Search

Endoderm specification in the nematode, C. elegans, occurs through a well-characterized pathway that is initiated by maternally provided SKN-1/Nrf, and with additional input from POP-1/TCF, which activates the GATA factor cascade MED-1,2 → END-1,3 → ELT-2,7. Orthologues of the MED and END factors, and ELT-7, are found only among nematodes of the Elegans Supergroup consisting of species closely related to C. elegans, which raises the question of how gut is specified in their absence. In this work, we investigate gut specification outside the Elegans Supergroup. We find that the C. angaria and C. portoensis orthologues of the elt-3 GATA factor gene are expressed in the early E lineage, just before their elt-2 orthologues. In C. angaria, both Can-pop-1(RNAi) and Can-elt-3(RNAi) result in a penetrant ‘gutless’ phenotype. Can-pop-1 is necessary for Can-elt-3 activation, showing that it acts upstream. When introduced into C. elegans as transgenes, overexpressed Can-elt-3 is sufficient to specify gut, while Can-elt-2 can rescue gut differentiation under the control of its own promoter. Our results demonstrate an ancestral mechanism for gut specification and differentiation in Caenorhabditis involving a simplified gene network consisting of POP-1 → ELT-3 → ELT-2.Summary statementSpecification of the gut progenitor E in a distant relative of C. elegans uses a different GATA factor, ELT-3, suggesting that the ancestral network was simpler.

Endoderm specification in Caenorhabditis elegans occurs through a network in which maternally provided SKN-1/Nrf, with additional input from POP-1/TCF, activates the GATA factor cascade MED-1,2→END-1,3→ELT-2,7. Orthologues of the MED, END and ELT-7 factors are found only among nematodes closely related to C. elegans, raising the question of how gut is specified in their absence in more distant species in the genus. We find that the C. angaria, C. portoensis and C. monodelphis orthologues of the GATA factor gene elt-3 are expressed in the early E lineage, just before their elt-2 orthologues. In C. angaria, Can-pop-1(RNAi), Can-elt-3(RNAi) and a Can-elt-3 null mutation result in a penetrant ‘gutless’ phenotype. Can-pop-1 is necessary for Can-elt-3 activation, showing that it acts upstream. Forced early E lineage expression of Can-elt-3 in C. elegans can direct the expression of a Can-elt-2 transgene and rescue an elt-7 end-1 end-3; elt-2 quadruple mutant strain to viability. Our results demonstrate an ancestral mechanism for gut specification and differentiation in Caenorhabditis involving a simpler POP-1→ELT-3→ELT-2 gene network.

The C. elegans gut descends from the E progenitor cell through a series of stereotyped cell divisions and morphogenetic events. Effects of perturbations of upstream cell specification on downstream organogenesis have not been extensively investigated. Here we have assembled an allelic series of strains that variably compromise specification of E by perturbing the activation of the gut-specifying end-1 and end-3 genes. Using a marker that allows identification of all E descendants regardless of fate, superimposed with markers that identify cells that have adopted a gut fate, we have examined the fate of E lineage descendants among hundreds of embryos. We find that when specification is partially compromised, the E lineage undergoes hyperplasia accompanied by stochastic and variable specification of gut fate among the E descendants. As anticipated by prior work, the activation of the gut differentiation factor elt-2 becomes delayed in these strains, although ultimate protein levels of a translational ELT-2::GFP reporter resemble those of the wild type. By comparing these effects among the various specification mutants, we find that the stronger the defect in specification (i.e. the fewer number of embryos specifying gut), the stronger the defects in the E lineage and delay in activation of elt-2. Despite the changes in the E lineage in these strains, we find that supernumerary E descendants that adopt a gut fate are accommodated into a relatively normal-looking intestine. Hence, upstream perturbation of specification dramatically affects the E lineage, but as long as sufficient descendants adopt a gut fate, organogenesis overcomes these effects to form a relatively normal intestine.

In C. elegans the 4-cell stage blastomere EMS is an endomesodermal precursor. Its anterior daughter, MS, makes primarily mesodermal cells, while its posterior daughter E generates the entire intestine. The gene regulatory network underlying specification of MS and E has been the subject of study for more than 15 years. A key component of the specification of the two cells is the involvement of the Wnt/β-catenin asymmetry pathway, which through its nuclear effector POP-1, specifies MS and E as different from each other. Loss of pop-1 function results in the mis-specification of MS as an E-like cell, because POP-1 directly represses the end-1 and end-3 genes in MS, which would otherwise promote an endoderm fate. A long-standing question has been whether POP-1 plays a role in specifying MS fate beyond repression of endoderm fate. This question has been difficult to ask because the only chromosomal lesions that remove both end-1 and end-3 are large deletions removing hundreds of genes. Here, we report the construction of bona fide end-1 end-3 double mutants. In embryos lacking activity of end-1, end-3 and pop-1 together, we find that MS fate is partially restored, while E expresses early markers of MS fate and adopts characteristics of both MS and C. Our results suggest that POP-1 is not critical for MS specification beyond repression of endoderm specification, and reveal that Wnt-modified POP-1 and END-1/3 further reinforce E specification by repressing MS fate in E. By comparison, a previous work suggested that in the related nematode C. briggsae, Cb-POP-1 is not required to repress endoderm specification in MS, in direct contrast with Ce-POP-1, but is critical for repression of MS fate in E. The findings reported here shed new light on the flexibility of combinatorial control mechanisms in endomesoderm specification in Caenorhabditis.

MED-1 is a member of a group of divergent GATA-type zinc finger proteins recently identified in several species of Caenorhabditis. The med genes are transcriptional regulators that are involved in the specification of the mesoderm and endoderm precursor cells in nematodes. Unlike other GATA-type zinc fingers that recognize the consensus sequence (A/C/T)GATA(A/G), the MED-1 zinc finger (MED1zf) binds the larger and atypical site GTATACT(T/C)3. We have examined the basis for this unusual DNA specificity using a range of biochemical and biophysical approaches. Most strikingly, we show that although the core of the MED1zf structure is similar to that of GATA-1, the basic tail C-terminal to the zinc finger unexpectedly adopts an α-helical structure upon binding DNA. This additional helix appears to contact the major groove of the DNA, making contacts that explain the extended DNA consensus sequence observed for MED1zf. Our data expand the versatility of DNA recognition by GATA-type zinc fingers and perhaps shed new light on the DNA-binding properties of mammalian GATA factors.

In C. elegans, many mesodermal cell types are made by descendants of the progenitor MS, born at the seven-cell stage of embryonic development. Descendants of MS contribute to body wall muscle and to the posterior half of the pharynx. We have previously shown that MS is specified by the activity of the divergent MED-1,2 GATA factors. We report that the MED-1,2 target gene tbx-35, which encodes a T-box transcription factor, specifies the MS fate. Embryos homozygous for a putative tbx-35-null mutation fail to generate MS-derived pharynx and body muscle, and instead generate ectopic PAL-1-dependent muscle and hypodermis, tissues normally made by the C blastomere. Conversely, overexpression of tbx-35 results in the generation of ectopic pharynx and muscle tissue. The MS and E sister cells are made different by transduction of a Wnt/MAPK/Src pathway signal through the nuclear effector TCF/POP-1. We show that in E, tbx-35 is repressed in a Wnt-dependent manner that does not require activity of TCF/POP-1, suggesting that an additional nuclear Wnt effector functions in E to repress MS development. Genes of the T-box family are known to function in protostomes and deuterostomes in the specification of mesodermal fates. Our results show that this role has been evolutionarily conserved in the early C. elegans embryo, and that a progenitor of multiple tissue types can be specified by a surprisingly simple gene cascade.

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.

Pap pili gene expression is controlled by a reversible OFF/ON phase switch that is orchestrated by binding of Lrp to pap pilin promoter proximal sites 1, 2, and 3 (OFF) or pap promoter distal sites 4, 5, and 6 (ON). Movement of Lrp between proximal and distal sites controls pap pilin transcription and is modulated by PapI and DNA adenine methylase. Here we show that activation of the environmentally responsive CpxAR two-component regulatory system inhibits Pap phase variation by generation of phosphorylated CpxR (CpxR-P). CpxR-P competes with Lrp for binding to both promoter proximal and distal pap DNA binding sites, inhibiting pap transcription in vitro and pili expression in vivo. In contrast to Lrp, CpxR-P is methylation insensitive and does not form DNA methylation patterns in vivo. CpxAR-dependent repression of pap transcription is also observed in response to alkaline growth conditions. These results provide insight into a mechanism for environmental control of epigenetically regulated gene expression.

The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. We report that the MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. We find that SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. Our results suggest that a broad tissue territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network.

To explore the role of neutrophil phagocytosis in host defense against Bordetella pertussis , bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum. In the absence of antibodies, FITC-labeled bacteria were located primarily on the surface of the neutrophils with few bacteria ingested. However, after opsonization, about seven times more bacteria were located intracellularly, indicating that antibodies promoted phagocytosis. In contrast, bacteria labeled intrinsically with GFP were not efficiently phagocytosed even in the presence of opsonizing antibodies, suggesting that FITC interfered with a bacterial defense. Because FITC covalently modifies proteins and could affect their function, we tested the effect of FITC on adenylate cyclase toxin activity, an important extracellular virulence factor. FITC-labeled bacteria had fivefold-less adenylate cyclase toxin activity than did unlabeled wild-type bacteria or GFP-expressing bacteria, suggesting that FITC compromised adenylate cyclase toxin activity. These data demonstrated that at least one extracellular virulence factor was affected by FITC labeling and that GFP is a more appropriate label for B. pertussis.

Nematode spermatozoa are highly specialized cells that lack flagella and, instead, extend a pseudopod to initiate motility. Crawling spermatozoa display classic features of amoeboid motility (e.g. protrusion of a pseudopod that attaches to the substrate and the assembly and disassembly of cytoskeletal filaments involved in cell traction and locomotion), however, cytoskeletal dynamics in these cells are powered exclusively by Major Sperm Protein (MSP) rather than actin and no other molecular motors have been identified. Thus, MSP-based motility is regarded as a simple locomotion machinery suitable for the study of plasma membrane protrusion and cell motility in general. This recent focus on MSP dynamics has increased the necessity of a standardized methodology to obtain C. elegans sperm extract that can be used in biochemical assays and proteomic analysis for comparative studies. In the present work we have modified a method to reproducibly obtain relative high amounts of proteins from C. elegans sperm extract. We show that these extracts share some of the properties observed in sperm extracts from the parasitic nematode Ascaris including Major Sperm Protein (MSP) precipitation and MSP fiber elongation. Using this method coupled to immunoblot detection, Mass Spectrometry identification, in silico prediction of functional domains and biochemical assays, our results indicate the presence of phosphorylation sites in MSP of Caenorhabditis elegans spermatozoa.

Matrotrophic fish in the genus Poeciliopsis (Poeciliidae) have a placenta-like structure used in postfertilization maternal provisioning of the developing embryo. To understand better the structure and function of the Poeciliopsis placenta, we derived cDNA libraries from the maternal follicular placenta of 2 matrotrophic Poeciliopsis sister species, P. turneri and P. presidionis. These species inherited their placenta from a common ancestor and represent one of 3 independent origins of placentas in Poeciliopsis. Expressed sequence tags (ESTs) were generated and putative function was determined using BLASTX homology searches and Gene Ontology (GO) annotation. Reverse transcription– polymerase chain reaction was used to verify placenta tissue expression of a putative candidate gene, alpha-2 macroglobulin. In total, 1956 (71.5% of the total submitted ESTs) and 924 (71.0% of the total submitted ESTs) unique transcripts were identified for the P. turneri and P. presidionis placenta, respectively. Homology search and GO annotation revealed putative genes whose products may be involved in specific transport functions of the maternal follicle. These putative genes are excellent candidates for future research on the evolution of the placenta. We discuss our results in light of the parent–offspring conflict theory of placental evolution and in terms of the Poeciliid placenta structure and function.

Detection of transcripts in situ is a rapid means by which gene expression can be characterized in many systems. In the nematode, Caenorhabditis elegans, the ease with which transgenics can be made and the general reliability of reporter fusion expression patterns, have made this technique comparatively less popular than in other systems. There are, however, still applications in which in situ hybridization is desired, such as for maternally expressed genes, or in related species without established transgene methods. The most frequently used method of in situ hybridization uses DNA probes and formaldehyde fixation. A newer approach that permits single-transcript detection has been reported and will not be described here (Raj and Tyagi, 2010). Rather, we describe an alternative protocol that uses RNA probes with a different fixative. This approach has been applied to C. elegans and related nematodes, providing reliable, sensitive detection of endogenous transcripts.

The C. elegans MS blastomere, born at the 7-cell stage of embryogenesis, generates primarily mesodermal cell types, including pharynx cells, body muscles and coelomocytes. A presumptive null mutation in the T-box factor gene tbx-35, a target of the MED-1 and MED-2 divergent GATA factors, was previously found to result in a profound decrease in the production of MS-derived tissues, although the tbx-35(-) embryonic arrest phenotype was variable. We report here that the NK-2 class homeobox gene ceh-51 is a direct target of TBX-35 and at least one other factor, and that CEH-51 and TBX-35 share functions. Embryos homozygous for a ceh-51 null mutation arrest as larvae with pharynx and muscle defects, although these tissues appear to be specified correctly. Loss of tbx-35 and ceh-51 together results in a synergistic phenotype resembling loss of med-1 and med-2. Overexpression of ceh-51 causes embryonic arrest and generation of ectopic body muscle and coelomocytes. Our data show that TBX-35 and CEH-51 have overlapping function in MS lineage development. As T-box regulators and NK-2 homeodomain factors are both important for heart development in Drosophila and vertebrates, our results suggest that these regulators function in a similar manner in C. elegans to specify a major precursor of mesoderm.

We have previously shown that the MED-1,2 divergent GATA factors act apparently zygotically to specify the fates of the MS (mesoderm) and E (endoderm) sister cells, born at the 7-cell stage of C. elegans embryogenesis. In the E cell, MED-1,2 activate transcription of the endoderm-promoting end-1 and end-3 genes. We demonstrate by in situ hybridization that med transcripts accumulate both in the EMS cell and in the maternal germline in a SKN-1-dependent manner. Removal of zygotic med function alone results in a weakly impenetrant loss of endoderm. However, med-1,2(−) embryos made by mothers in which germline med transcripts have been abrogated by transgene cosuppression fail to make endoderm 50% of the time, similar to the phenotype seen by RNAi. We also find that reduction of Med or End activity results in aberrant numbers of intestinal cells in embryos that make endoderm. We further show that regulation of the paralogous end-1 and end-3 genes consists of both shared and distinct inputs, and that END-3 activates end-1 expression. Our data thus reveal three new properties of C. elegans endoderm specification: both maternal and zygotic activities of the med genes act to specify endoderm, defects in endoderm specification also result in defects in gut cell number, and activation of the paralogous end-1 and end-3 genes differs qualitatively in the relative contributions of their upstream regulators.

Pap pili gene expression is controlled by a reversible OFF/ON phase switch that is orchestrated by binding of Lrp to pap pilin promoter proximal sites 1, 2, and 3 (OFF) or pap promoter distal sites 4, 5, and 6 (ON). Movement of Lrp between proximal and distal sites controls pap pilin transcription and is modulated by PapI and DNA adenine methylase. Here we show that activation of the environmentally responsive CpxAR two-component regulatory system inhibits Pap phase variation by generation of phosphorylated CpxR (CpxR-P). CpxR-P competes with Lrp for binding to both promoter proximal and distal pap DNA binding sites, inhibiting pap transcription in vitro and pili expression in vivo. In contrast to Lrp, CpxR-P is methylation insensitive and does not form DNA methylation patterns in vivo. CpxAR-dependent repression of pap transcription is also observed in response to alkaline growth conditions. These results provide insight into a mechanism for environmental control of epigenetically regulated gene expression.

In the nematode, C elegans, the divergent GATA-type transcription factors MED-1 and MED-2 are encoded by an unlinked, redundant pair of intronless genes. The med-1,2 genes are among the first to be activated in the embryo and are critical for the specification of the 7-cell stage MS (mesoderm) and E (endoderm) precursor cells. We have previously shown that the binding site recognized by MED-1 is a noncanonical RAGTATAC site that is not expected from the resemblance of its single C4-type zinc finger to those of other known GATA factors, which recognize the consensus HGATAR. To date, no MED-like zinc fingers have been described outside of C. elegans. In order to understand the evolution of these transcription factors, and the evolution of gene networks that specify early cell fates in Caenorhabditis, we have identified med sequence homologs in the related nematodes C. briggsae and C. remanei. While C. briggsae encodes two med-like genes similar to C. elegans, we find evidence for seven distinct med-like genes in C. remanei. Somewhat unexpectedly, the coding regions of all med genes appear to lack introns. We report that the med homologs have similar expression in their respective species. We further show that the C. briggsae homologs, and at least five of the seven C. remanei homologs, can fully complement the embryonic lethal phenotype of a C. elegans med-1,2(-) strain. We conclude that Med function and expression have been conserved over tens of millions of years of evolution, and that there may be a mechanism that selects against the acquisition of introns in these genes. (c) 2005 Elsevier Inc. All rights reserved.

In the nematode, C. elegans, the bZIP/homeodomain transcription factor SKN-1 and the Wnt effector TCF/POP-1 are central to the maternal specification of the endomesoderm prior to gastrulation. The 8-cell stage blastomere MS is primarily a mesodermal precursor, giving rise to cells of the pharynx and body muscle among others, while its sister E clonally generates the entire endoderm (gut). In C. elegans, loss of SKN-1 results in the absence of MS-derived tissues all of the time, and loss of gut most of the time, while loss of POP-1 results in a mis-specification of MS as an E-like cell, resulting in ectopic gut. We show that in C. briggsae, RNAi of skn-1 results in a stronger E defect but no apparent MS defect, while RNAi of pop-1 results in loss of gut and an apparent E to MS transformation, the opposite of the pop-1 knockdown phenotype seen in C. elegans. The difference in pop-1(−) phenotypes correlates with changes in how the endogenous endoderm-specifying end genes are regulated by POP-1 in the two species. Our results suggest that integration of Wnt-dependent and Wnt-independent cell fate specification pathways within the Caenorhabditis genus can occur in different ways.

© 2015 Elsevier Inc. The MED-1,2 GATA factors contribute to specification of E, the progenitor of the Caenorhabditis elegans endoderm, through the genes end-1 and end-3, and in parallel with the maternal factors SKN-1, POP-1 and PAL-1. END-1,3 activate elt-2 and elt-7 to initiate a program of intestinal development, which is maintained by positive autoregulation. Here, we advance the understanding of MED-1,2 in E specification. We find that expression of end-1 and end-3 is greatly reduced in med-1,2(-) embryos. We generated strains in which MED sites have been mutated in end-1 and end-3. Without MED input, gut specification relies primarily on POP-1 and PAL-1. 25% of embryos fail to make intestine, while those that do display abnormal numbers of gut cells due to a delayed and stochastic acquisition of intestine fate. Surviving adults exhibit phenotypes consistent with a primary defect in the intestine. Our results establish that MED-1,2 provide robustness to endoderm specification through end-1 and end-3, and reveal that gut differentiation may be more directly linked to specification than previously appreciated. The results argue against an "all-or-none" description of cell specification, and suggest that activation of tissue-specific master regulators, even when expression of these is maintained by positive autoregulation, does not guarantee proper function of differentiated cells.

SummaryMesoderm and endoderm in C. elegans arise from sister cells called MS and E, respectively. The identities of both of these mesendodermal progenitors are controlled by MED-1 and -2, members of the GATA factor family. In the E lineage, these factors activate a sequential cascade of GATA factors, beginning with their immediate targets, the endoderm-specifying end genes. We report that MED-1 binds invariant noncanonical sites in the end genes, revealing that the MEDs are atypical members of the GATA factor family that do not recognize GATA sequences. By searching the genome for clusters of these MED sites, we have identified 19 candidate MED targets. Based on their expression patterns, these define three distinct classes of MED-regulated genes: MS-specific, E-specific, and E plus MS-specific. Some MED targets encode transcription factors related to those that regulate mesendoderm development in other phyla, supporting the existence of an ancient metazoan mesendoderm gene regulatory network.