We used mRNA tagging to identify genes expressed in the intestine of C. elegans. Animals expressing an epitope-tagged protein that binds the poly-A tail of mRNAs (FLAG::PAB-1) from an intestine-specific promoter (ges-1) were used to immunoprecipitate FLAG::PAB-1/mRNA complexes from the intestine. A total of 1938 intestine-expressed genes (P<0.001) were identified using DNA microarrays. First, we compared the intestine-expressed genes with those expressed in the muscle and germline, and identified 510 genes enriched in all three tissues and 624 intestine-, 230 muscle-and 1135 germ line-enriched genes. Second, we showed that the 1938 intestine-expressed genes were physically clustered on the chromosomes, suggesting that the order of genes in the genome is influenced by the effect of chromatin domains on gene expression. Furthermore, the commonly expressed genes showed more chromosomal clustering than the tissue-enriched genes, suggesting that chromatin domains may influence housekeeping genes more than tissue-specific genes. Third, in order to gain further insight into the regulation of intestinal gene expression, we searched for regulatory motifs. This analysis found that the promoters of the intestine genes were enriched for the GATA transcription factor consensus binding sequence. We experimentally verified these results by showing that the GATA motif is required in cis and that GATA transcription factors are required in trans for expression of these intestinal genes.KEY WORDS: C. elegans, Intestine, Gene expression, GATA transcriptional regulation, Chromosomal clustering Development 133, 287-295 doi:10.1242 DEVELOPMENT 288The organogenesis of the C. elegans intestine has been detailed at the cellular level (Leung et al., 1999). It includes cytoplasmic polarization of cells in the intestinal primordium, intercalation of specific sets of cells, generation of an extracellular cavity within the primordium, and adherens junction formation. The adherens junctions present an ideal model with which to investigate epithelial cell polarity and several proteins involved in the process have been identified such as PAR-3, PAR-6, PKC-3, SMA-1, ERM-1, LET-413, DLG-1, AJM-1 and others (Knust and Bossinger, 2002). A molecular profile of the intestine would help to identify more genes involved in cell polarity and its development.By generating a profile of gene expression in the C. elegans intestine, we have identified the molecules that define intestinal function. The list of intestine-expressed genes includes genes of known and unknown function. The genes with known functions provide insight into mechanisms and pathways used in diverse intestinal functions, such as epithelial cell polarity, digestion, and resistance to pathogens and toxicity. The intestinal expression of genes with previously unknown function implies a role in intestinal processes.A genome-wide profile of intestinal gene expression can also be used to elucidate the regulatory networks that maintain intestinal differentiation. We have defined intestine-speci...

Although many cell fates differ between males and females, probably the most ancient type of sexual dimorphism is the decision of germ cells to develop as sperm or as oocytes. Genetic analyses of Caenorhabditis elegans suggest that fog-3 might directly control this decision. We used transformation rescue to clone the fog-3 gene and show that it produces a single major transcript of approximately 1150 nucleotides. This transcript is predicted to encode a protein of 263 amino acids. One mutation causes a frame shift at the sixth codon and is thus likely to define the null phenotype of fog-3. Although the carboxyl-terminus of FOG-3 is novel, the amino-terminal domain is similar to that of the Tob, BTG1, and BTG2 proteins from vertebrates, which might suppress proliferation or promote differentiation. This domain is essential for FOG-3 activity, since six of eight missense mutations map to this region. Furthermore, this domain of BTG1 and BTG2 interacts with a transcriptional regulatory complex that has been conserved in all eukaryotes. Thus, one possibility is that FOG-3 controls transcription of genes required for germ cells to initiate spermatogenesis rather than oogenesis. This model implies that FOG-3 is required throughout an animal's life for germ cells to initiate spermatogenesis. We used RNA-mediated interference to demonstrate that fog-3 is indeed required continuously, which is consistent with this model.

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In C. elegans, the zinc-finger protein TRA-1A is thought to be the final arbiter of somatic sexual identity. We show that fog-3, which is required for germ cells to become sperm rather than oocytes, is a target of TRA-1A. First, northern analyses and RT-PCR experiments indicate that expression of fog-3 is controlled by tra-1. Second, studies of double mutants show that this control could be direct. Third, the fog-3 promoter contains multiple sites that bind TRA-1A in gel shift assays, and mutations in these sites alter activity of fog-3 in vivo. These results establish fog-3 as one of the first known targets of transcriptional regulation by TRA-1A. Furthermore, they show that tra-1 controls a terminal regulator of sexual fate in germ cells, just as it is thought to do in the soma.

Rapid changes in sexual traits are ubiquitous in evolution. To analyze this phenomenon, we are studying species of the genus Caenorhabditis. These animals use one of two different mating systems—male/hermaphroditic, like the model organism Caenorhabditis elegans, or male/female, like C. remanei. Since hermaphrodites are essentially females that produce sperm for self-fertilization, elucidating the control of cell fate in the germ line in each species could provide the key to understanding how these mating systems evolved. In C. elegans, FOG-3 is required to specify that germ cells become sperm. Thus, we cloned its homologs from both C. remanei and C. briggsae. Each species produces a single homolog of FOG-3, and RNA-mediated interference indicates that FOG-3 functions in each species to specify that germ cells develop as sperm rather than as oocytes. What factors account for the different mating systems? Northern analyses and RT-PCR data reveal that the expression of fog-3 is always correlated with spermatogenesis. Since the promoters for all three fog-3 genes contain binding sites for the transcription factor TRA-1A and are capable of driving expression of fog-3 in C. elegans hermaphrodites, we propose that alterations in the upstream sex-determination pathway, perhaps acting through TRA-1A, allow spermatogenesis in C. elegans and C. briggsae XX larvae but not in C. remanei.