Kevin Eliceiri et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Swept-Field Confocal for Cell Biology Studies"

Kevin Eliceiri, Mike Szulczewski

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

We describe a novel instrument, The Swept-Field Confocal Microscope (SFC) that combines high resolution pinhole imaging with slit imaging. Unlike spinning disk confocal systems that have their pinhole apertures embedded in a spinning disc, the SFC's 32 pinhole array remains stationary. Galvonometric and piezo controlled mirrors sweep the image of the pinholes across the sample. The emission photons are de-scanned and focused through a complementary set of pinholes onto a CCD camera. This results in a high resolution image that can be collected at up to 100 frames/second in the pinhole mode and greater than 1000 frames per second in the slit mode. The ability for the scientist to match the optical recording with the temporal biological fluorescence response has great promise for cell and developmental biology studies where live dynamic events must be captured quickly in high resolution. In this poster we describe the optical path of the microscope and present some early data in collaboration with John White's group at the UW-Madison of using the SFC to image GFP constructs in C .elegans to investigate cell division in the early embryo.

Gattegno T et al. (1997) International C. elegans Meeting "A MUTATION AFFECTING CELL FUSIONS DURING ELONGATION OF THE EMBRYO"

Gattegno T, Podbilewicz B

[International C. elegans Meeting, 1997]

Cell fusion is a widespread process throughout development and evolution. Many organs in multicellular organisms contain multinucleate cells formed by cell fusion; e.g., in humans (placenta, bone and muscle) and in C. elegans (hypodermis, vulva, pharynx, uterus, and excretory gland). Enveloped viruses (e.g. HIV) fuse with cell membranes in order to invade target cells, and fertilization involves sperm-egg fusion. We have characterized the order of epithelial cell fusions in C. elegans (1) and have isolated mutations that disrupt cell fusions in the embryo. In our pilot screens we did not obtain any mutation with fewer fusions than in wild-type. The zygotic lethal mutation zu316 was isolated in a screen for mutations that arrest during embryonic elongation (2). In the strain JJ825 zu316/+; lin-2 (e1309) the dorsal hypodermal cells fail to fuse. This mutant was outcrossed and we are now characterizing zu316 by 4D microscopy and immunofluorescence using tissue-specific mAbs and confocal microscopy. The development of the mutant terminates between 1.5 to 2-fold stages of elongation but some embryos arrest earlier. The mutants appear normal up to the beginning of morphogenesis and then the dorsal hypodermal cells look round during elongation and do not get flattened as in N2. Using MH27 and anti-LIN26 antibodies (kindly provided by R. Waterston and M. Labouesse, respectively) we notice that in zu316 there are fewer cell fusions in the dorsal hyp6 and hyp7 than in wild-type embryos. 1. Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424 2. Costa M. and Priess J. (1995) Worm Meeting Abstract, p. 165 We thank Mike Costa and Jim Priess for sending JJ825 to us.

Ammar Hawasli et al. (2001) International C. elegans Meeting "Gain-of-function mutations in egl-30 produce resistance to volatile anesthetics"

Mike Nonet, C Michael Crowder, Stephen Hunt, Owais Saifee, Ammar Hawasli

[International C. elegans Meeting, 2001]

Because mutations in the neuronal syntaxin gene unc-64 alter volatile anesthetic (VA) sensitivity, we have screened for unc-64(rf) suppressors in hopes of identifying regulators of VA action. For different reasons, Owais Saifee in Mike Nonet's lab performed a similar screen. A total of 38,000 genomes were screened by the two labs, and 21 suppressor mutations were isolated. The suppressors were outcrossed from unc-64(rf) and fell into two phenotypic classes loopy and jerky that cosegregate with unc-64(rf) suppression. Two semidominant loopy suppressors mapped by their behavioral and VA resistant phenotypes to chromosome IL near egl-30. egl-30 was sequenced in the mutants, and one of the alleles js126 was found to have a missense mutation in the coding sequence. We did not find an egl-30 mutation in the other strain. egl-30(tg26gf) , kindly given to us by K. Iwasaki, was also loopy and resistant to VAs. Testing of strains transformed with a constitutively active egl-30 array (thanks to Carol Bastiani - Sternberg lab) were also resistant to VAs confirming that egl-30(gf) could indeed produce VA resistance. Furthermore, we treated N2 with phorbol-ester, which activates diacyl glycerol-binding proteins and effectively enhances EGL-30 signaling; treated worms were resistant to VAs. Finally, we found that egl-30(js126) , egl-30(tg26) , the egl-30(gf) array-containing strains, and phorbol ester treated strains were all aldicarb hypersensitive. Aldicarb hypersensitivity suggests that presynaptic release of acetylcholine is increased. Thus, consistent with our previous results for goa-1(rf) and unc-64(rf) that suggested a presynaptic mechanism of VA action, egl-30 regulates VA sensitivity, presumably by controlling transmitter release. Currently, we are constructing double mutants between various VA resistant and hypersensitive strains to place egl-30 in the VA sensitivity pathway.

Kemphues KJ et al. (1991) International C. elegans Meeting "par MUTANT EMBRYOS SHOW ALTERED EARLY EXPRESSION OF CeMyoD."

Dickens S, Kemphues KJ, Kirby CM, MacCaulay A, Shakes DC

[International C. elegans Meeting, 1991]

Mutations in the par genes lead to a maternal effect lethal phenotype characterized by alterations in cleavage pattern and timing, aberrant partitioning of P granules, and extensive alterations in cell fates. We have proposed that the par genes encode components of a maternal system for asymmetric intracellular localization in early embryos and that proper functioning of this system is required for specification of early embryonic cell fates. Here we provide evidence that the par gene functions are necessary to establish proper lineage- specific transcription patterns in early cleavage stage embryos. Recently, the CeMyoD gene was shown to be an early marker of lineage specific transcription, with its earliest expression limited to the 2- 4 cells of the MS lineages at the 24-28 cell stages of embryogenesis. ( Krause et al., Cell 63,907). Andy Fire and Mike Krause anticipated our interest in this marker and generously gave us prepublication access to their data and a transgenic strain carrying a CeMyoD- -gal transcription reporter construct. We have constructed strains that segregate par homozygous worms carrying the reporter gene and are presently assaying -gal activity in the mutant embryos. Conclusions thus far: 1 ) All pars thus far examined (par-2,3,5) show ectopic expression of the CeMyoD reporter at the 32-cell stage (equivalent chronologically to wild type 24-28 cell stage). 2) No expression is seen prior to the 32-cell stage. 3) At least part of the early ectopic expression in par-3 and par-5 is transient; while many 32-and 64-cell-stage embryos are -gal positive in all nuclei, all embryos past the 64-cell stage have significant numbers of -gal negative nuclei. 4) ectopic 13-gal expression in par-2 embryos is limited to the posterior half of most embryos.

Monique van der Voet et al. (2006) European Worm Meeting "The Function of LIN-5 in Chromosome Segregation and Asymmetric Division"

Matilde Galli, Christian Berends, Monique van der Voet, Justin Peters, Sander Van Den Heuvel, Mike Boxem, Marjolein Wildwater, Adri Thomas, Inge The

[European Worm Meeting, 2006]

Monique van der Voet1, Marjolein Wildwater1, Mike Boxem, Justin Peters1, Christian Berends1, Matilde Galli1, Inge The1, Adri Thomas1 and Sander van den Heuvel. The position of the spindle apparatus determines the plane of cell division. During asymmetric division, proper positioning of the spindle is needed in the generation of daughter cells that differ in size, cytoplasmic determinants and developmental fate. We have focused our attention on the C. elegans gene lin-5 to identify novel regulatory mechanisms and molecules that control spindle-mediated functions.. Our previous studies demonstrated that lin-5 is generally required for chromosome segregation and spindle positioning during meiotic and mitotic M phase. lin-5 encodes a coiled-coil protein that localizes to the cell cortex as well as spindle asters, and recruits the G protein regulators GPR-1/GPR-2 to the same locations. Results from our lab as well as others support that LIN-5 and GPR act in concert with the GOA-1/GPA-16 G?i/o subunits of heterotrimeric G proteins in an evolutionarily conserved pathway for spindle positioning. Many aspects of this process remain poorly understood. In particular, it is unclear how polarity determinants at the cortex affect LIN-5/GPR/G?i/o function to create asymmetry in the pulling forces that act on the spindle asters. In addition, the downstream targets of the LIN-5/GPR/G?i/o pathway remain unknown. To better understand the molecular mechanisms of these processes, we continue to use a combined genetic and biochemical approach. We have identified novel candidate partners of LIN-5 through immunoprecipitation from embryonic lysates followed by mass spectrometry. In addition, we identified multiple residues of LIN-5 and GPR that are phosphorylated in vivo. We are currently testing the in vivo relevance of these interactions and modifications and hope to present our results at the meeting.

S Hunt et al. (1999) International C. elegans Meeting "Suppressors of syntaxin reduction-of-function regulate general anesthetic sensitivity"

CM Crowder, C Liu, B van Swinderen, S Hunt, A Hawasli

[International C. elegans Meeting, 1999]

We have previously shown that mutations in the neuronal syntaxin gene unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of C. elegans . We found that two hypomorphic unc-64 alleles confer hypersensitivity to the VAs isoflurane and halothane but that a third unc-64 hypomorph is VA resistant. The difference between the isoflurane EC 50 's (the concentration where the effect on locomotion is half maximal) of the hypersensitive and resistant alleles is over 30-fold. In order to understand the molecular mechanisms of syntaxin's regulation of anesthetics, we are searching for genes that interact with the syntaxin gene. Thus, we initiated a screen for suppressors of the locomotion defect of unc-64(e246lf) . The F2 progeny of EMS treated unc-64(e246) were screened for better moving animals. We have screened 14,400 F1 genomes and have established seven independent strains that clearly move better than e246 . The suppressor strains moved between 6-8 times faster than e246 as measured from movies taken of them crawling on agar without food. Outcrossing of the seven strains revealed three segregating phenotypes, loopy movers (4 strains), jerky movers (1 strain), and long worms (1 strain); one strain segregated no obvious visible phenotype. We are currently determining whether these phenotypes are in fact those of the e246 suppressors. A similar screen has been performed by Owais Saifee in Mike Nonet's lab. They also isolated loopy e246 suppressors. We tested the anesthetic sensitivity of two of their loopy strains, js126 and js127e246 . Both strains were anesthetic resistant with EC 50s more than twice that of N2. This confirms that this is a reasonable approach towards finding "anesthesia genes". We are currently determining the anesthetic sensitivity of the suppressors isolated in our screen.

K Yook et al. (1999) International C. elegans Meeting "Components of the SNARE complex exhibit nonallelic noncomplementation"

K Yook, SR Proulx, E Jorgensen

[International C. elegans Meeting, 1999]

The failure of two recessive alleles to complement has been the established method of determining genetic identity. However, there are instances in which alleles of two separate genes exhibit noncomplementation. One interpretation of this genetic interaction is that the gene products of these noncomplementing loci are involved in the same process. Although nonallelic noncomplementation has potential use as a means to genetically dissect biological processes, attempts to use it as a strategy to identify interacting genes have had limited success. Defining the genetic principles behind nonallelic interactions may increase the success rate of this strategy. We have explored the rules of nonallelic noncomplementation by characterizing interactions among five synaptic function genes. Our results demonstrate that nonallelic noncomplementation requires the presence of an altered gene product. We tested the interactions among three synaptic function genes known to physically interact, unc-13, unc-64 and snb-1 (kindly provided by Mike Nonet). These three genes encode the C.elegans homologues of the exocytotic SNARE complex proteins Munc-13, syntaxin and synaptobrevin, respectively. We used resistance to an inhibitor of cholinesterase as an assay of synaptic efficacy. Null alleles of unc-13 and unc-64 fully complement one another implying that synaptic efficacy in C.elegans is not based on gene dosage. Hypomorphic alleles of unc-13, unc-64 and snb-1 exhibit robust nonallelic noncomplementation with one another. Surprisingly, in the presence of a hypomorphic synaptic function allele, the dosage of the other synaptic function gene becomes limiting. Moreover nonallelic noncomplementation may be specific to physically interacting gene products. Noncomplementation of unc-13(n2813) is not seen with alleles of unc-41, a protein likely to be involved in endocytosis (K.Grundahl and J.Rand per.com.) nor unc-17, the vesicular acetylcholine transporter. Both unc-41 and unc-17 are required in the synaptic vesicle cycle pathway during neurotransmission; however, they are not known to physically interact with unc-13. Therefore it is likely that nonallelic noncomplementation may be a phenomenon limited to interacting gene products. Results from our screen using this strategy support this hypothesis.

AF Dernburg et al. (1999) International C. elegans Meeting "Getting Intimate with the Right Partner"

AF Dernburg, AM Villeneuve

[International C. elegans Meeting, 1999]

A unique and ubiquitous feature of sexual reproduction is the pairing between homologous chromosomes that occurs during meiosis. This intimate association allows partner chromosomes to undergo genetic exchange, which is in turn essential for accurate chromosome segregation at the first meiotic division. Although this process has intrigued biologists for decades, many fundamental questions about its mechanism remain; e.g., how do chromosomes first encounter their homologs? Once physical contact is made, how is their homology recognized? Using FISH and high-resolution 3-D microscopy, we can visualize chromosome pairing at specific chromosomal loci within intact gonads. Since the entire temporal sequence of meiosis is present within each adult worm, this approach quickly revealed the stage at which meiotic pairing occurs. Combined with reverse genetics, these cytological methods have resolved another crucial question: Conflicting results from yeast and Drosophila had fueled a debate as to whether recombination is essential for meiotic chromosome pairing. By knocking out a gene, spo-11 , whose function is necessary to initiate recombination (in collaboration with Mike Dresser, Gary Moulder, and Bob Barstead) we abolished all meiotic exchange, but found that pairing and synapsis were unperturbed in this mutant. This study demonstrated pairing can occur in the absence of recombination in a metazoan organism, and that this paradigm is probably extremely widespread. We are now focusing on the role of "pairing centers" in meiotic chromosome pairing and synapsis. Cytological examination of worms lacking X -chromosome pairing centers has revealed that these distal regions are indeed essential for intimate association between the X s. We are testing whether synaptonemal complex components can associate with defective X chromosomes. Two additional approaches have enabled us to perturb normal homolog pairing: First, genetic screens for Him mutants have uncovered trans-acting factors involved in pairing (see MacQueen abstract). Another strategy is to study rearrangements, which require the pairing machinery to contend with chromosomes that do not share homology along their entire lengths. We are currently testing the long-standing assumption that balancer chromosomes suppress recombination by disrupting pairing between homologous sequences.

Edgar LG et al. (1991) International C. elegans Meeting "IS BODY WALL MUSCLE DETERMINATION CELL AUTONOMOUS?"

Edgar LG, Wood WB

[International C. elegans Meeting, 1991]

While maternal information appears to specify cell fate in the gut and germ line, early cell interactions may be required to specify cell fate in other lineages. Shifting blastomere position at the 6-cell stage to reverse left-right asymmetry also reverses left-right differences in fates of AB descendantsl. Laser ablation of some early blastomeres affects fates of others2. Certain early acting nonmaternal embryonic mutations that probably affect cell interactions cause changes in cell fates, for example, glp-l mutations affect pharyngeal cell specification3 and zen-l mutants appear to increase neuronal cells at the expense of muscle 4. We have found that some embryonic transcription is evident by 16-20 cells, confirming earlier results from run-on transcription assays in vitroS, which is consistent with the possibility that some embryonic patterning might depend on early acting genes regulated by cell interactions. We are using PD280, a CeMyoD-LacZ fusion strain generously given by Mike Krause, to assess whether isolated MS, C, and D blastomeres can express this gene (hlh-l) autonomously, and/or whether interaction with cells of other lineages is needed for correct expression of a mesodermal marker (as in frogs, for example). We have so far established that some descendants of isolated and cultured C, D, and MS blastomeres will express the fusion product in the absence of neighbors. Thus cell-autonomous signals appear sufficient for expression of the hlh-l gene, which presumably represents an early step in the commitment of these lineages to production of muscle. In general fewer cells appear to stain in the embryo fragments than would be expected if all body wall muscle precursors expressed the fusion construct, and fewer than in intact embryos. We are currently counting blue cells, and investigating the temporal pattern of LacZ expression in isolated MS and C blastomeres to see whether expression patterns are the same in isolated lineages and intact embryos.

Solari F et al. (1998) European Worm Meeting "A genetic enhancer of vab-7 is important for embryonic pattern formation and encodes an Mta1 homologue"

Ahringer JA, Solari F

[European Worm Meeting, 1998]

To find additional genes involved in posterior patterning in the embryo, we did a screen for mutants that enhance the phenotype of a weak vab-7 allele, ed6. After screening 900 chromosomes, 6 enhanced strains were found. One of the enhancers, named we3, has been further characterised. vab-7(ed6) mutants have either very mild posterior defects or appear wild-type. we3; vab-7(ed6) have very severe posterior defects and are often lethal. When separated from vab-7(ed6), we3 displays incompletely penetrant maternal effect defects, although we3 enhances the vab-7(ed6) phenotype zygotically. we3/we3 homozygotes descended from we3/+ mothers appear wild-type, but we3/we3 from we3/we3 mothers are often lethal and show variable morphological defects of the tail and of the mid-posterior body. we3 mutants have abnormally patterned muscle and hypodermal tissues as assayed by the expression of marker proteins. we3 appears to be a strong loss of function mutation, but is probably not null as we3/Df has a higher percentage lethality than we3/we3. We cloned the gene mutated in we3 animals and found it was encoded by CO4A2.2; the we3 mutation introduces a stop codon into the open reading frame. C04A2.2 has weak similarity to Mta1, a human gene whose expression is elevated in human metastasising mammary adenocarcinoma cell lines. The function of Mta1 is not known. Mike Herman reported (International Worm Meeting, 1998) that egl-27 mutants are rescued by a genomic fragment covering 2 predicted open reading frames: CO4A2.3 and C04A2.2. egl-27 hermaphrodites are partially egg-laying defective (as are we3 homozogotes), and males have abnormal tails, but they do not display the morphological defects of we3. Furthermore, we3 appears to complement egl-27(n170). This genetic complementation suggests that C04A2.2 may be a complex locus. We are currently analysing the expression of C04A2.2 and exploring the nature of the interaction between we3 and vab-7.