An important goal of developmental biology is the elucidation of the genetic programs that control organogenesis. Little is known about the genetic programs required for mesenchymal interactions with epithelial or neuronal cells in development, particularly as they relate to the formation of organs of the digestive system. Recent studies on Hlx, a gene encoding a homeobox transcription factor, suggest that this gene plays an important role in development and growth of the digestive system. Hlx is expressed in mesenchymal cells of the developing mouse gastrointestinal system. Targeted inactivation of this gene resulted in an embryonic lethal phenotype in which the intestine and liver are present but do not grow. The overarching hypothesis of this proposal is that the specific expression of Hlx protein in mesenchyme results in regulation of the development of the intestine and liver by regulating the expression of genes encoding proteins participating in interactions of mesenchyme with other tissues. In order to determine the role of Hlx in development of the intestine and liver, and to help define the genetic program controlling formation of these organs, three specific aims are proposed. First, it is proposed to identify cis-regulatory elements and trans-activating factors regulating Hlx expression in developing mesenchyme. Segments of the Hlx 5' flanking region which regulate reporter gene expression in cell lines in a manner expected for that of the Hlx promoter have already been identified. It is proposed to evaluate these regions further using reporter constructs (in cell lines and transgenic mice) and biochemical methods. The second specific aim is to test several candidate downstream target genes by comparing their expression in wild-type and Hlx knockout embryos. In the third specific aim, we propose to identify genes regulated by Hlx using Hlx-/-mesenchymal cell lines in which Hlx is present and absent. Hlx will be added to these cells using an inducible expression system or using a retroviral vector. Genes differentially expressed will be identified using microarray techniques. The principal investigator is a pediatric gastroenterologist with significant prior research experience (including a Ph.D.), who is developing a research program in the molecular developmental biology of the digestive system. A Research Career Award will provide the opportunity for mentored development of this program in a rigorous scientific environment. This program will allow additional training in molecular and cellular techniques, development of supervisory skills, and further instruction in the responsible conduct of research.
