Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy."

Papp, Andy, Aldrich, Chris, Bangalore, Srikanth, Perry, David

[International Worm Meeting, 2009]

The Green Fluorescent Protein (GFP) gene from the fluorescent jellyfish Aequorea victoria, and its variants (such as EGFP), are used extensively to study the location and timing of gene expression in transgenic animals and plants (Chalfie et al, 1994). Resultant GFP fusion proteins are especially well-suited to studying in vivo gene expression patterns in the transparent nematode, C. elegans and the transparent larva of the fly D. melanogaster. Perhaps one of the most significant limitations to its use in large-scale genetic screens is the high cost of equipment needed to observe GFP microscopically - namely the Fluorescence Dissecting Stereomicroscope for screening and picking mutants and upright and inverted epi-fluorescent compound microscopes for more detailed studies. Commercially available Fluorescence Dissecting Stereomicroscopes typically sell in the midrange between US$10,000 and US$20,000. They are offered by only the "high-end" microscope companies, based upon their most expensive dissecting scopes, and incorporate their premium-priced mercury arc-lamp illuminators, power supplies and epi-fluorescence modules. This leads us to wonder whether it is possible to cut corners without sacrificing utility, and which corners can be cut. Since the inception of GFP-based expression screens, a variety of researchers have produced custom-made and home-made GFP dissecting scopes. These include, for example, Welcome Bender (Harvard Medical School, pers. comm.) and Ian Chin-Sang (Chin-Sang, 2004). There are several possibilities to consider toward lowering the cost of a Fluorescence Dissecting Stereomicroscope. It may be possible to get sufficient light from relatively inexpensive, long-lived, lower-power-consuming Light Emitting Diodes (LEDs) vs. mercury arc lamps. Depending upon the spectral specificity of the LEDs, filters or dichroic mirrors might be omitted. Getting enough light intensity of the precise wavelengths that excite GFP fluorescence focused onto the sample is important. The exciting beam can be provided directly or focused backward through the microscope using "epi-illumination". We will explore these possibilities and report (and possibly demonstrate) the results. Chin-Sang, Ian. (2004) GFP Stereoscope Using LED light source. Queen''s University, Kingston, ON, Canada. http://130.15.90.245/gfp_stereoscope.htm.

Martin L Hudson et al. (2003) International Worm Meeting "Functions of ephrins in embryonic and post-embryonic development: exploring connections with Kallman syndrome protein and cadherins"

Andrew D Chisholm, Martin L Hudson

[International Worm Meeting, 2003]

During C. elegans gastrulation, embryonic cells rearrange to form tissue layers. Endodermal and mesodermal cells move into the interior of the embryo, leaving a transient ventral cleft that is sealed up by the short-range movements of ventral neuroblasts. These ventral neuroblasts and their progeny provide the substrate for the subsequent enclosure movements of the epidermis. Mutants with abnormal ventral neuroblast movements display enlarged ventral clefts that later result in epidermal enclosure defects. Previous work in our lab showed that signaling via the C. elegans Eph receptor VAB-1 and its ephrin ligand EFN-1/VAB-2 regulate neuroblast movements at the end of gastrulation (George et al. 1998; Chin-Sang et al. 1999). The divergent ephrin EFN-4 is also required for neuroblast movements but appears to function independently of the VAB-1 receptor (Chin-Sang et al. 2002). A third pathway is defined by the LAR-like receptor phosphotyrosine phosphatase PTP-3 (Harrington et al. 2002). These pathways appear to play related and partly redundant roles in ventral neuroblast movements.We have examined whether other mutants with epidermal morphogenesis phenotypes affect neuroblast movements and have used double mutant analysis to determine if these genes might define new pathways or can be placed in the VAB-1/EFN-4/PTP-3 pathways. KAL-1 is the C. elegans homolog of human Kallman syndrome protein, anosmin-1 (Rugarli et al. 2002). A kal-1 mutation displays low penetrance defects in epidermal morphogenesis. Using 4-D analysis, we find that kal-1 mutants display delayed closure of the ventral cleft. We also find that a kal-1 mutation enhances the morphogenetic phenotypes of all mutants tested (vab-1(null), efn null mutations and ptp-3), suggesting that KAL-1 may define a separate signaling pathway. We are also studying the post-embryonic functions of ephrins. EFN-4 is expressed in the primary cells of the developing vulva (Hudson and Chisholm, 2002 WCWM, abstract 144). efn-4 mutants display very low penetrance defects in vulval morphogenesis and induction. The cadherin CDH-3 is also expressed in vulval cells (Pettit et al. 1996). A cdh-3 mutation causes epidermal morphogenesis defects (6) reminiscent of those in weak efn-4 mutants. We find that the cdh-3 mutation does not enhance efn-4(null) mutant phenotypes but significantly enhances efn-1(null) mutant phenotypes, suggestive of a role for cdh-3 in efn-4 signaling. Further analysis of the phenotypes will be presented.

Vivegananthan U et al. (1997) International C. elegans Meeting "ANALYSIS OF FEM-3 PROTEIN INTERACTIONS"

Spence AM, Vivegananthan U

[International C. elegans Meeting, 1997]

Male development in C. elegans depends on the activities of the three fem genes. Conversely, female development requires that fem activity be negatively regulated. We are interested in how the products of the fem genes function and how their activity is regulated. We have previously presented evidence that the negative regulation of fem activity in XX somatic tissues depends upon a protein-protein interaction between FEM-3 and the cytoplasmic domain of the transmembrane protein, TRA-2A (Spence et al., 1995 C. elegans meeting). FEM-3 also associates with FEM-2, and we predict that this association is important for the specification of male fates (Chin-Sang and Spence, 1996. Genes Dev. 10: 2314-25.). We wish to define the domains in FEM-3 responsible for each interaction and to establish the significance of both interactions. Deletion analysis in the yeast two-hybrid system identifies two regions required for interaction with FEM-2 but not with TRA-2A. We have modified the yeast two-hybrid system to screen for fem-3 mutations that selectively disrupt the FEM-3/FEM-2 or the FEM-2/TRA-2A interaction. We will test the resulting mutant proteins for their ability to promote male development and their sensitivity to regulation by TRA-2A.

Chin-Sang ID et al. (1997) International C. elegans Meeting "FEM-2 PROTEIN TO THE RESCUE!"

Spence AM, Chin-Sang ID

[International C. elegans Meeting, 1997]

Male development in C. elegans requires the activities of the three fem genes. We previously reported that FEM-3 physically interacts with FEM-2 and that FEM-2 is a Type 2C protein phosphatase (Chin-Sang and Spence, 1996. Genes Dev. 10: 2314-25). Expression of fem-2 cDNA from the heat shock promoter rescues male somatic development in fem-2 mutants. To complement these experiments we injected bacterially expressed GST-FEM-2 fusion protein into fem-2 mutants and asked whether we could rescue the germline phenotype. fem-2(b245ts) dpy-1(e1) homozygous animals are self-fertile at 15C but develop as females at 25C. We injected GST-FEM-2 protein (1mg/ml) into the gonad arms of adult fem-2(b245ts)dpy-1(e1) hermaphrodites raised at the permissive temperature. These animals were allowed to recover at 15C for 1-3 hours and then shifted to 25C so their F1 progeny developed at the non permissive temperature. Of 19 animals injected, 7 of them produced F1 progeny (20/141 F1s) that developed as hermaphrodites at 25C. The average brood size or these rescued animals was 34 +/-22 (n=20) and all of their F2 progeny developed as females. The progeny of uninjected animals or those injected with GST alone never developed as hermaphrodites at 25C (n>600). These experiments provide us with a simple assay to test the affects of wild-type and phosphatase inactive forms of GST-FEM-2 in the germline.

Vivegananthan U et al. (1998) Midwest Worm Meeting "ANALYSIS OF FEM-3 PROTEIN INTERACTIONS"

Spence AM, Vivegananthan U

[Midwest Worm Meeting, 1998]

Male development in C. elegans depends on the activities of the three fem genes. Conversely, female development requires that fem activity be negatively regulated. We are interested in how the products of the fem genes function and how their activities are regulated. FEM-3 physically associates with FEM-2 and we predict that this association is important for the specification of male fates (Chin-Sang and Spence, 1996. Genes and Dev. 10: 2314-25). FEM-3 also binds to the predicted cytoplasmic domain of the transmembrane protein TRA-2A and we have evidence that the negative regulation of fem activity in XX somatic tissues depends upon this interaction (Mehra et. al., in preparation). To test our hypotheses about the biological significance of the interactions involving FEM-3, we have mutagenized a fem-3 cDNA in vitro and used a modified version of the yeast two-hybrid system to screen for mutations that selectively disrupt one interaction or the other. Our screen identified several mutations of each class. We are testing their effects on sex determination in vivo. We predict that FEM-2 non-binding mutants of FEM-3 will be incapable of supporting male development. In contrast, we predict that FEM-3 mutants that are unable to interact with TRA-2A will be insensitive to negative regulation in XX animals and hence, will cause dominant masculinization. Tests of these predictions are underway.

Sarah L Moseley et al. (2002) West Coast Worm Meeting "The roles of EFN-4 in embryonic morphogenesis and oogenesis"

Sarah L Moseley, Andrew D Chisholm

[West Coast Worm Meeting, 2002]

Loss of function in the ephrin EFN-4 causes defects in neuroblast migrations during closure of the ventral gastrulation cleft and in subsequent epidermal morphogenesis (Chin-Sang + Chisholm, 2001 Intl Worm Meeting). Synergistic interactions between efn-4 mutations and mutations in the Eph receptor VAB1 suggest that EFN-4 has functions independent of the Eph receptor. We have investigated the relative contributions of the four ephrin ligands to VAB-1 signaling during morphogenesis. First, if we reduce vab-1 expression by RNAi in an efn-1 efn-2 efn-3triple null mutant background we do not observe enhancement of the triple mutant phenotype (as determined by 4D microscopy). This is consistent with previous data suggesting that most of the VAB-1 signaling in morphogenesis is provided by EFN-1, EFN-2 and EFN-3. Second, we generated quadruple ephrin mutant animals using efn-1 RNAi in an efn-2 efn-3 efn-4triple null mutant. Quadruply mutant embryos laid by these animals display severe defects in gastrulation cleft closure and a fully penetrant embryonic arrest. Thus, efn-4(RNAi) enhances the phenotypes of the efn-1,-2-,3 triple mutant. Taken together, our data suggest that EFN-4 functions independently of EFNs 1, 2 and 3 and the VAB-1 receptor during morphogenesis. In addition to signaling through Eph receptors, ephrins can also transduce signals to cells expressing them, a process known as reverse signaling. An interesting possibility is that EFN-4 may be a constitutively active reverse signaling ephrin.

U Vivegananthan et al. (1999) International C. elegans Meeting "Protein-Protein Interactions Involving the Sex Determining Protein, FEM-3"

AM Spence, U Vivegananthan

[International C. elegans Meeting, 1999]

Male development in C. elegans depends on the activities of the three fem genes. Conversely, female development requires that fem activity be negatively regulated. We are interested in how the sex determining protein FEM-3 functions and how its activity is regulated. We believe that a physical interaction between FEM-3 and FEM-2 is required to promote the adoption of male cell fates (Chin-Sang and Spence, 1996. Genes and Dev. 10 : 2314-25). We also have evidence that an interaction between FEM-3 and the C-terminus of TRA-2A is required for the negative regulation of fem activity in XX somatic tissues (Mehra, A., Gaudet, J., Heck, L., Kuwabara, P. and Spence, A. submitted). We want to use genetic assays to test our hypotheses about the biological significance of the two known FEM-3 interactions. We screened in vitro mutagenized fem-3 cDNAS, in a modified version of the two-hybrid system, to look for FEM-3 mutants that are able to bind to FEM-2, or to TRA-2A, but not both. We have identified two mutations that selectively disrupt the FEM-3/FEM-2 interaction and five mutations that selectively disrupt the FEM-3/TRA-2A interaction. We are currently testing the effects of these mutants on sex determination in vivo . We predict that FEM-2 non-binding mutants of FEM-3 will be incapable of supporting male development. In contrast, we predict that the TRA-2A non-binding mutants of FEM-3 will dominantly masculinize the XX soma, irrespective of the level of TRA-2A activity.

SE George et al. (1999) International C. elegans Meeting "Still Searching for components of the VAB-1 pathways"

AD Chisholm, SE George

[International C. elegans Meeting, 1999]

We are identifying the mechanisms by which the C. elegans Eph receptor tyrosine kinase, VAB-1, signals in neural and epithelial morphogenesis. VAB-1 has both kinase-dependent and kinase-independent functions during C. elegans development (George et al, Cell 92:633). The kinase-dependent (forward signaling) function of VAB-1 is mediated by its cytoplasmic kinase domain, possibly through tyrosine phosphorylation of the juxtamembrane domain. Proteins that interact with the cytoplasmic domain of Eph receptors have been identified in vertebrates using the yeast two-hybrid system. Candidate molecules include a low molecular weight protein tyrosine phosphatase and adaptor proteins such as Nck, Grb2, and Grb10. We are using a modified yeast two-hybrid screen to identify genes that function in VAB-1 forward signaling. Initial progress and methodology of the screen will be presented. The nature of the VAB-1 kinase-independent function is unknown but may involve the extracellular domain. Our genetic and biochemical studies indicate that VAB-2 (a C. elegans ephrin) is a ligand for VAB-1 and that it appears to mediate the kinase-independent function of VAB-1 (see Chin-Sang et al abstract). vab-2 mutations display synthetic lethality with vab-1 kinase mutations. We also observe synthetic lethality between vab-1 kinase mutations and a mutation in the receptor protein tyrosine phosphatase, PTP-1, suggesting that PTP-1 may be acting in a parallel pathway to the VAB-1 kinase activity (see Harrington et al abstract). To identify genes that are acting in the VAB-1 kinase-independent pathway or in parallel pathways we are performing screens to identify mutations that are synthetic lethal with vab-1 . Progress of the screen will be presented.

PE Mains (1999) International C. elegans Meeting "A Role for fem-2 During Embryonic Elongation"

PE Mains

[International C. elegans Meeting, 1999]

An actin-mediated contraction of the epidermal cells elongates the spherical embryo into a long, thin worm 1 . let-502 mutants fail to elongate while mel-11 mutant embryos hypercontract 2,3 . Genetic results and analogies with smooth muscle contraction suggest that myosin phosphatase (MEL-11) inhibits contraction. At the appropriate time, Rho-binding kinase (LET-502) inactivates MEL-11, allowing elongation to proceed. We found that fem-2 enhances let-502 and suppresses mel-11 , suggesting a role for fem-2 during morphogenesis. Effects of fem-2 on embryonic elongation were unexpected. However, when fem-2(0) hermaphrodites were selfed, 4% of the progeny arrested as unelongated embryos and another 20% had morphological phenotypes (Dpy, Vab, or Rol) suggestive of embryonic morphogenesis defects (because of maternal-effects, first generation fem-2 homozygotes are self-fertile). fem-2 function during elongation is likely independent of its role in sex determination since genetic interactions were not observed when let-502 or mel-11 were combined with fem-1, fem-3, tra-1, tra-3 or fog-2 . We noticed no changes in sexual phenotype with let-502 or mel-11 alone. fem-2 and mel-11 encode subunits of unrelated types of phosphatases 2,4,5 . These phosphatases cannot be interchangeable because while fem-2(+) potentiates morphogenesis, mel-11(+) inhibits it. The triple mutant suggests that fem-2 is part of a parallel system that can elongate the embryo in the absence of the let-502/mel-11 pathway: while let-502;mel-11 double mutants undergo near normal morphogenesis, let-502; mel-11; fem-2 triple mutants fail to elongate. 1 Priess & Hirsh (1986) Dev. Biol. 117:156; 2 Wissmann et al. (1997) Genes Dev. 11:409; 3 Wissmann et al.(1999) Dev. Biol. (in press); 4 Pilgrim et al. (1995) Mol. Biol. Cell 6:1159; 5 Chin-Sang & Spence (1996) Genes Develop. 10:2314

Mandy Smith et al. (2001) International C. elegans Meeting "Expression of a C. elegans Mucin-Like Gene"

Samuel M Politz, Mandy Smith

[International C. elegans Meeting, 2001]

Based on the amino acid sequence of a cDNA encoding the major larval surface protein of the parasitic nematode Toxocara canis 1 , plus studies of T. canis surface proteins, it is likely that nematode surface proteins are structurally similar to the mucins that line and protect epithelial cell layers in vertebrate animals. Mucins have two characteristic features: 1. domains rich in serine and threonine, which are extensively O-glycosylated; 2. domains rich in cysteine, which form inter- or intra-molecular disulfide bonds. These features confer special physical and biochemical properties on mucins, such as high solution viscosity and resistance to proteolysis. The epitope recognized by monoclonal antibody M38 is detected on the surface of live C. elegans L1s, and the timing of its expression is altered in srf-6 mutants, which display it in stages L1 through L4. We demonstrated that the M38 epitope is carried by a 30 kD antigen detected in extracts of C. elegans L1s 2 . The antigenicity of this protein is destroyed by pretreatment with O-glycanase, suggesting that it is an O-linked glycoprotein, like the T. canis TES-120 surface antigen. A nematode-specific gene family in C. elegans has been described, members of which encode adjacent serine-threonine-rich and cysteine-rich (SXC or six-cysteine) domains 3 . Because of the likelihood that these encode surface proteins, we have begun to characterize their expression by RT-PCR. For initial studies, we have chosen the predicted genomic coding sequence F41G3.10, which contains 10 exons, each of which encodes a ser-thr-rich sequence followed by an SXC domain. In initial experiments using gene-specific primers, we have amplified a small cDNA fragment spanning 3' terminal exons 9 and 10 in total RNA from mixed stages of C. elegans. We plan to study the stage-specificity of expression of this RNA, as well as map a full-length cDNA representing this transcript. 1 Gems, D. and Maizels, R.M. (1996) Proc. Natl. Acad. Sci. USA (1996) 93: 1665-1670. 2 Hemmer, R.M., Donkin, S.G., Chin, K.J., Grenache, D.G., Bhatt, H., and Politz, S.M. (1991) J. Cell Biol. 115: 1237-1247. 3 Blaxter, M. (1999) Science 282: 2041-2046.