Several mouse genes containing the homeobox domain have been shown to exhibit precisely regulated patterns of expression in the embryo and in certain tissues of the adult, suggesting that these genes may be important in development and differentiation in mammals. We have recently demonstrated that abnormal development of the gut and enteric nervous system, resulting in a condition resembling congenital megacolon, is associated with overexpression of the homeobox-containing gene Hox-1.4 in transgenic mice. Preliminary morphological analysis indicates that the mice are hypoganglionic in the distal region of the colon and that the affected region exhibits other structural alterations involving abnormal innervation. We want to determine the cellular and molecular mechanisms responsible for this mutant phenotype in order to elucidate the potential functions of homeobox-containing genes in development of the mammalian gut. We will begin by comparing the morphology of neural elements, smooth muscle, and extracellular matrix of the normal and abnormal segments of the colon of three lines of the transgenic mice which exhibit varying degrees of severity of the megacolon phenotype. Both adults and fetuses will be examined using immunocytochemistry and electron microscopy. The results will be compared to the observations made on mice that are homozygous for the lethal spotted (ls) mutation and those heterozygous for Dominant megacolon (Dom). To begin to identify the cells responsible for the abnormal phenotype, we will determine if normal neural crest-derived cells can colonize the presumptive abnormal gut of the transgenic mice and can differentiate into neurons appropriate for the enteric nervous system in vitro, or conversely, to determine if transgenic crest-derived cells can colonize a segment of bowel from control animals. Finally, we will address the functional redundancy of murine Hox genes, particularly with respect to gut development, by producing transgenic mice carrying multiple copies of the Hox-2 paralogue of Hox-1.4, namely Hox-2.6, under the Hox-1.4 promoter. These studies will provide new insight into the regulation of expression and function of homeobox-containing genes during mammalian development, their potential role in regulating the complex pathways responsible for proper differentiation and innervation of the mammalian gut, and their potential role in the etiology of congenital megacolon.
