The Caenorhabditis elegans synthetic multivulva genes prevent ras pathway activation by tightly repressing global ectopic expression of lin-3 EGF

The Caenorhabditis elegans class A and B synthetic multivulva (synMuv) genes redundantly antagonize an EGF/Ras pathway to prevent ectopic vulval induction. We identify a class A synMuv mutation in the promoter of the lin-3 EGF gene, establishing that lin-3 is the key biological target of the class A synMuv genes in vulval development and that the repressive activities of the class A and B synMuv pathways are integrated at the level of lin-3 expression. Using FISH with single mRNA molecule resolution, we find that lin-3 EGF expression is tightly restricted to only a few tissues in wild-type animals, including the germline. In synMuv double mutants, lin-3 EGF is ectopically expressed at low levels throughout the animal. Our findings reveal that the widespread ectopic expression of a growth factor mRNA at concentrations much lower than that in the normal domain of expression can abnormally activate the Ras pathway and alter cell fates. These results suggest hypotheses for the mechanistic basis of the functional redundancy between the tumor-suppressor-like class A and B synMuv genes: the class A synMuv genes either directly or indirectly specifically repress ectopic lin-3 expression; while the class B synMuv genes might function similarly, but alternatively might act to repress lin-3 as a consequence of their role in preventing cells from adopting a germline-like fate. Analogous genes in mammals might function as tumor suppressors by preventing broad ectopic expression of EGF-like ligands.     

The C. elegans Mi-2 chromatin-remodelling proteins function in vulval cell fate determination

The Mi-2 protein is the central component of the recently isolated NuRD nucleosome remodelling and histone deacetylase complex. Although the NuRD complex has been the subject of extensive biochemical analyses, little is known about its biological function. Here we show that the two C. elegans Mi-2 homologues, LET-418 and CHD-3, play essential roles during development. The two proteins possess both shared and unique functions during vulval cell fate determination, including antagonism of the Ras signalling pathway required for vulval cell fate induction and the proper execution of the 2 degrees cell fate of vulval precursor cells, a process under the control of LIN-12 Notch signalling.     

Protruding vulva mutants identify novel loci and Wnt signaling factors that function during Caenorhabditis elegans vulva development

The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.     

Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes

The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.     

Control and integration of cell signaling pathways during C. Elegans vulval development

Vulval development in the Caenorhabditis elegans hermaphrodite represents a simple, genetically tractable system for studying how cell signaling events control cell fata decisions. Current models suggest that proper specification of vulval cell fates relies on the integration of multiple signaling systems, including one that involves a receptor tyrosine kinase (RTK)→Ras→mitogen activated protein kinase (MAPK) cascade and one that involves a LIN-12/Notch family receptor. In this review, we first discuss how genetic strategies are being used to identify and analyze components that control vulval cell fate decisions. We then describe the different signaling systems that have been elucidated and how they relate to one another. Finally, we highlight several recently characterized genes that encode positive regulators, negative regulators or potential targets of the RTK→Ras→MAPK cascade involved in vulval induction.     

Multiple levels of redundant processes inhibit Caenorhabditis elegans vulval cell fates

Many mutations cause obvious abnormalities only when combined with other mutations. Such synthetic interactions can be the result of redundant gene functions. In Caenorhabditis elegans, the synthetic multivulva (synMuv) genes have been grouped into multiple classes that redundantly inhibit vulval cell fates. Animals with one or more mutations of the same class undergo wild-type vulval development, whereas animals with mutations of any two classes have a multivulva phenotype. By varying temperature and genetic background, we determined that mutations in most synMuv genes within a single synMuv class enhance each other. However, in a few cases no enhancement was observed. For example, mutations that affect an Mi2 homolog and a histone methyltransferase are of the same class and do not show enhancement. We suggest that such sets of genes function together in vivo and in at least some cases encode proteins that interact physically. The approach of genetic enhancement can be applied more broadly to identify potential protein complexes as well as redundant processes or pathways. Many synMuv genes are evolutionarily conserved, and the genetic relationships we have identified might define the functions not only of synMuv genes in C. elegans but also of their homologs in other organisms.     

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

Background

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

Results

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

Conclusion

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.     

The two steps of vulval induction in Oscheius tipulae CEW1 recruit common regulators including a MEK kinase

The cell interactions that specify the spatial pattern of vulval precursor cell (VPC) fates differ between the nematodes Oscheius tipulae CEW1 and Caenorhabditis elegans. In the former, the centered pattern of fates is obtained by two successive inductions from the gonadal anchor cell, whereas in the latter, a single inductive step by the anchor cell (EGF-Ras-MAP kinase pathway) can act as a morphogen and is reinforced by lateral signaling between the vulval precursors (Notch pathway). We performed a genetic screen for vulva mutants in O. tipulae CEW1. Here we present the mutants that specifically affect the vulval induction mechanisms. Phenotypic and epistatic analyses of these mutants show that both vulval induction steps share common components, one of which appears to be MEK kinase(s). Moreover, the inductive pathway (including MEK kinase) influences the competence of the vulval precursor cells and more strikingly their division pattern as well, irrespective of their vulval fate. Finally, a comparison of vulval mutant phenotypes obtained in C. elegans and O. tipulae CEW1 highlights the evolution of vulval induction mechanisms between the two species.