Nonet ML et al. (1991) International C. elegans Meeting "sdc-1 and a change of pace."

Nonet ML, Meyer BJ

[International C. elegans Meeting, 1991]

The sdc-1 gene is required in hermaphrodite embryos, but not in males, to express their sex-specific developmental fate and sex- specific mode of dosage compensation. sdc-l mutant XX animals fail to properly regulate X-linked transcript levels and often show transformation towards the male fate. In the past year we have cloned and molecularly characterized the sdc1 gene. Our results indicate that sdc-l encodes a large protien containing seven zinc fingers. In contrast to most previously decribed multiple zinc finger proteins, which have their fingers arranged in a regularily spaced tandem array, the sdc-l protein has its zinc fingers arranged in widely spaced clusters of single and paired motifs. It seems likely that sdc-l is a maternally-supplied embryonically-acting transcription factor. Developmental northern analysis demonstrates that sdc-l transcript levels are high in embryos, decrease through the larval stages and then increase in adults. We have recently created an sdc :: lacZ translational fusion. Incorporated into a rol-6(sulO06J array, this fusion is highly expressed in most cells from approximately the 50-100 cell stage, through the L1 larval stage, and is expressed more irregularly during the L2 stage. Hereafter the only expression is a variable, very low level expression in some neurons in the head and tail and a few cells in the pharynx. The fusion is not expressed in the germline. We are presently integrating the fusion before carefully characterizing the staining pattern . We are interested in genes which are essential for the proper development and/or function of the nervous system. We have started with the premise that the very uncoordinated, pharyngeal pumping defective L1 lethal terminal phenotype of unc-104(nullJ and cha-1(null) , is a representative phenotype of one class of such nervous system defects. With this phenotype as a guide we are isolating L1 lethals in both unbalanced and balanced screens. We subsequently hope to sort this class of lethal mutations into smaller groups by looking for defects by Nomarski optics, nervous system specific antibodies (anti- GABA, etc), dye filling, B-gal staining after crossing in B-gal fusions which express in the nervous system. On a more focused note, we have initiated a more detailed characterization of unc-62. unc-62 is represented by two alleles, the stronger of which is an embryonic lethal, the other a slow, irregular moving Unc with abnormalities in VD and DD commissures and significant associated lethality (,60%, both dead eggs and uncoordinated L1 lethals). We are isolating additional alleles of unc-62 to determine the null phenotype of the gene and more carefully examine the defects associated with unc-62 lesions.

Nonet ML et al. (1993) International C. elegans Meeting "Characterizing the presynaptic nerve terminal."

Meyer BJ, Nonet ML, Rand JB

[International C. elegans Meeting, 1993]

Nonet ML et al. (1993) International C. elegans Meeting "Synaptotagmin-deficient mutants of C. elegans."

Meyer BJ, Grundahl K, Rand JB, Nonet ML, McManus JR

[International C. elegans Meeting, 1993]

Nonet ML et al. (1995) International C. elegans Meeting "unc-64 and snb-1 encode SNAP receptors"

Nonet ML, Potter A, Rand JB

[International C. elegans Meeting, 1995]

The SNAP receptors VAMP and syntaxin are thought to be involved in vesicle docking and fusion at synaptic terminals. Biochemical experiments have documented that VAMP, located on vesicles, and syntaxin, located at the active zone, interact with high affinity. We have been characterizing mutants in C. elegans to determine if these components are required for vesicle fusion and to determine the extent of their roles in synaptic release and other processes. Rescue experiments have provided evidence that unc-64 encodes syntaxin. unc-64 is represented by several alleles with phenotypes ranging from mild uncoordination to relatively severe paralysis. snb-1 encodes VAMP and is represented by a single allele. snb-1(md247) animals are lethargic and jerky and are phenotypically similar to weaker alleles of unc-64. We are presently isolating null alleles in both unc-64 and snb-1 in non-complementation screens. A variety of evidence suggests that both snb-1 and unc-64 have defects in synaptic function. unc-64 and snb-1 mutants are resistant to aldicarb and accumulate elevated levels of acetylcholine suggesting they release reduced levels of transmitter. These mutants are members of a gang of five with similar phenotypes (ric-4, snb-1, unc-13, unc-18, and unc-64) which are all likely to be defective in vesicle docking and/or fusion. As many double mutant combinations among these five loci have lethal phenotypes, we suspect that some of these molecules may play additional roles in nervous system function. Consistent with this hypothesis syntaxin antibodies have revealed that this molecule is not localized to transmitter release sites, but is found ubiquitously in neuronal membrane.

Kesheng Liu et al. (2002) West Coast Worm Meeting "Functional characterization of a structural motif in UNC-11 that has been implicated in phosphoinositide binding."

Ravit Golan, Kondury Prasad, Kesheng Liu, Aixa Alfonso, Suping Jin, Eileen M Lafer, Andrea M Holgado

[West Coast Worm Meeting, 2002]

In C. elegans, the unc-11 gene encodes a family of proteins with structural and functional homology to the mammalian monomeric clathrin adaptor protein AP180 (Nonet et al., 1999). In the nematode these proteins have a role in the retrieval and sorting of the integral synaptic vesicle protein synaptobrevin from the plasma membrane and to synaptic vesicles (Nonet et al., 1999).

Schaefer AM et al. (1996) West Coast Worm Meeting "ANALYZING SYNAPSE FORMATION BY THE TOUCH SENSORY NEURONS"

Nonet ML, Schaefer AM

[West Coast Worm Meeting, 1996]

C. elegans has a characteristic response to touch: When touched on the tail, the animal moves forward; when touched on the head, the animal moves backward. The neuronal circuitry underlying this response has been described, and is mediated by the sensory neurons ALM (L/R), AVM, PLM (L/R) and PVM (the "touch cells"). These sensory neurons form gap junctions or synapses with each of the interneurons AVD, PVC, AVA, and AVB. Using a GFP construct fused to VAMP and the mec-7 promoter ("pmec-7::VAMP::GFP"), we can visualize the cell bodies, and likely synapses of the touch cells. The synapses appear as clusters of GFP fluorescence in the nerve ring, and as two relatively long "patches" of fluorescence along the ventral nerve cord. By focusing on these patches, we intend to study the genetic and molecular mechanisms controlling formation of the synapse. The pmec-7:VAMP::GFP integrated sequence indicates that the patches form in late L1 or L2, and that both PLML and PLMR are the pre-synaptic partners in these synapses. We have looked at these patches in several mutant backgrounds relating to function and/or development of the touch cells, and find that the patches appear normal when the PLMs are present. Laser ablation studies indicate that when either PLML or PLMR is eliminated early in L1, prior to formation of the patches, only one patch will develop. If both PLMs are ablated, no patches will form. Using laser ablation as well as a genetic approach, we currently are attempting to confirm the identity of the post-synaptic partners, and to determine the role of the post-synaptic partner in the formation of the synapse. The patches appear relatively normal in mutants with synaptic function deficits, such as snt-1 and unc-13, suggesting that formation of the synapses is independent of synaptic function. We hope to identify genes that are necessary for synapse development, by mutagenizing pmec-7:VAMP::GFP animals, and looking for aberrations in the location, size, presence, or number of the patches. A preliminary mutagenesis screen revealed a mutation that generates ectopic patches, and a mutation that results in the absence of patches. A more rigorous screen should uncover additional relevant mutations.

Schaefer AM et al. (1997) International C. elegans Meeting "SYNAPSE FORMATION BY THE TOUCH SENSORY NEURONS"

Nonet ML, Schaefer AM

[International C. elegans Meeting, 1997]

We are using the neuronal connections of the touch circuit to investigate genetic and molecular mechanisms controlling formation of the synapse. An integrated construct which contains GFP fused to VAMP and the mec-7 promoter enables us to visualize two prominent and stereotypical accumulations or !patches! of GFP along the ventral cord, just posterior to the vulva, which arise at late L1/early L2. These patches likely correspond to chemical synapses formed by the PLMs, as determined by prior EM reconstructions as well as mutant analysis and laser ablation. When the PLMs are absent, due to either genetic mutation or laser ablation in early L1, the patches are not seen. Further, only one patch forms along the ventral cord if either PLML or PLMR is ablated prior to synapse formation. The PLMs form synapses with at least six different cells. Two of these, AVA and AVD, are interneurons that mediate the worm!s movement in response to touch. We currently are determining the effects of laser ablating PLM!s post-synaptic partners either before or after synapse formation. We will report these results. We have looked at the GFP pattern in the background of several known synaptic function mutants, and find that synapse formation appears to be independent of activity. We are conducting a mutagenesis screen to identify genes that may be involved in synapse formation and synaptic specificity. So far, we have identified single gene mutations that generate ectopic patches/synapses, as well as mutations which cause an absence of the patches. Some of these mutants have visible phenotypes, including sterility and maternal effect lethality. We will report the screening results at the meeting.

Zhao HJ et al. (1997) International C. elegans Meeting "REDUCTION OF ACTIVITY MEDIATES AXONAL SPROUTING AND DE NOVO SYNAPSE FORMATION IN SAB MOTOR NEURONS"

Zhao HJ, Nonet ML

[International C. elegans Meeting, 1997]

Synaptic varicosities along each of the four cholinergic SAB motor axons were visualized via a punc-4-VAMP-GFP fusion construct. In the synaptic transmission mutants unc-13, unc-18, snb-1 and rab-3, those motor axons sprouted and formed ectopic synapses onto an unidentified target. The percentage of axons with sprouts reaching 88% in unc-18 depends upon the extent of the reduction of neurotransmitter release. These results suggest that reducing pre-synaptic activity mediates the sprouting of SAB motor axons and de novo synapse formation. This sprouting phenotype is specific to cholinergic transmission because sprouts were observed when blocking either ACh synthesis using a temperature-sensitive allele of cha-1 or ACh transport using a unc-17 mutant. In contrast, unc-25 which causes a GABA deficiency has no effect on these axons. Furthermore, this isn!t an effect of VAMP-GFP expression because the same sprouting phenotype was observed in cha-1 by staining axons with anti-synaptotagmin antibody. The TSP of sprouting extended to L3. When cha-1(ts) was shifted at L2, 52% of the axons had sprouted after 12 hours and 15% of them later retracted. Interestingly, we also found that disruption of the levamisole ACh receptor using a unc-29 mutant caused the same sprouting phenotype as cha-1, suggesting that the muscle cell and its activity, is involved in the process of sprouting. We are currently trying to rescue the sprouting phenotype by expressing wild type UNC-29 specifically in muscle cells. We are also performing a non-clonal screen for mutations suppressing sprouting in unc-18.

Staunton J et al. (1995) International C. elegans Meeting "RAB-3 AND SYNAPTIC VESICLE TRANSPORT IN C. elegans"

Nonet ML, Staunton J

[International C. elegans Meeting, 1995]

Rab-3 is a member of a family of small GTP binding proteins and has been implicated in the trafficking of synaptic vesicles. Rab family members and the related p21ras cycle between GTP- and GDP-bound forms. Each Rab has been associated with a distinct type of vesicle-mediated transport, suggesting possible regulatory functions. However, as the actual role of Rab proteins remains unclear, we are using genetics to study Rab-3 function in C. elegans. Previously, a single C. elegans rab-3 homolog was cloned by PCR and further examination has revealed no additional isoforms. The predicted product of the C. elegans rab-3 homolog shows 75% amino acid identity to the human Rab3A. In situ immunofluorescence microscopy demonstrates that C. elegans Rab-3 is localized to synaptic vesicles. Two recessive, aldicarb resistant mutants, y250 and y251, have mild defects in coordination, chemotaxis and egg-laying. Ultrastructural examination of mutant synaptic terminals reveals a reduction in both the number of synaptic vesicles and the concentration of vesicles at the active zone (E. Jorgensen, pers. comm.) Each recessive allele confers a single amino acid substitution in a putative GTP binding region, and the mutants behave genetically as nulls. However, as we cannot be certain that these mutants reflect the null phenotype, we are screening for definitive null alleles. Dominant aldicarb resistant mutants were generated with transgenic arrays. One of the dominant mutants exhibits strong behavioral defects, which has inspired us to screen for dominant chromosomal mutations. In order to identify genes which modulate Rab-3 activity, we are using the sensitized background of the recessive mutants to screen for enhancers of the mild behavioral phenotype, as well as screening for suppressors of the dominant phenotype.

Richter A et al. (1995) International C. elegans Meeting "VISUALIZATION OF PRESYNAPTIC TERMINALS USING GFP"

Nonet ML, Richter A

[International C. elegans Meeting, 1995]

We have created a fusion protein between VAMP, an integral synaptic vesicle membrane protein, and GFP, and have an integrated array (jsIs1) of it in nematodes. jsIs1 worms show bright fluorescence in the nerve ring, ventral cord, and dorsal cord. Sublateral bundles are visible as chains of discrete bright spots. Commissures and the amphid dendritic bundles are not normally visible. Some neuronal cell bodies fluoresce faintly. Several lines of evidence imply that this fluorescence is primarily due to VAMP-GFP localizing to synaptic vesicles. First, VAMP antibody staining in wildtype worms shows that VAMP-GFP fluorescence parallels normal VAMP localization. Second, in unc-104; jsIs1 animals, fluorescence is concentrated in cell bodies and absent from the dorsal cord entirely. This suggests that axonal membrane does not contain significant amounts of VAMP-GFP. Third, in unc-5; jsIs1 animals, patchy fluorescence is seen in lateral and sublateral positions, where ectopic synapses have been seen by Hedgecock, et. al. Fourth, in strains where VAMP-GFP is expressed only in specific subclasses of neurons using neuronal specific promoters such as mec-7, we see fluorescence where synapses have been described by the ultrastructural studies of White, et. al. We have performed a pilot screen for synaptogenesis mutants using jsIs1 and have isolated mutants with axonal elongation or migration problems, with accumulation of label in cell bodies, and with inappropriate patches of fluorescence. To increase our ability to see subtle changes in synaptic label, we are developing several VAMP-GFP constructs under control of promoters for specific neuronal subtypes. We are also exploring the plasticity properties of the nervous system using ablation studies in combination with VAMP-GFP.