Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans

Signaling by fibroblast growth factors (FGFs) and their receptors has been previously implicated in control of cell proliferation, differentiation and migration. Here we report a novel role for signaling by the EGL-15 FGFR of Caenorhabditis elegans in controlling protein degradation in differentiated muscle. Activation of EGL-15, by means of a reduction of function mutation (clr-1) affecting an inhibitory phosphatase, triggers protein degradation in adult muscle cells using a pre-existing proteolytic system. This activation is not suppressed by mutation in either of the known genes encoding FGF ligands (egl-17 or let-756) but is well suppressed when both are mutated, indicating that either ligand is sufficient and at least one is necessary for FGFR activation. Activity of the Ras pathway through mitogen-activated protein kinase (MAPK) is required to trigger protein degradation. This is the first report that degradation of intracellular protein can be triggered by a growth factor receptor using an identified signal transduction pathway. The data raise the possibility that FGF-triggered proteolysis may be relevant to muscle remodeling or dedifferentiation.     

Functional phylogeny relates LET-756 to fibroblast growth factor 9

Fibroblast growth factors (FGFs) are secreted regulatory proteins involved in various developmental processes. In vertebrates, the FGF superfamily comprises 22 members. In non-vertebrates, six FGF genes have been identified in Ciona intestinalis, three in Drosophila melanogaster, and two (let-756 and egl-17) in Caenorhabditis elegans. The core of LET-756 shares a 30-50% sequence identity with the various members of the superfamily. The relationships between vertebrate and non-vertebrate FGFs are not clear. We made chimeric FGFs by replacing the core region of LET-756 by the cores of various mammalian, fly, and worm FGFs. LET-756 deleted in its core region was no longer able to rescue the lethal phenotype of a let-756 null mutant, and only chimeras containing the cores of FGFs 9, 16, and 20 showed rescue capacity. This core contains an internal motif of six amino acid residues (EFISIA) whose deletion or mutation abolished both the rescue activity and FGF secretion in the supernatant of transfected COS-1 cells. Chimera containing the core of C. intestinalis FGF9/16/20, a potential ortholog of FGF9 lacking the complete EFISIA motif, was not able to rescue the lethal phenotype or be secreted. However, the introduction of the EFISIA motif restored both activities. The data show that the EFISIA motif in the core of LET-756 is essential for its biological activity and that FGFs 9, 16, and 20, which contain that motif, are functionally close to LET-756 and may be evolutionary related. This non-classical mode of secretion using an internal motif is conserved throughout evolution.     

The Caenorhabditis elegans spalt-like gene sem-4 restricts touch cell fate by repressing the selector Hox gene egl-5 and the effector gene mec-3

Members of the spalt (sal) gene family encode zinc-finger proteins that are putative tumor suppressors and regulate anteroposterior (AP) patterning, cellular identity, and, possibly, cell cycle progression. The mechanism through which sal genes carry out these functions is unclear. The Caenorhabditis elegans sal gene sem-4 controls the fate of several different cell types, including neurons, muscle and hypodermis. Mutation of sem-4 transforms particular tail neurons into touch-neuron-like cells. In wild-type C. elegans, six touch receptor neurons mediate the response of the worm to gentle touch. All six touch neurons normally express the LIM homeobox gene mec-3. A subset, the two PLM cells, also express the Hox gene egl-5, an Abdominal-B homolog, which we find is required for correct mec-3 expression in these cells. The abnormal touch-neuron-like-cells in sem-4 animals express mec-3; we show that a subset also express egl-5. We report: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. We conclude that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes, that sem-4 indirectly modulates mec-3 expression through its repression of egl-5 and that this negative regulation is required for proper determination of neuronal fates. We suggest that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes.     

Muscle arm development in Caenorhabditis elegans

In several types of animals, muscle cells use membrane extensions to contact motor axons during development. To better understand the process of membrane extension in muscle cells, we investigated the development of Caenorhabditis elegans muscle arms, which extend to motor axons and form the postsynaptic element of the neuromuscular junction. We found that muscle arm development is a highly regulated process: the number of muscle arms extended by each muscle, the shape of the muscle arms and the path taken by the muscle arms to reach the motor axons are largely stereotypical. We also investigated the role of several cytoskeletal components and regulators during arm development, and found that tropomyosin (LEV-11), the actin depolymerizing activity of ADF/cofilin (UNC-60B) and, surprisingly, myosin heavy chain B (UNC-54) are each required for muscle arm extension. This is the first evidence that UNC-54, which is found in thick filaments of sarcomeres, can also play a role in membrane extension. The muscle arm phenotypes produced when these genes are mutated support a 'two-phase' model that distinguishes passive muscle arm development in embryogenesis from active muscle arm extension during larval development.     

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.     

FGF and PI3 kinase signaling pathways antagonistically modulate sex muscle differentiation in C. elegans

Myogenesis in vertebrate myocytes is promoted by activation of the phosphatidyl-inositol 3'-kinase (PI3 kinase) pathway and inhibited by fibroblast growth factor (FGF) signaling. We show that hyperactivation of the Caenorhabditis elegans FGF receptor, EGL-15, similarly inhibits the differentiation of the hermaphrodite sex muscles. Activation of the PI3 kinase signaling pathway can partially suppress this differentiation defect, mimicking the antagonistic relationship between these two pathways known to influence vertebrate myogenesis. When ectopically expressed in body wall muscle precursor cells, hyperactivated EGL-15 can also interfere with the proper development of the body wall musculature. Hyperactivation of EGL-15 has also revealed additional effects on a number of fundamental processes within the postembryonic muscle lineage, such as cell division polarity. These studies provide important in vivo insights into the contribution of FGF signaling events to myogenesis.     

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.     

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.     

The adaptor protein soc-1/Gab1 modifies growth factor receptor output in Caenorhabditis elegans

Previous genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK)-stimulated Ras/Map kinase signaling through the recruitment of csw/ptp-2/Shp2. Using sensitized assays in Caenorhabditis elegans for let-23/Egfr and daf-2/InsR (insulin receptor-like) signaling, it is shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K)-mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional as the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling that were examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K-mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma-mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK-signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma-mediated signaling.     

Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans

We designed an automatic system to measure body length, diameters and volume of a C. elegans worm. By using this system, mutants with an increased body volume exceeding 50% were isolated. Four of them are grossly normal in morphology and development, grow longer to be almost twice as big, and have weak egg-laying defects and extended lifespan. All the four mutants have a mutation in the egl-4 gene. We show that the egl-4 gene encodes cGMP-dependent protein kinases. egl-4 promoter::gfp fusion genes are mainly expressed in head neurons, hypodermis, intestine and body wall muscles. Procedures to analyze morphology and volume of major organs were developed. The results indicate that volumes of intestine, hypodermis and muscle and cell volumes in intestine and muscle are increased in the egl-4 mutants, whereas cell numbers are not. Experiments on genetic interaction suggest that the cGMP-EGL-4 signaling pathway represses body size and lifespan through DBL-1/TGF-beta and insulin pathways, respectively.     

Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs

BACKGROUND: The let-7 and lin-4 microRNAs belong to a class of temporally expressed, noncoding regulatory RNAs that function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the rapid evolution of new morphologies. let-7 is highly conserved and in Drosophila is temporally coregulated with the lin-4 homolog, miR-125. Little is known, however, about their requirement outside the nematode or whether they universally control the timing of developmental processes. RESULTS: We report the generation of a Drosophila mutant that lacks let-7 and miR-125 activities and that leads to a pleiotropic phenotype arising during metamorphosis. We focus on two defects and demonstrate that loss of let-7 and miR-125 results in temporal delays in two distinct metamorphic processes: the terminal cell-cycle exit in the wing and maturation of neuromuscular junctions (NMJs) at adult abdominal muscles. We identify the abrupt (ab) gene, encoding a nuclear protein, as a bona fide let-7 target and provide evidence that let-7 governs the maturation rate of abdominal NMJs during metamorphosis by regulating ab expression. CONCLUSIONS: Drosophila Iet-7 and miR-125 mutants exhibit temporal misregulation of specific metamorphic processes. As in C. elegans, Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit, indicating that its function as a heterochronic microRNA is conserved. The ab gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. Our results suggest that let-7 and miR-125 serve as conserved regulators of events necessary for the transition from juvenile to adult life stages.     

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.     

β-Catenin stabilization in skeletal muscles, but not in motor neurons, leads to aberrant motor innervation of the muscle during neuromuscular development in mice

β-Catenin, a key component of the Wnt signaling pathway, has been implicated in the development of the neuromuscular junction (NMJ) in mice, but its precise role in this process remains unclear. Here we use a β-catenin gain-of-function mouse model to stabilize β-catenin selectively in either skeletal muscles or motor neurons. We found that β-catenin stabilization in skeletal muscles resulted in increased motor axon number and excessive intramuscular nerve defasciculation and branching. In contrast, β-catenin stabilization in motor neurons had no adverse effect on motor innervation pattern. Furthermore, stabilization of β-catenin, either in skeletal muscles or in motor neurons, had no adverse effect on the formation and function of the NMJ. Our findings demonstrate that β-catenin levels in developing muscles in mice are crucial for proper muscle innervation, rather than specifically affecting synapse formation at the NMJ, and that the regulation of muscle innervation by β-catenin is mediated by a non-cell autonomous mechanism.     

Characterization of seven genes affecting Caenorhabditis elegans hindgut development.

We have identified and characterized 12 mutations in seven genes that affect the development of the Caenorhabditis elegans hindgut. We find that the mutations can disrupt the postembryonic development of the male-specific blast cells within the hindgut, the hindgut morphology in both males and hermaphrodites, and in some cases, the expression of a hindgut marker in hermaphrodite animals. Mutations in several of the genes also affect viability. On the basis of their mutant phenotypes, we propose that the genes fall into four distinct classes: (1) egl-5 is required for regional identity of the tail; (2) sem-4 is required for a variety of ectodermal and mesodermal cell types, including cells in the hindgut; (3) two genes, lin-49 and lin-59, affect development of many cells, including hindgut; and (4) three genes, mab-9, egl-38, and lin-48, are required for patterning fates within the hindgut, making certain hindgut cells different from others. We also describe a new allele of the Pax gene egl-38 that is temperature sensitive and affects the conserved beta-hairpin of the EGL-38 paired domain. Our results suggest that a combination of different factors contribute to normal C. elegans hindgut development.     

ARK-1 inhibits EGFR signaling in C. elegans

A screen for synthetic enhancers of sli-1 identified ark-1 (forAck-related tyrosine kinase), a novel inhibitor of let-23 EGFR signaling in C. elegans. An ark-1 mutation synergizes with mutations in other negative regulators of let-23, resulting in increased RAS signaling. Genetic analysis suggests that ARK-1 acts upstream of RAS and is dependent upon SEM-5. ARK-1 inhibits LET-23-mediated ovulation, a RAS-independent function. ARK-1 physically interacts with SEM-5 in the yeast two-hybrid assay. We find that sem-5 also has a negative function in let-23-mediated ovulation and suggest that this negative function is mediated by the recruitment of inhibitors such as ARK-1.     

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.     

Caenorhabditis elegans Galphaq regulates egg-laying behavior via a PLCbeta-independent and serotonin-dependent signaling pathway and likely functions both in the nervous system and in muscle

egl-30 encodes the single C. elegans ortholog of vertebrate Galphaq family members. We analyzed the expression pattern of EGL-30 and found that it is broadly expressed, with highest expression in the nervous system and in pharyngeal muscle. We isolated dominant, gain-of-function alleles of egl-30 as intragenic revertants of an egl-30 reduction-of-function mutation. Using these gain-of-function mutants and existing reduction-of-function mutants, we examined the site and mode of action of EGL-30. On the basis of pharmacological analysis, it has been determined that egl-30 functions both in the nervous system and in the vulval muscles for egg-laying behavior. Genetic epistasis over mutations that eliminate detectable levels of serotonin reveals that egl-30 requires serotonin to regulate egg laying. Furthermore, pharmacological response assays strongly suggest that EGL-30 may directly couple to a serotonin receptor to mediate egg laying. We also examined genetic interactions with mutations in the gene that encodes the single C. elegans homolog of PLCbeta and mutations in genes that encode signaling molecules downstream of PLCbeta. We conclude that PLCbeta functions in parallel with egl-30 with respect to egg laying or is not the major effector of EGL-30. In contrast, PLCbeta-mediated signaling is likely downstream of EGL-30 with respect to pharyngeal-pumping behavior. Our data indicate that there are multiple signaling pathways downstream of EGL-30 and that different pathways could predominate with respect to the regulation of different behaviors.     

FGF signaling functions in the hypodermis to regulate fluid balance in C. elegans

Signaling by the Caenorhabditis elegans fibroblast growth factor receptor EGL-15 is activated by LET-756, a fibroblast growth factor, and attenuated by CLR-1, a receptor tyrosine phosphatase. Hyperactive EGL-15 signaling results in a dramatic Clr phenotype characterized by the accumulation of clear fluid within the pseudocoelomic space, suggesting that regulated EGL-15 signaling is essential for fluid homeostasis in C. elegans. To determine the cellular focus of EGL-15 signaling, we identified an enhancer element (e15) within the egl-15 promoter, which is both necessary for the promoter activity and sufficient when duplicated to drive either egl-15 or clr-1 rescue activity. This enhancer drives GFP expression in hypodermal cells. Consistent with this finding, immunofluorescence studies of EGL-15 indicate that EGL-15 is expressed in hypodermal cells, and hypodermal promoters can drive full clr-1 and egl-15 rescue activity. Moreover, a mosaic analysis of mpk-1, which acts downstream of egl-15, suggests that its suppression of Clr (Soc) function is required in the hypodermis. These results suggest that EGL-15 and CLR-1 act in the hypodermis to regulate fluid homeostasis in worms.