The GATA factor ELT-3 specifies endoderm in Caenorhabditis angaria in an ancestral gene network.

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.

Feedforward regulatory logic controls the specification-to-differentiation transition and terminal cell fate during Caenorhabditis elegans endoderm development.

The architecture of gene regulatory networks determines the specificity and fidelity of developmental outcomes. We report that the core regulatory circuitry for endoderm development in Caenorhabditis elegans operates through a transcriptional cascade consisting of six sequentially expressed GATA-type factors that act in a recursive series of interlocked feedforward modules. This structure results in sequential redundancy, in which removal of a single factor or multiple alternate factors in the cascade leads to a mild or no effect on gut development, whereas elimination of any two sequential factors invariably causes a strong phenotype. The phenotypic strength is successfully predicted with a computational model based on the timing and levels of transcriptional states. We found that one factor in the middle of the cascade, END-1, which straddles the distinct events of specification and differentiation, functions in both processes. Finally, we reveal roles for key GATA factors in establishing spatial regulatory state domains by repressing other fates, thereby defining boundaries in the digestive tract. Our findings provide a paradigm that could account for the genetic redundancy observed in many developmental regulatory systems.

Gut development in C. elegans.

The midgut (intestine) of the nematode, C. elegans, is a tube consisting of 20 cells that arises from a single embryonic precursor. Owing to its comparatively simple anatomy and the advantages inherent to the C. elegans system, the gut has been used as a model for organogenesis for more than 25 years. In this review, the salient features of C. elegans gut development are described from the E progenitor through to the 20-cell intestine. The core gene regulatory network that drives specification of the gut, and other genes with roles in organogenesis, lumen morphogenesis and the cell cycle, are also described. Questions for future work are posed.

The C. elegans intestine: organogenesis, digestion, and physiology.

The comparatively simple Caenorhabditis elegans intestine fulfills many of the complex functions of the mammalian digestive tract, liver, and fat tissues, while also having roles in pathogen defense, immunity, and longevity. In this review, we describe the structure of the C. elegans gut and how it develops from the embryonic precursor E. We examine what is currently known about how the animal's microbial diet is moved through the intestinal lumen, and how its enzymatic functions contribute to physiology and metabolism. The underlying gene regulatory networks behind both development and physiology are also described. Finally, we consider recent studies that examine metabolism and digestion and describe emerging areas for future work.

Partially compromised specification causes stochastic effects on gut development in C. elegans.

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.

MED GATA factors promote robust development of the C. elegans endoderm.

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.

Developmental robustness in the Caenorhabditis elegans embryo.

Developmental robustness is the ability of an embryo to develop normally despite many sources of variation, from differences in the environment to stochastic cell-to-cell differences in gene expression. The nematode Caenorhabditis elegans exhibits an additional level of robustness: Unlike most other animals, the embryonic pattern of cell divisions is nearly identical from animal to animal. The endoderm (gut) lineage is an ideal model for studying such robustness as the juvenile gut has a simple anatomy, consisting of 20 cells that are derived from a single cell, E, and the gene regulatory network that controls E specification shares features with developmental regulatory networks in many other systems, including genetic redundancy, parallel pathways, and feed-forward loops. Early studies were initially concerned with identifying the genes in the network, whereas recent work has focused on understanding how the endoderm produces a robust developmental output in the face of many sources of variation. Genetic control exists at three levels of endoderm development: Progenitor specification, cell divisions within the developing gut, and maintenance of gut differentiation. Recent findings show that specification genes regulate all three of these aspects of gut development, and that mutant embryos can experience a "partial" specification state in which some, but not all, E descendants adopt a gut fate. Ongoing studies using newer quantitative and genome-wide methods promise further insights into how developmental gene-regulatory networks buffer variation.

Gene expression profiling of gastrocnemius of "minimuscle" mice.

Few studies have investigated heterogeneity of selection response in replicate lines subjected to equivalent selection. We developed four replicate lines of mice based on high levels of voluntary wheel running (high runner or HR lines) while also maintaining four nonselected control lines. This led to the unexpected discovery of the HR minimuscle (HRmini) phenotype, recognized by a 50% reduction in hindlimb muscle mass, which became fixed in 1 of the four HR selected lines. Here, we report genome-wide expression profiling describing transcriptome differences between HRnormal and HRmini medial gastrocnemius. Consistent with the known reduction of type IIB fibers in HRmini, Myh4 gene expression was -8.82-fold less (P = 0.0001) in HRmini, which was closely associated with differences in the "calcium signaling" canonical pathway, including structural genes (e.g., Mef2c, twofold greater in HRmini, P = 0.0003) and myogenic factors (e.g., Myog, 3.8-fold greater in HRmini, P = 0.0026) associated with slow-type myofibers. The gene that determines the HRmini phenotype is known to reside in a 2.6335-Mb interval on mouse chromosome 11 and 7 genes (Myh10, Chrnb1, Acadvl, Senp3, Gabarap, Eif5a, and Clec10a) from this region were differentially expressed. Verification by real-time PCR confirmed 1.5-fold greater (P < 0.05) expression of very long chain acyl-CoA dehydrogenase (Acadvl) in HRmini. Ten other genes associated with fatty acid metabolism were also upregulated in HRmini, suggesting differences in the ability to metabolize fatty acids in HRnormal and HRmini muscles. This work provides a resource for understanding differences in muscle phenotypes in populations exhibiting high running capacity.

The long isoform of the C. elegans ELT-3 GATA factor can specify endoderm when overexpressed.

The C. elegans elt-3 gene encodes a GATA transcription factor that is expressed in the hypodermis and has roles in hypodermal specification and regulation of collagen and stress response genes. The gene encodes short and long isoforms, ELT-3A and ELT-3B respectively, that differ upstream of their DNA-binding domains. Previous work showed that ELT-3A can specify hypodermal cell fates when forcibly overexpressed throughout early embryos. We recently showed that the ELT-3B orthologue from the distantly related species C. angaria can specify endodermal fates when forcibly overexpressed in C. elegans. Here, we show that C. elegans ELT-3B can also specify endoderm.

Evolutionary Change in Gut Specification in Caenorhabditis Centers on the GATA Factor ELT-3 in an Example of Developmental System Drift.

Cells in a developing animal embryo become specified by the activation of cell-type-specific gene regulatory networks. The network that specifies the gut in the nematode Caenorhabditis elegans has been the subject of study for more than two decades. In this network, the maternal factors SKN-1/Nrf and POP-1/TCF activate a zygotic GATA factor cascade consisting of the regulators MED-1,2 → END-1,3 → ELT-2,7, leading to the specification of the gut in early embryos. Paradoxically, the MED, END, and ELT-7 regulators are present only in species closely related to C. elegans, raising the question of how the gut can be specified without them. Recent work found that ELT-3, a GATA factor without an endodermal role in C. elegans, acts in a simpler ELT-3 → ELT-2 network to specify gut in more distant species. The simpler ELT-3 → ELT-2 network may thus represent an ancestral pathway. In this review, we describe the elucidation of the gut specification network in C. elegans and related species and propose a model by which the more complex network might have formed. Because the evolution of this network occurred without a change in phenotype, it is an example of the phenomenon of Developmental System Drift.

The NK-2 class homeodomain factor CEH-51 and the T-box factor TBX-35 have overlapping function in C. elegans mesoderm development.

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.

Effects of residual antibiotics in groundwater on Salmonella typhimurium: changes in antibiotic resistance, in vivo and in vitro pathogenicity.

An outbreak-causing strain of Salmonella enterica serovar Typhimurium was exposed to groundwater with residual antibiotics for up to four weeks. Representative concentrations (0.05, 1, and 100 µg L(-1)) of amoxicillin, tetracycline, and a mixture of several other antibiotics (1 µg L(-1) each) were spiked into artificially prepared groundwater (AGW). Antibiotic susceptibility analysis and the virulence response of stressed Salmonella were determined on a weekly basis by using human epithelial cells (HEp2) and soil nematodes (C. elegans). Results have shown that Salmonella typhimurium remains viable for long periods of exposure to antibiotic-supplemented groundwater; however, they failed to cultivate as an indication of a viable but nonculturable state. Prolonged antibiotics exposure did not induce any changes in the antibiotic susceptibility profile of the S. typhimurium strain used in this study. S. typhimurium exposed to 0.05 and 1 µg L(-1) amoxicillin, and 1 µg L(-1) tetracycline showed hyper-virulent profiles in both in vitro and in vivo virulence assays with the HEp2 cells and C. elegans respectively, most evident following 2nd and 3rd weeks of exposure.

Endoderm development in Caenorhabditis elegans: the synergistic action of ELT-2 and -7 mediates the specification→differentiation transition.

The transition from specification of cell identity to the differentiation of cells into an appropriate and enduring state is critical to the development of embryos. Transcriptional profiling in Caenorhabditis elegans has revealed a large number of genes that are expressed in the fully differentiated intestine; however, no regulatory factor has been found to be essential to initiate their expression once the endoderm has been specified. These gut-expressed genes possess a preponderance of GATA factor binding sites and one GATA factor, ELT-2, fulfills the expected characteristics of a key regulator of these genes based on its persistent expression exclusively in the developing and differentiated intestine and its ability to bind these regulatory sites. However, a striking characteristic of elt-2(0) knockout mutants is that while they die shortly after hatching owing to an obstructed gut passage, they nevertheless contain a gut that has undergone complete morphological differentiation. We have discovered a second gut-specific GATA factor, ELT-7, that profoundly synergizes with ELT-2 to create a transcriptional switch essential for gut cell differentiation. ELT-7 is first expressed in the early endoderm lineage and, when expressed ectopically, is sufficient to activate gut differentiation in nonendodermal progenitors. elt-7 is transcriptionally activated by the redundant endoderm-specifying factors END-1 and -3, and its product in turn activates both its own expression and that of elt-2, constituting an apparent positive feedback system. While elt-7 loss-of-function mutants lack a discernible phenotype, simultaneous loss of both elt-7 and elt-2 results in a striking all-or-none block to morphological differentiation of groups of gut cells with a region-specific bias, as well as reduced or abolished gut-specific expression of a number of terminal differentiation genes. ELT-2 and -7 synergize not only in activation of gene expression but also in repression of a gene that is normally expressed in the valve cells, which immediately flank the termini of the gut tube. Our results point to a developmental strategy whereby positive feedback and cross-regulatory interactions between two synergistically acting regulatory factors promote a decisive and persistent transition of specified endoderm progenitors into the program of intestinal differentiation.

Roles of the Wnt effector POP-1/TCF in the C. elegans endomesoderm specification gene network.

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/beta-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.

Cell fate specification in the C. elegans embryo.

Cell specification requires that particular subsets of cells adopt unique expression patterns that ultimately define the fates of their descendants. In C. elegans, cell fate specification involves the combinatorial action of multiple signals that produce activation of a small number of "blastomere specification" factors. These initiate expression of gene regulatory networks that drive development forward, leading to activation of "tissue specification" factors. In this review, the C. elegans embryo is considered as a model system for studies of cell specification. The techniques used to study cell fate in this species, and the themes that have emerged, are described.

Knockdown of SKN-1 and the Wnt effector TCF/POP-1 reveals differences in endomesoderm specification in C. briggsae as compared with C. elegans.

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.

Structure and evolution of the C. elegans embryonic endomesoderm network.

The specification of the Caenorhabditis elegans endomesoderm has been the subject of study for more than 15 years. Specification of the 4-cell stage endomesoderm precursor, EMS, occurs as a result of the activation of a transcription factor cascade that starts with SKN-1, coupled with input from the Wnt/beta-catenin asymmetry pathway through the nuclear effector POP-1. As development proceeds, transiently-expressed cell fate factors are succeeded by stable, tissue/organ-specific regulators. The pathway is complex and uses motifs found in all transcriptional networks. Here, the regulators that function in the C. elegans endomesoderm network are described. An examination of the motifs in the network suggests how they may have evolved from simpler gene interactions. Flexibility in the network is evident from the multitude of parallel functions that have been identified and from apparent changes in parts of the corresponding network in Caenorhabditis briggsae. Overall, the complexities of C. elegans endomesoderm specification build a picture of a network that is robust, complex, and still evolving.

The noncanonical binding site of the MED-1 GATA factor defines differentially regulated target genes in the C. elegans mesendoderm.

Mesoderm 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.

Evolutionary Dynamics of the SKN-1 → MED → END-1,3 Regulatory Gene Cascade in Caenorhabditis Endoderm Specification.

Gene regulatory networks and their evolution are important in the study of animal development. In the nematode, Caenorhabditis elegans, the endoderm (gut) is generated from a single embryonic precursor, E. Gut is specified by the maternal factor SKN-1, which activates the MED → END-1,3 → ELT-2,7 cascade of GATA transcription factors. In this work, genome sequences from over two dozen species within the Caenorhabditis genus are used to identify MED and END-1,3 orthologs. Predictions are validated by comparison of gene structure, protein conservation, and putative cis-regulatory sites. All three factors occur together, but only within the Elegans supergroup, suggesting they originated at its base. The MED factors are the most diverse and exhibit an unexpectedly extensive gene amplification. In contrast, the highly conserved END-1 orthologs are unique in nearly all species and share extended regions of conservation. The END-1,3 proteins share a region upstream of their zinc finger and an unusual amino-terminal poly-serine domain exhibiting high codon bias. Compared with END-1, the END-3 proteins are otherwise less conserved as a group and are typically found as paralogous duplicates. Hence, all three factors are under different evolutionary constraints. Promoter comparisons identify motifs that suggest the SKN-1, MED, and END factors function in a similar gut specification network across the Elegans supergroup that has been conserved for tens of millions of years. A model is proposed to account for the rapid origin of this essential kernel in the gut specification network, by the upstream intercalation of duplicate genes into a simpler ancestral network.

Evolution of Developmental GATA Factors in Nematodes.

GATA transcription factors are found in animals, plants, and fungi. In animals, they have important developmental roles in controlling specification of cell identities and executing tissue-specific differentiation. The Phylum Nematoda is a diverse group of vermiform animals that inhabit ecological niches all over the world. Both free-living and parasitic species are known, including those that cause human infectious disease. To date, GATA factors in nematodes have been studied almost exclusively in the model system C. elegans and its close relatives. In this study, we use newly available sequences to identify GATA factors across the nematode phylum. We find that most species have fewer than six GATA factors, but some species have 10 or more. Comparisons of gene and protein structure suggest that there were at most two GATA factors at the base of the phylum, which expanded by duplication and modification to result in a core set of four factors. The high degree of structural similarity with the corresponding orthologues in C. elegans suggests that the nematode GATA factors share similar functions in development.