Caenorhabditis elegans male tail is decorated by nine pairs of sensory rays. Each ray consists of three cellular processes, one from the structural cell and two from neuronal cells, surrounded by a hypodermal sheath and the outermost cuticle. To identify the dramatis personae acting in ray development, a genome-wide RNAi screen was conducted to query 16,000 genes in C. elegans genome for their functions in ray development. The screen identified irx-1, an Iroquois homeodomain-containing transcription factor implicated in organogenesis in multiple species from invertebrates to vertebrates, to be required for ray development. irx-1(RNAi) animals display ray missing phenotype reminiscent of the ray assembly defects displayed by the mutants of two other transcription factors ceh-43 and mab-2...[ Read more ]

Caenorhabditis elegans male tail is decorated by nine pairs of sensory rays. Each ray consists of three cellular processes, one from the structural cell and two from neuronal cells, surrounded by a hypodermal sheath and the outermost cuticle. To identify the dramatis personae acting in ray development, a genome-wide RNAi screen was conducted to query 16,000 genes in C. elegans genome for their functions in ray development. The screen identified irx-1, an Iroquois homeodomain-containing transcription factor implicated in organogenesis in multiple species from invertebrates to vertebrates, to be required for ray development. irx-1(RNAi) animals display ray missing phenotype reminiscent of the ray assembly defects displayed by the mutants of two other transcription factors ceh-43 and mab-22. By tracing the developmental process throughout L4 stage, irx-1 was shown to be required for papillae formation whereby the structural cells and hypodermal cells attach to the cuticle. irx-1 transcription reporters revealed its expression in hypodermis and ray neuronal A cells. This expression profile suggests that it might control expression of junctional components. Since a comprehensive list of Iroquois target genes have not been defined in the literature, I aimed to identify irx-1 direct targets and to assess their functions in ray assembly. Phylogenetic footprinting was used to predict an i̲n s̲ilico d̲irect r̲e̲g̲u̲l̲o̲m̲e̲ (isDiregulome) of irx-1. A combination of in silico analysis and experimental biology was employed to select relevant genes: (1) Intersection of the potential target genes with ray phenome to identify irx-1 target genes from ray developmental gene pool; (2) paralogous pairing followed by co-expression profiling to identify potential functionally redundant paralogs; and (3) integration of different data sets (e.g., phenome, interactome, expression) to identify functional gene modules regulated by irx-1 and to generate hypothesis about their genetic interactions. Several potential irx-1 target genes were identified, the spectrum spanning transcription factors, cytoskeleton/membrane/matrix components and modifying enzymes/scaffold/structural components. While irx-1 regulation on these target genes has yet to be confirmed, characterization of the expression patterns of the targets revealed a significant number of targets with hypodermal expression overlapping that of irx-1.

The integration of computational techniques and experimental biology has allowed efficient selection of potential transcription factor target genes and has a broad application in studying transcription factors targets in ray development. The workflow proposed in this study could be adopted to ultimately delineate the gene regulatory networks in ray development.