Colavita A et al. (1995) International C. elegans Meeting "MUTATIONS THAT SUPPRESS UNC-5-INDUCED REORIENTATION OF THE TOUCH CELL AXONS"

Colavita A, Culotti JG

[International C. elegans Meeting, 1995]

The UNC-5 guidance receptor, in response to the laminin- related UNC-6 path cue molecule, orients growing axons in a dorsal direction on the C. elegans epidermis. When ectopically expressed in a set of touch receptor neurons, which normally grow ventrally or longitudinally, UNC-5 is able to reorient their axons towards the dorsal side (Hamelin et al. Nature 364: 327-330, 1993). This UNC-5 induced reorientation is suppressed by mutations in unc-6, which is known from genetic evidence to act in the same pathway as unc-5. We have screened for mutations that act like unc-6 mutations in their ability to suppress UNC-5-induced reorientation of the touch cell axons. Theoretically, these suppressor mutations may identify other axon guidance genes that act in the UNC-5/UNC-6 pathway. So far we have identified mutations in approximately nine genes, including unc-6 (as expected), unc-40 (another axon guidance gene required for ventral and dorsal guidance, Hedgecock et al. Neuron 4: 61-85, 1990), unc-34, and unc-44 (a C. elegans ankyrin, A. Otsuka, personal communication). The other mutations are being further characterized genetically and phenotypically for axon guidance errors.

Antonio Colavita et al. (2001) International C. elegans Meeting "GENETIC ANALYSIS OF AXON BRANCHING IN C. ELEGANS"

Marc Tessier-Lavigne, Antonio Colavita

[International C. elegans Meeting, 2001]

Neurons innervate multiple targets by sprouting secondary axon collaterals from a primary axon and/or by bifurcation of a primary axon during process outgrowth or on reaching post-synaptic targets (arborization). Little is known about the molecular identity of molecules that are involved in the formation, stabilization, and elongation of axon branches. We have undertaken a genetic screen in C. elegans to try to identify some of these components. Among the C. elegans neurons that display branching morphologies are the six VC (VC1-6) and two HSN motor neurons that synapse onto vulval muscles to control egg laying behavior. The VC neurons are evenly spaced along the ventral nerve cord (VNC) and send axons toward the developing vulva (situated between VC4 and VC5) where they sprout dorsally extending branches that innervate the vulval muscles. The HSNs, situated on the lateral body wall near the vulva, each send an axon ventrally which then turns and extends anteriorly along the VNC. At the vulva the HSN frequently (but not always) extends a dorsally directed branch. In both VC and HSN neurons branching has been shown to require external cues from vulval cells as branches are not observed in Lin (lineage defective) mutants that do not have vulvae (Li and Chalfie,1990; Garriga et al.,1993). To identify new genes involved in axon branching we have primarily focused on VC4 and VC5 as they display the most branched axon morphologies. VC4 and VC5 were visualized using a GFP transcriptional fusion to the vesicular monoamine transporter (VMAT) encoded by the cat-1 gene (Duerr et al.,1999). We have performed a visual genetic screen for mutants that display defects in VC4/5 branching, including reduced or excessive branching as well as elongation or guidance defects. In an initial screen of approximately 2,000 EMS mutagenized genomes we have identified several mutants that display branching defects. One of these, bam-1 (Branching AbnorMal) displays a highly penetrant excess branching or arborization defect in VC4 and VC5. We have cloned bam-1 and shown that it encodes a peptide:N-glycanase (PNGase).

Colavita A et al. (1997) International C. elegans Meeting "Suppressors of Ectopic UNC-5 Expression Identify a Novel TGF-b Involved in Axon Guidance in C. elegans."

Zheng H, Culotti JG, Krishna S, Padgett RW, Colavita A

[International C. elegans Meeting, 1997]

UNC-5 encodes a transmembrane receptor that guides migrating cells and pioneer axons away from ventral sources of secreted UNC-6 netrin guidance molecules in C. elegans. When ectopically expressed in the touch receptor neurons, which don!t normally respond to UNC-6, UNC-5 is able to confer UNC-6 responsiveness to their growth cones. This result forms the basis of a screen to look for mutations in components of the UNC6/UNC-5 signaling cascade - defects in components of this pathway should suppress the ability of ectopic UNC-5 to steer growth cones in response to UNC-6. This screen yielded mutants of 8 genes, one of which, unc-129, is a new uncoordinated mutant with axon guidance defects like those of unc-5 and unc-6 mutants. unc-129 was found to encode a novel member of the TGF-b family of secreted signaling proteins. unc-129-reporter transgenes are expressed in motorneurons and in dorsal muscles. Promoter bashing results indicate that the muscle expression is required for motor axon guidance; we are not yet certain whether motorneuron expression is also required. Ectopic expression of this TGF-b indicates that its spatial patterning is important for guiding cell and growth cone migrations. We are examining whether unc-129 acts on the UNC-6/UNC-5 signaling cascade as anticipated by the rationale for the suppressor screen, or whether it acts by an independent mechanism.

B Nash et al. (1999) International C. elegans Meeting "unc-13 is required to maintain the correct expression pattern of unc-129 in C. elegans."

JG Culotti, B Nash

[International C. elegans Meeting, 1999]

unc-130 is required for the dorsoventral guidance of pioneer growth cones and distal tip cells during development in C. elegans . The gene encodes a putative transcription factor with a highly conserved forkhead domain. In order to characterize the role of unc-130 in circumferential guidance, we have constructed double mutants between unc-130 and other mutants known to be required for this process. unc-5 , unc-6 and unc-40 are all required for dorsalward guidance of distal tip cells and pioneer growth cones (Hedgecock et al,1990. Neuron 4, 61-85). Double mutants between null alleles of unc-130 and either unc-5 or unc-6 both have more penetrant dorsalward guidance defects than the respective single mutants, suggesting that unc-130 acts, at least partially, in a parallel pathway. unc-129 , a TGF-beta, is also required to guide pioneer growth cones along the dorsoventral axis (Colavita et al, 1998. Science 281, 706-709). unc-129 mutants do not, however, have defects in the dorsalward migration of the distal tip cell. Strikingly, unc-129 ; unc-130 double mutants have much less penetrant distal tip cell migration defects than the unc-130 alleles. unc-129 ::GFP is expressed in dorsal body wall muscle but not ventral muscle. This pattern of expression is important for unc-129 function (Colavita et al). However, in unc-130 mutants unc-129 ::GFP can be detected in both dorsal and ventral muscles. Thus, forkhead UNC-130 appears to be required to maintain the correct spatial expression pattern of the TGF-beta UNC-129. We have recently discovered that mutations in unc-130 also disrupt the morphology of sensory rays in the male tail. The defects are most similar to those seen in mab-21 mutants. The male tail defects appear to act through a separate pathway, as unc-129 mutants do not suppress this phenotype. An unc-130 ::GFP fusion construct is expressed in hypodermal cells, intestinal cells and in ventral body wall muscle. We are presently investigating where unc-130 is required by mosaic analysis.

Carr, D. et al. (2011) International Worm Meeting "A prkl-1suppressor screen to identify new planar cell polarity components involved in neuronal polarity."

Visanuvimol, J., Sanchez-Alvarez, L., Colavita, A., Carr, D.

[International Worm Meeting, 2011]

The planar cell polarity (PCP) pathway is responsible for organizing cell polarity in the plane of the epithelium. We have recently shown that the core PCP pathway components Van Gogh (vang-1), Prickle (prkl-1), and Dishevelled (dsh-1) are required in a subset of peripheral motor neurons (VC neurons) to restrict neurite emergence to a specific tissue axis. In loss-of-function mutants, VC4 and VC5 neurons display ectopic neurites that extend abnormally along the anteroposterior (AP) body axis. The unc-4 promoter is expressed in the VA, DA and VC motor neurons. unc-4 driven over-expression of PRKL-1 in the VC neurons is sufficient to suppress neurite growth. These transgenic animals also displayed backwards locomotion impairment; a movement controlled by the VA and DA neurons. This inability to move backwards is suppressed by vang-1 null mutants, which are known to interact with prkl-1 in the PCP pathway. We used these observations as the basis of a genetic screen to identify other suppressors that can restore backwards locomotion. Our preliminary findings include the identification of several suppressor mutants including at least 8 new alleles of vang-1 and at least one allele of fntb-1, the beta subunit of farnesyltransferase. Farnesyltransferases post translationally modifies proteins by attaching a 15-carbon isoprenoid lipid, farnesyl, to the cysteine residue of a C-terminal CAAX motif. The addition of a farnesyl group facilitates membrane association of the protein due to the hydrophobic nature of the lipid. We believe that FNTB-1 prenylates PRKL-1 at its C-terminal CAAX motif to promote membrane association and interaction in the PCP pathway. We will be describing the characterisation of fntb-1 and genetic interactions with prkl-1 as well as further findings from the genetic screen.

Zheng H et al. (2000) East Coast Worm Meeting "seu-1 Encodes Novel Nuclear Proteins Required for the Function of Ectopic UNC-5"

Colavita A, Zheng H, Merz DC, Culotti JG

[East Coast Worm Meeting, 2000]

The UNC-6/netrin guidance cue and its receptors UNC-5 and UNC-40/DCC form a highly conserved system for circumferential guidance of migrating cells and growth cones. In C. elegans, ectopic expression of the UNC-5 receptor causes repulsion of touch neuron growth cones away from sources of UNC-6. A genetic suppressor screen identified 8 genes required for this repulsion: unc-6, unc-34, unc-40, unc-44, unc-129, seu-1, seu-2, and seu-3. We have cloned seu-1 and report that it encodes 2 novel proteins (SEU-1A and SEU-1B), which differ through alternative splicing at their C-termini. Both SEU-1 isoforms contain acidic and proline-rich regions and nuclear localization sequences, but no recognizable DNA- or RNA-binding domains. The seu-1(ev520) allele contains an early nonsense mutation and is likely to be a null allele. seu-1::GFP is nuclear localized and dynamically expressed throughout development in neural and non-neural tissues. Similar to unc-5 reporter constructs, expression in the hermaphrodite distal tip cells was observed only at the time of the circumferential second migration phase. Transgenic expression of the combination of SEU-1A and SEU-1B in the touch neurons (using the mec-7 promoter) was sufficient to restore the function of UNC-5 in the touch neurons in a seu-1 mutant background, suggesting a cell autonomous function. Neither the SEU-1A nor the SEU-1B isoform alone had rescuing ability. Although seu-1 mutations alone have subtle defects in axonal morphology, over-expression in neurons caused more severe cell migration and axon guidance defects. We propose that SEU-1 in neurons regulates growth cone responses to guidance cues. We are currently examining genetic interactions between seu-1 and unc-5,6,40.

Noblett, N. et al. (2017) International Worm Meeting "A role for dip-2 (Disco-Interacting Protein-2 homolog) in neuronal migration and the maintenance of neuronal morphology."

Flibotte, S., Roenspies, T., Noblett, N., Colavita, A., Ding, Z.

[International Worm Meeting, 2017]

Mechanisms that inhibit inappropriate neurite outgrowth in the mature nervous system are likely to play important roles in maintaining normal neuronal function. A genetic screen for ectopic neurites in the VC motor neurons identified mutations in nde-5 (neurite outgrowth defective-5) (1). We now show that nde-5 encodes the worm orthologue of Disco Interacting Protein-2 (Dip2) and has thus been renamed dip-2. DIP2 proteins belong to a highly conserved protein family that contain an N-terminal DNA methyltransferase-associated protein 1 (DMAP1) binding domain and two adenylate-forming enzyme family domains. In addition to VC defects, we found that dip-2 mutants show morphology defects in several other neurons. Most prominently in the touch neurons where morphology defects show an age-dependent increase suggestive of a role in neuronal maintenance. dip-2 mutants also show HSN neuronal migration defects. A CRISPR/Cas9-mediated functional GFP insertion into the native dip-2 locus revealed expression in many neurons including the touch neurons and HSN as well as epidermal cells. DIP-2 localization in neurons was found to be predominantly cytoplasmic. In contrast, DIP-2 in epidermal cells was predominantly plasma membrane localized. Cell specific rescue experiments are consistent with a cell autonomous role in neurons to promote proper morphology and migration. These findings indicate that DIP2 proteins are important for the development and maintenance of neuronal connections. (1) D. Carr et al., 2016 Plos One.

Shah, P.K. et al. (2017) International Worm Meeting "PCP and SAX-3/Robo pathways cooperate to regulate convergent extension-based nerve cord assembly."

Shah, P.K., Colavita, A., Rankin, A., Kovacevic, I., Tanner, M., Bao, Z.

[International Worm Meeting, 2017]

At hatching DD, DA and DB motor neurons are stereotypically ordered relative to each other and distributed as a single track along the anterior-posterior (AP) axis. During embryogenesis, these neurons arise from left and right lineages, move towards the midline and intercalate to assemble the presumptive VNC. This morphogenetic process, in particular how neurons intercalate to assemble the VNC, has not been well characterized. We performed 3D fluorescence time-lapse imaging to show that intercalation of VNC neurons into a linear structure that extends the length of the AP axis involves a rosette-mediated convergent extension (CE) process. The planar cell polarity (PCP) pathway plays a highly conserved role in CE-based movements. Using the 6 DD neurons which are evenly spaced along the AP axis and therefore a convenient marker of VNC assembly, we found that mutations in the core PCP components vang-1/Vang Gogh and prkl-1/Prickle caused DD neurons to be anteriorly displaced in newly hatched worms. In the embryo, these mutants displayed defects in cell neighbor exchange and rosette resolution. Interestingly, we found that mutations in sax-3/Robo, a gene better known for roles in axon guidance, displayed similar defects in DD positioning and embryonic cell-neighbor exchanges. VANG-1 and SAX-3 are localized to contracting edges and rosette foci. Exploring further, we found that PCP/sax-3 double mutants display a significantly shortened, anteriorly displaced distribution of VNC neurons compared to single mutants. This simultaneous loss of both PCP and sax-3/Robo correlated with a severe disruption of CE in the embryo suggesting that these pathways act in parallel during VNC assembly. Our results indicate that rosette-based convergent extension is conserved in central nerve cord assembly and reveals a novel role for sax-3/Robo in this process.

Hwai-Jong Cheng et al. (2001) International C. elegans Meeting "A GENETIC SCREEN FOR DA/DB AXON GUIDANCE MUTANTS IN C. ELEGANS"

Marc Tessier-Lavigne, Hwai-Jong Cheng, Xun Huang, Yishi Jin

[International C. elegans Meeting, 2001]

In order to identify novel factors involved in axonal guidance, we have taken a genetic approach and used C. elegans to identify mutant animals that have defects in axonal projections in a subset of motoneurons which normally project their axons dorsally, the DA and DB neurons. These axons are already known to be repelled by the netrin UNC-6 (Ishii et al., Neuron 9:873-881, 1992), and also require the TGF-beta family member UNC-129 derived from dorsal muscle for proper guidance (Colavita et al., Science 281:706-709, 1998). These neurons actually also express unc-129, so that their axons can be visualized by driving GFP expression from the unc-129 promoter. We have mutagenized unc-129::gfp worms with EMS and screened for mutants that have defects in projections of DA and DB axons. Reasoning that most of severely uncoordinated mutants with global axon guidance defects might have been isolated in previous screens, we focused our screen on mutants with minimal uncoordinated phenotypes and less severe axon guidance defects. We have isolated mutants in this screen, and named these mutants "max" for motor axon guidance defect. We have characterized and mapped one of the mutants, max-1.

Tanner, M. et al. (2013) International Worm Meeting "A PCP-like pathway acts to position or properly space (tile) embryonic motor neurons along the ventral nerve cord."

Tanner, M., Slatculescu, C., Perkins, T., Colavita, A., Huang, B., Ghadban, A.

[International Worm Meeting, 2013]

Planar Cell Polarity (PCP)-like signaling has recently been linked to multiple aspects of nervous system development and wiring. We have found that a PCP-like pathway involving PRKL-1 and VANG-1 is required for DD motor neuron position along the AP axis. The six DD neurons (DD1-6) are normally positioned equidistant from each other in the ventral nerve cord. In prkl-1 and, to a lesser extent, vang-1 mutants, DD neurons are shifted anteriorly and display distinctive cell spacing defects. These positioning defects can be rescued by prkl-1 or vang-1 expression from a pan-neuronal promoter, suggesting cell-autonomous roles. Conversely, over-expression of neuronally-expressed prkl-1 results in posteriorly shifted DD neurons. Interestingly, the strong anterior shift in prkl-1 mutants can be suppressed by simultaneous loss of vang-1, suggesting that PRKL-1 normally antagonizes VANG-1 function in DD neurons. A candidate screen of known Wnt and PCP mutants revealed prkl-1 and vang-1-like DD positioning and spacing phenotypes in several other genes including mom-2/WNT and let-502/ROCK. Ectopic expression of MOM-2, which is normally expressed in the posterior embryo, from the more anteriorly expressing cwn-1 promoter was sufficient to induce DD positioning defects similar to those of loss-of-function mutants. Taken together, these data suggest a role for a PCP-like pathway in regulating neuronal cell body placement or spacing (tiling) along the ventral nerve cord in the embryo, a process which may involve an instructive role for Wnts along the AP axis.