New genes that interact with lin-35 Rb to negatively regulate the let-60 ras pathway in Caenorhabditis elegans

Previous studies have shown that a synthetic multivulva phenotype results from mutations in genes that antagonize the ras-mediated intercellular signaling system responsible for vulval induction in Caenorhabditis elegans. Synthetic multivulva mutations define two classes of genes, A and B, and a mutation in a gene of each class is required to produce the multivulva phenotype. The ectopic vulval tissue in multivulva animals is generated by vulval precursor cells that in the wild type do not generate vulval tissue. One of the class B synthetic multivulva genes, lin-35, encodes a protein similar to the retinoblastoma (Rb) protein. In this article, we describe the isolation and characterization of 50 synthetic multivulva mutations, the identification of new components of both the class A and class B lin-35 Rb pathways, and the cloning of lin-52, a class B gene that may have a conserved role in Rb-mediated signaling.     

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 gene lin-9,which acts in an Rb-related pathway, is required for gonadal sheath cell development and encodes a novel protein

The Caenorhabditis elegans gene lin-9 functions in an Rb-related pathway that acts antagonistically to a receptor tyrosine kinase/Ras signal transduction pathway controlling vulval induction. We show that lin-9 is also required for the development of the sheath cells in the hermaphrodite gonad and for the development of the male spicule, rays and gonad. lin-9 is transcribed as two alternatively spliced 2.4kb messages, which use two distinct polyadenylation sites and are SL1 trans-spliced. The conceptual translation of lin-9 cDNA sequences predicts proteins of 642 and 644 amino acids with a significant similarity to predicted Drosophila and vertebrate proteins. We suggest that lin-9 is the founding member of a new protein family that functions in Rb-related pathways in many species.     

sem-4 promotes vulval cell-fate determination in Caenorhabditis elegans through regulation of lin-39 Hox

Vulval cell-fate determination in Caenorhabditis elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39. sem-4 encodes a zinc finger protein with previously characterized roles in fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans (M. Basson and R. Horvitz, 1996, Genes Dev. 10, 1953-1965). By characterizing three new alleles of sem-4 that we identified in a screen for vulval-defective mutants, we determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. We analyzed sem-4 interactions with other genes involved in vulval differentiation and determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. We demonstrate that sem-4 regulates lin-39 expression and propose that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals.     

The C. elegans LIM homeobox gene lin-11 specifies multiple cell fates during vulval development

LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lin-11 have previously been shown to alter the cell division pattern of a subset of the 2 degrees lineage vulval cells. We demonstrate multiple functions of lin-11 during vulval development. We examined the fate of vulval cells in lin-11 mutant animals using five cellular markers and found that lin-11 is necessary for the patterning of both 1 degrees and 2 degrees lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, we show that lin-11 is initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2 degrees lineage cells. Using a conditional RNAi approach, we show that lin-11 regulates vulval morphogenesis. Finally, we show that LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning.     

Caenorhabditis elegans lin-45 raf is essential for larval viability, fertility and the induction of vulval cell fates

The protein kinase Raf is an important signaling protein. Raf activation is initiated by an interaction with GTP-bound Ras, and Raf functions in signal transmission by phosphorylating and activating a mitogen-activated protein (MAP) kinase kinase named MEK. We identified 13 mutations in the Caenorhabditis elegans lin-45 raf gene by screening for hermaphrodites with abnormal vulval formation or germline function. Weak, intermediate, and strong loss-of-function or null mutations were isolated. The phenotype caused by the most severe mutations demonstrates that lin-45 is essential for larval viability, fertility, and the induction of vulval cell fates. The lin-45(null) phenotype is similar to the mek-2(null) and mpk-1(null) phenotypes, indicating that LIN-45, MEK-2, and MPK-1 ERK MAP kinase function in a predominantly linear signaling pathway. The lin-45 alleles include three missense mutations that affect the Ras-binding domain, three missense mutations that affect the protein kinase domain, two missense mutations that affect the C-terminal 14-3-3 binding domain, three nonsense mutations, and one small deletion. The analysis of the missense mutations indicates that Ras binding, 14-3-3-binding, and protein kinase activity are necessary for full Raf function and suggests that a 14-3-3 protein positively regulates Raf-mediated signaling during C. elegans development.     

Sli-1, a Negative Regulator of Let-23-Mediated Signaling in C. Elegans

By screening for suppressors of hypomorphic mutations of let-23, a receptor tyrosine kinase necessary for vulval induction in Caenorhabditis elegans, we recovered >/=12 mutations defining the sli-1 (suppressor of lineage defect) locus. sli-1 mutations suppress four of five phenotypes associated with hypomorphic alleles of let-23 but do not suppress let-23 null alleles. Thus, a sli-1 mutation does not bypass the requirement for functional let-23 but rather allows more potent LET-23-dependent signaling. Mutations at the sli-1 locus are otherwise silent with respect to vulval differentiation and cause only a low-penetrance abnormal head phenotype. Mutations at sli-1 also suppress the vulval defects but not other defects associated with mutations of sem-5, whose product likely interacts with LET-23 protein during vulval induction. Mutations at sli-1 suppress lin-2, lin-7 and lin-10 mutations but only partially suppress lin-3 and let-60 mutations and do not suppress a lin-45 mutation. The sli-1 locus displays dosage sensitivity: severe reduction of function alleles of sli-1 are semidominant suppressors; a duplication of the sli-1 (+) region enhances the vulvaless phenotype of hypomorphic mutations of let-23. We propose that sli-1 is a negative regulator that acts at or near the LET-23-mediated step of the vulval induction pathway. Our analysis suggests that let-23 can activate distinct signaling pathways in different tissues: one pathway is required for vulval induction; another pathway is involved in hermaphrodite fertilty and is not regulated by sli-1.     

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.