We are interested in understanding the genetic control of spatial form during the development of an organism. One class of genes that have been shown to be important in controlling pattern in developmental systems are those containing homeoboxes. The long term goal of this project is to focus on the development of a simple structure, the regenerating newt limb, to identify all of the homeobox containing genes involved in that process and to elucidate the role each plays in its regulation. such roles may include specifying the unique morphology of a particular limb (forelimb or hindlimb) or determining the basic limb axes (proximal/distal, dorsal/ventral and anterior/posterior). Several genes related to the drosophila Antp class of homeobox have already been cloned by low stringency hybridization. Two are specific to the forelimb, one to the hindlimb. Other classes of homeobox-containing genes will be isolated using a set of degenerate oligonucleotides. Other functionally related genes have been identified by a subtractive library screen. These cDNA clones will then be used to isolate genomic sequences. Both will be mapped. The clones will be further characterized by northern blots and in situ hybridization to determine their temporal and spatial expression patterns. Using different experimental manipulations, retinoic acid can be used to reprogram each of the regenerating limb axes. Expression patterns of the cloned genes will therefore be investigated after different retinoic acid treatments to test their possible roles in axis specification. Regenerating cells can be transferred to tissue culture, induced to take up DNA either by lipofection or utilizing VSV-pseudotyped retroviral vectors, and then reintroduced into the limb while retaining the capacity for participating in regeneration. This technology will be used to misexpress the homeobox cDNA clones in inappropriate spatial and temporal locations. Homologous recombination techniques will be developed to allow these genes to be inactivated as well. In vitro gene transfer followed by RNAse protection will test whether these genes trans-regulate each others expression. Attaching the promotor regions of genomic clones to marker genes such as lac Z (encoding beta-galactosidase) will enable the dissection of cis-acting elements that determine the genes specific expression patterns. Such patterns may in part be determined by lineage relationships. Lineages of regenerating clones will be investigated by infecting regenerating limbs with VSV-pseudotyped retroviral vectors carrying lac Z, and histologically staining for beta-galactosidase- containing clones at later stages.
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