Valuable insight into normal and abnormal embryonic development may be gained from the study of genes and gene products that control these processes. Current knowledge on the structure, evolution and expression of mammalian homeobox genes identifies these as excellent candidates for loci that regulate mammalian development. We propose experiments that explore the regulation of the homeobox genes Hox 1.3 and Hox 5.1 and the function of Hox 1.3. 1. The cis-acting elements responsible for the spatial expression of Hox 1.3/lacZ and Hox 5.1/lacZ gene fusions in the central nervous system of transgenic mice will be defined. The cis-acting elements will be used as affinity tools in order to characterize the trans-acting factors involved. 2. Previous results suggest that the developing spinal cord may be subdivided into discrete rostrocaudal regions based on the activity of particular Hox genes. Therefore the brachial and cervical CNS cells from Hox 1.3/lacZ and Hox 5.1/lacZ mice will be purified using a protocol based on fluorescence activated cell sorting of lacZ+ cells. It will then be determined whether the brachial and cervical CNS domain express different combinations of active genes, including Hox genes. This analysis will be done with purified cells using immunoblots, RNase mapping and subtractive cDNA cloning. Genetic ablation of brachial cells will also be performed in transgenic mice expressing a Hox 1.3/diphtheria toxin A fusion gene. The resulting phenotype will be described. 3. Transgenic mice that express ectopically the Hox 1.3 coding region under the control of the ubiquitous GRP70 promoter or the specific Hox 5.1 promoter will be produced and analyzed with respect to several molecular and morphological parameters. 4. Vectors that allow for enrichment of ES cells containing a Hox 1.3 gene activated by homologous recombination will be used to isolate such cells. Chimeric mice will be produced from these ES cells and bred to generate mice heterozygous and homozygous for the inactivated Hox 1.3 gene.
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