Establishment of spatially defined cell fate patterns is required for organogenesis. These events, in turn, depend on complex functional interconnections between regulatory genes and their targets, which should account for the morphological differences seen between species. Dissecting these gene regulatory networks entails characterizing the trans-acting factors that regulate gene expression and analyzing cis-regulatory sequences that respond to these diverse inputs. The C. elegans vulva invariantly consists of seven distinct cell types (vulA, vulB1, vulB2, vulC, vulD, vulE and vulF), each with its own unique gene expression profile. Diverse spatial and temporal cell fate markers, reverse genetics (RNAi), bioinformatics and the ease of manipulation at the single cell level make this a particularly attractive system for studying gene expression programs acting in organogenesis. By looking for sequence conservation in three Caenorhabditis species, we found vulval enhancers in over 15 genes. We then identified transcription factors that bind some of these elements through yeast one hybrid studies: for example, F53F8.1 protein, an ortholog of human WT1, interacts with the vulval enhancer of
sqv-4 (sugarless/UGDH). Also, Cistematic (see abstract by Schwarz/Mortazavi) successfully predicted four cis-regulatory motifs in
zmp-1, which were then tested by site-directed mutagenesis. Three of these motifs have demonstrable function: two positive, one negative. Meanwhile, using RNAi screens of over 500 C. elegans transcription factors, vulval specific factors were independently discovered by their effects on the expression of various vulval reporter gene constructs. For example,
nhr-67 (a TAILLESS ortholog) is required to inhibit the expression of
ceh-2 (EMS) and
egl-17 (FGF) in vulE and vulF cells. We find that some of these transcription factors interact with each other to specify different properties of the vulval cells. For example, both
cog-1 and
nhr-67 are mutually antagonistic and autoinhibitory in vulE and vulF cells. We also find that interconnections of these regulatory factors vary among the seven vulval cell types, which may partly account for how these cells acquire a precise pattern of fates. After integrating all the data from the above studies, we can now present a general overview of the regulatory network architecture for each of the seven vulval cell types.