positional information along the anteroposterior embryonic axis. One class of homeobox genes that is involved in the determination of posterior axial structures is the caudal family of genes. There are three members of the caudal family in mammals, namely Cdx1, 2 and 4. Loss of function mutations in Cdx1 and 2 genes results in several skeletal abnormalities including homeotic transformations. Our understanding of the regulation of Cdx genes is poor. As a first step towards understanding the regulation of Cdx genes, a cis-regulatory analysis of the Cdx2 gene was performed using reporter gene assay in transgenic mouse embryos. Critical regulatory elements that direct the expression of the lacZ reporter gene to the posterior neural tube and paraxial mesoderm of the developing embryos were identified. Elements present within the 10 Kb Cdx2 genomic region are sufficient to recapitulate temporal and spatial patterns of the endogenous Cdx2 in ectodermal and mesodermal tissues. Deletion analysis reveals that Cdx2 regulation is complex involving sets of positive and negative elements. Major elements responsible for embryonic neural tube expression are delimited to a 1 Kb fragment located approximately 2.5 Kb from the Cdx2 coding region. Surprisingly, a second enhancer activity was identified within the first intron. This enhancer directs robust lacZ staining to the posterior two-third of the developing embryo. This ectopic expression, where lacZ is directed to regions outside of endogenous Cdx2 domains, is remarkably silent when additional sequences within the 10 Kb genomic fragment are present. Although the significance of the intronic enhancer activity to the regulation of Cdx2 is not clear, a high degree of sequence conservation is observed with human orthologous sequences which suggests functional significance.;In addition to the studies on Cdx2 regulation, I also present studies on the cis-regulatory analysis of mouse Hoxc8. Hoxc8 is likely to be a downstream target of Cdx2 and is involved in specifying thoracic structures in the mouse. Consistent with this hypothesis, one of the regulatory regions of Hoxc8, termed Hoxc8 early enhancer contains two potential Cdx2 binding sites. The Hoxc8 early enhancer has been delimited to a 200 by fragment. Both comparative studies and mutation analysis identify nearly a dozen elements that control the early embryonic expression of Hoxc8. This well-characterized compact enhancer region shows remarkable plasticity among organisms representing diverse morphologies. Comparative approaches presented in this study support the hypothesis that changes occurring in the cis-regulatory region can contribute to changes in Hox gene expression leading to patterning diversity.