CARDIAC DEVELOPMENT DURING SITUS INVERSUS EMBRYOGENESIS

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Critical events in early development include the initiation of looping in the embryonic heart tube, and the concomitant establishment of left-right asymmetry. Although the molecular events that determine the directions of looping remain unidentified, a number of genes have recently been identified that exhibit left-right asymmetric expression prior to the onset of heart looping. However, these asymmetrically expressed genes do not appear to be responsible for the initial specification of left-right asymmetry. Developmental studies of transgenic mice that carry the inv (inversion of embryonic rotation) insertional mutation suggest that the inv gene is essential for the earliest stages of left-right axis specification. Homozygous inv embryos exhibit a consistent reversal of morphological and molecular left-right asymmetry. In order to begin to characterize the inv locus, the transgenic integration site was cloned and characterized. Mapping studies revealed that integration of the transgene was accompanied by a significant genomic deletion and an intrachromosomal duplication on mouse chromosome 4. Single-copy genomic sequences flanking the integration site was used to identify a YAC clone (GO571) that spanned the deleted region. This YAC was purified and used for microinjection to generate transgenic mice. Six founder mice that had integrated both arms of the YAC were identified. Breeding studies revealed that the G0571 YAC can cure the inv mutant phenotype. Therefore this YAC contains the coding and regulatory elements of the inv gene. In order to further characterize this gene and its role in establishing left-right polarity, the following specific aims are proposed: 1. To identify the coding sequences for the inv gene (by exon trapping and cDNA library screening). 2. To determine the embryonic patterns of expression of the candidate inv mRNA and protein. 3. To characterize changes in expression of asymmetrically expressed genes (Nodal, lefty, Snail, flectin and dHAND) in YAC-cured embryos, in chimeric embryos, and in double mutant (iv/iv;inv/inv) embryos. 4. To construct an inv minigene and to use the minigenes to identify and characterize the functional domains in the protein. These experiments should provide molecular details about the earliest steps in left-right axis specification and the control of cardiac morphogenesis.