This proposal is concerned with the two bHLH proteins, dHAND and eHAND, and their role in cardiac morphogenesis. The investigator was responsible for their cloning and showed that expression of eHAND is seen at 9.5dpc and dHAND at 8.5dpc in the embryonic heart. They are co-expressed in a variety of neural crest derived tissues and in the pharyngeal arches. eHAND is basically embryonic specific whilst dHAND is expressed at low levels in a number of adult tissues. In mice with a disruption of the endothelin-1 gene which exhibit heart malformations, the HAND genes were downregulated in the heart but not in the non-neural crest derived tissues.
The first aim of this proposal, based upon the previous studies of the investigator, involve the generation of null mutants and examination of the morphological consequences. Initially, an emphasis will be placed upon dHAND null mutants since eHAND expression occurs in the extraembryonic membranes and could lead to embryonic lethality.
The second aim will delineate the domains of dHAND and eHAND that are responsible for their binding to DNA and their further characterization to identify which amino acids are necessary for the interaction. Also, after identification of the binding site, promoters of genes expressed in the lateral mesoderm and neural crest will be examined as potential targets. The second part of this aim will attempt to define the transactivation domain of the genes using their known interaction with E12 which will be fused to the GAL4 DNA binding domain. Various truncated versions of the HAND genes will be tested for activation of a CAT reporter gene downstream of the GAL4 DNA binding sites. The final portion of this aim will determine which portions of the HAND proteins are necessary for protein-protein interactions using the CAT reporter system as for the transactivation experiments. The third and final aim will define the cis-acting sequences that regulate the expression of the HAND genes in tissue culture cells and transgenic mice.
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