This grant application describes a new experimental approach to the analysis of axis formation in vertebrates. Anterior-posterior axis formation is governed by the Hox genes that are organized into four clusters in mammals. Each cluster contains subsets of thirteen primordial cognate genes organized within a span of approximately 100 kb. The clusters are estimated to have existed over a period of at least 500 million years, suggesting an intimate and biological adaptive interplay among the clustered genes. The Ruddle laboratory has studied the role of the Hoxc8 gene intensively with respect to its contributions to axis formation in the brachial and thoracic regions of the developing mouse embryo from molecular, genetic, developmental, and evolutionary points of view. The PI has identified enhancers that regulate the transcriptional activity of Hoxc8 and regulate its expression temporally, control its spatial distribution on the A/P axis, and target its expression in spedtic organ rudiments. One of these enhancers, the early enhancer, has been analysed in considerable detail. It is highly conserved in amniotes, extends over a 200 bp domain, and contains at least ten transcription factor binding motifs. The PI has assembled evidence for the identities of the transcription factors interacting with this regulatory unit and have shown that the factors interact cooperatively in a manner similar to that of an enhanceosome. Much of this work has been accomplished using normal and mutated forms of the early enhancer in reporter constructs that are introduced into the early embryo by transgenesis. While highly informative, reporter experiments do not provide direct knowledge of enhancer behavior in the context of the intact gene cluster. The PI now seeks to specifically modify the endogenous enhancer element by homologous recombination using embryonal stem cell methodologies. In this way, he expects to obtain unambiguous information on the influence of individual enhancer motifs on developmental patterning, the effect of the early enhancer on cis regulation of genes throughout the Hoxc cluster, and the interplay between the early enhancer and other enhancer elements in the Hoxc8 region of the C cluster. The PI believes that this new line of analysis will greatly deepen understanding of the means by which the Hox genes regulate pattern formation.
