DORSOVENTRAL PATTERNING OF THE MAMMALIAN CLOACA

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DESCRIPTION (provided by applicant): Approximately one out of 2500 gut malformations occur in the hindgut region. The most common birth defects of this region are persistent cloaca and imperforate anus, both of which result from improper closure between the urogenital sinus and anorectal sinus. During hindgut development the urogenital and anorectal sinuses are initially one organ, the cloaca. Subsequent development leads to septation of the cloaca by the urorectal septum and further morphogenesis produces the bladder, genital organs and rectum. It is known that the epithelium lining the mature bladder, genital organs and rectum are morphologically and functionally different. However, it is not known how the early specification of anorectal (dorsal cloaca) versus urogenital (ventral cloaca) cell fate takes place in the embryonic cloaca. We ask the question does epithelial differentiation require proper septation to occur and are there signaling centers for epithelial differentiation. In order to determine if septation is a prerequisite for differentiation of these two cell lineages, I will first investigate the timing of cloaca differentiation and the molecular mechanisms that initiate these early cell fates. To determine whether the embryonic cloaca is dorsoventrally polarized before septation, I will compare gene expression profiles using deep sequencing of the ventral and dorsal epithelial cells of the undivided embryonic cloaca and the recently divided cloaca in mice. Cloacae will be dissected from embryonic mice and divided into dorsal and ventral sections. The cells will then be sequenced to determine their genetic expression profile. Genes found to be dorsoventrally expressed will be mapped for their domains and boundaries of gene expression in the cloaca using in situ hybridization. To then determine whether cloacal septation is required for dorsoventral polarity, I will examine molecular markers of dorsal and ventral identity in mice with persistent cloaca. The genes identified during sequencing will be examined in Bmp7 mutant embryos using in situ hybridization. To determine if surrounding tissues influence cloacal epithelial differentiation, I will co-culture the embryonic cloaca with and without the notochord and allantois. Cultures will be assayed for morphological and molecular markers of dorsal and ventral fate. Identification of the initial molecular genetic steps in the induction of anorectal and urogenital tissues from a common pool of cloacal progenitor cells, will have significant clinical implications by uncovering which mechanisms are open to perturbation in children with urogenital and anorectal malformations. Additionally, knowing the genetic signals that direct progenitor cells to differentiate into either urothelial or anorectal issues will provide an essential foundation for tissue engineering.