The primary goal of this project is to understand the genetic differences that underlie morphological variation between mammalian species. Dr. Behringer hypothesizes that many of these genetic differences reside in DNA sequences that regulate the transcription of developmental control genes. He has chosen to focus upon the classic developmental biology paradigm of vertebrate limb development. He proposes to make functional comparisons between mouse and bat limb morphogenesis because compared to mouse, bats have highly modified limbs. The developmental control gene that he proposes to study, Prx1, encodes a paired homeodomain transcription factor that is required in mice for limb and craniofacial development. He has localized a discreet <600 bp DNA segment encoding a transcriptional enhancer that directs limb expression in a Prx1-specific pattern during mouse development. He has isolated the Prx1 orthologue from bat (Carollia perspicillata) libraries that we have generated. He is characterizing the expression pattern of this gene in bat embryos by whole-mount and section RNA in situ hybridization. He has identified the putative limb enhancer of the bat Prx1 gene by virtue of its conserved DNA sequence and genomic position relative to mouse and human orthologues. He will test the activity of this putative limb enhancer using a lacZ reporter assay in transgenic mice. He will then replace the endogenous limb enhancer of the mouse Prx1 gene with the bat Prx1 limb enhancer by gene targeting in mouse embryonic stem (ES) cells. Thus, mice will be generated where the mouse Prx1 gene is expressed under the control of the bat Prx1 limb enhancer. He will assess the morphological consequences of this regulatory element alteration in the limbs. He proposes that this type of highly focused in vivo test will provide important insights into the genetic basis of morphological divergence. In addition, his studies should further our understanding of how regulatory diversity can modulate organogenesis.
