The DEP domain-containing protein TOE-2 promotes apoptosis in the Q lineage of C. elegans through two distinct mechanisms.

Neuroblast divisions in the nematode Caenorhabditis elegans often give rise to a larger neuron and a smaller cell that dies. We have previously identified genes that, when mutated, result in neuroblast divisions that generate daughter cells that are more equivalent in size. This effect correlates with the survival of daughter cells that would normally die. We now describe a role for the DEP domain-containing protein TOE-2 in promoting the apoptotic fate in the Q lineage. TOE-2 localized at the plasma membrane and accumulated in the cleavage furrow of the Q.a and Q.p neuroblasts, suggesting that TOE-2 might position the cleavage furrow asymmetrically to generate daughter cells of different sizes. This appears to be the case for Q.a divisions where loss of TOE-2 led to a more symmetric division and to survival of the smaller Q.a daughter. Localization of TOE-2 to the membrane is required for this asymmetry, but, surprisingly, the DEP domain is dispensable. By contrast, loss of TOE-2 led to loss of the apoptotic fate in the smaller Q.p daughter but did not affect the size asymmetry of the Q.p daughters. This function of TOE-2 required the DEP domain but not localization to the membrane. We propose that TOE-2 ensures an apoptotic fate for the small Q.a daughter by promoting asymmetry in the daughter cell sizes of the Q.a neuroblast division but by a mechanism that is independent of cell size in the Q.p division.

Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

Autonomous and nonautonomous regulation of Wnt-mediated neuronal polarity by the C. elegans Ror kinase CAM-1.

Wnts are a conserved family of secreted glycoproteins that regulate various developmental processes in metazoans. Three of the five Caenorhabditis elegans Wnts, CWN-1, CWN-2 and EGL-20, and the sole Wnt receptor of the Ror kinase family, CAM-1, are known to regulate the anterior polarization of the mechanosensory neuron ALM. Here we show that CAM-1 and the Frizzled receptor MOM-5 act in parallel pathways to control ALM polarity. We also show that CAM-1 has two functions in this process: an autonomous signaling function that promotes anterior polarization and a nonautonomous Wnt-antagonistic function that inhibits anterior polarization. These antagonistic activities can account for the weak ALM phenotypes displayed by cam-1 mutants. Our observations suggest that CAM-1 could function as a Wnt receptor in many developmental processes, but the analysis of cam-1 mutants may fail to reveal CAM-1's role as a receptor in these processes because of its Wnt-antagonistic activity. In this model, loss of CAM-1 results in increased levels of Wnts that act through other Wnt receptors, masking CAM-1's autonomous role as a Wnt receptor.

MIG-15 and ERM-1 promote growth cone directional migration in parallel to UNC-116 and WVE-1.

Neurons require precise targeting of their axons to form a connected network and a functional nervous system. Although many guidance receptors have been identified, much less is known about how these receptors signal to direct growth cone migration. We used Caenorhabditis elegans motoneurons to study growth cone directional migration in response to a repellent UNC-6 (netrin homolog) guidance cue. The evolutionarily conserved kinase MIG-15 [homolog of Nck-interacting kinase (NIK)] regulates motoneuron UNC-6-dependent repulsion through unknown mechanisms. Using genetics and live imaging techniques, we show that motoneuron commissural axon morphology defects in mig-15 mutants result from impaired growth cone motility and subsequent failure to migrate across longitudinal obstacles or retract extra processes. To identify new genes acting with mig-15, we screened for genetic enhancers of the mig-15 commissural phenotype and identified the ezrin/radixin/moesin ortholog ERM-1, the kinesin-1 motor UNC-116 and the actin regulator WVE-1 complex. Genetic analysis indicates that mig-15 and erm-1 act in the same genetic pathway to regulate growth cone migration and that this pathway functions in parallel to the UNC-116/WVE-1 pathway. Further, time-lapse imaging of growth cones in mutants suggests that UNC-116 might be required to stimulate protrusive activity at the leading edge, whereas MIG-15 and ERM-1 maintain low activity at the rear edge. Together, these results support a model in which the MIG-15 kinase and the UNC-116-WVE-1 complex act on opposite sides of the growth cone to promote robust directional migration.

NMY-2, TOE-2 and PIG-1 regulate Caenorhabditis elegans asymmetric cell divisions.

Asymmetric cell division is an important mechanism that generates cellular diversity during development. Not only do asymmetric cell divisions produce daughter cells of different fates, but many can also produce daughters of different sizes, which we refer to as Daughter Cell Size Asymmetry (DCSA). In Caenorhabditis elegans, apoptotic cells are frequently produced by asymmetric divisions that exhibit DCSA, where the smaller daughter dies. We focus here on the divisions of the Q.a and Q.p neuroblasts, which produce larger surviving cells and smaller apoptotic cells and divide with opposite polarity using both distinct and overlapping mechanisms. Several proteins regulate DCSA in these divisions. Previous studies showed that the PIG-1/MELK and TOE-2 proteins regulate DCSA in both the Q.a and Q.p divisions, and the non-muscle myosin NMY-2 regulates DCSA in the Q.a division but not the Q.p division. In this study, we examined endogenously tagged NMY-2, TOE-2, and PIG-1 reporters and characterized their distribution at the cortex during the Q.a and Q.p divisions. In both divisions, TOE-2 localized toward the side of the dividing cell that produced the smaller daughter, whereas PIG-1 localized toward the side that produced the larger daughter. As previously reported, NMY-2 localized to the side of Q.a that produced the smaller daughter and did not localize asymmetrically in Q.p. We used temperature-sensitive nmy-2 mutants to determine the role of nmy-2 in these divisions and were surprised to find that these mutants only displayed DCSA defects in the Q.p division. We generated double mutant combinations between the nmy-2 mutations and mutations in toe-2 and pig-1. Because previous studies indicate that DCSA defects result in the transformation of cells fated to die into their sister cells, the finding that the nmy-2 mutations did not significantly alter the Q.a and Q.p DCSA defects of toe-2 and pig-1 mutants but did alter the number of daughter cells produced by Q.a and Q.p suggests that nmy-2 plays a role in specifying the fates of the Q.a and Q.p that is independent of its role in DCSA.

Caenorhabditis elegans flamingo cadherin fmi-1 regulates GABAergic neuronal development.

In a genetic screen for regulators of synaptic morphology, we identified the single Caenorhabditis elegans flamingo-like cadherin fmi-1. The fmi-1 mutants exhibit defective axon pathfinding, reduced synapse number, aberrant synapse size and morphology, as well as an abnormal accumulation of synaptic vesicles at nonsynaptic regions. Although FMI-1 is primarily expressed in the nervous system, it is not expressed in the ventral D-type (VD) GABAergic motorneurons, which are defective in fmi-1 mutants. The axon and synaptic defects of VD neurons could be rescued when fmi-1 was expressed exclusively in non-VD neighboring neurons, suggesting a cell nonautonomous action of FMI-1. FMI-1 protein that lacked its intracellular domain still retained its ability to rescue the vesicle accumulation defects of GABAergic motorneurons, indicating that the extracellular domain was sufficient for this function of FMI-1 in GABAergic neuromuscular junction development. Mutations in cdh-4, a Fat-like cadherin, cause similar defects in GABAergic motorneurons. The cdh-4 is expressed by the VD neurons and seems to function in the same genetic pathway as fmi-1 to regulate GABAergic neuron development. Thus, fmi-1 and cdh-4 cadherins might act together to regulate synapse development and axon pathfinding.

C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless.

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.

The Flamingo ortholog FMI-1 controls pioneer-dependent navigation of follower axons in C. elegans.

Development of a functional neuronal network during embryogenesis begins with pioneer axons creating a scaffold along which later-outgrowing axons extend. The molecular mechanism used by these follower axons to navigate along pre-existing axons remains poorly understood. We isolated loss-of-function alleles of fmi-1, which caused strong axon navigation defects of pioneer and follower axons in the ventral nerve cord (VNC) of C. elegans. Notably follower axons, which exclusively depend on pioneer axons for correct navigation, frequently separated from the pioneer. fmi-1 is the sole C. elegans ortholog of Drosophila flamingo and vertebrate Celsr genes, and this phenotype defines a new role for this important molecule in follower axon navigation. FMI-1 has a unique and strikingly conserved structure with cadherin and C-terminal G-protein coupled receptor domains and could mediate cell-cell adhesion and signaling functions. We found that follower axon navigation depended on the extracellular but not on the intracellular domain, suggesting that FMI-1 mediates primarily adhesion between pioneer and follower axons. By contrast, pioneer axon navigation required the intracellular domain, suggesting that FMI-1 acts as receptor transducing a signal in this case. Our findings indicate that FMI-1 is a cell-type dependent axon guidance factor with different domain requirements for its different functions in pioneers and followers.

Losing their minds: Mena/VASP/EVL triple knockout mice.

A recent Neuron paper by Kwiatkowski et al. that analyzes the phenotypes of mouse embryos lacking all members of the Ena/VASP family reveals new roles for these molecules in cortical development.

Abl kinase inhibits the engulfment of apoptotic [corrected] cells in Caenorhabditis elegans.

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor) proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.

The role of C. elegans Ena/VASP homolog UNC-34 in neuronal polarity and motility.

Ena/VASP proteins mediate the effects of guidance cues on the actin cytoskeleton. The single C. elegans homolog of the Ena/VASP family of proteins, UNC-34, is required for the migrations of cells and growth cones. Here we show that unc-34 mutant alleles also interact genetically with Wnt mutants to reveal a role for unc-34 in the establishment of neuronal polarity along the C. elegans anterior-posterior axis. Our mutant analysis shows that eliminating UNC-34 function results in neuronal migration and polarity phenotypes that are enhanced at higher temperatures, revealing a heat-sensitive process that is normally masked by the presence of UNC-34. Finally, we show that the UNC-34 protein is expressed broadly and accumulates in axons and at the apical junctions of epithelial cells. While most mutants lacked detectable UNC-34, three unc-34 mutants that contained missense mutations in the EVH1 domain produced full-length UNC-34 that failed to localize to apical junctions and axons, supporting the role for the EVH1 domain in localizing Ena/VASP family members.

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

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

C. elegans VAB-8 and UNC-73 regulate the SAX-3 receptor to direct cell and growth-cone migrations.

During nervous system development, a small number of conserved guidance cues and receptors regulate many axon trajectories. How could a limited number of cues and receptors regulate such complex projection patterns? One way is to modulate receptor function. Here we show that the Caenorhabditis elegans kinesin-related protein VAB-8L, which is necessary and sufficient for posterior cell and growth-cone migrations, directs these migrations by regulating the levels of the guidance receptor SAX-3 (also known as robo). Genetic experiments indicate that VAB-8L and the Rac guanine nucleotide exchange factor activity of UNC-73 (trio) increase the ability of the SLT-1 (slit) and UNC-6 (netrin) guidance pathways to promote posterior guidance. The observations of higher SAX-3 receptor abundance in animals with increasing amounts of VAB-8L, and of physical interactions between UNC-73 and both VAB-8L and the intracellular domain of the SAX-3, support a model whereby VAB-8L directs cell and growth-cone migrations by promoting localization of guidance receptors to the cell surface.

CDK-5 regulates the abundance of GLR-1 glutamate receptors in the ventral cord of Caenorhabditis elegans.

The proline-directed kinase Cdk5 plays a role in several aspects of neuronal development. Here, we show that CDK-5 activity regulates the abundance of the glutamate receptor GLR-1 in the ventral cord of Caenorhabditis elegans and that it produces corresponding changes in GLR-1-dependent behaviors. Loss of CDK-5 activity results in decreased abundance of GLR-1 in the ventral cord, accompanied by accumulation of GLR-1 in neuronal cell bodies. Genetic analysis of cdk-5 and the clathrin adaptin unc-11 AP180 suggests that CDK-5 functions prior to endocytosis at the synapse. The scaffolding protein LIN-10/Mint-1 also regulates GLR-1 abundance in the nerve cord. CDK-5 phosphorylates LIN-10/Mint-1 in vitro and bidirectionally regulates the abundance of LIN-10/Mint-1 in the ventral cord. We propose that CDK-5 promotes the anterograde trafficking of GLR-1 and that phosphorylation of LIN-10 may play a role in this process.

The T-box gene tbx-2, the homeobox gene egl-5 and the asymmetric cell division gene ham-1 specify neural fate in the HSN/PHB lineage.

Understanding how neurons adopt particular fates is a fundamental challenge in developmental neurobiology. To address this issue, we have been studying a Caenorhabditis elegans lineage that produces the HSN motor neuron and the PHB sensory neuron, sister cells produced by the HSN/PHB precursor. We have previously shown that the novel protein HAM-1 controls the asymmetric neuroblast division in this lineage. In this study we examine tbx-2 and egl-5, genes that act in concert with ham-1 to regulate HSN and PHB fate. In screens for mutants with abnormal HSN development, we identified the T-box protein TBX-2 as being important for both HSN and PHB differentiation. TBX-2, along with HAM-1, regulates the migrations of the HSNs and prevents the PHB neurons from adopting an apoptotic fate. The homeobox gene egl-5 has been shown to regulate the migration and later differentiation of the HSN. While mutations that disrupt its function show no obvious role for EGL-5 in PHB development, loss of egl-5 in a ham-1 mutant background leads to PHB differentiation defects. Expression of EGL-5 in the HSN/PHB precursor but not in the PHB neuron suggests that EGL-5 specifies precursor fate. These observations reveal a role for both EGL-5 and TBX-2 in neural fate specification in the HSN/PHB lineage.

The Enigmatic Canal-Associated Neurons Regulate Caenorhabditis elegans Larval Development Through a cAMP Signaling Pathway.

Caenorhabditis elegans larval development requires the function of the two Canal-Associated Neurons (CANs): killing the CANs by laser microsurgery or disrupting their development by mutating the gene ceh-10 results in early larval arrest. How these cells promote larval development, however, remains a mystery. In screens for mutations that bypass CAN function, we identified the gene kin-29, which encodes a member of the Salt-Inducible Kinase (SIK) family and a component of a conserved pathway that regulates various C. elegans phenotypes. Like kin-29 loss, gain-of-function mutations in genes that may act upstream of kin-29 or growth in cyclic-AMP analogs bypassed ceh-10 larval arrest, suggesting that a conserved adenylyl cyclase/PKA pathway inhibits KIN-29 to promote larval development, and that loss of CAN function results in dysregulation of KIN-29 and larval arrest. The adenylyl cyclase ACY-2 mediates CAN-dependent larval development: acy-2 mutant larvae arrested development with a similar phenotype to ceh-10 mutants, and the arrest phenotype was suppressed by mutations in kin-29 ACY-2 is expressed predominantly in the CANs, and we provide evidence that the acy-2 functions in the CANs to promote larval development. By contrast, cell-specific expression experiments suggest that kin-29 acts in both the hypodermis and neurons, but not in the CANs. Based on our findings, we propose two models for how ACY-2 activity in the CANs regulates KIN-29 in target cells.

The Caenorhabditis elegans gene ham-1 regulates daughter cell size asymmetry primarily in divisions that produce a small anterior daughter cell.

C. elegans cell divisions that produce an apoptotic daughter cell exhibit Daughter Cell Size Asymmetry (DCSA), producing a larger surviving daughter cell and a smaller daughter cell fated to die. Genetic screens for mutants with defects in apoptosis identified several genes that are also required for the ability of these divisions to produce daughter cells that differ in size. One of these genes, ham-1, encodes a putative transcription factor that regulates a subset of the asymmetric cell divisions that produce an apoptotic daughter cell. In a survey of C. elegans divisions, we found that ham-1 mutations affect primarily anterior/posterior divisions that produce a small anterior daughter cell. The affected divisions include those that generate an apoptotic cell as well as those that generate two surviving cells. Our findings suggest that HAM-1 primarily promotes DCSA in a certain class of asymmetric divisions.

Size Matters: How C. elegans Asymmetric Divisions Regulate Apoptosis.

Apoptosis is a form of programmed cell death used by metazoans to eliminate abnormal cells, control cell number, and shape the development of organs. The use of the nematode Caenorhabditis elegans as a model for the study of apoptosis has led to important insights into how cells die and how their corpses are removed. Eighty percent of these apoptotic cell deaths occur during nervous system development and in daughters of neuroblasts that divide asymmetrically. Pioneering work defined a conserved apoptosis pathway that is initiated in C. elegans by the BH3-only protein EGL-1 and that leads to the activation of the caspase CED-3. While the execution of the apoptotic fate is well understood, much less is known about the mechanisms that specify the apoptotic fate of particular cells. In some cells fated to die, this regulation occurs at the level of the egl-1 gene transcription, and investigators have identified several lineage-specific transcription factors that both positively and negatively regulate egl-1. In this review, we focus on a second set of molecules that appear to influence apoptosis by controlling the position of the cleavage plane in divisions that produce apoptotic cells.

Caenorhabditis elegans WASP and Ena/VASP proteins play compensatory roles in morphogenesis and neuronal cell migration.

We report here that WASP and Ena/VASP family proteins play overlapping roles in C. elegans morphogenesis and neuronal cell migration. Specifically, these studies demonstrate that UNC-34/Ena plays a role in morphogenesis that is revealed only in the absence of WSP-1 function and that WSP-1 has a role in neuronal cell migration that is revealed only in the absence of UNC-34/Ena activity. To identify additional genes that act in parallel to unc-34/ena during morphogenesis, we performed a screen for synthetic lethals in an unc-34 null mutant background utilizing an RNAi feeding approach. To our knowledge, this is the first reported RNAi-based screen for genetic interactors. As a result of this screen, we identified a second C. elegans WASP family protein, wve-1, that is most homologous to SCAR/WAVE proteins. Animals with impaired wve-1 function display defects in gastrulation, fail to undergo proper morphogenesis, and exhibit defects in neuronal cell migrations and axon outgrowth. Reducing wve-1 levels in either unc-34/ena or wsp-1 mutant backgrounds also leads to a significant enhancement of the gastrulation and morphogenesis defects. Thus, unc-34/ena, wsp-1, and wve-1 play overlapping roles during embryogenesis and unc-34/ena and wsp-1 play overlapping roles in neuronal cell migration. These observations show that WASP and Ena/VASP proteins can compensate for each other in vivo and provide the first demonstration of a role for Ena/VASP proteins in gastrulation and morphogenesis. In addition, our results provide the first example of an in vivo role for WASP family proteins in neuronal cell migrations and cytokinesis in metazoans.

The Caenorhabditis elegans Ror RTK CAM-1 inhibits EGL-20/Wnt signaling in cell migration.

During Caenorhabditis elegans development, the HSN neurons and the right Q neuroblast and its descendants undergo long-range anteriorly directed migrations. Both of these migrations require EGL-20, a C. elegans Wnt homolog. Through a canonical Wnt signaling pathway, EGL-20/Wnt transcriptionally activates the Hox gene mab-5 in the left Q neuroblast and its descendants, causing the cells to migrate posteriorly. In this report, we show that CAM-1, a Ror receptor tyrosine kinase (RTK) family member, inhibits EGL-20 signaling. Excess EGL-20, like loss of cam-1, caused the HSNs to migrate too far anteriorly. Excess CAM-1, like loss of egl-20, shifted the final positions of the HSNs posteriorly and caused the left Q neuroblast descendants to migrate anteriorly. The reversal in the migration of the left Q neuroblast and its descendants resulted from a failure to express mab-5, an egl-20 mutant phenotype. Our data suggest that CAM-1 negatively regulates EGL-20.