Morck, Catarina et al. (2011) International Worm Meeting "Soulless nematodes: KIN-29 mediates the essential function of the CAN neurons."

Garriga, Gian, Morck, Catarina

[International Worm Meeting, 2011]

The canal-associated neurons (CANL/R) are two bilaterally symmetric neurons that are born in the head and migrate to the middle of the embryo. Each neuron extends two processes: one process grows to the head and one to the tail (1). The CANs are essential for viability. Sulston and Hodgkin originally discovered that killing the CANs with laser microsurgery resulted in a starved appearance and larval arrest, leading them to propose that the CANs were the nematode soul (2). Similarly, ceh-10(gm58) mutants lack CANs and die as larvae (3). In vab-8(e1017) mutants, the CAN neurons fail to migrate, and their posterior processes only extend a short distance. As a consequence, the part of the body that lacks CAN processes becomes much thinner and the worms exhibit a withered tail phenotype (4). We identified a mutation that suppresses the withered tail phenotype of vab-8 mutants and the larval lethality of ceh-10 mutants. Surprisingly, the double mutants still retain defective CANs, which indicates that the suppressor mutation bypasses the requirement for CAN function. The suppressor encodes kin-29, a serine threonine kinase most homologous to members of the ELKL motif kinase (EMK) family and salt-induced kinase family (5). KIN-29 and its homologs phosphorylate and inhibit class II histone deacetylases. In C. elegans, KIN-29 inhibits HDA-4, which acts with the MADS domain transcription factor MEF-2 to regulate chemoreceptor gene expression (6). Loss of HDA-4 or MEF-2 is not lethal, indicating that KIN-29 must have other targets that mediate CAN function. The pseudocoelom fills with fluid in animals with compromised CAN function. This phenotype and the association of the CAN processes with the excretory canals led us to test the model where the CANs essential function is to regulate the activity of the excretory canals. The analysis of CAN and excretory canal mutants suggest that the CANs regulate other cells, and we are currently attempting to identify CAN target cells by addressing where KIN-29 functions. REFERENCES: 1.Sulston, J.E., E.Schierenberg, J.G. White and Thomson, J.N. (1983) Dev. Biol. 100: 64-119 2.Sulston, J.E. and Hodgkin, J.A. Worm Breeder's Gazette 5(1): 19 3.Forrester, W.C., Garriga, G. (1997) Development 124(9): 1831-43. 4.Manser, J. Wood, W.B. (1990) Dev Gen 11:49-64 5.Lanjuin, A., Sengupta, P. (2002) Neuron 33(3): 369-81 6.van der Linden, A.M., Nolan, K., Sengupta, P. (2007) EMBO. 26(2): 358-70.

DEVKOTA, RANJAN et al. (2015) International Worm Meeting "Novel regulators of the C. elegans UNC-6/Netrin pathway."

Morck, Catarina, CHIEN, JASON, DEVKOTA, RANJAN, Garriga, Gian

[International Worm Meeting, 2015]

Precise targeting of axons is essential for the proper functioning of the nervous system. Axonal growth cones respond to various guidance cues and navigate through a complex environment to reach their synaptic targets1. Even though many guidance cues and receptors have been identified, there is less knowledge of how axons respond to different cues in different phases of their growth. The aim of this project is to enhance the understanding of axon guidance by focusing on the development of the bilaterally symmetric Hermaphrodite Specific Neurons (HSNs). Each HSN extends a single axon to the ventral nerve cord, which then turns anteriorly and extends to the nerve ring2. The HSNs innervate vulval muscles and the AVF neurons to stimulate hermaphrodite egg laying and locomotion, respectively3,4. We have isolated three mutants whose HSN axons fail to extend ventrally. Our preliminary mapping, complementation and genetic interaction data suggest that these mutations define three genes that function together to antagonize the conserved UNC-6/Netrin signaling pathway. Our initial goal is to identify the underlying mutations with whole genome sequencing and rescue experiments. The identification of these genes will shed light on how the UNC-6/Netrin pathway is modulated for proper axon guidance along the dorsal-ventral axis.References:1. Dickson, B. J. Molecular mechanisms of axon guidance. Science 298, 1959-1964(2002).2. Garriga, G., Desai, C. & Horvitz, H. R. Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans. Development 117, 1071-1087(1993).3. Desai, C., Garriga, G., McIntire, S. L. & Horvitz, H. R. A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons. Nature 336, 638-646(1988).4. Hardaker, L. A., Singer, E., Kerr, R., Zhou, G. & Schafer, W. R. Serotonin modulates locomotory behavior and coordinates egg-laying and movement in Caenorhabditis elegans. J. Neurobiol. 49, 303-313(2001). .

Ikegami, Richard et al. (2011) International Worm Meeting "New methods to study nerve bundle organization."

Garriga, Gian, Ikegami, Richard

[International Worm Meeting, 2011]

Recent findings have shown that mammalian nerve bundles are organized and that this organization facilitates topographic targeting1,2. John White and colleagues observed that nerve bundles in C. elegans are also organized3, suggesting to us that the C. elegans ventral nerve cord (VNC) could be a useful model for understanding how organization within bundles develop. Sets of neurons have axons run adjacent to one another over long distances in C. elegans nerve bundles. Neighbors of a particular axon presumably reflect the path that guided the neuron's growth cone along the nerve bundle during development. We developed approaches to study the organization of the HSN motor neurons in the VNC. These are the last neurons to extend axons along an otherwise, fully developed VNC. Until now, only EM analysis of serially sectioned worms provided the resolution to monitor axon positions within nerve bundles3. We adapted the membrane-localized split-GFP (GRASP)4 to label the interface between the HSN axons and those of the PVP and PVQ neurons that guide HSN fasciculation in the VNC5. The labeled interfaces reproduce the fasciculation pattern predicted from EM analysis. Earlier in development, the PVP axon provides a track for the PVQ growth cone6. PVP/PVQ GRASP along the VNC is continuous, which differs from the EM analysis. We suspect that GRASP mediated adhesion maintains interactions between axons that would normally be disrupted during postembryonic development. This discrepancy has important implications for the use of GRASP and how the VNC develops. In order to further investigate how the VNC develops, we have developed several additional techniques. These include a heterologous cell adhesion system to drive changes in HSN fasciculation, an RNAi system to target HSN fasciculation before the HSN axons extend but after the VNC has developed and strains that are missing one of the redundant cells involved in HSN fasciculation. These techniques will be used to re-evaluate the roles of molecules implicated in HSN fasciculation as well as identify new molecules. Furthermore, these techniques are easily adapted for use with other neurons. 1Imai T. et al. (2009) Science 325(5940):585-90. 2Plas D.T. et al. (2005) J Comp Neurol. 491(4):305-19. 3White J.G. et al. (1986) Phil. Trans. R. Soc. Lond. B. 314(1165):1-340. 4Feinberg E.H. et al. (2008) Neuron 57(3):353-63. 5Garriga G. et al. (1993) Development 117(3):1071-87. 6Durbin R. (1987) Ph.D. Dissertation, Cambridge University, England.

Teuliere, Jerome et al. (2013) International Worm Meeting "Cortical HAM-1 positions the cleavage furrow in myosin-dependent asymmetric neuroblast divisions that generates apoptotic cells."

Teuliere, Jerome, Hawkins, Nancy, Garriga, Gian

[International Worm Meeting, 2013]

While the mechanisms that execute apoptosis in C. elegans are understood, less is known about how somatic cells choose the apoptotic fate. Cell divisions that produce apoptotic cells are asymmetric, generating a larger cell that lives and a smaller cell that dies. Two examples are the anterior (Q.a) and posterior (Q.p) daughters of the Q cell. Distinct mechanisms, however, shift the position of the cleavage furrow in these two divisions to produce an anterior apoptotic cell (Q.aa) division and a posterior apoptotic cell (Q.pp) 3. Protrusion of the posterior membrane and anterior localization of myosin II produce an anteriorly-shifted Q.a cleavage furrow. Protrusion of the anterior membrane and posterior displacement of the mitotic spindle produce a posteriorly-shifted Q.p furrow. We find that the winged-helix protein HAM-1 specifically controls the Q.a furrow position. Q.a divided with a reversed polarity in ham-1 mutants to produce a larger anterior and a smaller posterior daughter, a Q.p division pattern. As in the wild-type Q.p division, the anterior Q.a membrane protrudes and the spindle is displaced posteriorly. The asymmetric distribution of myosin II remained asymmetric in QR.a, indicating that while myosin asymmetry is required for an anteriorly positioned Q.a cleavage furrow 3, it is not sufficient in QR.a. A functional GFP::HAM-1 transgene had a Q.a-specific expression, consistent with the ham-1 phenotype. Although HAM-1 antibodies only detected cortical HAM-1 in dividing cells 4, we detected HAM-1::GFP at the cortex and in the nucleus of both embryonic cells and in the Q lineage. Misexpression of HAM-1 in Q.p led to daughter size asymmetry defects. We observed this defect with either wild-type HAM-1 or a mutant that does not accumulate in the nucleus, suggesting that HAM-1 acts at the cortex to position the cleavage furrow. The spectrum of ham-1 mutant defects suggests that HAM-1 primarily regulates a subset of divisions that produce anterior cells fated to die. 3 Ou, G et al. Science 330, 677-680 (2010) 4 Guenther, C & Garriga, G. Development 122, 3509-3518 (1996).

Gurling, Mark et al. (2009) International Worm Meeting "Frizzled receptor antagonism in neuronal polarity."

Pan, Chun-Liang, Gurling, Mark, Garriga, Gian

[International Worm Meeting, 2009]

Wnt glycoproteins signal through Frizzled receptors to regulate many aspects of neural development such as neuronal migration and polarity1,2,3,4. The two Frizzled receptors LIN-17 and MIG-1 appear to play antagonistic roles in certain developmental contexts3,5. In worms mutant for the Frizzled receptor LIN-17, the polarity of the PLM mechanosensory neuron is reversed. Mutations in lin-44, which encodes one of the Wnt proteins, caused similar PLM phenotypes. Synapses made by the anterior process in wild-type animals are now made by the posterior process of mutant PLMs1,2. A similar phenotype is seen when another Frizzled, MIG-1, is over expressed in the PLM suggesting an antagonistic relationship between lin-17 and mig-1. Consistent with this hypothesis, loss of MIG-1 suppresses the polarity reversal of lin-17 and lin-44 mutants. We propose two explanations for how excess MIG-1 may cause a reversal of PLM polarity. In model 1, increased MIG-1 signaling antagonizes LIN-17 signaling. In model 2, excess MIG-1 competes with LIN-17 for the Wnt ligand LIN-44, thereby reducing signaling through LIN-17, and possibly another Wnt receptor. Model 1 predicts that loss of the MIG-1 Wnt ligand should suppress the PLM polarity defect caused by excess MIG-1. Loss of cwn-1, cwn-2 or mom-2 does not ameliorate the polarity phenotype. This lack of a genetic interaction suggests that MIG-1 might bind LIN-44 to prevent it from interacting with LIN-17, inconsistent with a MIG-1 signaling role proposed in model 1. We are attempting to distinguish between these two models by determining which domains of MIG-1 are necessary and sufficient to generate the PLM polarity defect. 1. Prasad BC, Clark SG. (2006) Development 133: 1757-1766 2. Hilliard MA, Bargmann CI. (2006) Developmental Cell 10: 379-390 3. Pan C-L, et al. (2006) Developmental Cell 10: 367-377 4. Maloof JN, Whangbo J, et al. (1999) Development 126: 37-49 5. Forrester, W.C., Kim, C., Garriga, G. (2004) Genetics 168: 1951-1962.

Chien, Shih-Chieh et al. (2011) International Worm Meeting "C. elegans orthologs of LKB1, STRAD and MO25 regulate asymmetric cell division of the Q.p lineage."

Garriga, Gian, Chien, Shih-Chieh

[International Worm Meeting, 2011]

Many C. elegans neuroblasts, including the Q.p neuroblasts, divide to produce a larger neuronal precursor and a smaller cell that dies. Mutations in some of the genes that regulate these divisions result in daughter cells that are more equivalent in size and transform the fate of the apoptotic cell to that of its sister, resulting in the production of extra neurons. One such gene is pig-1, which encodes a protein orthologous to vertebrate MELK and belongs to a family of serine/threonine kinases that include PAR-1, SAD-1 and AMPK [1]. This group of kinases can be phosphorylated and activated by the polarity-regulating kinase LKB1. LKB1 kinase, along with its binding partners STRAD and MO25, are master regulators of polarity in many different contexts, and we find that C. elegans orthologs of LKB1 (PAR-4), STRAD (STRD-1) and MO25 (MOP-25.2) regulate the Q.p division. Mutations in par-4 or strd-1 generated Q.p daughter cells that are more symmetric in size, and in a sensitized background, par-4 or strd-1 mutations or mop-25.2(RNAi) resulted in extra neurons, suggesting that PAR-4/STRD-1/MOP-25.2 activates PIG-1. Consistent with this hypothesis, par-4 and strd-1 mutations enhance the extra-neuron phenotype of a weak but not a null pig-1 mutant. LKB1 activates AMPK family kinases by phosphorylating a conserved threonine residue in their activation loops [2]. By contrast, MELK is autophosphorylated at this residue [3], and mutating it to alanine abolishes kinase activity [2, 3]. To test whether the threonine residue in the activation loop (T169) of PIG-1 is equally essential for its activity, we generated a non-phosphorylatable form, PIG-1(T169A), and a phosphomimetic form, PIG-1 (T169D). Transgenes expressing PIG-1(T169A) failed to rescue the extra-neuron phenotype of a pig-1 mutant, indicating that the threonine residue is important for PIG-1 activity. We observed a partial rescue of the extra-neuron phenotype from transgenes expressing PIG-1(T169D). PIG-1(T169D) also induced extra neurons in the wildtype background, suggesting that the phosphomimetic form possessed deregulated PIG-1 activity. [1] Cordes, S. et al. (2006). Development 133, 2747-2756. [2] Lizcano, J. M. et al. (2004). Embo J 23, 833-843. [3] Beullens, M. et al. (2005). J Biol Chem 280, 40003-40011.

Chien, Shih-Chieh et al. (2013) International Worm Meeting "Autonomous and nonautonomous regulation of Wnt-mediated neuronal polarity by the C. elegans Ror kinase CAM-1."

Garriga, Gian, Gurling, Mark, Chien, Shih-Chieh

[International Worm Meeting, 2013]

Wnts are a conserved family of secreted glycoproteins that regulate various developmental processes in metazoans. Three of the five C. 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.1,2,3 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 fails 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. 1: Hilliard & Bargmann (2006) Dev Cell 10(3):379-90 2: Prasad & Clark (2006) Development 133(9):1757-66 3: Babu et al. (2011) Neuron 71(1):103-16.

Teuliere, Jerome et al. (2013) International Worm Meeting "Several ArfGEFs regulate the apoptotic fate in Q neuroblast asymmetric cell divisions."

Garriga, Gian, Teuliere, Jerome, Cordes, Shaun

[International Worm Meeting, 2013]

Arf guanine nucleotide exchange factors (GEFs) can regulate cell adhesion, actin cytoskeletal dynamics, and membrane trafficking through the activation of the Arf small GTPases. We previously found that an Arf GTPase Activating Protein (GAP), CNT-2, was essential in controlling cell size and the cell death fate in the Q neuroblast lineage (Singhvi et al, 2011). We report here that several ArfGEFs control the asymmetric divisions of the Q cell daughters. Q.a and Q.p are both neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. Loss of the cytohesin ArfGEF homolog GRP-1 resulted in the production of daughter cells that are more similar in size and in the transformation of the apoptotic daughter into its sister, resulting in the production of extra neurons. GRP-1's GEF activity, mediated by its SEC7 domain, is necessary for its role in the Q.p neuroblast. The loss of GRP-1, however, resulted in a phenotype that was less severe than the loss of CNT-2, suggesting that additional ArfGEFs remained to be identified. By screening for grp-1 enhancers, we indeed identified two more ArfGEFs, EFA-6 and M02B7.5/Schizo/IQSEC, that are necessary for the Q.p division. Functional GFP-tagged GRP-1 proteins localized primarily to the nucleus, but targeting the GRP-1 SEC7 domain to different cellular compartments suggests that GRP-1 acts at the cell cortex. EFA-6 localized at the cell cortex, suggesting a functions there as well. Surprisingly, loss of GRP-1 and CNT-2 resulted in identical Q.a phenotypes, and neither efa-6 nor M02B7.5 appears to function in this division. We will propose models that explain the differential involvement of these ArfGEFs in these two asymmetric divisions. Singhvi et al., Curr Biol. 2011 Jun 7;21(11):948-54.

Pandey, Amita et al. (2009) International Worm Meeting "UNC-53 antagonizes VAB-8 function in posterior migrations of neuronal cell bodies and growth cones."

Pandey, Amita, Garriga, Gian, Wolf, Fred W.

[International Worm Meeting, 2009]

Nervous system development involves the migrations of neuronal cell bodies and their axons along the dorsal-ventral and anterior-posterior axes of C. elegans in response to various navigational cues. The kinesin-like protein VAB-8 is both necessary and sufficient for the posterior migrations of cells and neuronal growth cones. VAB-8 appears to act through the Rac GEF activity of UNC-73 to promote guidance receptor activity that guides these migrations toward the tail 1,2. While VAB-8 and UNC-73 promote the function of these receptors, we have identified a molecule, UNC-53, that appears to inhibit their function. UNC-53 is a conserved protein that has an actin binding domain, a SH3 binding domain, a putative microtubule binding domain and an AAA domain. UNC-53 antagonizes VAB-8 function in the ALM and CAN neurons. Ectopic expression of VAB-8 reverses the polarity of the ALM, causing it to extend its anterior process toward the posterior. A reduction in unc-53 function enhances the frequency of the ALM polarity reversals. Similarly, UNC-53 also suppresses the CAN migration defects of vab-8 partial loss-of-function but not null mutants. We found that the mutation identified originally as enu-1(ev419) (enhancer of Unc) by Joe Culotti is an allele of unc-53. Unlike other unc-53 alleles, the ev419 mutation did not cause an Unc phenotype. But like the other alleles, ev419 enhanced the ALM reversal phenotype and suppressed the CAN migration defects of vab-8 mutants, suggesting that this allele defines an UNC-53 function that specifically antagonizes VAB-8. The ev419 mutation affects RNA splicing of the unc-53 transcript and appears to generate a protein that is lacking 23 amino acids. unc-53 was identified in a genome-wide RNAi screen for genes involved in endocytosis of yolk protein by oocytes3. We also found that ev419 and other unc-53 mutants are defective in oocyte endocytosis, suggesting that UNC-53''s function in promoting endocytosis antagonizes VAB-8. 1Strumpf, N.C and Culotti, J.G. (2007) Nature Neurosci. 10: 161-168. 2Watari-Goshima, N et al. (2007) Nature Neurosci. 10: 169-176. 3Balklava, Z. et al. (2007) Nature Cell Biology 9: 1066-1073. A.

Williams, Falina J et al. (2013) International Worm Meeting "Wnts and VANG-1/Van Gogh control cell fates in the Q lineage."

Williams, Falina J, Garriga, Gian, Teuliere, Jerome

[International Worm Meeting, 2013]

Asymmetric cell division (ACD) is a process that generates cell diversity. Both intrinsic and extrinsic mechanisms can distribute developmental potential asymmetrically to generate daughter cells of different fates and position the cleavage furrow asymmetrically to generate cells of different sizes. Wnts are evolutionarily conserved secreted glycoproteins that are utilized throughout development and play a role in ACD.1 During C. elegans development, Wnts regulate asymmetric divisions by controlling the distributions of the b-catenin SYS-1 and the LEF/TCF POP-1.2 They can also regulate the orientation of the spindle.3 In other organisms, Wnts can also regulate cytoskeletal dynamics via the Planar Cell Polarity (PCP) pathway, which allows polarization of neighboring cells along an axis orthogonal to the apical-basal axis within an epithelial sheet.4 The ACDs of the Q.a and Q.p neuroblasts to give rise to a larger daughter that lives and a smaller cell destined to die. We find that two Frizzled homologs LIN-17 and MOM-5 together are necessary for both apoptotic fates, for the asymmetric distribution of POP-1 in the Q.a and Q.p daughter cells, and for the asymmetric position of the Q.a and Q.p furrows that produce daughter cells of different sizes. Reduction of Wnt signaling, however, fails to generate the same robust disturbance of POP-1 distribution and does not affect the furrow localization. Instead, reduction of Wnt signaling can result in a reversal of POP-1 asymmetry, a phenotype that is enhanced by loss of the Van Gogh homolog VANG-1, a component of the PCP pathway. 1.Munro and Bowerman. CSH Perspectives. 2009. 2.Mizumoto and Sawa. Trends in Cell Biology. 2007. 3.Walston and Hardin. Seminars in Cell and Developmental Biology. 2006. 4.Segalen and Bellaiche. Seminars in Cell and Developmental Biology. 2009.