Morris F. Maduro

Restriction of Mesendoderm to a Single Blastomere by the Combined Action of SKN-1 and a GSK-3β Homolog Is Mediated by MED-1 and -2 in C. elegans

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.

A POP-1 repressor complex restricts inappropriate cell type-specific gene transcription during Caenorhabditis elegans embryogenesis

In Caenorhabditis elegans, histone acetyltransferase CBP‐1 counteracts the repressive activity of the histone deacetylase HDA‐1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box‐containing protein, POP‐1, through an unknown mechanism. In this study, we show that CBP‐1, HDA‐1 and POP‐1 converge on end‐1, an initial endoderm‐determining gene. In the E lineage, an essential function of CBP‐1 appears to be the activation of end‐1 transcription. We further identify a molecular mechanism for the endoderm‐suppressive effect of POP‐1 in the MS lineage by demonstrating that POP‐1 functions as a transcriptional repressor that inhibits inappropriate end‐1 transcription. We provide evidence that POP‐1 represses transcription via the recruitment of HDA‐1 and UNC‐37, the C.elegans homolog of the co‐repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate‐determining gene during development. Furthermore, they identify a strategy by which concerted actions of histone deacetylases and other co‐repressors ensure maximal repression of inappropriate cell type‐specific gene transcription.

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.

Specification of the C. elegans MS blastomere by the T-box factor TBX-35

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.

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.

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.

The UNC-119 family of neural proteins is functionally conserved between humans, Drosophila and C. elegans.

C. elegans animals mutant for the unc-119 gene exhibit movement, sensory and behavioral abnormalities. Consistent with a nervous system role, unc-119 reporter genes are expressed throughout the C. elegans nervous system. The UNC-119 protein has strong sequence similarity to the predicted protein from a human gene, HRG4/HsUNC-119, whose transcript is abundant in the retina. Using these similarities, we have identified a Drosphila homolog, DmUNC-119, which is expressed in the Drosophila nervous system. The predicted C. elegans, human and Drosophila gene products are conserved across two domains. Expression of portions of HRG4/HsUNC-119 or DmUNC-119, directed by the unc-119 promoter, can fully rescue the C. elegans unc-119 mutant phenotype. We tested the ability of portions of HRG4/HsUNC-119 to rescue, and found that its function in C. elegans requires the conserved carboxyl terminus, while the dissimilar amino terminus is dispensable. UNC-119, HRG4 and DmUNC-119 constitute members of a new class of neural genes whose common function has been maintained through metazoan evolution.

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. Developmental Dynamics 239:1315–1329, 2010.

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.

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.