HEX EXPRESSION AND REGULATION IN DEVELOPING LUNG

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The research goals of this proposal are to elucidate the molecular mechanisms involved in lung development. Homeobox genes, which encode proteins which function as sequence-specific DNA binding proteins, are expressed in specific temporo-spatial patterns in the developing mouse and have been shown to direct patterning along the anterior-posterior axis and in specifying regional identity. The divergent homeobox gene Hex is expressed in the developing lung but nothing is known about its temporo- spatial pattern of expression or its regulation. The specific aims of this project are: 1. to identify, in the developing mouse, the specific regions and cell types that express Hex and to determine how that pattern of expression changes with advancing gestational age; 2. to identify lung- specific enhancers for Hex by reporter gene analysis in transgenic mice; and 3. to characterize trans-acting transcription factors interacting with the lung-specific enhancer(s) of Hex. The methodology to be used to complete these specific aims includes: In situ hybridization, isolation and restriction mapping of genomic clones for Hex; identification of enhancer elements using reporter gene analysis in transgenic mice; characterization of lung-specific enhancer elements by deletional analysis, including the use of a novel shuttle vector to excise specific regions of PI clones containing Hex genomic sequence by homologous recombination; and characterization of trans-acting factors interacting with lung-specific enhancers by in vitro DNA-protein binding studies including identification of consensus binding sites for possible DNA binding proteins and examination of the specificity of DNA-protein binding by competition, mutational analysis and supershift assays using specific antibodies. The ability of any identified transcription factors to activate Hex lung enhancers will be tested by cotransfection in tissue culture cells. Ultimately, potential regulators of Hex will be ectopically expressed in transgenic mice to test the ability of these elements to regulate Hex lung expression in vivo. The laboratory research will be conducted by the principal investigator, Dr. Clifford W. Bogue, in conjunction with a didactic program under the guidance of two sponsors, Dr. Frank H. Ruddle and Dr. Harris C. Jacobs and an advisory committee that will meet regularly with the P.I. to offer guidance and to assess progress. This proposal will allow the candidate to further develop technical expertise in the areas of molecular genetics and developmental biology as well as enhancing his fundamental knowledge and refining his intellectual scientific approach to problems in these areas. The additional training provided in this proposal will prepare the candidate for an independent and productive career in the biology of lung development.