Cell-specific Gene Expression: Pylorus Morphogenesis and Hedgehog-regulated Enhancers.

Abstract

The precise spatiotemporal control of gene expression is integral to the survival of all organisms. Inappropriate gene expression can lead to developmental defects in newborns, such as Infantile Hypertrophic Pyloric Stenosis, in which hypertrophy of pyloric sphincter smooth muscle leads to gastric outlet obstruction. This thesis work analyzes the mechanisms and consequences of cell-specific gene expression in three systems: establishment of the epithelial gastro-duodenal (pyloric) border, development of smooth muscle structures at the pylorus, and transcriptional response to Hedgehog (Hh) signaling in Drosophila.

Microarray is used to characterize the antral, pyloric, and duodenal transcriptomes at embryonic days (E) 14.5 and 16.5. At E16.5, hundreds of genes are upregulated specifically in duodenal epithelium. This event is termed intestinalization because the activated genes are associated with intestinal function. Several transcription factors (i.e., Tcfec, Creb3l3, and Hnf4gamma) are upregulated in duodenal epithelium and levels of Hh signaling are downregulated in duodenal mesenchyme. In addition, novel pyloric genes are identified, including Gata3, which encodes a zinc finger transcription factor.

A role for Gata3 during pylorus development is elucidated using a genetic model of Gata3 insufficiency. Gata3 and the homeodomain transcription factor Nkx2-5 co-localize with molecular markers of pyloric smooth muscle and are expressed in novel bilateral smooth muscle structures at the pylorus (i.e., the ventral pyloric cords). Loss of Gata3 alters the shape of the pylorus and attenuates the pyloric constriction. The ventral pyloric cords and outer longitudinal smooth muscle at the pylorus are absent in Gata3 null embryos. Gata3 does not control Nkx2-5 expression at the pylorus.

An in silico approach identifies Hh-regulated enhancers in Drosophila. Binding sites for the Hh transcriptional effector cubitus interruptus (Ci) are significantly clustered in the genomes of two divergent Drosophila species, but mutant Ci sites are not. Putative Hh-regulated enhancers are identified by the comparison of orthologous regions of significant Ci clustering. Two of these enhancers (inv and rdx) are active in Hh-responsive cells of the Drosophila larval imaginal wing disc.

These studies reveal novel gene expression patterns during pylorus morphogenesis and suggest an approach to identifying direct transcriptional targets of signaling pathways.