Ian D. Chin-Sang

The divergent C. elegans ephrin EFN-4 functions inembryonic morphogenesis in a pathway independent of the VAB-1 Eph receptor

The C. elegans genome encodes a single Eph receptor tyrosine kinase, VAB-1, which functions in neurons to control epidermal morphogenesis. Four members of the ephrin family of ligands for Eph receptors have been identified in C. elegans. Three ephrins (EFN-1/VAB-2, EFN-2 and EFN-3) have been previously shown to function in VAB-1 signaling. We show that mutations in the gene mab-26 affect the fourth C. elegans ephrin, EFN-4. We show that efn-4 also functions in embryonic morphogenesis, and that it is expressed in the developing nervous system. Interestingly, efn-4 mutations display synergistic interactions with mutations in the VAB-1 receptor and in the EFN-1 ephrin, indicating that EFN-4 may function independently of the VAB-1 Eph receptor in morphogenesis. Mutations in the LAR-like receptor tyrosine phosphatase PTP-3 and in the Semaphorin-2A homolog MAB-20 disrupt embryonic neural morphogenesis. efn-4 mutations synergize with ptp-3 mutations, but not with mab-20 mutations, suggesting that EFN-4 and Semaphorin signaling could function in a common pathway or in opposing pathways in C. elegans embryogenesis.

A Caenorhabditis elegans allatostatin/galanin-like receptor NPR-9 inhibits local search behavior in response to feeding cues

Movement in Caenorhabditis elegans is the result of sensory cues creating stimulatory and inhibitory output from sensory neurons. Four interneurons (AIA, AIB, AIY, and AIZ) are the primary recipients of this information that is further processed en route to motor neurons and muscle contraction. C. elegans has >1,000 G protein-coupled receptors (GPCRs), and their contribution to sensory-based movement is largely undefined. We show that an allatostatin/galanin-like GPCR (NPR-9) is found exclusively in the paired AIB interneuron. AIB interneurons are associated with local search/pivoting behavior. npr-9 mutants display an increased local search/pivoting that impairs their ability to roam and travel long distances on food. With impaired roaming behavior on food npr-9 mutants accumulate more intestinal fat as compared with wild type. Overexpression of NPR-9 resulted in a gain-of-function phenotype that exhibits enhanced forward movement with lost pivoting behavior off food. As such the animal travels a great distance off food, creating arcs to return to food. These findings indicate that NPR-9 has inhibitory effects on the AIB interneuron to regulate foraging behavior, which, in turn, may affect metabolic rate and lipid storage.

A Role for C. elegans Eph RTK Signaling in PTEN Regulation

PTEN is one of the most commonly lost tumor suppressors in human cancer and is known to inhibit insulin signaling. Eph receptor tyrosine kinases (RTKs) have also been implicated in cancer formation and progression, and they have diverse functions, including nervous and vascular system development. We show that in C. elegans, the VAB-1 Eph kinase domain physically interacts with and phosphorylates PTEN (DAF-18), diminishing its protein levels and function. vab-1 mutants show increased longevity and sensitivity to dauer conditions, consistent with increased DAF-18/PTEN activity and decreased insulin-like signaling. Moreover, daf-18 mutations suppress vab-1 oocyte maturation phenotypes independent of PI3K signaling. We also present evidence that DAF-18 has protein phosphatase activity to antagonize VAB-1 action. Possible implications for human cancers are discussed, based on the idea that mutually inhibitory interactions between PTEN and Eph RTKs may be conserved.

The VAB-1 Eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function together during C. elegans embryonic morphogenesis.

Mutations that affect the single C. elegans Eph receptor tyrosine kinase VAB-1 cause defects in cell movements during embryogenesis. Here, we provide genetic and molecular evidence that the VAB-1 Eph receptor functions with another neuronal receptor, SAX-3/Robo, for proper embryogenesis. Our analysis of sax-3 mutants shows that SAX-3/Robo functions with the VAB-1 Eph receptor for gastrulation cleft closure and ventral epidermal enclosure. In addition, SAX-3 functions autonomously for epidermal morphogenesis independently of VAB-1. A double-mutant combination between vab-1 and slt-1 unmasks a role for the SLT-1 ligand in embryogenesis. We provide evidence for a physical interaction between the VAB-1 tyrosine kinase domain and the juxtamembrane and CC1 region of the SAX-3/Robo receptor. Gene dosage, non-allelic non-complementation experiments and co-localization of the two receptors are consistent with a model in which these two receptors form a complex and function together during embryogenesis.

A Unified Nomenclature and Amino Acid Numbering for Human PTEN

With the discovery of an isoform based on an alternative translation start site, PTEN nomenclature needs an update. The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.

The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

The FGLamide-Allatostatins Influence Foraging Behavior in Drosophila melanogaster

Allatostatins (ASTs) are multifunctional neuropeptides that generally act in an inhibitory fashion. ASTs were identified as inhibitors of juvenile hormone biosynthesis. Juvenile hormone regulates insect metamorphosis, reproduction, food intake, growth, and development. Drosophila melanogaster RNAi lines of PheGlyLeu-amide-ASTs (FGLa/ASTs) and their cognate receptor, Dar-1, were used to characterize roles these neuropeptides and their respective receptor may play in behavior and physiology. Dar-1 and FGLa/AST RNAi lines showed a significant reduction in larval foraging in the presence of food. The larval foraging defect is not observed in the absence of food. These RNAi lines have decreased for transcript levels which encodes cGMP- dependent protein kinase. A reduction in the for transcript is known to be associated with a naturally occuring allelic variation that creates a sitter phenotype in contrast to the rover phenotype which is caused by a for allele associated with increased for activity. The sitting phenotype of FGLa/AST and Dar-1 RNAi lines is similar to the phenotype of a deletion mutant of an AST/galanin-like receptor (NPR-9) in Caenorhabditis elegans. Associated with the foraging defect in C. elegans npr-9 mutants is accumulation of intestinal lipid. Lipid accumulation was not a phenotype associated with the FGLa/AST and Dar-1 RNAi lines.

Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster

The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation.

C. elegans as a model to study PTEN's regulation and function.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) has important roles in tumor suppression, metabolism, and development, yet its regulators, effectors, and functions are not fully understood. DAF-18 is the PTEN ortholog in Caenorhabditis elegans. DAF-18s role is highly conserved to human PTEN, and can be functionally replaced by human PTEN. Thus C. elegans provides a valuable model to study PTEN. This review assesses current and emerging methods to study DAF-18s regulators and functions in C. elegans. We propose genetic modify screens to identify genes that interact with daf-18/PTEN. These genes are potential targets for anticancer drug therapies. We also provide a review on the roles DAF-18/PTEN has during C. elegans development and how studying these physiological roles can provide mechanistic insight on DAF-18/PTEN function.

The C. elegans nck-1 gene encodes two isoforms and is required for neuronal guidance

The NCK adaptor proteins are composed entirely of SH3 and SH2 domains and serve as protein interaction bridges for several receptors during signal transduction events. Here we report the molecular and genetic analysis of the Caenorhabditis elegans nck-1 gene. C. elegans nck-1 encodes two isoforms: NCK-1A and a shorter isoform that lacks the first SH3 domain, NCK-1B. C. elegans nck-1 mutants exhibit defects in axon guidance and neuronal cell position, as well as defects in the excretory canal cell, gonad, and male mating. NCK-1 is broadly expressed in neurons and epithelial cells with NCK-1B being the most abundant isoform. NCK-1A and NCK-1B share a similar expression pattern in parts of the nervous system, but also have independent expression patterns in other tissues. Interestingly, NCK-1B is localized to the nuclei of many cells. Genetic rescue experiments show that NCK-1 functions cell autonomously and, in general, either NCK-1A or NCK-1B is sufficient to function in axon guidance. However, there appears to be specific roles for each isoform, for example NCK-1B is required for HSN cell migration while NCK-1A is required for efficient male mating. Genetic epistasis experiments show that NCK-1 functions redundantly with the LAR Receptor Tyrosine Phosphatase, PTP-3, and the Netrin receptor UNC-40.