A Multipartite Approach to Mapping the Gene Network Directing Caenorhabditis elegans Vulval Organogenesis

Abstract

The complex interactions of signaling molecules, transcription factors, and target genes that direct the development of diverse organisms and their component parts are known as gene regulatory networks (GRNs). The Caenorhabditis elegans vulva, with its invariant lineage, 22 cells of seven types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), and availability of 30 reporter constructs exhibiting a variety of differentiated vulval cell expression patterns, provides us with an elegant system for studying a GRN directing organogenesis. I took several approaches for identifying additional aspects of the vulval gene network. First, I conducted a screen in a zmp-1::GFP (zinc metalloproteinase) background by disrupting activity of 836 transcription factors by RNA interference (RNAi). zmp-1 is a readout for differentiation of the vulA, vulD, and vulE cells. This screen identified nhr-67 (ortholog of Drosophila tailless) and nhr-113 (orphan nuclear hormone receptor) as positive regulators of zmp-1 expression in the vulA cells. nhr-113 appears to have a narrow role in vulval organogenesis; perhaps only as a partial regulator of vulA cell differentiation. Furthering the analysis of zmp-1, I utilized a phylogenetic footprinting approach for analyzing the upstream region of C. elegans zmp-1, and its homologs in the related species C. briggsae and C. remanei. This analysis identified four conserved motifs upstream of the ZMP-1 translational start site. Characterization of these motifs, by deleting them from a zmp-1::GFP transgene, revealed that three of these four motifs exhibit cis-regulatory activity. In addition to investigation of zmp-1 regulation, we undertook a parallel study where we performed sequence alignment analysis on 17 genes with reported vulval expression in combination with in vivo testing. This identified nine ~ 200-bp vulva-specific enhancer elements associated with six of these genes. Yeast one-hybrid analysis of six of these enhancer elements isolated six protein-DNA interactions. Further characterization of these interactions uncovered the role of the zinc finger transcription factor, ZTF-16, in vulval induction and differentiation. These results show that by taking multiple approaches, including the use of post-genome technologies, we can expand our understanding of a gene regulatory network.