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.     

Caenorhabditis elegans lin-13, a member of the LIN-35 Rb class of genes involved in vulval development, encodes a protein with zinc fingers and an LXCXE motif

The SynMuv genes appear to be involved in providing a signal that inhibits vulval precursor cells from adopting vulval fates in Caenorhabditis elegans. One group of SynMuv genes, termed class B, includes genes encoding proteins related to the tumor suppressor Rb and RbAp48, a protein that binds Rb. Here, we provide genetic evidence that lin-13 behaves as a class B SynMuv gene. We show that null alleles of lin-13 are temperature sensitive and maternally rescued, resulting in phenotypes ranging in severity from L2 arrest (when both maternal and zygotic activities are removed at 25 degrees ), to sterile Multivulva (when only zygotic activity is removed at 25 degrees ), to sterile non-Multivulva (when both maternal and zygotic activities are removed at 15 degrees ), to wild-type/class B SynMuv (when only zygotic activity is removed at 15 degrees ). We also show that LIN-13 is a nuclear protein that contains multiple zinc fingers and a motif, LXCXE, that has been implicated in Rb binding. These results together suggest a role for LIN-13 in Rb-mediated repression of vulval fates.     

lin-35/Rb cooperates with the SWI/SNF complex to control Caenorhabditis elegans larval development

Null mutations in lin-35, the Caenorhabditis elegans ortholog of the mammalian Rb protein, cause no obvious morphological defects. Using a genetic approach to identify genes that may function redundantly with lin-35, we have isolated a mutation in the C. elegans psa-1 gene. lin-35; psa-1 double mutants display severe developmental defects leading to early larval arrest and adult sterility. The psa-1 gene has previously been shown to encode a C. elegans homolog of yeast SWI3, a critical component of the SWI/SNF complex, and has been shown to regulate asymmetric cell divisions during C. elegans development. We observed strong genetic interactions between psa-1 and lin-35 as well as a subset of the class B synMuv genes that include lin-37 and lin-9. Loss-of-function mutations in lin-35, lin-37, and lin-9 strongly enhanced the defects of asymmetric T cell division associated with a psa-1 mutation. Our results suggest that LIN-35/Rb and a certain class B synMuv proteins collaborate with the SWI/SNF protein complex to regulate the T cell division as well as other events essential for larval growth.     

Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.     

C. elegans class B synthetic multivulva genes act in G(1) regulation

The single C. elegans member of the retinoblastoma gene family, lin-35 Rb, was originally identified as a synthetic Multivulva (synMuv) gene [1]. These genes form two redundant classes, A and B, that repress ectopic vulval cell fate induction. Recently, we demonstrated that lin-35 Rb also acts as a negative regulator of G(1) progression and likely is the major target of cyd-1 Cyclin D and cdk-4 CDK4/6. Here, we describe G(1) control functions for several other class B synMuv genes. We found that efl-1 E2F negatively regulates cell cycle entry, while dpl-1 DP appeared to act both as a positive and negative regulator. In addition, we identified a negative G(1) regulatory function for lin-9 ALY, as well as lin-15B and lin-36, which encode novel proteins. Inactivation of lin-35 Rb, efl-1, or lin-36 allowed S phase entry in the absence of cyd-1/cdk-4 and increased ectopic cell division when combined with cki-1 Cip/Kip RNAi. These data are consistent with lin-35 Rb, efl-1, and lin-36 acting in a common pathway or complex that negatively regulates G(1) progression. In contrast, lin-15B appeared to act in parallel to lin-35. Our results demonstrate the potential for genetic identification of novel G(1) regulators in C. elegans.     

LIN-14 inhibition of LIN-12 contributes to precision and timing of C. elegans vulval fate patterning

Studies of C. elegans vulval development have illuminated mechanisms underlying cell fate specification and elucidated intercellular signaling pathways [1]. The vulval precursor cells (VPCs) are spatially patterned during the L3 stage by the EGFR-Ras-MAPK-mediated inductive signal and the LIN-12/Notch-mediated lateral signal. The pattern is both precise and robust [2] because of crosstalk between these pathways [3]. Signaling is also regulated temporally, because constitutive activation of the spatial patterning pathways does not alter the timing of VPC fate specification [4, 5]. The heterochronic genes, including the microRNA lin-4 and its target lin-14, constitute a temporal control mechanism used in different contexts [6-8]. We find that lin-4 specifically controls the activity of LIN-12/Notch through lin-14, but not other known targets, and that persistent lin-14 blocks LIN-12 activity without interfering with the key events of LIN-12/Notch signal transduction. In the L2 stage, there is sufficient lin-14 activity to inhibit constitutive lin-12. Our results suggest that lin-4 and lin-14 contribute to spatial patterning through temporal gating of LIN-12. We propose that in the L2 stage, lin-14 sets a high threshold for LIN-12 activation to help prevent premature activation of LIN-12 by ligands expressed in other cells in the vicinity, thereby contributing to the precision and robustness of VPC fate patterning.     

Structural requirements for the tissue-specific and tissue-general functions of the Caenorhabditis elegans epidermal growth factor LIN-3.

Caenorhabditis elegans lin-3 encodes a homolog of the epidermal growth factor (EGF) family of growth factors. LIN-3 is the inductive signal for hermaphrodite vulval differentiation, and it is required for animal viability, hermaphrodite fertility, and the specification of anterior cell fates in the male B cell lineage. We describe the cloning of a lin-3 homolog from C. briggsae, sequence comparison of C. elegans lin-3 with C. briggsae lin-3, and the determination of molecular lesions in alleles of C. elegans lin-3, including three new alleles. We also analyzed the severity of phenotypes caused by the new and existing alleles of lin-3. Correlation of mutant phenotypes and their molecular lesions, as well as sequence comparison between two species, reveal that the EGF motif and the N-terminal portion of the cytoplasmic domain are important for the functions of LIN-3 in all tissues, while the C-terminal portion of the cytoplasmic domain is involved in the tissue-specific functions of lin-3. We discuss how the structure of lin-3 contributes to its functions in multiple developmental processes.     

LET-23-mediated signal transduction during Caenorhabditis elegans development

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming α-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the “vulvaless” phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the “multivulva” phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development—larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways. © 1995 wiley-Liss, Inc.     

Identification of cis-regulatory elements from the C. elegans Hox gene lin-39 required for embryonic expression and for regulation by the transcription factors LIN-1, LIN-31 and LIN-39

Expression of the Caenorhabditis elegans Hox gene lin-39 begins in the embryo and continues in multiple larval cells, including the P cell lineages that generate ventral cord neurons (VCNs) and vulval precursor cells (VPCs). lin-39 is regulated by several factors and by Wnt and Ras signaling pathways; however, no cis-acting sites mediating lin-39 regulation have been identified. Here, we describe three elements controlling lin-39 expression: a 338-bp upstream fragment that directs embryonic expression in P5-P8 and their descendants in the larva, a 247-bp intronic region sufficient for VCN expression, and a 1.3-kb upstream cis-regulatory module that drives expression in the VPC P6.p in a Ras-dependent manner. Three trans-acting factors regulate expression via the 1.3-kb element. A single binding site for the ETS factor LIN-1 mediates repression in VPCs other than P6.p; however, loss of LIN-1 decreases expression in P6.p. Therefore, LIN-1 acts both negatively and positively on lin-39 in different VPCs. The Forkhead domain protein LIN-31 also acts positively on lin-39 in P6.p via this module. Finally, LIN-39 itself binds to this element, suggesting that LIN-39 autoregulates its expression in P6.p. Therefore, we have begun to unravel the cis-acting sites regulating lin-39 Hox gene expression and have shown that lin-39 is a direct target of the Ras pathway acting via LIN-1 and LIN-31.     

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.