[Show abstract][Hide abstract] ABSTRACT: The GATA-type transcription factor ELT-2 is the major regulator of genes involved in differentiation, maintenance and function of the C. elegans intestine from the early embryo to the mature adult. The elt-2 gene responds to over-expression of the two GATA transcription factors END-1 and END-3 that specify the intestine, as well as to over-expression of the two GATA factors that are normally involved in intestinal differentiation, ELT-7 and ELT-2 itself. However, little is known about the molecular mechanisms that underlie these interactions, the general mechanisms by which ELT-2 levels are maintained throughout development, or how such systems respond to developmental perturbations. Here, we analyze regulation of the elt-2 gene through transgenic reporter assays, ELT-2 chromatin-immunoprecipitation and characterization of in vivo DNA-protein interactions. Our results lead to a model in which the elt-2 gene is controlled by three discrete regulatory regions conserved between C. elegans and C. briggsae and spanning >4 kb of 5'-flanking sequence. Although superficially the three regions are interchangeable, they have quantitatively different enhancer properties, and their combined activities indicate inter-region synergies. The regulatory activity of each region is mediated by a small number of conserved TGATAA sites that are also largely interchangeable and interact with different endodermal GATA factors with only modest differences in affinity. The redundant molecular mechanism that forms the elt-2 regulatory network is robust and flexible, as demonstrated by the fact that loss of the end-3 gene lowers ELT-2 levels by two-fold in the early embryo but ELT-2 returns to wildtype levels by hatching, several hours later. Finally, we report that when ELT-2 is expressed under the control of end-1 regulatory elements in addition to its own endogenous promoter, ELT-2 is able to replace the complete set of endoderm-specific GATA factors: END-1, END-3, ELT-7 (as well as the probably non-functional ELT-4). Thus, in addition to controlling gene expression during differentiation, ELT-2 is also capable of specifying the entire C. elegans endoderm.
[Show abstract][Hide abstract] ABSTRACT: Germ cells in animals are highly specialized to preserve the genome. A distinct set of chromatin structures must be properly established in germ cells to maintain cell fate and genome integrity. We describe DNA-surface interactions in activated Caenorhabditis elegans oocytes that are revealed through the activity of an endogenous nuclease ('endocleavage').
Our analysis began with an unexpected observation that a majority (>50%) of DNA from ovulated but unfertilized C. elegans oocytes can be recovered in fragments of approximately 500 base pairs or shorter, cleaved at regular intervals (10 to 11 nt) along the DNA helix. In some areas of the genome, DNA cleavage patterns in these endoreduplicated oocytes appear consistent from cell-to-cell, indicating coherent rotational positioning of the DNA in chromatin. Particularly striking in this analysis are arrays of sensitive sites with a periodicity of approximately 10 bp that persist for several hundred base pairs of genomic DNA, longer than a single nucleosome core. Genomic regions with a strong bias toward a 10-nt periodic occurrence of A(n)/T(n) (so-called PATC regions) appear to exhibit a high degree of rotational constraint in endocleavage phasing, with a strong tendency for the periodic A(n)/T(n) sites to remain on the face of the helix protected from nuclease digestion.
The present analysis provides evidence for an unusual structure in C. elegans oocytes in which genomic DNA and associated protein structures are coherently linked.
Preview · Article · Nov 2013 · Epigenetics & Chromatin
[Show abstract][Hide abstract] ABSTRACT: The transcriptional regulatory hierarchy that controls development of the Caenorhabditis elegans endoderm begins with the maternally provided SKN-1 transcription factor, which determines the fate of the EMS blastomere of the four-cell embryo. EMS divides to produce the posterior E blastomere (the clonal progenitor of the intestine) and the anterior MS blastomere, a major contributor to mesoderm. This segregation of lineage fates is controlled by an intercellular signal from the neighboring P2 blastomere and centers on the HMG protein POP-1. POP-1 would normally repress the endoderm program in both E and MS but two consequences of the P2-to-EMS signal are that POP-1 is exported from the E-cell nucleus and the remaining POP-1 is converted to an endoderm activator by complexing with SYS-1, a highly diverged β-catenin. In the single E cell, a pair of genes encoding small redundant GATA-type transcription factors, END-1 and END-3, are transcribed under the combined control of SKN-1, the POP-1/SYS-1 complex, as well as the redundant pair of MED-1/2 GATA factors, themselves direct zygotic targets of SKN-1 in the EMS cell. With the expression of END-1/END-3, the endoderm is specified. END-1 and END-3 then activate transcription of a further set of GATA-type transcription factors that drive intestine differentiation and function. One of these factors, ELT-2, appears predominant; a second factor, ELT-7, is partially redundant with ELT-2. The mature intestine expresses several thousand genes, apparently all controlled, at least in part, by cis-acting GATA-type motifs. WIREs Dev Biol 2013, 2:347–367. doi: 10.1002/wdev.93
For further resources related to this article, please visit the WIREs website.
No preview · Article · May 2013 · Wiley Interdisciplinary Reviews: Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: I wish to thank the editors of Mechanisms of Aging and Development for the opportunity to respond to this second letter from Drs. S. Kim, Y. Budovskaya and T. Johnson (hereafter referred to as KBJ#2, both for convenience and to distinguish it from KBJ, their previous letter (Kim et al., 2012)). As I will describe below, any issues raised by KBJ#2 are minor and should not distract us from the fact that they appear unable to replicate their published results, specifically those shown in Fig. 1c of Budovskaya et al. (2008), as were we (McGhee, 2012; Tonsaker et al., 2012). The following brief comments should help to keep even the minor issues raised by KBJ#2 in the correct perspective.
No preview · Article · Dec 2012 · Mechanisms of ageing and development
[Show abstract][Hide abstract] ABSTRACT: Budovskaya et al. (Cell. 134, 291-303, 2008) have proposed that the ELT-3 GATA factor regulates somatic aging genes, including those expressed in the intestine, and participates in a transcription factor circuit that "guides Caenorhabditis elegans aging". We have re-investigated two key features of this proposal: (i) expression of elt-3 in the C. elegans adult intestine where the majority of somatic aging genes are expressed, and; (ii) the ability of elt-3 loss-of-function to revert the extended lifespan of daf-2(e1370) mutants. We find that: (i) in agreement with our previously published results, ELT-3 expression is largely hypodermal and is not expressed at significant levels in the adult C. elegans intestine, and; (ii) the elt-3(vp1) zinc-finger deletion mutant does not significantly influence the extended lifespan of daf-2(e1370) mutants. We thus suggest that the role of ELT-3 in C. elegans aging should be re-evaluated.
No preview · Article · Jan 2012 · Mechanisms of ageing and development
[Show abstract][Hide abstract] ABSTRACT: Erlins are highly conserved proteins associated with lipid rafts within the endoplasmic reticulum (ER). Biochemical studies in mammalian cell lines have shown that erlins are required for ER associated protein degradation (ERAD) of activated inositol-1,4,5-trisphosphate receptors (IP3Rs), implying that erlin proteins might negatively regulate IP3R signalling. In humans, loss of erlin function appears to cause progressive intellectual disability, motor dysfunction and joint contractures. However, it is unknown if defects in IP3R ERAD are the underlying cause of this disease phenotype, whether ERAD of activated IP3Rs is the only function of erlin proteins, and what role ERAD plays in regulating IP3R-dependent processes in the context of an intact animal or embryo. In this study, we characterize the erlin homologue of the nematode Caenorhabditis elegans and examine erlin function in vivo. We specifically set out to test whether C. elegans erlin modulates IP3R-dependent processes, such as egg laying, embryonic development and defecation rates. We also explore the possibility that erlin might play a more general role in the ERAD pathway of C. elegans.
We first show that the C. elegans erlin homologue, ERL-1, is highly similar to mammalian erlins with respect to amino acid sequence, domain structure, biochemical properties and subcellular location. ERL-1 is present throughout the C. elegans embryo; in adult worms, ERL-1 appears restricted to the germline. The expression pattern of ERL-1 thus only partially overlaps with that of ITR-1, eliminating the possibility of ERL-1 being a ubiquitous and necessary regulator of ITR-1. We show that loss of ERL-1 does not affect overall phenotype, or alter brood size, embryonic development or defecation cycle length in either wild type or sensitized itr-1 mutant animals. Moreover we show that ERL-1 deficient worms respond normally to ER stress conditions, suggesting that ERL-1 is not an essential component of the general ERAD pathway.
Although loss of erlin function apparently causes a strong phenotype in humans, no such effect is seen in C. elegans. C. elegans erlin does not appear to be a ubiquitous major modulator of IP3 receptor activity nor does erlin appear to play a major role in ERAD.
[Show abstract][Hide abstract] ABSTRACT: Oocytes were purified from the temperature-sensitive fertilization-defective fer-1(b232ts) mutant of the nematode Caenorhabditis elegans and used for comprehensive mass spectrometric analysis. Using stringent criteria, 1165 C. elegans proteins were identified; at lower stringency, an additional 288 proteins were identified. We validate the high degree of sample purity and evaluate several possible sources of bias in the proteomic data. We compare the classes of proteins identified in the current oocyte proteome with protein classes identified in our previously determined oocyte transcriptome. The oocyte proteome appears enriched in proteins likely to be needed immediately upon fertilization, whereas the transcriptome appears enriched in molecules and processes needed later in embryogenesis. The current study provides fundamental background information for future more detailed studies of oocyte biology.
No preview · Article · Apr 2011 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: The C. elegans digestive tract (pharynx, intestine, and rectum) contains only approximately 100 cells but develops under the control of the same types of transcription factors (e.g. FoxA and GATA factors) that control digestive tract development in far more complex animals. The GATA-factor dominated core regulatory hierarchy directing development of the homogenous clonal intestine from oocyte to mature organ is now known with some degree of certainty, setting the stage for more biochemical experiments to understand developmental mechanisms. The FoxA-factor dominated development of the pharynx (and rectum) is less well understood but is beginning to reveal how transcription factor combinations produce unique cell types within organs.
Preview · Article · Aug 2010 · Current opinion in genetics & development
[Show abstract][Hide abstract] ABSTRACT: We review recent studies that have advanced our understanding of the molecular mechanisms regulating transcription in the nematode C. elegans. Topics covered include: (i) general properties of C. elegans promoters; (ii) transcription factors and transcription factor combinations involved in cell fate specification and cell differentiation; (iii) new roles for general transcription factors; (iv) nucleosome positioning in C. elegans "chromatin"; and (v) some characteristics of histone variants and histone modifications and their possible roles in controlling C. elegans transcription.
Preview · Article · May 2010 · Developmental Dynamics
[Show abstract][Hide abstract] ABSTRACT: Starting with SAGE-libraries prepared from C. elegans FAC-sorted embryonic intestine cells (8E-16E cell stage), from total embryos and from purified oocytes, and taking advantage of the NextDB in situ hybridization data base, we define sets of genes highly expressed from the zygotic genome, and expressed either exclusively or preferentially in the embryonic intestine or in the intestine of newly hatched larvae; we had previously defined a similarly expressed set of genes from the adult intestine. We show that an extended TGATAA-like sequence is essentially the only candidate for a cis-acting regulatory motif common to intestine genes expressed at all stages. This sequence is a strong ELT-2 binding site and matches the sequence of GATA-like sites found to be important for the expression of every intestinal gene so far analyzed experimentally. We show that the majority of these three sets of highly expressed intestinal-specific/intestinal-enriched genes respond strongly to ectopic expression of ELT-2 within the embryo. By flow-sorting elt-2(null) larvae from elt-2(+) larvae and then preparing Solexa/Illumina-SAGE libraries, we show that the majority of these genes also respond strongly to loss-of-function of ELT-2. To test the consequences of loss of other transcription factors identified in the embryonic intestine, we develop a strain of worms that is RNAi-sensitive only in the intestine; however, we are unable (with one possible exception) to identify any other transcription factor whose intestinal loss-of-function causes a phenotype of comparable severity to the phenotype caused by loss of ELT-2. Overall, our results support a model in which ELT-2 is the predominant transcription factor in the post-specification C. elegans intestine and participates directly in the transcriptional regulation of the majority (>80%) of intestinal genes. We present evidence that ELT-2 plays a central role in most aspects of C. elegans intestinal physiology: establishing the structure of the enterocyte, regulating enzymes and transporters involved in digestion and nutrition, responding to environmental toxins and pathogenic infections, and regulating the downstream intestinal components of the daf-2/daf-16 pathway influencing aging and longevity.
Full-text · Article · Dec 2008 · Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: The med-1 and med-2 genes encode small, highly similar proteins related to GATA-type transcription factors and have been proposed as necessary for specification of both the mesoderm and the endoderm of Caenorhabditis elegans. However, we have previously presented evidence that neither maternal nor zygotic expression of the med-1/2 genes is necessary to specify the C. elegans endoderm. Contradicting our conclusions, a recent report presented evidence, based on presumed transgene-induced cosuppression, that the med-1/2 genes do indeed show an endoderm-specifying maternal effect. In this article, we reinvestigate med-2(-); med-1(-) embryos using a med-2- specific null allele instead of the chromosomal deficiences used previously and confirm our previous results: the large majority (approximately 84%) of med-2(-); med-1(-) embryos express gut granules. We also reinvestigate the possibility of a maternal med-1/2 effect by direct injection of med dsRNA into sensitized (med-deficient) hermaphrodites using the standard protocol known to be effective in ablating maternal transcripts, but again find no evidence for any significant maternal med-1/2 effect. We do, however, show that expression of gut granules in med-1/2-deficient embryos is exquisitely sensitive to RNAi against the vacuolar ATPase-encoding unc-32 gene [present on the same multicopy med-1(+)-containing transgenic balancer used in support of the maternal med-1/2 effect]. We thus suggest that the experimental evidence for a maternal med-1/2 effect should be reexamined and may instead reflect cosuppression caused by multiple transgenic unc-32 sequences, not med sequences.
[Show abstract][Hide abstract] ABSTRACT: A SAGE library was prepared from hand-dissected intestines from adult Caenorhabditis elegans, allowing the identification of >4000 intestinally-expressed genes; this gene inventory provides fundamental information for understanding intestine function, structure and development. Intestinally-expressed genes fall into two broad classes: widely-expressed "housekeeping" genes and genes that are either intestine-specific or significantly intestine-enriched. Within this latter class of genes, we identified a subset of highly-expressed highly-validated genes that are expressed either exclusively or primarily in the intestine. Over half of the encoded proteins are candidates for secretion into the intestinal lumen to hydrolyze the bacterial food (e.g. lysozymes, amoebapores, lipases and especially proteases). The promoters of this subset of intestine-specific/intestine-enriched genes were analyzed computationally, using both a word-counting method (RSAT oligo-analysis) and a method based on Gibbs sampling (MotifSampler). Both methods returned the same over-represented site, namely an extended GATA-related sequence of the general form AHTGATAARR, which agrees with experimentally determined cis-acting control sequences found in intestine genes over the past 20 years. All promoters in the subset contain such a site, compared to <5% for control promoters; moreover, our analysis suggests that the majority (perhaps all) of genes expressed exclusively or primarily in the worm intestine are likely to contain such a site in their promoters. There are three zinc-finger GATA-type factors that are candidates to bind this extended GATA site in the differentiating C. elegans intestine: ELT-2, ELT-4 and ELT-7. All evidence points to ELT-2 being the most important of the three. We show that worms in which both the elt-4 and the elt-7 genes have been deleted from the genome are essentially wildtype, demonstrating that ELT-2 provides all essential GATA-factor functions in the intestine. The SAGE analysis also identifies more than a hundred other transcription factors in the adult intestine but few show an RNAi-induced loss-of-function phenotype and none (other than ELT-2) show a phenotype primarily in the intestine. We thus propose a simple model in which the ELT-2 GATA factor directly participates in the transcription of all intestine-specific/intestine-enriched genes, from the early embryo through to the dying adult. Other intestinal transcription factors would thus modulate the action of ELT-2, depending on the worm's nutritional and physiological needs.
Full-text · Article · Feb 2007 · Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: The intestine is one of the major organs in C. elegans and is largely responsible for food digestion and assimilation as well as the synthesis and storage of macromolecules. In addition, the intestine is emerging as a powerful experimental system in which to study such universal biological phenomena as vesicular trafficking, biochemical clocks, stress responses and aging. The present chapter describes some of these many and varied properties of the C. elegans intestine: the embryonic cell lineage, intestine morphogenesis, structure and physiology of the intestinal cell and, finally, the transcription factor network controlling intestine development and function.
[Show abstract][Hide abstract] ABSTRACT: We fed adult Caenorhabditis elegans fluorescent microspheres mixed with their Escherichia coli food and then measured the total fluorescence of expelled faeces as a function of time after transfer to unlabelled bacteria. Intestinal clearance obeys a simple first-order decay or dilution curve: we estimate that 43 ± 10% of the maximum intestinal volume is expelled in each defecation and the average residence time of a bead (by inference, a bacterium) is less than 2 min. Our results raise questions how bacteria can be sufficiently digested in this brief period to provide energy and material to sustain the high rate of C. elegans oocyte production.
[Show abstract][Hide abstract] ABSTRACT: Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in a variety of functions in vertebrates, especially angiogenesis. Here we identify and characterize a PDGF/VEGF-like factor (named PVF-1) from the nematode C. elegans. We show that PVF-1 has biochemical properties similar to vertebrate PDGF/VEGF growth factors. More important, PVF-1 binds to the human receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) and is able to induce angiogenesis in two model systems derived from vertebrates. Our results highlight the widespread evolutionary conservation of this important class of growth factors and raise the possibility that C. elegans can provide a simple experimental system in which to investigate how these factors function.
Full-text · Article · Mar 2006 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: We identified the gob-1 (gut-obstructed) gene in a forward genetic screen for intestinal defects in the nematode Caenorhabditis elegans. gob-1 loss of function results in early larval lethality, at least in part because of a blocked intestinal lumen and consequent starvation. The gob-1 gene is first expressed in the 8E cell stage of the embryonic intestine, and the GATA factor ELT-2 is sufficient but not necessary for this early phase of gob-1 expression; gob-1 expression later becomes widespread in embryos, larvae, and adults. GOB-1 is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. Trehalose is a glucose disaccharide found in bacteria, fungi, plants, insects, and nematodes but not in mammals. Trehalose plays a number of critical roles such as providing flexible energy reserves and contributing to thermal and osmotic stress resistance. In budding yeast and in plants, the intermediate in trehalose synthesis, trehalose-6-phosphate, has additional critical but less well-defined roles in controlling glycolysis and carbohydrate metabolism. Strong loss-of-function mutants in the C. elegans tps-1 and tps-2 genes (which encode the two trehalose phosphate synthases responsible for trehalose-6-phosphate synthesis) completely suppress the lethality associated with gob-1 loss of function. The suppression of gob-1 lethality by ablation of TPS-1 and TPS-2, the upstream enzymes in the trehalose synthesis pathway, suggests that gob-1 lethality results from a toxic build-up of the intermediate trehalose-6-phosphate, not from an absence of trehalose. GOB-1 is the first trehalose-6-phosphate phosphatase to be identified in nematodes and, because of its associated lethality and distinctive sequence properties, provides a new and attractive target for anti-parasitic drugs.
Preview · Article · Dec 2005 · Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: The med-1 and med-2 genes encode a pair of essentially identical GATA factor-related transcription factors that have been proposed to be necessary for specification of the C. elegans endoderm (intestine or E lineage) as well as part of the C. elegans mesoderm. med-1 and med-2 are proposed to be the direct downstream targets and the principal effectors of the maternally provided SKN-1 transcription factor; med-1 and med-2 would thus occupy the pivotal interface between maternal and zygotic control of gene expression. The conclusion that med-1 and med-2 are necessary for C. elegans endoderm specification was based on a partially penetrant (approximately 50%) loss of endoderm markers produced by RNA-mediated interference (RNAi). To determine whether this partial penetrance reflects: (i) inefficient RNAi against early zygotic transcripts, (ii) experimental uncertainty in the expected level of endoderm loss in skn-1 nulls, or (iii) additional redundancy in the pathway of endoderm specification, we constructed worm strains that segregate embryos lacking both the med-1 gene (because of a gene-specific deletion) and the med-2 gene (using either of two chromosomal deficiencies). Contrary to expectations, we observe that only approximately 3-20% of med-2(-); med-1(-) embryos do not express markers of endoderm differentiation. Furthermore, we found no evidence for a maternal contribution of the med genes to endoderm specification. We conclude that the major pathway(s) for endoderm specification in C. elegans must be independent of the med-1 and med-2 genes.
[Show abstract][Hide abstract] ABSTRACT: We have previously described an acid phosphatase enzyme, PHO-1, present at the lumenal surface of all but the anterior six cells of the Caenorhabditis elegans intestine. In the present paper, we identify the pho-1 structural gene, which encodes a histidine acid phosphatase showing highest similarity to human prostatic acid phosphatase. The pho-1 5'-flanking DNA is capable of directing reporter gene expression that is both gut specific, correctly timed and correctly "patterned", that is, not expressed in the gut anterior. Furthermore, this anterior-posterior patterning of pho-1 expression responds to the C. elegans Wnt pathway as if pho-1 is repressed (directly or indirectly) by high levels of the HMG effector protein POP-1. Transgenic analysis of the pho-1 promoter shows that gut expression is critically dependent on a single WGATAR site. The gut-specific GATA factor ELT-2 binds to this site in vitro and removal of ELT-2 from the embryo destroys expression of the pho-1 reporter. Thus, all our results indicate that pho-1 is a direct downstream target of ELT-2. Finally, the pho-1 loss-of-function mutation shows an interesting and unexpected phenotype for a somatically-expressed hydrolytic enzyme: loss of pho-1 causes arrest of the majority of embryos but this lethality is a maternal effect. We suggest that pho-1 is required by the maternal intestine to assimilate some nutrient or cleavage product that is subsequently provided to the next generation of embryos.
[Show abstract][Hide abstract] ABSTRACT: We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.
Full-text · Article · May 2004 · Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: We describe the elt-4 gene from the nematode Caenorhabditis elegans. elt-4 is predicted to encode a very small (72 residues, 8.1 kD) GATA-type zinc finger transcription factor. The elt-4 gene is located approximately 5 kb upstream of the C. elegans elt-2 gene, which also encodes a GATA-type transcription factor; the zinc finger DNA-binding domains are highly conserved (24/25 residues) between the two proteins. The elt-2 gene is expressed only in the intestine and is essential for normal intestinal development. This article explores whether elt-4 also has a role in intestinal development. Reporter fusions to the elt-4 promoter or reporter insertions into the elt-4 coding regions show that elt-4 is indeed expressed in the intestine, beginning at the 1.5-fold stage of embryogenesis and continuing into adulthood. elt-4 reporter fusions are also expressed in nine cells of the posterior pharynx. Ectopic expression of elt-4 cDNA within the embryo does not cause detectable ectopic expression of biochemical markers of gut differentiation; furthermore, ectopic elt-4 expression neither inhibits nor enhances the ectopic marker expression caused by ectopic elt-2 expression. A deletion allele of elt-4 was isolated but no obvious phenotype could be detected, either in the gut or elsewhere; brood sizes, hatching efficiencies, and growth rates were indistinguishable from wild type. We found no evidence that elt-4 provided backup functions for elt-2. We used microarray analysis to search for genes that might be differentially expressed between L1 larvae of the elt-4 deletion strain and wild-type worms. Paired hybridizations were repeated seven times, allowing us to conclude, with some confidence, that no candidate target transcript could be identified as significantly up- or downregulated by loss of elt-4 function. In vitro binding experiments could not detect specific binding of ELT-4 protein to candidate binding sites (double-stranded oligonucleotides containing single or multiple WGATAR sequences); ELT-4 protein neither enhanced nor inhibited the strong sequence-specific binding of the ELT-2 protein. Whereas ELT-2 protein is a strong transcriptional activator in yeast, ELT-4 protein has no such activity under similar conditions, nor does it influence the transcriptional activity of coexpressed ELT-2 protein. Although an elt-2 homolog was easily identified in the genomic sequence of the related nematode C. briggsae, no elt-4 homolog could be identified. Analysis of the changes in silent third codon positions within the DNA-binding domains indicates that elt-4 arose as a duplication of elt-2, some 25-55 MYA. Thus, elt-4 has survived far longer than the average duplicated gene in C. elegans, even though no obvious biological function could be detected. elt-4 provides an interesting example of a tandemly duplicated gene that may originally have been the same size as elt-2 but has gradually been whittled down to its present size of little more than a zinc finger. Although elt-4 must confer (or must have conferred) some selective advantage to C. elegans, we suggest that its ultimate evolutionary fate will be disappearance from the C. elegans genome.