The Roles of Multiple UNC-40 (DCC) Receptor-Mediated Signals in Determining Neuronal Asymmetry Induced by the UNC-6 (Netrin) Ligand

The polarization of post-mitotic neurons is poorly understood. Preexisting spatially asymmetric cues, distributed within the neuron or as extracellular gradients, could be required for neurons to polarize. Alternatively, neurons might have the intrinsic ability to polarize without any preestablished asymmetric cues. In Caenorhabditis elegans, the UNC-40 (DCC) receptor mediates responses to the extracellular UNC-6 (netrin) guidance cue. For the HSN neuron, an UNC-6 ventral-dorsal gradient asymmetrically localizes UNC-40 to the ventral HSN surface. There an axon forms, which is ventrally directed by UNC-6. In the absence of UNC-6, UNC-40 is equally distributed and the HSN axon travels anteriorly in response to other cues. However, we find that a single amino acid change in the UNC-40 ectodomain causes randomly oriented asymmetric UNC-40 localization and a wandering axon phenotype. With UNC-6, there is normal UNC-40 localization and axon migration. A single UNC-6 amino acid substitution enhances the mutant phenotypes, whereas UNC-6 second-site amino acid substitutions suppress the phenotypes. We propose that UNC-40 mediates multiple signals to polarize and orient asymmetry. One signal triggers the intrinsic ability of HSN to polarize and causes randomly oriented asymmetry. Concurrently, another signal biases the orientation of the asymmetry relative to the UNC-6 gradient. The UNC-40 ectodomain mutation activates the polarization signal, whereas different forms of the UNC-6 ligand produce UNC-40 conformational changes that allow or prohibit the orientation signal.A major challenge for developmental neuroscience has been to understand how axons are able to detect and follow molecular gradients of different extracellular guidance cues. Attractive guidance cues are proposed to stimulate cytoplasmic signaling pathways that promote actin polymerization (Huber et al. 2003). Thus the direction of axon outgrowth is directly linked to the extracellular gradient of the guidance cue; i.e., there is greater extension on the side of the neuron that is closest to the source of the cue. Netrins are bifunctional guidance cues that are attractive to some axons but repulsive to others. Studies have shown that the axon response to netrin is determined by the composition of netrin receptors on the cell surface and the internal state of the growth cone (Round and Stein 2007). The UNC-6 (netrin) guidance cue in Caenorhabditis elegans interacts with the UNC-40 (DCC) receptor to mediate attraction (Hedgecock et al. 1990; Ishii et al. 1992; Chan et al. 1996). The AVM and HSN neurons are useful for studying UNC-40-mediated responses to UNC-6. The cell bodies of these neurons are situated on the lateral body wall and send a single axon ventrally during larval development.In AVM and HSN, a signaling module comprising UNC-6, UNC-40, phosphoinositide 3-kinase (PI3K), Rac, and MIG-10 (lamellipodin) is thought to transmit the directional information provided by the graded distribution of extracellular guidance cues to the internal cellular machinery that promotes directed outgrowth (Adler et al. 2006; Chang et al. 2006; Quinn et al. 2006, 2008). MIG-10 appears to provide an important link because this family of proteins can interact with proteins that promote actin polymerization, and it is associated with asymmetric concentrations of f-actin and microtubules in turning growth cones (Krause et al. 2004; Quinn et al. 2008). MIG-10 is observed as asymmetrically localized to the ventral site of axon outgrowth in developing HSN neurons. This MIG-10 localization is sensitive to the source of UNC-6. Normally, the source of UNC-6 is ventral; in the absence of UNC-6, there is an equal distribution of MIG-10 along the cell surface, whereas ectopic UNC-6 expression from dorsal muscles causes dorsal MIG-10 localization (Adler et al. 2006). The UNC-40 receptor is also asymmetrically localized in HSN, and this localization is also dependent on UNC-6 (Adler et al. 2006). UNC-40 signaling activates Rac GTPase, and MIG-10 interacts specifically with the activated Rac (Quinn et al. 2008). Therefore, the asymmetric activation of Rac through UNC-40 recruits asymmetric MIG-10 localization.By activating or directing components to the surface nearest the UNC-6 source, the asymmetric distribution of UNC-6 could polarize the neuron. However, an alternative idea is suggested from studies of chemotaxing cells. This model predicts that chemoattractant signaling involves two different elements: one that activates the intrinsic ability of cells to generate asymmetry and another that biases the orientation of the asymmetry (Wedlich-Soldner and Li 2003). The polarization signal does not depend on the spatial information provided by the chemoattractant gradient, whereas the orientation signal does. The asymmetric localization of the UNC-40 and MIG-10 signaling complex is suggestive of the segregation of signaling components into separate “front” and “rear” regions during chemotactic cell migration (Weiner 2002; Mortimer et al. 2008). It is hypothesized that this segregation is accomplished through short-range positive feedback mechanisms that promote the local production or recruitment of signaling molecules. In addition, a long-range inhibition mechanism globally increases the degradation of these molecules. Together such mechanisms could strongly amplify the asymmetric distribution of molecules needed for directed movement. This model has been put forth to explain why chemotactic cells polarize and move in a random direction when encountering a uniform chemoattractant concentration. Although the chemoattractant receptors may be uniformly stimulated across the surface of the cells, randomly oriented asymmetry can be established through these mechanisms.If the AVM and HSN neurons behave similarly to chemotactic cells, then uniformly stimulating UNC-40 receptors might similarly cause nonspecific asymmetric UNC-40 localization and axon migrations in varying directions. However, this is difficult to test in vivo. Unlike exposing chemotactic cells to a uniform concentration of a chemotractant in vitro, there is no reliable way to ensure that a neuron in vivo is exposed to a uniform concentration of UNC-6. The pseudocoelomic cavity of C. elegans is fluid filled, and UNC-6 expression patterns are spatially and temporally complex (Wadsworth et al. 1996). How the distribution of UNC-6 is affected by interactions with the extracellular matrix and cell surfaces is unknown.Using a genetic approach, we have found an UNC-40 mutation that triggers randomly oriented neuronal asymmetry. On the basis of the models proposed for chemotactic cells, we suggest that there is an UNC-6/UNC-40-mediated signal that specifically induces the neuron''s intrinsic ability to polarize. The UNC-40 mutation activates this signal; however, a second signal, which normally would concurrently orient asymmetry relative to the UNC-6 gradient, is not activated. Single amino acid changes within the UNC-6 ligand can enhance or suppress the randomly oriented asymmetry phenotype caused by the UNC-40 mutation. This suggests that specific UNC-40 conformations uncouple the activation of the different signals.     

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/VAChT and synaptobrevin.     

Reversal of Salt Preference Is Directed by the Insulin/PI3K and Gq/PKC Signaling in Caenorhabditis elegans

Animals search for foods and decide their behaviors according to previous experience. Caenorhabditis elegans detects chemicals with a limited number of sensory neurons, allowing us to dissect roles of each neuron for innate and learned behaviors. C. elegans is attracted to salt after exposure to the salt (NaCl) with food. In contrast, it learns to avoid the salt after exposure to the salt without food. In salt-attraction behavior, it is known that the ASE taste sensory neurons (ASEL and ASER) play a major role. However, little is known about mechanisms for learned salt avoidance. Here, through dissecting contributions of ASE neurons for salt chemotaxis, we show that both ASEL and ASER generate salt chemotaxis plasticity. In ASER, we have previously shown that the insulin/PI 3-kinase signaling acts for starvation-induced salt chemotaxis plasticity. This study shows that the PI 3-kinase signaling promotes aversive drive of ASER but not of ASEL. Furthermore, the G_q signaling pathway composed of G_qα EGL-30, diacylglycerol, and nPKC (novel protein kinase C) TTX-4 promotes attractive drive of ASER but not of ASEL. A putative salt receptor GCY-22 guanylyl cyclase is required in ASER for both salt attraction and avoidance. Our results suggest that ASEL and ASER use distinct molecular mechanisms to regulate salt chemotaxis plasticity.ANIMALS show various behaviors in response to environmental cues and modulate behaviors according to previous experience. To understand neuronal plasticity underlying learning, it is important to dissect neurons and molecules for sensing environmental stimuli, storing memory, and executing learned behaviors.The nematode Caenorhabditis elegans has only 302 neurons and functions of sensory neurons are well characterized (White et al. 1986; Bargmann 2006). C. elegans is attracted to odorants sensed by the AWC olfactory neurons or to salts sensed by the ASE gustatory neurons (Bargmann and Horvitz 1991; Bargmann et al. 1993). The ASE neuron class consists of a bilaterally symmetrical pair, ASE-left (ASEL) and ASE-right (ASER), which sense different sets of ions including Na⁺ and Cl⁻, respectively (Pierce-Shimomura et al. 2001; Suzuki et al. 2008; Ortiz et al. 2009). ASEL is activated by an increase in salt concentration, whereas ASER is activated by a decrease in salt concentration (Suzuki et al. 2008). In the ASE gustatory neurons, a cyclic GMP (cGMP) signaling pathway mediates sensory transduction (Komatsu et al. 1996; Suzuki et al. 2008; Ortiz et al. 2009). ASEL and ASER express different sets of receptor-type guanylyl cyclases (gcys) (Ortiz et al. 2006). Of these, gcy-22, which is specifically expressed in ASER, is important for attraction to ASER-sensed ions such as Cl⁻ (Ortiz et al. 2009).Preference for salts changes according to previous experience (known as gustatory plasticity or salt chemotaxis learning) (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). When worms are grown on a medium that contains sodium chloride (NaCl) and food (Escherichia coli), they show attraction to NaCl by using ASE neurons (Bargmann and Horvitz 1991; Suzuki et al. 2008). In contrast, after exposure to the salt under starvation conditions, they show reduced attraction to or even avoid the salt (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). In C. elegans, it was proposed that preference for a sensory cue is defined by the sensory neuron that detects the cue (Troemel et al. 1997). ASE neurons play a major role for salt attraction (Bargmann and Horvitz 1991; Suzuki et al. 2008; Ortiz et al. 2009). However, little is known about sensory neurons that drive the learned salt avoidance; it remains unclear whether ASE neurons act as salt receptors for the learned avoidance.We have previously shown that an insulin/PI 3-kinase signaling pathway is essential for salt chemotaxis learning (Tomioka et al. 2006). In C. elegans, the insulin-like signaling is composed of daf-2, age-1, and akt-1, which encode homologs of insulin receptor, PI 3-kinase, and protein kinase B, respectively (Morris et al. 1996; Kimura et al. 1997; Paradis and Ruvkun 1998). Mutants of daf-2, age-1, and akt-1 show attraction to salt even after starvation/NaCl conditioning (Tomioka et al. 2006).daf-18 encodes a homolog of phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten), which dephosphorylates phosphatidylinositol (3,4,5)-triphosphate and counteracts the insulin/PI 3-kinase signaling (Ogg and Ruvkun 1998; Gil et al. 1999; Mihaylova et al. 1999; Rouault et al. 1999; Solari et al. 2005). Mutants of daf-18, in which the PI 3-kinase signaling is activated, show reduced attraction to NaCl even without conditioning. Since the insulin/PI 3-kinase signaling acts in ASER, we proposed that the insulin/PI 3-kinase signaling attenuates the attractive drive of ASER (Tomioka et al. 2006).In C. elegans, diacylglycerol (DAG) regulates functions of motor neurons and sensory neurons. egl-30, which encodes the α-subunit of heterotrimeric G-protein G_q, facilitates production of DAG and enhances locomotory movements (Brundage et al. 1996; Lackner et al. 1999). In the AWC olfactory neurons, a novel protein kinase C-ɛ/η (nPKC-ɛ/η) ortholog TTX-4 (also known as PKC-1), which is one of DAG targets, plays an essential role in attraction behavior to AWC-sensed odors (Okochi et al. 2005; Tsunozaki et al. 2008). GOA-1 G_oα regulates olfactory adaptation by antagonizing G_qα–DAG signaling (Matsuki et al. 2006).This study investigated the involvement of the ASE taste receptor neurons in the starvation-induced salt avoidance. We show that both ASEL and ASER contribute to salt chemotaxis learning. Activation of the PI 3-kinase signaling and the G_q/DAG/PKC signaling acted antagonistically in reversal of ASER function, whereas these signaling pathways did not have prominent effects on ASEL function. In ASER, GCY-22 was required for both salt attraction and avoidance. These results suggest that ASE neurons are important for bidirectional chemotaxis and also suggest that distinct molecular mechanisms regulate functions of ASEL and ASER in salt chemotaxis learning.     

Genetics of Lipid-Storage Management in Caenorhabditis elegans Embryos

Lipids play a pivotal role in embryogenesis as structural components of cellular membranes, as a source of energy, and as signaling molecules. On the basis of a collection of temperature-sensitive embryonic lethal mutants, a systematic database search, and a subsequent microscopic analysis of >300 interference RNA (RNAi)–treated/mutant worms, we identified a couple of evolutionary conserved genes associated with lipid storage in Caenorhabditis elegans embryos. The genes include cpl-1 (cathepsin L–like cysteine protease), ccz-1 (guanine nucleotide exchange factor subunit), and asm-3 (acid sphingomyelinase), which is closely related to the human Niemann-Pick disease–causing gene SMPD1. The respective mutant embryos accumulate enlarged droplets of neutral lipids (cpl-1) and yolk-containing lipid droplets (ccz-1) or have larger genuine lipid droplets (asm-3). The asm-3 mutant embryos additionally showed an enhanced resistance against C band ultraviolet (UV-C) light. Herein we propose that cpl-1, ccz-1, and asm-3 are genes required for the processing of lipid-containing droplets in C. elegans embryos. Owing to the high levels of conservation, the identified genes are also useful in studies of embryonic lipid storage in other organisms.     

A Ubiquitin E2 Variant Protein Acts in Axon Termination and Synaptogenesis in Caenorhabditis elegans

In the developing nervous system, cohorts of events regulate the precise patterning of axons and formation of synapses between presynaptic neurons and their targets. The conserved PHR proteins play important roles in many aspects of axon and synapse development from C. elegans to mammals. The PHR proteins act as E3 ubiquitin ligases for the dual-leucine-zipper-bearing MAP kinase kinase kinase (DLK MAPKKK) to regulate the signal transduction cascade. In C. elegans, loss-of-function of the PHR protein RPM-1 (Regulator of Presynaptic Morphology-1) results in fewer synapses, disorganized presynaptic architecture, and axon overextension. Inactivation of the DLK-1 pathway suppresses these defects. By characterizing additional genetic suppressors of rpm-1, we present here a new member of the DLK-1 pathway, UEV-3, an E2 ubiquitin-conjugating enzyme variant. We show that uev-3 acts cell autonomously in neurons, despite its ubiquitous expression. Our genetic epistasis analysis supports a conclusion that uev-3 acts downstream of the MAPKK mkk-4 and upstream of the MAPKAPK mak-2. UEV-3 can interact with the p38 MAPK PMK-3. We postulate that UEV-3 may provide additional specificity in the DLK-1 pathway by contributing to activation of PMK-3 or limiting the substrates accessible to PMK-3.CHEMICAL synapses are specialized cellular junctions that enable neurons to communicate with their targets. An electrical impulse causes calcium channel opening and consequently stimulates synaptic vesicles in the presynaptic terminals to fuse at the plasma membrane. Neurotransmitter activates receptors on the postsynaptic membrane and triggers signal transduction in the target cell. For this communication to occur efficiently, the organization of the proteins within these juxtaposed pre- and postsynaptic terminals must be tightly regulated (Jin and Garner 2008). Previous studies in Caenorhabditis elegans have identified RPM-1, a member of the conserved PHR (Pam/Highwire/RPM-1) family of proteins, as an important regulator for the synapse (Schaefer et al. 2000; Zhen et al. 2000). Recent functional studies of other PHR proteins have shown that they are also required for a number of steps during nervous system development including axon guidance, growth, and termination (Wan et al. 2000; D''souza; et al. 2005; Bloom et al. 2007; Grill et al. 2007; Lewcock et al. 2007; Li et al. 2008).The signaling cascades regulated by the PHR proteins have been identified using genetic modifier screens (Diantonio et al. 2001; Liao et al. 2004; Nakata et al. 2005; Collins et al. 2006) and biochemical approaches (Grill et al. 2007; Wu et al. 2007). These studies reveal that a major function of PHR proteins is to act as ubiquitin E3 ligases (Jin and Garner 2008). In C. elegans, RPM-1 (Regulator of Presynaptic Morphology-1) regulates the abundance of its substrate, the dual-leucine-zipper-bearing MAP kinase kinase kinase (DLK MAPKKK), and controls the activity of the MAP kinase cascade composed of three additional kinases, MAPKK MKK-4, p38 MAPK PMK-3, and MAPKAPK MAK-2 (Nakata et al. 2005; Yan et al. 2009). This signaling cascade further regulates the activity of the CCAAT/enhancer binding protein (C/EBP), CEBP-1, via a mechanism involving 3′-UTR-mediated mRNA decay.Signal transduction involving MAP kinases can be fine tuned using multiple mechanisms to ensure optimal signaling outputs (Raman et al. 2007). For example, scaffold proteins for MAP kinases can provide spatial regulation of kinase activation in response to different stimuli (Remy and Michnick 2004; Whitmarsh 2006). Small protein tags such as ubiquitin have also been shown to control the activation of kinases. Specifically, in the IKK pathway ubiquitination via Lys63 chain formation catalyzed by the Ubc13/Uev1a E2 complex and TRAF6 E3 ligase is required for TAK1 kinase activation (Skaug et al. 2009).To further the understanding of the DLK-1 pathway in the development of the nervous system, we characterized a new complementation group of rpm-1(lf) suppressors. These mutations affect the gene uev-3, a ubiquitin E2 conjugating (UBC) enzyme variant (UEV). UEV proteins belong to the UBC family, but lack the catalytic active cysteine necessary for conjugating ubiquitin (Sancho et al. 1998). The best characterized UEV proteins are yeast Mms2 and mammalian Uev1A, both of which act as the obligatory partner for the active E2 Ubc13 and function in DNA repair and IKB pathways, respectively (Deng et al. 2000; Hurley et al. 2006). In addition, UEV proteins, such as Tsg101, can also regulate endosomal trafficking (Babst et al. 2000). We find that similar to other members of the DLK-1 pathway, uev-3 functions cell autonomously in neurons. uev-3 genetically acts downstream of mkk-4 and upstream of mak-2. UEV-3 can bind PMK-3 in heterologous protein interaction assays. We hypothesize that UEV-3 may add specificity to the DLK-1 pathway by binding to PMK-3 for its activation or for selecting specific downstream targets.     

toca-1 Is in a Novel Pathway That Functions in Parallel with a SUN-KASH Nuclear Envelope Bridge to Move Nuclei in Caenorhabditis elegans

Moving the nucleus to an intracellular location is critical to many fundamental cell and developmental processes, including cell migration, differentiation, fertilization, and establishment of cellular polarity. Bridges of SUN and KASH proteins span the nuclear envelope and mediate many nuclear positioning events, but other pathways function independently through poorly characterized mechanisms. To identify and characterize novel mechanisms of nuclear migration, we conducted a nonbiased forward genetic screen for mutations that enhanced the nuclear migration defect of unc-84, which encodes a SUN protein. In Caenorhabditis elegans larvae, failure of hypodermal P-cell nuclear migration results in uncoordinated and egg-laying–defective animals. The process of P-cell nuclear migration in unc-84 null animals is temperature sensitive; at 25° migration fails in unc-84 mutants, but at 15° the migration occurs normally. We hypothesized that an additional pathway functions in parallel to the unc-84 pathway to move P-cell nuclei at 15°. In support of our hypothesis, forward genetic screens isolated eight emu (enhancer of the nuclear migration defect of unc-84) mutations that disrupt nuclear migration only in a null unc-84 background. The yc20 mutant was determined to carry a mutation in the toca-1 gene. TOCA-1 functions to move P-cell nuclei in a cell-autonomous manner. TOCA-1 is conserved in humans, where it functions to nucleate and organize actin during endocytosis. Therefore, we have uncovered a player in a previously unknown, likely actin-dependent, pathway that functions to move nuclei in parallel to SUN-KASH bridges. The other emu mutations potentially represent other components of this novel pathway.     

Genome sequence and description of Aeromicrobium massiliense sp. nov.

Aeromicrobium massiliense strain JC14^Tsp. nov. is the type strain of Aeromicrobium massiliense sp. nov., a new species within the genus Aeromicrobium. This strain, whose genome is described here, was isolated from the fecal microbiota of an asymptomatic patient. Aeromicrobium massiliense is an aerobic rod-shaped gram-positive bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,322,119 bp long genome contains 3,296 protein-coding and 51 RNA genes.     

Complete genome sequence of the sulfate-reducing firmicute Desulfotomaculum ruminis type strain (DLT)

Desulfotomaculum ruminis Campbell and Postgate 1965 is a member of the large genus Desulfotomaculum which contains 30 species and is contained in the family Peptococcaceae. This species is of interest because it represents one of the few sulfate-reducing bacteria that have been isolated from the rumen. Here we describe the features of D. ruminis together with the complete genome sequence and annotation. The 3,969,014 bp long chromosome with a total of 3,901 protein-coding and 85 RNA genes is the second completed genome sequence of a type strain of the genus Desulfotomaculum to be published, and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2009.Keywords : anaerobic, motile, sporulating, mesophilic, sulfate-reducer, hydrogen sulfide, incomplete oxidizer, mixotrophic, CSP 2009, Peptococcaceae, Clostridiales     

The Jaw of the Worm: GTPase-activating Protein EAT-17 Regulates Grinder Formation in Caenorhabditis elegans

Constitutive transport of cellular materials is essential for cell survival. Although multiple small GTPase Rab proteins are required for the process, few regulators of Rabs are known. Here we report that EAT-17, a novel GTPase-activating protein (GAP), regulates RAB-6.2 function in grinder formation in Caenorhabditis elegans. We identified EAT-17 as a novel RabGAP that interacts with RAB-6.2, a protein that presumably regulates vesicle trafficking between Golgi, the endoplasmic reticulum, and plasma membrane to form a functional grinder. EAT-17 has a canonical GAP domain that is critical for its function. RNA interference against 25 confirmed and/or predicted RABs in C. elegans shows that RNAi against rab-6.2 produces a phenotype identical to eat-17. A directed yeast two-hybrid screen using EAT-17 as bait and each of the 25 RAB proteins as prey identifies RAB-6.2 as the interacting partner of EAT-17, confirming that RAB-6.2 is a specific substrate of EAT-17. Additionally, deletion mutants of rab-6.2 show grinder defects identical to those of eat-17 loss-of-function mutants, and both RAB-6.2 and EAT-17 are expressed in the terminal bulb of the pharynx where the grinder is located. Collectively, these results suggest that EAT-17 is a specific GTPase-activating protein for RAB-6.2. Based on the conserved function of Rab6 in vesicular transport, we propose that EAT-17 regulates the turnover rate of RAB-6.2 activity in cargo trafficking for grinder formation.     

Functional Genomic Identification of Genes Required for Male Gonadal Differentiation in Caenorhabditis elegans

The Caenorhabditis elegans somatic gonad develops from a four-cell primordium into a mature organ that differs dramatically between the sexes in overall morphology (two arms in hermaphrodites and one in males) and in the cell types comprising it. Gonadal development in C. elegans is well studied, but regulation of sexual differentiation, especially later in gonadal development, remains poorly elucidated. To identify genes involved in this process, we performed a genome-wide RNAi screen using sex-specifically expressed gonadal GFP reporters. This screen identified several phenotypic classes, including ∼70 genes whose depletion feminized male gonadal cells. Among the genes required for male cell fate specification are Wnt/β-catenin pathway members, cell cycle regulators, and genes required for mitotic spindle function and cytokinesis. We find that a Wnt/β-catenin pathway independent of extracellular Wnt ligand is essential for asymmetric cell divisions and male differentiation during gonadal development in larvae. We also find that the cell cycle regulators cdk-1 and cyb-3 and the spindle/cytokinesis regulator zen-4 are required for Wnt/β-catenin pathway activity in the developing gonad. After sex is determined in the gonadal primordium the global sex determination pathway is dispensable for gonadal sexual fate, suggesting that male cell fates are promoted and maintained independently of the global pathway during this period.THE Caenorhabditis elegans gonad derives from a simple primordium of four cells that coalesces during embryogenesis and contains two somatic gonad precursors (SGPs), Z1 and Z4, flanking two germline precursors, Z2 and Z3 (Kimble and Hirsh 1979). The SGPs undergo very different developmental programs in each sex, involving sexually dimorphic cell lineages and migrations and sex-specific cellular differentiation. The result is a two-armed bilaterally symmetrical gonad in the adult hermaphrodite or a single-armed asymmetric gonad in the adult male. The high degree of sexual dimorphism of the mature organ and variety of cellular events that occur sex specifically during its development make the C. elegans gonad an outstanding model for sex-specific organogenesis.Development of the somatic gonad occurs in two phases. The early phase defines the gonadal axes and establishes the precursors of the major gonadal cell types. This takes place during the first larval stage (L1), beginning shortly after hatching with the first division of the SGPs. In both sexes SGP division is asymmetric in terms of both the sizes and the fates of the daughter cells, and establishes the proximal/distal axis of the gonad (Hirsh et al. 1976; Kimble and Hirsh 1979). The global sex determination pathway establishes the future sex of the gonad around the time of hatching (Klass et al. 1976; Nelson et al. 1978), and sexual dimorphism is already apparent when the SGPs divide: the size asymmetry of the SGP daughters is much more pronounced in males than hermaphrodites. In both sexes the asymmetry of the first SGP division requires a Wnt/β-catenin pathway. Mutations compromising this pathway cause a “symmetrical sisters” phenotype in which both daughters adopt the same fate (Miskowski et al. 2001; Siegfried and Kimble 2002; Phillips and Kimble 2009). Sex specificity is imposed on the SGPs by the global sex determining gene tra-1 (Hodgkin 1987) and the gonad-specific sex determining gene fkh-6 (Chang et al. 2004). These genes play opposing roles in SGP sex determination, with tra-1 feminizing and fkh-6 masculinizing the somatic gonad, and they also act redundantly to promote mitotic proliferation of the SGP lineage (Chang et al. 2004). SGP sex determination is linked to cell cycle progression by cyclin D, which is required to overcome repression of fkh-6 expression in the SGPs by E2F (Tilmann and Kimble 2005).The later phase of gonadal development involves the elongation of the gonad, together with cellular proliferation and differentiation, and lasts from L2 to adulthood. During L2 the somatic cells enlarge and leader cells (distal tip cells in the hermaphrodite, linker cell in the male) begin long-range migrations that extend the gonad. During L3, somatic gonad cell division resumes in both sexes, leading to the formation of differentiated somatic cell types by the end of L3 or beginning of L4. Gonadal morphogenesis is completed and gametogenesis begins during L4 (Kimble and Hirsh 1979).Although SGP division and much of hermaphrodite gonadal development have been well studied (Hubbard and Greenstein 2000), sexual cell fate specification in the somatic gonad is more poorly understood, particularly after the L1 stage. Despite the importance of fkh-6 in promoting male differentiation, it is expressed in males only during early L1 and null mutants have incomplete gonadal sex reversal. We have therefore performed a genome-wide RNAi screen to identify additional genes required after hatching for gonadal development in each sex. Among the advantages of this approach is the ability to identify gonadal regulators that also are essential for embryonic development. To our knowledge this is the first functional genomic study of gonadal sex differentiation.The screen identified many genes whose depletion disrupts gonadogenesis in each sex and nearly 70 genes whose depletion causes gonadal feminization in males. Prominent among this latter class were components of a Wnt/β-catenin pathway, cell cycle regulators, and genes involved in mitotic spindle function and cytokinesis. We find that Wnt/β-catenin activity continues in both sexes after SGP division and is required for male cell fate commitment in the gonad. We also find that the cyclin-dependent kinase cdk-1 and its cognate cyclin cyb-3 as well as the mitotic spindle regulator zen-4 are required for gonadal Wnt/β-catenin pathway activity, providing a potential new link between the cell cycle, asymmetric division, and sexual differentiation. The feminization caused by depletion of Wnt/β-catenin pathway components or cdk-1 is independent of the global sex determination pathway, suggesting that sexual fates in the male gonad remain plastic after the primary sex determination decision.     

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase’s role in germline development in higher eukaryotes.     

Systematic Analyses of rpm-1 Suppressors Reveal Roles for ESS-2 in mRNA Splicing in Caenorhabditis elegans

The PHR (Pam/Highwire/RPM-1) family of ubiquitin E3 ligases plays conserved roles in axon patterning and synaptic development. Genetic modifier analysis has greatly aided the discovery of the signal transduction cascades regulated by these proteins. In Caenorhabditis elegans, loss of function in rpm-1 causes axon overgrowth and aberrant presynaptic morphology, yet the mutant animals exhibit little behavioral deficits. Strikingly, rpm-1 mutations strongly synergize with loss of function in the presynaptic active zone assembly factors, syd-1 and syd-2, resulting in severe locomotor deficits. Here, we provide ultrastructural evidence that double mutants, between rpm-1 and syd-1 or syd-2, dramatically impair synapse formation. Taking advantage of the synthetic locomotor defects to select for genetic suppressors, previous studies have identified the DLK-1 MAP kinase cascade negatively regulated by RPM-1. We now report a comprehensive analysis of a large number of suppressor mutations of this screen. Our results highlight the functional specificity of the DLK-1 cascade in synaptogenesis. We also identified two previously uncharacterized genes. One encodes a novel protein, SUPR-1, that acts cell autonomously to antagonize RPM-1. The other affects a conserved protein ESS-2, the homolog of human ES2 or DGCR14. Loss of function in ess-2 suppresses rpm-1 only in the presence of a dlk-1 splice acceptor mutation. We show that ESS-2 acts to promote accurate mRNA splicing when the splice site is compromised. The human DGCR14/ES2 resides in a deleted chromosomal region implicated in DiGeorge syndrome, and its mutation has shown high probability as a risk factor for schizophrenia. Our findings provide the first functional evidence that this family of proteins regulate mRNA splicing in a context-specific manner.