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.     

Two homologous regulatory genes, lin-12 and glp-1, have overlapping functions.

Two homologous genes, lin-12 and glp-1, encode transmembrane proteins required for regulatory cell interactions during C. elegans development. Based on their single mutant phenotypes, each gene has been thought to govern a distinct set of cell fates. We show here that lin-12 and glp-1 are functionally redundant during embryogenesis: Unlike either single mutant, the lin-12 glp-1 double mutant dies soon after hatching. Numerous cellular defects can be observed in these Lag (for lin-12 and glp-1) double mutants. Furthermore, we have identified two genes, lag-1 and lag-2, that appear to be required for both lin-12 and glp-1-mediated cell interactions. Strong loss-of-function lag mutants are phenotypically indistinguishable from the lin-12 glp-1 double; weak lag mutants have phenotypes typical of lin-12 and glp-1 single mutants. We speculate that the lin-12 and glp-1 proteins are biochemically interchangeable and that their divergent roles in development may rely largely on differences in gene expression.     

Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis.

We have used Tn917lacZ to mutagenize the Bacillus subtilis chromosome and have isolated mutants that are defective in chemotaxis and motility. Mapping of the transposon inserts identified two new loci. Mutations in one of these loci generated mutants that had paralyzed flagella. Accordingly, we designate this a mot locus. The other locus is closely linked to the first and encodes proteins specifying chemotaxis functions. This locus is designated the cheX locus. Both the mot and cheX loci map close to ptsI. An additional transposon insert that maps in the hag locus was obtained. The pattern of beta-galactosidase expression from some of the transposons suggested that the mot locus is regulated by sigD, a minor sigma factor of B. subtilis. The cheX locus appeared to be under the control of vegetative sigA. Four transposon inserts were mapped to a previously characterized che locus near spcB. These mutants did not produce flagellin and were defective in the methylation of the methyl-accepting chemotaxis proteins. This locus probably encodes proteins required for flagellum biosynthesis and other proteins that are required for the methylation response.     

Tissue-specific regulation of the LIM homeobox gene lin-11 during development of the Caenorhabditis elegans egg-laying system

The egg-laying system of Caenorhabditis elegans hermaphrodites requires development of the vulva and its precise connection with the uterus. This process is regulated by LET-23-mediated epidermal growth factor signaling and LIN-12-mediated lateral signaling pathways. Among the nuclear factors that act downstream of these pathways, the LIM homeobox gene lin-11 plays a major role. lin-11 mutant animals are egg-laying defective because of the abnormalities in vulval lineage and uterine seam-cell formation. However, the mechanisms providing specificity to lin-11 function are not understood. Here, we examine the regulation of lin-11 during development of the egg-laying system. Our results demonstrate that the tissue-specific expression of lin-11 is controlled by two distinct regulatory elements that function as independent modules and together specify a wild-type egg-laying system. A uterine pi lineage module depends on the LIN-12/Notch signaling, while a vulval module depends on the LIN-17-mediated Wnt signaling. These results provide a unique example of the tissue-specific regulation of a LIM homeobox gene by two evolutionarily conserved signaling pathways. Finally, we provide evidence that the regulation of lin-11 by LIN-12/Notch signaling is directly mediated by the Su(H)/CBF1 family member LAG-1.     

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.     

Evolution of discrete Notch-like receptors from a distant gene duplication in Caenorhabditis

SUMMARY Caenorhabditis elegans possesses two Notch-like receptors, LIN-12 and GLP-1, which have both overlapping and individual biological functions. We examined the lin-12 and glp-1 genes in closely related nematodes to learn about their evolution. Here we report molecular and functional analyses of lin-12 orthologs from two related nematodes, C. briggsae (Cb) and C. remanei (Cr). In addition, we compare these lin-12 findings with similar studies of Cb-glp-1 and Cr-glp-1 orthologs. Cb-LIN-12 and Cr-LIN-12 retain the same number and order of motifs as Ce-LIN-12. Intriguingly, we find that LIN-12 conservation differs from that of GLP-1 in two respects. First, individual motifs are conserved to a different degree for the two receptors. For example, the transmembrane domain is 16-32% identical among LIN-12 orthologs but 65-70% identical among GLP-1 orthologs. Second, certain amino acids are conserved in a receptor-specific manner, a phenomenon most prevalent in the CC-linker. We suggest that LIN-12 and GLP-1 have been molded by selective constraints that are receptor specific and that the two proteins may not be entirely interchangeable. To analyze the functions of the lin-12 orthologs, we used RNA-mediated interference (RNAi). Cb-lin-12(RNAi) or Cr-lin-12(RNAi) progeny are nearly 100% Lag, a larval lethality typical of C. elegans lin-12 glp-1 double mutants, but not the primary defect observed in Ce-lin-12 null mutants or Ce-lin-12(RNAi). Therefore, LIN-12 functions are similar, but not identical, among the Caenorhabditis species. We suggest that ancestral functions may have been divided between LIN-12 and GLP-1 receptors in a process contributing to the retention of both genes after gene duplication (i.e., subfunctionalization).     

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.     

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.     

The C. elegans heterochronic gene lin-46 affects developmental timing at two larval stages and encodes a relative of the scaffolding protein gephyrin

The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. We identified a new heterochronic gene, lin-46, from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. Our analysis indicates that lin-46 acts at a step immediately downstream of lin-28, affecting both the regulation of the heterochronic gene pathway and execution of stage-specific developmental events at two stages: the third larval stage and adult. We also show that lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. Our findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development.     

Genetic and Phenotypic Studies of Hypomorphic Lin-12 Mutants in Caenorhabditis Elegans

The lin-12 gene of Caenorhabditis elegans is thought to encode a receptor for intercellular signals that specify certain cell fates during development. We describe several alleles of lin-12 that reduce but do not eliminate lin-12 activity (hypomorphic alleles). These alleles cause a novel egg-laying defective (Egl) phenotype in hermaphrodites as well as incompletely penetrant cell fate transformations seen with high penetrance in lin-12 null mutants. Characterization of the Egl phenotype revealed additional roles of lin-12 in the development of the egg-laying system that were not apparent from studying lin-12 null mutants: lin-12 activity is required for proper early vulval morphogenesis as well as for some unknown later aspect of egg-laying system development. Reversion of the Egl phenotype caused by one lin-12 hypomorphic allele was used to identify potential interacting genes as described in the accompanying paper.     

Point mutations identify a conserved region of the saccharomyces cerevisiae AFR1 gene that is essential for both the pheromone signaling and morphogenesis functions

Mating pheromone receptors activate a G protein signal pathway that leads to the conjugation of the yeast Saccharomyces cerevisiae. This pathway also induces the production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required to form pointed projections of new growth that become the site of cell fusion during mating. Afr1p lacks strong similarity to any well-characterized proteins to help predict how it acts. Therefore, we investigated the relationship between the different functions of Afr1p by isolating and characterizing seven mutants that were defective in regulating pheromone signaling. The AFR1 mutants were also defective when expressed as fusions to STE2, the alpha-factor receptor, indicating that the mutant Afr1 proteins are defective in function and not in co-localizing with receptors. The mutant genes contained four distinct point mutations that all occurred between codons 254 and 263, identifying a region that is critical for AFR1 function. Consistent with this, we found that the corresponding region is very highly conserved in the Afr1p homologs from the yeasts S. uvarum and S. douglasii. In contrast, there were no detectable effects on pheromone signaling caused by deletion or overexpression of YER158c, an open reading frame with overall sequence similarity to Afr1p that lacks this essential region. Interestingly, all of the AFR1 mutants showed a defect in their ability to form mating projections that was proportional to their defect in regulating pheromone signaling. This suggests that both functions may be due to the same action of Afr1p. Thus, these studies identify a specific region of Afr1p that is critical for its function in both signaling and morphogenesis.     

Multiple mechanisms are involved in regulating the expression of the developmental timing regulator lin-28 in Caenorhabditis elegans

The timing of postembryonic developmental programs in Caenorhabditis elegans is regulated by a set of so-called heterochronic genes, including lin-28 that specifies second larval programs. lin-66 mutations described herein cause delays in vulval and seam cell differentiation, indicating a role for lin-66 in timing regulation. A mutation in daf-12/nuclear receptor or alg-1/argonaute dramatically enhances the retarded phenotypes of the lin-66 mutants, and these phenotypes are suppressed by a lin-28 null allele. We further show that the LIN-28 protein level is upregulated in the lin-66 mutants and that this regulation is mediated by the 3'UTR of lin-28. We have also identified a potential daf-12-response element within lin-28 3'UTR and show that two microRNA (miRNA) (lin-4 and let-7)-binding sites mediate redundant inhibitory activities that are likely lin-66-independent. Quantitative PCR data suggest that the lin-28 mRNA level is affected by lin-14 and miRNA regulation, but not by daf-12 and lin-66 regulation. These results suggest that lin-28 expression is regulated by multiple independent mechanisms including LIN-14-mediated upregulation of mRNA level, miRNAs-mediated RNA degradation, LIN-66-mediated translational inhibition and DAF-12-involved translation promotion.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

Identification of heterochronic mutants in Caenorhabditis elegans. Temporal misexpression of a collagen::green fluorescent protein fusion gene.

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 specify the timing of lateral hypodermal seam cell terminal differentiation in Caenorhabditis elegans. We devised a screen to identify additional genes involved in this developmental timing mechanism based on identification of mutants that exhibit temporal misexpression from the col-19 promoter, a downstream target of the heterochronic gene pathway. We fused the col-19 promoter to the green fluorescent protein gene (gfp) and demonstrated that hypodermal expression of the fusion gene is adult-specific in wild-type animals and temporally regulated by the heterochronic gene pathway. We generated a transgenic strain in which the col-19::gfp fusion construct is not expressed because of mutation of lin-4, which prevents seam cell terminal differentiation. We have identified and characterized 26 mutations that restore col-19::gfp expression in the lin-4 mutant background. Most of the mutations also restore other aspects of the seam cell terminal differentiation program that are defective in lin-4 mutant animals. Twelve mutations are alleles of three previously identified genes known to be required for proper timing of hypodermal terminal differentiation. Among these are four new alleles of lin-42, a heterochronic gene for which a single allele had been described previously. Two mutations define a new gene, lin-58. When separated from lin-4, the lin-58 mutations cause precocious seam cell terminal differentiation and thus define a new member of the heterochronic gene pathway.     

The Caenorhabditis Elegans Sel-1 Gene, a Negative Regulator of Lin-12 and Glp-1, Encodes a Predicted Extracellular Protein

The Caenorhabditis elegans lin-12 and glp-1 genes encode members of the LIN-12/NOTCH family of receptors. The sel-1 gene was identified as an extragenic suppressor of a lin-12 hypomorphic mutant. We show in this report that the sel-1 null phenotype is wild type, except for an apparent elevation in lin-12 and glp-1 activity in sensitized genetic backgrounds, and that this genetic interaction seems to be lin-12 and glp-1 specific. We also find that sel-1 encodes a predicted extracellular protein, with a domain sharing sequence similarity to predicted proteins from humans and yeast. SEL-1 may interact with the LIN-12 and GLP-1 receptors and/or their respective ligands to down-regulate signaling.     

The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when nutrients are limiting. Defective fruiting-body formation and sporulation result from mutations in the sasA locus, which encodes the wzm wzt wbgA (formerly rfbABC ) lipopolysaccharide (LPS) O-antigen biosynthesis genes. Mutants carrying these same sasA mutations are defective in social motility and form small glossy colonies. We report here that the developmental and motility phenotypes of four mutants each containing different Tn 5 insertions in LPS O-antigen biosynthesis genes are similar to those of the original sasA locus mutants. All of the LPS O-antigen mutants tested exhibited defective developmental aggregation and sporulated at only 0.02–15% of the wild-type level. In addition, all of the LPS O-antigen mutants were determined by genetic analyses to be wild type for adventurous motility and defective in social motility, indicating that the LPS O-antigen is necessary for normal development and social motility. The two previously identified cell-surface components required for social motility, type IV pili and the protein-associated polysaccharide material termed fibrils, were detected on the surfaces of all of the LPS O-antigen mutants. This indicates that LPS O-antigen is a third cell-surface component required for social motility.