Moseley-Alldredge, Melinda et al. (2019) International Worm Meeting "SAX-7/L1CAM genetically interact with MPK-1/Erk to coordinate locomotion."

Moseley-Alldredge, Melinda, Yoo, Hayoung, O'Keefe, Calvin, Richmond, Janet, chen, lihsia, Sheoran, Seema

[International Worm Meeting, 2019]

L1CAMs are immunoglobulin cell adhesion molecules that are linked to diverse neurological conditions, including intellectual disability, autism, and schizophrenia. Studies in C. elegans and mice revealed L1CAM developmental roles that include axon guidance and dendrite morphogenesis, as well as maintenance of neural integrity. We recently uncovered a non-developmental role for the C. elegans L1CAM encoded by the sax-7 gene in promoting coordinated locomotion. sax-7 genetically interacts with rab-3, unc-13, and snb-1, genes that function in the synaptic vesicle cycle, resulting in synthetic or synergistic uncoordinated (Unc) locomotion. To determine the basis of the synthetic Unc, we performed a genetic suppressor screen and identified loss-of-function mutations in ksr-1, which encodes for a scaffold that facilitates MPK-1/Erk activation. Indeed, we provide data showing that loss of MPK-1 also suppresses rab-3; sax-7 synthetic Unc locomotion. Further analyses reveal that loss of ksr-1 suppresses the abnormal response rab-3; sax-7 animals display to an acetylcholinesterase inhibitor, consistent with defects in the synaptic vesicle cycle.In addition to these genetic and behavioral data, we conducted electrophysiological and high-pressure electron microscopy analyses to examine how SAX-7 and MPK-1/Erk signaling promote coordinated locomotion. These functional data provide new insights into the interpretation of the behavioral phenotypes and the underlying circuitry involved.

Dong, Binyun et al. (2013) International Worm Meeting "The LAD-2/L1CAM functions in EFN-4/ephrin-mediated axon guidance."

chen, lihsia, Moseley-Alldredge, Melinda, Dong, Binyun

[International Worm Meeting, 2013]

The ephrin-Eph signaling pathway plays important roles in many diverse developmental processes. In C. elegans, the ephrin-Eph signaling pathway functions in axon guidance and cell movements critical during embryogenesis and male tail morphogenesis, requiring the function of the sole Eph receptor, VAB-1, and one or more of the four GPI-linked ephrins, EFN-1, 2, 3, and 4. In this study, we present data that demonstrates a role for EFN-4 in axon guidance that requires the function for SUP-17/Kuzbanian ADAM metalloprotease, suggesting the importance of ectodomain shedding of EFN-4. Expression of an engineered secreted form of EFN-4 can rescue efn-4(bx80) null axon guidance defects, raising the possibility that EFN-4 can function as a diffusible guidance cue. Of the four ephrins, EFN-4 is thus far shown to have roles that are independent of VAB-1. Consistent with EFN-4 functioning via an alternative receptor, we provide evidence that EFN-4 functions in the same pathway as LAD-2, a transmembrane protein belonging to the L1 family of cell adhesion molecules (L1CAMs). We had previously uncovered a role for LAD-2 as a co-receptor for MAB-20/semaphorin in directing axon migration. Our preliminary results reveal LAD-2 and EFN-4 can form a biochemical complex. Taken together, these results suggest LAD-2 may act as a potential receptor or regulatory co-receptor in EFN-4-mediated axon guidance.

Waltman, Michael et al. (2019) International Worm Meeting "SAX-7/L1CAM genetically interacts with LET-60/RAS to promote proper vulval development and viability."

Waltman, Michael, Love, Caitland, Somia, Nirali, chen, lihsia, Alley, Divya, Moseley-Alldredge, Melinda

[International Worm Meeting, 2019]

L1 cell adhesion molecules (L1CAMs) are transmembrane immunoglobulin-like proteins that play important roles in nervous system development. Genes encoding L1CAMs - L1, NrCAM, CHL1, and Neurofascin - have been highly associated with genetically complex neurological disorders including addiction, schizophrenia, autism, and Hirschsprung's disease. In addition, mutations in the L1 gene directly underlie the neurological X-linked L1 Syndrome. Interestingly, siblings with the same L1 allele can show highly variable penetrance and severity of symptoms, consistent with the presence of L1-interacting genes. C. elegans has a single gene, sax-7, which encodes a canonical L1CAM with major hallmarks of mammalian L1CAMs. We have identified a novel interaction between sax-7 and the Ras-mitogen activated protein kinase (MAPK) signaling pathway. The Ras GTPase, encoded by the let-60 gene in C. elegans, has been identified to play key roles in development, cell signaling, and cancer. In C. elegans, LET-60 is key for proper development of several tissues, notably of the vulva. We discovered that loss of sax-7 function enhances let-60 gain-of-function (gf) multivulval phenotype. Loss of sax-7 function also synergizes with let-60 (gf), resulting in synthetic Clr phenotypes, hypo-osomotic sensitivity, and lethality. In addition to identifying novel roles for sax-7, these findings also reveal a link between SAX-7 and intracellular signaling that likely have implications on disease manifestation. Here, we provide a more detailed study of these phenotypes and use the well-established vulva model to dissect the interplay between L1CAMs and Ras.

Chin-Sang ID et al. (2000) West Coast Worm Meeting "mab-26 encodes the C. elegans ephrin EFN-4"

Chisholm AD, Chin-Sang ID, Ding M, George SE, Holcomb T

[West Coast Worm Meeting, 2000]

In C. elegans, Eph signaling functions in embryonic neuroblast movements after gastrulation, and in epidermal enclosure. The C. elegans genome encodes one Eph receptor, VAB-1, and four ephrins: VAB-2/EFN-1, EFN-2, EFN-3, and EFN-4. mab-26 mutants have defects in male tail morphogenesis (Chow and Emmons 1994). The predicted ephrin gene efn-4 (aka F56A11.3), was located in the same region as mab-26. A genomic clone containing F56A11.3 rescued Mab-26 phenotypes in transgenic arrays. These data combined with identification of mutant alleles demonstrate that mab-26 and efn-4 are the same gene. The EFN-4 protein encodes a divergent ephrin. EFN-4 overall is most similar to murine Ephrin-B2 (27% identity and 45% similarity). Like other worm ephrins, EFN-4 is predicted to be GPI-anchored. Unlike all other ephrins EFN-4 contains a 24 amino acid insert in the putative receptor-binding domain. The mutation bx80 is a deletion of second exon of efn-4, and thus is likely a null mutation. Three other alleles from the Cambridge collection cause alterations in efn-4: e36 causes an early nonsense mutation, and the weak alleles e660 and e1746 (provided by Jonathan Hodgkin) cause missense alterations in the receptor binding domain. The phenotypes of efn-4 mutants indicate that like other eph signaling proteins it is required for embryonic morphogenesis. Genetic interactions with the other ephrins and vab-1 are inconsistent with EFN-4 acting only in the VAB-1 pathway (see abstract by Moseley and Chisholm). These observations have led us to examine the the EFN-4/VAB-1 binding interaction using cell culture experiments. Previously the EFN-1 ephrin was shown to bind VAB-1::AP fusion proteins with high affinity in mammalian cell culture assays (Chin-Sang et al 1999). We are using parallel approaches to test EFN-4/VAB-1 interactions. An EFN-4::GFP reporter gene is expressed in a subset of neurons. This expression pattern suggests that EFN-4/VAB-1 signaling in the embryo occurs between neurons and is thus required non-autonomously for epidermal development. We will test this idea using genetic mosaic analysis and tissue-specific rescue experiments.

Ian Chin-Sang et al. (2001) International C. elegans Meeting "The Ephrin EFN-4 may function independently of the VAB-1 Eph Receptor in morphogenesis"

Ian Chin-Sang, Andrew D Chisholm

[International C. elegans Meeting, 2001]

Eph receptor tyrosine kinases bind membrane bound ligands (ephrins) to regulate a variety of processes, including embryonic pattern formation, angiogenesis, and axon guidance. The C. elegans genome encodes one Eph receptor, VAB-1, and four ephrins, EFN-1 to EFN-4. We showed previously that EFN-1 is a ligand for VAB-1 and functions in neurons to regulate epithelial and neuronal morphogenesis (Chin-Sang et al., Cell 99: 781). We reported previously that mutations in the mab-26 gene, initially identified as a ray morphology mutant (Chow and Emmons, Development 120:2579), affect the fourth ephrin, EFN-4. Although VAB-1 encodes the only Eph receptor in the worm genome, several lines of evidence suggest that EFN-4 does not function only through the VAB-1 receptor. First, we tested whether cell-surface expressed EFN-4 binds VAB-1 using a soluble VAB-1-AP fusion protein as an affinity reagent. Our preliminary results suggest that the affinity of EFN-4 for VAB-1-AP is extremely low compared with the EFN-1/VAB-1 affinity. Second, the embryonic and larval phenotypes of efn-4 mutants differ from those of vab-1 and efn-1 mutants, although they show some overlap (see abstract by Moseley and Chisholm). This overlap in phenotype is reflected in the efn-4 expression pattern. Using EFN-4-GFP transgenes and anti-EFN-4 antibodies we have found that EFN-4 is expressed in the developing nervous system in a pattern that appears to overlap both VAB-1 and EFN-1 expression domains. Finally, efn-4 alleles display strong synthetic lethality with either vab-1 or efn-1 mutations. vab-1; efn-4 double mutants display fully penetrant embryonic lethality, a phenotype that is stronger than the vab-1 null phenotype. These data are inconsistent with efn-4 acting exclusively in a VAB-1 dependent pathway and suggest that EFN-4 may act independently of the VAB-1 receptor. To identify genes that may be involved in this independent pathway we have begun suppressor screens of the vab-1;efn-4 synthetic lethality and have also screened for mutations that show synthetic lethality with weak vab-1 alleles. Characterization of our suppressors and enhancers will be presented.

Sarah L Moseley et al. (2003) International Worm Meeting "Ephrin signaling and oocyte size"

Andrew D Chisholm, Sarah L Moseley

[International Worm Meeting, 2003]

Signaling via the VAB-1 Eph receptor and the ephrins EFN-1 through EFN-4 is is required for neuroblast migration during embryonic morphogenesis (Chin-Sang and Moseley, et al., 2002, Development 129: 5499). In the course of analyzing an ephrin quadruple mutant we uncovered a new role for ephrins in oogenesis. efn-2 efn-3 efn-4 triple mutants injected with dsRNA for efn-1 lay eggs of very variable size. Wild type eggs are ~35 microns in length, whereas ephrin quadruple mutant eggs range from 25 to 65 microns in length. Quadruple mutant embryos all arrest with grossly defective morphogenesis. The large eggs develop from oocytes that are enlarged at least by the diakinesis stage of oogenesis. VAB-1 and EFN-2 have been shown to inhibit MAP kinase activation during oocyte maturation (Miller et al., 2003, Genes Dev 17: 187). However, vab-1 and efn-2 mutants have normally sized eggs. Thus, the requirement for ephrin signaling in oocyte size control prior to diakinesis is distinct from the function of ephrin signaling in oocyte maturation. Ephrin double and triple mutants display very weak deregulation of egg size, suggesting that all four ephrins function partly redundantly in oocyte size control. The determinants of oocyte size are not well understood, but likely include time spent in pachytene of meiosis I. Activity of the MAP kinase MPK-1 appears to act as a rate-limiting factor in progression through pachytene (Hajnal and Berset 2002). By analogy to oocyte maturation, Eph signaling might modulate MAPK activation during or after pachytene. Loss of ephrin function could result in enhanced or reduced MAPK activation, thus longer or shorter time spent in pachytene and thus larger or smaller oocytes. In efn-1 efn-2 efn-3 triple mutant gonads the pattern of MAPK activation appears normal, as assayed using anti-diphospho-MAPK antibodies. In contrast, efn-2 efn-3 efn-4 triple mutant gonads display elevated levels of activated MAPK, suggesting that ephrins redundantly repress MAPK activation in oogenesis. Where are ephrins required for oocyte size control? Functional EFN-4::GFP and EFN-1::GFP transgenes are expressed in somatic gonad sheath cells and thus are in the right time and place to influence oogenesis. VAB-1 and EFN-2 appear to be expressed at least in the germline. A simple model envisages ephrin signaling from somatic sheath cells to germline-expressed VAB-1 which then regulates MAPK in pachytene cells. However, any model must account for the role of EFN-4, which does not appear to signal via VAB-1. We speculate that bidirectional Eph signaling between the germline and somatic gonad may be involved in monitoring progress through the meiotic cell cycle