CARDIAC DEVELOPMENT DURING SITUS INVERSUS EMBRYOGENESIS

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As part of the normal pattern of embryonic cardiac development, the heart establishes a left-right polarity through cardiac looping. The factor(s) that coordinate cardiac looping and thereby establish a left-sided heart in mammals have yet to be identified. One strategy to gain information about such factors is the generation and characterization of mutations that alter the patter of cardiac development. We have developed an efficient strategy to screen for insertional mutations in transgenic mice, and have recently identified a family with a recessive mutation that causes situs inversus. In this family, 100% of the homozygous transgenic mice have a mirror-image reversal of the left-right polarity of the heart as well as the other major internal organs, including lung, liver, spleen, pancreas and stomach. The new mutation is not allelic to the previously identified iv mutation in mice. Since the new mutation is an insertional mutation, the transgenic insert provides a molecular probe that can be used to isolate the genomic region where the inactivated gene is located. In order to clone the new situs inversus gene and the characterize its role in the regulation of cardiac/embryonic development, the specific aims of this proposal are to: 1) establish the chromosomal location and physical map of the new situs inversus mutation; 2) determine the structure and predicted product of this situs inversus gene and verify the function of the gene by targeted mutagenesis in ES cells; 3) define the pattern of expression of the situs inversus gene product, particularly during establishment of the left-right polarity of the heart, and characterize the intra- or extra-cellular localization of the encoded protein; and 4) investigate cardiac morphogenesis in chimeric and double mutant embryos. Since the mutated gene plays an essential role in defining the polarity of normal cardiac development, the identification of the encoded protein and the characterization of its pattern of expression during embryogenesis will provide valuable molecular information about a factor that coordinates cardiac morphogenesis.