Identification of Caenorhabditis elegans genes required for neuronal differentiation and migration.

To understand the mechanisms that guide migrating cells, we have been studying the embryonic migrations of the C. elegans canal-associated neurons (CANs). Here, we describe two screens used to identify genes involved in CAN migration. First, we screened for mutants that died as clear larvae (Clr) or had withered tails (Wit), phenotypes displayed by animals lacking normal CAN function. Second, we screened directly for mutants with missing or misplaced CANs. We isolated and characterized 30 mutants that defined 14 genes necessary for CAN migration. We found that one of the genes, ceh-10, specifies CAN fate. ceh-10 had been defined molecularly as encoding a homeodomain protein expressed in the CANs. Mutations that reduce ceh-10 function result in Wit animals with CANs that are partially defective in their migrations. Mutations that eliminate ceh-10 function result in Clr animals with CANs that fail to migrate or express CEH-23, a CAN differentiation marker. Null mutants also fail to express CEH-10, suggesting that CEH-10 regulates its own expression. Finally, we found that ceh-10 is necessary for the differentiation of AIY and RMED, two additional cells that express CEH-10.     

Control of cell migration during Caenorhabditis elegans development.

In Caenorhabditis elegans, cell migration is guided by localized cues, including molecules such as EGL-17/FGF and UNC-6/netrin. These external cues are linked to an intracellular response to migrate, at least in part, by CED-5, a homolog of DOCK180/MBC, and MIG-2, a Rac-like GTPase. In addition, metalloproteases are required for a cell migration that controls organ shape.     

Different isoforms of the C. elegans FGF receptor are required for attraction and repulsion of the migrating sex myoblasts

The Caenorhabditis elegans FGF receptor, EGL-15, is alternatively-spliced to yield two major isoforms that differ in their extracellular domains. The EGL-15(5A) isoform is necessary for the gonadal chemoattraction of the migrating sex myoblasts (SMs), while the EGL-15(5B) isoform is required for viability. Here we show that 5A is predominantly expressed in the M lineage, which gives rise to the migrating SMs and their sex muscle descendants, while 5B is predominantly expressed in the hypodermis. Tissue-specific expression, however, explains only part of the functional differences between these two receptor isoforms. 5A can carry out the reciprocal essential function of 5B when expressed in the hypodermis, but 5B is incapable of carrying out SM chemoattraction. Our data, therefore, indicate that the structural differences in these two isoforms contribute to their functional differences. Two lines of evidence indicate that the 5B isoform also plays a role in SM migration, implicating it in the repulsion that is observed when the chemoattraction is compromised. Thus, structural differences in the extracellular domains of these two isoforms can specify either attraction to or repulsion from the gonad.     

The Caenorhabditis elegans EGL-15 Signaling Pathway Implicates a DOS-Like Multisubstrate Adaptor Protein in Fibroblast Growth Factor Signal Transduction

EGL-15 is a fibroblast growth factor receptor in the nematode Caenorhabditis elegans. Components that mediate EGL-15 signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. We have isolated a gain-of-function allele of let-60 ras that confers a Clr phenotype and implicated both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-15 signaling by their Soc phenotype. Epistasis analysis indicates that the gene soc-1 functions in EGL-15 signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 was found to be involved in EGL-15 signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-15 and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-15.     

Alternative splicing affecting a novel domain in the C. elegans EGL-15 FGF receptor confers functional specificity

Fibroblast growth factor (FGF) receptors trigger a wide variety of cellular responses as diverse as cell migration, cell proliferation and cell differentiation. However, the molecular basis of the specificity of these responses is not well understood. The C. elegans FGF receptor EGL-15 similarly mediates a number of different responses, including transducing a chemoattractive signal and mediating an essential function. Analysis of the migration-specific alleles of egl-15 has identified a novel EGL-15 isoform that provides a molecular explanation for the different phenotypic effects of lesions at this locus. Alternative splicing yields two EGL-15 proteins containing different forms of a domain located within the extracellular region of the receptors immediately after the first IG domain. Neither of these two domain forms is found in any other FGF receptor. We have tested the roles of these EGL-15 receptor isoforms and their two FGF ligands for their signaling specificity. Our analyses demonstrate different physiological functions for the two receptor variants. EGL-15(5A) is required for the response to the FGF chemoattractant that guides the migrating sex myoblasts to their final positions. By contrast, EGL-15(5B) is both necessary and sufficient to elicit the essential function mediated by this receptor.     

UNC-52/perlecan affects gonadal leader cell migrations in C. elegans hermaphrodites through alterations in growth factor signaling

The unc-52 gene of Claenorhabditis elegans encodes a homologue of the basement membrane heparan sulfate proteoglycan perlecan. Viable alleles reduce the abundance of UNC-52 in late larval stages and increase the frequency of distal tip cell (DTC) migration defects caused by mutations disrupting the UNC-6/netrin guidance system. These unc-52 alleles do not cause circumferential DTC migration defects in an otherwise wild-type genetic background. The effects of unc-52 mutations on DTC migrations are distinct from effects on myofilament organization and can be partially suppressed by mutations in several genes encoding growth factor-like molecules, including EGL-17/FGF, UNC-129/TGF-beta, DBL-1/TGF-beta, and EGL-20/WNT. We propose that UNC-52 serves dual roles in C. elegans larval development in the maintenance of muscle structure and the regulation of growth factor-like signaling pathways.     

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.     

Identification of genes that regulate a left-right asymmetric neuronal migration in Caenorhabditis elegans

In C. elegans, cells of the QL and QR neuroblast lineages migrate with left-right asymmetry; QL and its descendants migrate posteriorly whereas QR and its descendants migrate anteriorly. One key step in generating this asymmetry is the expression of the Hox gene mab-5 in the QL descendants but not in the QR descendants. This asymmetry appears to be coupled to the asymmetric polarizations and movements of QL and QR as they migrate and relies on an asymmetric response to an EGL-20/Wnt signal. To identify genes involved in these complex layers of regulation and to isolate targets of mab-5 that direct posterior migrations, we screened visually for mutants with cell migration defects in the QL and QR lineages. Here, we describe a set of new mutants (qid-5, qid-6, qid-7, and qid-8) that primarily disrupt the migrations of the QL descendants. Most of these mutants were defective in mab-5 expression in the QL lineage and might identify genes that interact directly or indirectly with the EGL-20/Wnt signaling pathway.     

The cooperation of FGF receptor and Klotho is involved in excretory canal development and regulation of metabolic homeostasis in Caenorhabditis elegans

FGFs have traditionally been associated with cell proliferation, morphogenesis, and development; yet, a subfamily of FGFs (FGF19, -21, and -23) functions as hormones to regulate glucose, lipid, phosphate, and vitamin D metabolism with impact on energy balance and aging. In mammals, Klotho and beta-Klotho are type 1 transmembrane proteins that function as obligatory co-factors for endocrine FGFs to bind to their cognate FGF receptors (FGFRs). Mutations in Klotho/beta-Klotho or fgf19, -21, or -23 are associated with a number of human diseases, including autosomal dominant hypophosphatemic rickets, premature aging disorders, and diabetes. The Caenorhabditis elegans genome contains two paralogues of Klotho/beta-Klotho, klo-1, and klo-2. klo-1 is expressed in the C. elegans excretory canal, which is structurally and functionally paralogous to the vertebrate kidney. KLO-1 associates with EGL-15/FGFR, suggesting a role for KLO-1 in the fluid homeostasis phenotype described previously for egl-15/fgfr mutants. Altered levels of EGL-15/FGFR signaling lead to defects in excretory canal development and function in C. elegans. These results suggest an evolutionarily conserved function for the FGFR-Klotho complex in the development of excretory organs such as the mammalian kidney and the worm excretory canal. These results also suggest an evolutionarily conserved function for the FGFR-Klotho axis in metabolic regulation.     

A normally attractive cell interaction is repulsive in two C. elegans mesodermal cell migration mutants.

In wild-type Caenorhabditis elegans hermaphrodites, two bilaterally symmetric sex myoblasts (SMs) migrate anteriorly to flank the precise center of the gonad, where they divide to generate the muscles required for egg laying (J. E. Sulston and H. R. Horvitz (1977) Devl Biol. 56, 110-156). Although this migration is largely independent of the gonad, a signal from the gonad attracts the SMs to their precise final positions (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041-1052). Here we show that mutations in either of two genes, egl-15 and egl-17, cause the premature termination of the migrations of the SMs. This incomplete migration is caused by the repulsion of the SMs by the same cells in the somatic gonad that are the source of the attractive signal in wild-type animals.     

Caenorhabditis elegans SOS-1 is necessary for multiple RAS-mediated developmental signals

Vulval induction in Caenorhabditis elegans has helped define an evolutionarily conserved signal transduction pathway from receptor tyrosine kinases (RTKs) through the adaptor protein SEM-5 to RAS. One component present in other organisms, a guanine nucleotide exchange factor for Ras, has been missing in C.ELEGANS: To understand the regulation of this pathway it is crucial to have all positive-acting components in hand. Here we describe the identification, cloning and genetic characterization of C.ELEGANS: SOS-1, a putative guanine nucleotide exchanger for LET-60 RAS. RNA interference experiments suggest that SOS-1 participates in RAS-dependent signaling events downstream of LET-23 EGFR, EGL-15 FGFR and an unknown RTK. We demonstrate that the previously identified let-341 gene encodes SOS-1. Analyzing vulval development in a let-341 null mutant, we find an SOS-1-independent pathway involved in the activation of RAS signaling. This SOS-1-independent signaling is not inhibited by SLI-1/Cbl and is not mediated by PTP-2/SHP, raising the possibility that there could be another RasGEF.     

Clathrin interaction and subcellular localization of Ce-DAB-1, an adaptor for protein secretion in Caenorhabditis elegans

Growth factors must be secreted appropriately to co-ordinate cell proliferation, specification and movement during development and to control cell numbers and migrations in adult animals. Previous results showed that the secretion of the Caenorhabditis elegans fibroblast growth factor homologue, EGL-17, from vulval precursor cells in vivo involves the cytoplasmic adaptor protein Ce-DAB-1 and two lipoprotein receptors that bind Ce-DAB-1 and EGL-17. Here, we confirm the Ce-DAB-1 requirement for EGL-17 secretion using mutant animals. In vitro, Ce-DAB-1 binds to clathrin and APT-4, the C. elegans homologue of the alpha-adaptin subunit of adaptor protein 2 (AP2), and weakly to the gamma-appendage domains of APT-1 (AP1gamma-adaptin) and APT-9 (GGA protein). In tissue-culture cells, Ce-DAB-1 localizes to various compartments, including AP2-containing vesicles near the cell surface and perinuclear vesicles that contain AP1. The latter also contain Rab8, but not Rab5 or Rab11, as well as proteins en route from the trans Golgi network (TGN) to the surface. In vivo, EGL-17 secretion was inhibited by depletion of apt-1, apt-9 or ce-rab-8 and partially inhibited by RNAi of ce-rab-5, consistent with an important role for these proteins in the secretion of EGL-17 in vivo. These results suggest that Ce-DAB-1 might co-ordinate the assembly of endocytic or secretory vesicles in vivo and may mediate EGL-17 secretion directly, by recruiting clathrin to lipoprotein receptors at the TGN, or indirectly, by affecting lipoprotein receptor endocytosis and recycling.     

Multiple Wnts and frizzled receptors regulate anteriorly directed cell and growth cone migrations in Caenorhabditis elegans

A set of conserved molecules guides axons along the metazoan dorsal-ventral axis. Recently, Wnt glycoproteins have been shown to guide axons along the anterior-posterior (A/P) axis of the mammalian spinal cord. Here, we show that, in the nematode Caenorhabditis elegans, multiple Wnts and Frizzled receptors regulate the anterior migrations of neurons and growth cones. Three Wnts are expressed in the tail, and at least one of these, EGL-20, functions as a repellent. We show that the MIG-1 Frizzled receptor acts in the neurons and growth cones to promote their migrations and provide genetic evidence that the Frizzleds MIG-1 and MOM-5 mediate the repulsive effects of EGL-20. While these receptors mediate the effects of EGL-20, we find that the Frizzled receptor LIN-17 can antagonize MIG-1 signaling. Our results indicate that Wnts play a key role in A/P guidance in C. elegans and employ distinct mechanisms to regulate different migrations.     

Mutations affecting embryonic cell migrations in Caenorhabditis elegans

Four recessive mutations that affect long-range embryonic migration of the two canal-associated neurons (CANs) in C. elegans were isolated and characterized with the goal of identifying genes involved in control of directed cell movement. Mutant animals were identified initially by their "withered" tails, a phenotype associated with abnormal CAN migration; the mutants were then analyzed for abnormal cell migrations by Nomarski microscopy. Based on genetic complementation tests, the mutations were assigned to four different loci, two new (mig-10 III, mig-11 III) and two previously identified (unc-39 V, vab-8 V). Mutations at all four loci affect CAN migration with high to moderate penetrance (the percentage of mutant animals that exhibit the phenotype). In addition, two other bilaterally symmetric pairs of neurons (ALM and HSN), the mesoblast M, and a pair of coelomocyte mother cells are affected by one or more of the mutations, generally with lower penetrance. With the exceptions of HSN and the right coelomocyte mother cell, which occasionally migrate beyond their normal destinations, the cells affected appear to migrate either incompletely or not at all. All the migration phenotypes show incomplete penetrance and variable expressively, although genetic tests suggest that mutations at mig-10 and vab-8 result in complete or nearly complete loss of gene function. The variability in mutant phenotypes allowed tests for interdependence of several of the affected migrations; all those analyzed appeared independent of one another. The possible nature of the mutant defects and possible roles of these four loci in cell migration are discussed.     

Differential functions of the C. elegans FGF receptor in axon outgrowth and maintenance of axon position

Wiring of the nervous system requires that axons navigate to their targets and maintain their correct positions in axon fascicles after termination of axon outgrowth. We show here that the C. elegans fibroblast growth factor receptor (FGFR), EGL-15, affects both processes in fundamentally distinct manners. FGF-dependent activation of the EGL-15 tyrosine kinase and subsequently the GTPase LET-60/ras is required within epidermal cells, the substratum for most outgrowing axon, for appropriate outgrowth of specific axon classes to their target area. In contrast, genetic elimination of the FGFR isoform EGL-15(5A), defined by the inclusion of an alternative extracellular interimmunoglobulin domain, has no consequence for axon outgrowth but leads to a failure to postembryonically maintain axon position within defined axon fascicles. An engineered, secreted form of EGL-15(5A) containing only its ectodomain is sufficient for maintenance of axon position, thus providing novel insights into receptor tyrosine kinase function and the process of maintaining axon position.     

The conserved kinase UNC-51 acts with VAB-8 and UNC-14 to regulate axon outgrowth in C. elegans

Directional cues guide growth cones. While molecules like UNC-6/netrin direct migrations along the dorsoventral axis of many organisms, it is unclear how anteroposterior guidance is achieved. We describe a physical interaction between VAB-8, a protein both necessary and sufficient for posteriorly directed migrations in C. elegans, and UNC-51, a conserved serine/threonine kinase that functions generally in axon outgrowth. We show that both proteins function in the CAN neurons to direct their axons posteriorly. Expression in the CANs of peptides predicted to interfere with interactions between UNC-51 and both VAB-8 and UNC-14, a second protein that interacts physically with UNC-51, disrupts CAN axon outgrowth. We provide genetic evidence that VAB-8 functions in an UNC-51 pathway for posteriorly directed CAN axon guidance and show that VAB-8 and UNC-14 can be targets of UNC-51 kinase activity. Taken together, our results suggest that VAB-8 and UNC-14 are substrates that mediate the function of UNC-51 in axon outgrowth.     

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.     

A Wnt signaling system that specifies two patterns of cell migration in C. elegans

In C. elegans, a bilateral pair of neuroblasts, QL and QR, give rise to cells that migrate in opposite directions along the anteroposterior (A/P) body axis. QL and its descendants migrate posteriorly whereas QR and its descendants migrate anteriorly. We find that a Wnt family member, EGL-20, acts in a dose-dependent manner to specify these opposite migratory behaviors. High levels of EGL-20 promote posterior migration by activating a canonical Wnt signal transduction pathway, whereas low levels promote anterior migration by activating a separate, undefined pathway. We find that the two Q cells respond differently to EGL-20 because they have different response thresholds. Thus, in this system two distinct dose-dependent responses are specified not by graded levels of the Wnt signal, but instead by left-right asymmetrical differences in the cellular responsiveness to Wnt signaling.