Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans.

BACKGROUND: Parkinson's disease is a disabling neurodegenerative movement disorder characterized by dopaminergic neuron loss induced by α-synuclein oligomers. There is an urgent need for disease-modifying therapies for Parkinson's disease, but drug discovery is challenged by lack of in vivo models that recapitulate early stages of neurodegeneration. Invertebrate organisms, such as the nematode worm Caenorhabditis elegans, provide in vivo models of human disease processes that can be instrumental for initial pharmacological studies. METHODS: To identify early motor impairment of animals expressing α-synuclein in dopaminergic neurons, we first used a custom-built tracking microscope that captures locomotion of single C. elegans with high spatial and temporal resolution. Next, we devised a method for semi-automated and blinded quantification of motor impairment for a population of simultaneously recorded animals with multi-worm tracking and custom image processing. We then used genetic and pharmacological methods to define the features of early motor dysfunction of α-synuclein-expressing C. elegans. Finally, we applied the C. elegans model to a drug repurposing screen by combining it with an artificial intelligence platform and cell culture system to identify small molecules that inhibit α-synuclein oligomers. Screen hits were validated using in vitro and in vivo mammalian models. RESULTS: We found a previously undescribed motor phenotype in transgenic α-synuclein C. elegans that correlates with mutant or wild-type α-synuclein protein levels and results from dopaminergic neuron dysfunction, but precedes neuronal loss. Together with artificial intelligence-driven in silico and in vitro screening, this C. elegans model identified five compounds that reduced motor dysfunction induced by α-synuclein. Three of these compounds also decreased α-synuclein oligomers in mammalian neurons, including rifabutin which has not been previously investigated for Parkinson's disease. We found that treatment with rifabutin reduced nigrostriatal dopaminergic neurodegeneration due to α-synuclein in a rat model. CONCLUSIONS: We identified a C. elegans locomotor abnormality due to dopaminergic neuron dysfunction that models early α-synuclein-mediated neurodegeneration. Our innovative approach applying this in vivo model to a multi-step drug repurposing screen, with artificial intelligence-driven in silico and in vitro methods, resulted in the discovery of at least one drug that may be repurposed as a disease-modifying therapy for Parkinson's disease.

Sensory regulated Wnt production from neurons helps make organ development robust to environmental changes in C. elegans.

Long-term survival of an animal species depends on development being robust to environmental variations and climate changes. We used C. elegans to study how mechanisms that sense environmental changes trigger adaptive responses that ensure animals develop properly. In water, the nervous system induces an adaptive response that reinforces vulval development through an unknown backup signal for vulval induction. This response involves the heterotrimeric G-protein EGL-30//Gαq acting in motor neurons. It also requires body-wall muscle, which is excited by EGL-30-stimulated synaptic transmission, suggesting a behavioral function of neurons induces backup signal production from muscle. We now report that increased acetylcholine during liquid growth activates an EGL-30-Rho pathway, distinct from the synaptic transmission pathway, that increases Wnt production from motor neurons. We also provide evidence that this neuronal Wnt contributes to EGL-30-stimulated vulval development, with muscle producing a parallel developmental signal. As diverse sensory modalities stimulate motor neurons via acetylcholine, this mechanism enables broad sensory perception to enhance Wnt-dependent development. Thus, sensory perception improves animal fitness by activating distinct neuronal functions that trigger adaptive changes in both behavior and developmental processes.

The directed migration of gonadal distal tip cells in Caenorhabditis elegans requires NGAT-1, a ß1,4-N-acetylgalactosaminyltransferase enzyme.

Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted ß1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC). An amino-terminal partial deletion of ngat-1 causes a similar but lower penetrance ts phenotype. The existence of multiple ts alleles with distinctly different molecular DNA lesions, neither of which is likely to encode a ts protein, indicates that NGAT-1 normally prevents innate temperature sensitivity for phase 2 DTC pathfinding. Temperature shift analyses indicate that the ts period for the ngat-1 mutant defect ends by the beginning of post-embryonic development-nearly 3 full larval stages prior to the defective phase 2 migration affected by ngat-1 mutations. NGAT-1 homologs generate glycan-terminal GalNAc-ß1-4GlcNAc, referred to as LacdiNAc modifications, on glycoproteins and glycolipids. We also found that the absence of the GnT1/Mgat1 activity [UDP-N-acetyl-D-glucosamine:α-3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase 1 (encoded by C. elegans gly-12, gly-13, and gly-14 and homologous to vertebrate GnT1/Mgat1)], causes a similar spectrum of DTC phenotypes as ngat-1 mutations-primarily affecting posterior DTC phase 2 migration and preventing manifestation of the same innate ts period as ngat-1. GnT1/Mgat1 is a medial Golgi enzyme known to modify mannose residues and initiate N-glycan branching, an essential step in the biosynthesis of hybrid, paucimannose and complex-type N-glycans. Quadruple mutant animals bearing putative null mutations in ngat-1 and the three GnT genes (gly-12, gly-13, gly-14) were not enhanced for DTC migration defects, suggesting NGAT-1 and GnT1 act in the same pathway. These findings suggest that GnTI generates an N-glycan substrate for NGAT-1 modification, which is required at restrictive temperature (25°C) to prevent, stabilize, reverse or compensate a perinatal thermo-labile process (or structure) causing late larval stage DTC phase 2 migration errors.

Forty-five years of cell-cycle genetics.

In the early 1970s, studies in Leland Hartwell's laboratory at the University of Washington launched the genetic analysis of the eukaryotic cell cycle and set the path that has led to our modern understanding of this centrally important process. This 45th-anniversary Retrospective reviews the steps by which the project took shape, the atmosphere in which this happened, and the possible morals for modern times. It also provides an up-to-date look at the 35 original CDC genes and their human homologues.

The Wnt Frizzled Receptor MOM-5 Regulates the UNC-5 Netrin Receptor through Small GTPase-Dependent Signaling to Determine the Polarity of Migrating Cells.

Wnt and Netrin signaling regulate diverse essential functions. Using a genetic approach combined with temporal gene expression analysis, we found a regulatory link between the Wnt receptor MOM-5/Frizzled and the UNC-6/Netrin receptor UNC-5. These two receptors play key roles in guiding cell and axon migrations, including the migration of the C. elegans Distal Tip Cells (DTCs). DTCs migrate post-embryonically in three sequential phases: in the first phase along the Antero-Posterior (A/P) axis, in the second, along the Dorso-Ventral (D/V) axis, and in the third, along the A/P axis. Loss of MOM-5/Frizzled function causes third phase A/P polarity reversals of the migrating DTCs. We show that an over-expression of UNC-5 causes similar DTC A/P polarity reversals and that unc-5 deficits markedly suppress the A/P polarity reversals caused by mutations in mom-5/frizzled. This implicates MOM-5/Frizzled as a negative regulator of unc-5. We provide further evidence that small GTPases mediate MOM-5's regulation of unc-5 such that one outcome of impaired function of small GTPases like CED-10/Rac and MIG-2/RhoG is an increase in unc-5 function. The work presented here demonstrates the existence of cross talk between components of the Netrin and Wnt signaling pathways and provides further insights into the way guidance signaling mechanisms are integrated to orchestrate directed cell migration.

Netrins and Wnts function redundantly to regulate antero-posterior and dorso-ventral guidance in C. elegans.

Guided migrations of cells and developing axons along the dorso-ventral (D/V) and antero-posterior (A/P) body axes govern tissue patterning and neuronal connections. In C. elegans, as in vertebrates, D/V and A/P graded distributions of UNC-6/Netrin and Wnts, respectively, provide instructive polarity information to guide cells and axons migrating along these axes. By means of a comprehensive genetic analysis, we found that simultaneous loss of Wnt and Netrin signaling components reveals previously unknown and unexpected redundant roles for Wnt and Netrin signaling pathways in both D/V and A/P guidance of migrating cells and axons in C. elegans, as well as in processes essential for organ function and viability. Thus, in addition to providing polarity information for migration along the axis of their gradation, Wnts and Netrin are each able to guide migrations orthogonal to the axis of their gradation. Netrin signaling not only functions redundantly with some Wnts, but also counterbalances the effects of others to guide A/P migrations, while the involvement of Wnt signaling in D/V guidance identifies Wnt signaling as one of the long sought mechanisms that functions in parallel to Netrin signaling to promote D/V guidance of cells and axons. These findings provide new avenues for deciphering how A/P and D/V guidance signals are integrated within the cell to establish polarity in multiple biological processes, and implicate broader roles for Netrin and Wnt signaling--roles that are currently masked due to prevalent redundancy.

C. elegans PVF-1 inhibits permissive UNC-40 signalling through CED-10 GTPase to position the male ray 1 sensillum.

Graded distributions of netrin and semaphorin guidance cues convey instructive polarity information to migrating cells and growth cones, but also have permissive (i.e. non-polarity determining) functions in mammalian development and repair. The permissive functions of these cues are largely uncharacterised at a molecular level. We found previously that UNC-6 (netrin) signals permissively through UNC-40 (DCC) and UNC-5 receptors to prevent anterior displacement of the ray 1 sensillum in the C. elegans male tail. UNC-6/UNC-40 signalling functions in parallel with SMP-1 (semaporin 1)/PLX-1 (plexin) signalling to prevent this defect. Here, we report that a deletion allele of pvf-1, which encodes a VEGF-related protein, causes no ray 1 defects, but enhances ray 1 defects of a plx-1 mutant, and unexpectedly also suppresses unc-6(ev400)-null mutant ray 1 defects. These mutant ray 1 inductive and suppressive effects are mimicked by the ability of unc-40(+) and ced-10(gain-of-function) multi-copy transgene arrays to induce ray 1 defects or suppress unc-6 mutant ray 1 defects, depending on their dosage, suggesting the pvf-1 mutation causes UNC-40 overactivity that interferes with signalling but is partially sensitive to UNC-6. Additional data suggest PVF-1 functions through four VEGF receptor-related proteins and inhibits only CED-10 (a GTPase), but not MIG-2-dependent UNC-40 activity, even though UNC-40 functions through both GTPases to position ray 1. pvf-1 and receptor mutant ray 1 defects are rescued by transgenes expressing mouse VEGF164 and human VEGF receptors, respectively. These data report the first case of VEGF-induced inhibition of the netrin signalling and a molecular conservation of VEGF function from worms to humans.

Semaphorin-1 and netrin signal in parallel and permissively to position the male ray 1 sensillum in Caenorhabditis elegans.

Netrin and semaphorin axon guidance cues have been found to function in the genesis of several mammalian organs; however, little is known about the underlying molecular mechanisms involved. A genetic approach could help to reveal the underpinnings of these mechanisms. The most anterior ray sensillum (ray 1) in the Caenorhabditis elegans male tail is frequently displaced anterior to its normal position in smp-1/semaphorin-1a and plexin-1/plx-1 mutants. Here we report that UNC-6/netrin and its UNC-40/DCC receptor signal in parallel to SMP-1/semaphorin-1a and its PLX-1/plexin-1 receptor to prevent the anterior displacement of ray 1 and that UNC-6 plus SMP-1 signaling can account entirely for this function. We also report that mab-20/semaphorin-2a mutations, which prevent the separation of neighboring rays and cause ray fusions, suppress the anterior displacements of ray 1 caused by deficiencies in SMP-1 and UNC-6 signaling and this is independent of the ray fusion phenotype, whereas overexpression of UNC-40 and PLX-1 cause ray fusions. This suggests that for ray 1 positioning, a balance is struck between a tendency of SMP-1 and UNC-6 signaling to prevent ray 1 from moving away from ray 2 and a tendency of MAB-20/semaphorin-2a signaling to separate all rays from each other. Additional evidence suggests this balance involves the relative adhesion of the ray 1 structural cell to neighboring SET and hyp 7 hypodermal cells. This finding raises the possibility that changes in ray 1 positioning depend on passive movements caused by attachment to the elongating SET cell in opposition to the morphologically more stable hyp 7 cell. Several lines of evidence indicate that SMP-1 and UNC-6 function permissively in the context of ray 1 positioning.

Semaphorin and Eph receptor signaling guide a series of cell movements for ventral enclosure in C. elegans.

BACKGROUND: In the last stage of the Caenorhabditis elegans body wall closure, an open pocket in the epidermis is closed by the migration of marginal epidermal P/pocket cells to the ventral midline. The cellular and molecular mechanisms of this closure remain unknown. RESULTS: Cells within the pocket align to form a bridge for migration of contralateral P cell pair P9/10 L,R (and neighboring P cells) to the midline. Bridge formation involves rearrangement of five sister pairs of PLX-2/plexin and VAB-1/Eph receptor expressing "plexin band" cells, of which three pairs form a scaffold for bridge assembly and two pairs form the bridge. Bridge formation requires VAB-1 kinase-dependent extension of presumptive bridge cells over scaffold cells toward the ventral midline. An unassembled vab-1 null mutant bridge obstructs P cell migration, which is largely overcome by plexin band expression of VAB-1 or VAB-1(delC) (a kinase deletion of VAB-1). VAB-1 also functions redundantly with MAB-20/semaphorin to prevent perdurant gaps between sister plexin band cells that block P cell migration. CONCLUSIONS: The Eph receptor mediates cellular extensions required for bridge formation, independently facilitates P cell migration to the midline, and functions redundantly with PLX-2/plexin to prevent gaps in the bridge used for P9/10 cell migration in body wall closure.

Genetics of extracellular matrix remodeling during organ growth using the Caenorhabditis elegans pharynx model.

The organs of animal embryos are typically covered with an extracellular matrix (ECM) that must be carefully remodeled as these organs enlarge during post-embryonic growth; otherwise, their shape and functions may be compromised. We previously described the twisting of the Caenorhabditis elegans pharynx (here called the Twp phenotype) as a quantitative mutant phenotype that worsens as that organ enlarges during growth. Mutations previously known to cause pharyngeal twist affect membrane proteins with large extracellular domains (DIG-1 and SAX-7), as well as a C. elegans septin (UNC-61). Here we show that two novel alleles of the C. elegans papilin gene, mig-6(et4) and mig-6(sa580), can also cause the Twp phenotype. We also show that overexpression of the ADAMTS protease gene mig-17 can suppress the pharyngeal twist in mig-6 mutants and identify several alleles of other ECM-related genes that can cause or influence the Twp phenotype, including alleles of fibulin (fbl-1), perlecan (unc-52), collagens (cle-1, dpy-7), laminins (lam-1, lam-3), one ADAM protease (sup-17), and one ADAMTS protease (adt-1). The Twp phenotype in C. elegans is easily monitored using light microscopy, is quantitative via measurements of the torsion angle, and reveals that ECM components, metalloproteinases, and ECM attachment molecules are important for this organ to retain its correct shape during post-embryonic growth. The Twp phenotype is therefore a promising experimental system to study ECM remodeling and diseases.

Genetic association of the GDNF alpha-receptor genes with schizophrenia and clozapine response.

GDNF (glial-cell-line derived neurotrophic factor) is a potent neurotrophic factor for dopaminergic neurons. Neuropsychiatric diseases and their treatments are associated with alterations in the levels of both GDNF and its receptor family (GDNF family receptor alpha or GFRA). GFRA1, GFRA2 and GFRA3 are located in chromosomal regions with suggestive linkage to schizophrenia. In this study we analyzed polymorphisms located in all four known GFRA genes and examined association with schizophrenia and clozapine response. We examined SNPs across the genes GFRA1-4 in 219 matched case-control subjects, 85 small nuclear families and 140 schizophrenia patients taking clozapine for 6months. We observed that GFRA3 rs11242417 and GFRA1 rs11197557 variants were significantly associated with schizophrenia after combining results from both schizophrenia samples. Furthermore, we found an overtransmission of the G-C GFRA1 rs7920934-rs730357 haplotype to subjects with schizophrenia and association of A-T-G-G GFRA3 rs10036665-rs10952-rs11242417-rs7726580 with schizophrenia in the case-control sample. On the other hand, GFRA2 variants were not associated with schizophrenia diagnosis but subjects carrying T-G-G rs1128397-rs13250096-rs4567028 haplotype were more likely to respond to clozapine treatment. The statistical significance of results survived permutation testing but not Bonferroni correction. We also found nominally-significant evidence for interactions between GFRA1, 2 and 3 associated with schizophrenia and clozapine response, consistent with the locations of these three genes within linkage regions for schizophrenia.

Dopamine counteracts octopamine signalling in a neural circuit mediating food response in C. elegans.

Animals assess food availability in their environment by sensory perception and respond to the absence of food by changing hormone and neurotransmitter signals. However, it is largely unknown how the absence of food is perceived at the level of functional neurocircuitry. In Caenorhabditis elegans, octopamine is released from the RIC neurons in the absence of food and activates the cyclic AMP response element binding protein in the cholinergic SIA neurons. In contrast, dopamine is released from dopaminergic neurons only in the presence of food. Here, we show that dopamine suppresses octopamine signalling through two D2-like dopamine receptors and the G protein Gi/o. The D2-like receptors work in both the octopaminergic neurons and the octopamine-responding SIA neurons, suggesting that dopamine suppresses octopamine release as well as octopamine-mediated downstream signalling. Our results show that C. elegans detects the absence of food by using a small neural circuit composed of three neuron types in which octopaminergic signalling is activated by the cessation of dopamine signalling.

C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV.

The gonad arms of C. elegans hermaphrodites acquire invariant shapes by guided migrations of distal tip cells (DTCs), which occur in three phases that differ in the direction and basement membrane substrata used for movement. We found that mig-6 encodes long (MIG-6L) and short (MIG-6S) isoforms of the extracellular matrix protein papilin, each required for distinct aspects of DTC migration. Both MIG-6 isoforms have a predicted N-terminal papilin cassette, lagrin repeats and C-terminal Kunitz-type serine proteinase inhibitory domains. We show that mutations affecting MIG-6L specifically and cell-autonomously decrease the rate of post-embryonic DTC migration, mimicking a post-embryonic collagen IV deficit. We also show that MIG-6S has two separable functions - one in embryogenesis and one in the second phase of DTC migration. Genetic data suggest that MIG-6S functions in the same pathway as the MIG-17/ADAMTS metalloproteinase for guiding phase 2 DTC migrations, and MIG-17 is abnormally localized in mig-6 class-s mutants. Genetic data also suggest that MIG-6S and non-fibrillar network collagen IV play antagonistic roles to ensure normal phase 2 DTC guidance.

UNC-129 regulates the balance between UNC-40 dependent and independent UNC-5 signaling pathways.

The UNC-5 receptor mediates axon repulsion from UNC-6/netrin through UNC-40 dependent (UNC-5 + UNC-40) and independent (UNC-5 alone) signaling pathways. It has been shown that UNC-40-dependent signaling is required for long-range repulsion of UNC-6/netrin; however, the mechanisms used to regulate distinct UNC-5 signaling pathways are poorly understood. We found that the C. elegans transforming growth factor beta (TGF-beta) family ligand UNC-129, graded opposite to UNC-6/netrin, functions independent of the canonical TGF-beta receptors to regulate UNC-5 cellular responses. Our observations indicates that UNC-129 facilitates long-range repulsive guidance of UNC-6 by enhancing UNC-5 + UNC-40 signaling at the expense of UNC-5 alone signaling through interaction with the UNC-5 receptor. This increases the set point sensitivity of growth cones to UNC-6/netrin as they simultaneously migrated up the UNC-129 gradient and down the UNC-6 gradient. Similar regulatory interactions between oppositely graded extracellular cues may be a common theme in guided cell and axon migrations.

Dopamine suppresses octopamine signaling in C. elegans: possible involvement of dopamine in the regulation of lifespan.

Amine neurotransmitters, such as dopamine, serotonin, and noradrenaline, play important roles in the modulation of behaviors and metabolism of animals. InC. elegans, it has been shown that serotonin and octopamine, an invertebrate equivalent of noradrenaline, also regulate lifespan through a mechanism related to food deprivation-mediated lifespan extension. We have shown recently that dopamine signaling, activated by the tactile perception of food, suppresses octopamine signaling and that the cessation of dopamine signaling in the absence of food leads to activation of octopamine signaling. Here, we discuss the apparent conservation of neural and molecular mechanisms for dopamine regulation of octopamine/noradrenaline signaling and a possible role for dopamine in lifespan regulation.

Examination of neurons in wild type and mutants of Caenorhabditis elegans using antibodies to horseradish peroxidase.

Antibodies to horseradish peroxidase (HRP) recognize 27 of 302 neurons and several non-neuronal cells in adult hermaphrodites of the soil nematode Caenorhabditis elegans and can be used to label these cells for cytological analysis in whole animals. The antibodies bind to the anterior members, but not to the posterior members of a set of mechanosensory neurons in wild type animals. Binding to one of the posterior mechanosensory neurons (PVM) occurs when this neuron migrates to an abnormal anterior position in mab-5 mutant animals, suggesting that expression of the epitope recognized by these antibodies is position dependent or that mab-5 mutations transform PVM into AVM intrinsically. The antibodies were used to characterize morphologies of two pairs of lumbar neurons (PHC and PVN) in uncoordinated mutants representing 95 unc genes. PHC and PVN morphologies were normal in most of the unc mutants examined, however, in mutants of 9 unc genes (unc-6, unc-13, unc-33, unc-44, unc-51, unc-61, unc-71, unc-73, and unc-98), misdirected PHC and/or PVN processes were observed at a high frequency. The morphologies of 2 other lumbar neurons, PHA and PHB, were determined previously in these mutants (Hedgecock et al., 1985). Mutations in most, but not all of these 9 unc genes affect the growth of the embryonic lumbar neurons PHA and PHB differently than they affect the growth of the postembryonic lumbar neurons PHC and PVN, indicating that these neurons require different, but overlapping sets of genes for different stages of normal growth and guidance.

The slit receptor EVA-1 coactivates a SAX-3/Robo mediated guidance signal in C. elegans.

The SAX-3/roundabout (Robo) receptor has SLT-1/Slit-dependent and -independent functions in guiding cell and axon migrations. We identified enhancer of ventral-axon guidance defects of unc-40 mutants (EVA-1) as a Caenorhabditis elegans transmembrane receptor for SLT-1. EVA-1 has two predicted galactose-binding ectodomains, acts cell-autonomously for SLT-1/Slit-dependent axon migration functions of SAX-3/Robo, binds to SLT-1 and SAX-3, colocalizes with SAX-3 on cells, and provides cell specificity to the activation of SAX-3 signaling by SLT-1. Double mutants of eva-1 or slt-1 with sax-3 mutations suggest that SAX-3 can (when slt-1 or eva-1 function is reduced) inhibit a parallel-acting guidance mechanism, which involves UNC-40/deleted in colorectal cancer.

C. elegans seu-1 encodes novel nuclear proteins that regulate responses to UNC-6/netrin guidance cues.

In C. elegans, ectopic expression of the UNC-5 netrin receptor is sufficient to cause repulsion of growth cones and cells away from ventral sources of the UNC-6/netrin guidance cue. A genetic suppressor screen identified the seu-1 gene as required for repulsion of touch neuron growth cones ectopically expressing unc-5. We report here that seu-1 mutations also enhance the frequency of distal tip cell migrations of unc-5 or unc-40 mutants. The seu-1 gene encodes two novel proteins (SEU-1A and SEU-1B) containing a charged central domain and several regions of low amino acid complexity. Transgenic rescue experiments indicate that seu-1 can act cell autonomously in the touch neurons and distal tip cells and that SEU-1 function requires both the SEU-1A and SEU-1B isoforms. A GFP fusion construct was expressed in a dynamic pattern throughout development and localized in the nuclei of neuronal and non-neuronal cells, including gonadal leader cells. These results implicate nuclear SEU-1 in the interpretation of UNC-6/netrin directional information by migrating growth cones and cells.

VAB-8, UNC-73 and MIG-2 regulate axon polarity and cell migration functions of UNC-40 in C. elegans.

One of the most intriguing features of axons is their ability to pioneer precise paths to their targets. How guidance-cue information is interpreted and integrated to form intricate neuronal networks has not been fully deciphered. Using Caenorhabditis elegans, we show that highly conserved receptors that guide pioneer axons along the dorsoventral axis, such as UNC-40 and SAX-3 (receptors for UNC-6 and SLT-1 guidance cues, respectively), can be co-opted to affect axon and cell migrations along the anterior-posterior axis. We further identify the kinesin-related VAB-8 protein as an upstream regulator of UNC-40, illuminating VAB-8's mechanism of action in determining the polarity of cell and axon migration. Finally, we show that UNC-73 and its target MIG-2 function with VAB-8 as upstream regulators of UNC-40 and that MIG-2 activity specifies UNC-40 subcellular localization. These data are indicative of previously unidentified regulatory roles for VAB-8 and small GTPases, which act together to regulate guidance receptor functions.

The UNC-73/Trio RhoGEF-2 domain is required in separate isoforms for the regulation of pharynx pumping and normal neurotransmission in C. elegans.

In both Caenorhabditis elegans and Drosophila, UNC-73/Trio functions in axon guidance by signaling through the Rac GTPase to regulate cytoskeletal rearrangements necessary for growth cone migrations. Here, we show that the complex C. elegans unc-73 gene encodes at least eight differentially expressed UNC-73 intracellular protein isoforms. Previously reported mutations affecting UNC-73 isoforms encoding the Rac-specific RhoGEF-1 domain cause uncoordinated movement, correlating with defects in axon guidance. Mutations in isoforms encoding the Rho-specific RhoGEF-2 domain, which we describe here, result in L1 stage larval lethality with no associated axon guidance defects. Isoform-specific rescue experiments reveal separate functions for the various RhoGEF-2-containing UNC-73 isoforms, which would not likely be discovered by conventional genetic screening. UNC-73 D1 and D2 appear to function redundantly in pharynx muscle to regulate the rate and strength of pharynx pumping, and in the HSN neurons and vulval muscles to control egg laying. Isoforms C1, C2, E, and F act redundantly within the nervous system to regulate the speed of locomotion. The multiple UNC-73 isoforms containing Rac- and Rho-specific RhoGEF domains therefore have distinct physiological functions. In addition to its previously identified role involving RhoGEF-1 in migrating cells and growth cones, our data indicate that UNC-73 signals through RhoGEF-2 to regulate pharynx and vulva musculature and to modulate synaptic neurotransmission.