Ferreira, D.W. et al. (2015) International Worm Meeting "Germline defects in mitochondrial cholesterol transporter C. elegans mutants following bisphenol A or low cholesterol exposures."

Allard, P., Ferreira, D.W.

[International Worm Meeting, 2015]

Bisphenol A (BPA) treatment results in abnormal oocyte development in mammalian species as well as in the nematode C. elegans. In C. elegans, these defects are prevented by exposing the worms to cholesterol. We have therefore begun to investigate whether there is an interaction between BPA and normal cholesterol homeostasis in the germline. Previously, we have shown that mammalian homologs to cholesterol transporters (Steroid Acute Regulator Protein; StAR, 18kDa translocator protein; TSPO, and StAR related lipid transfer protein 3; StarD3) mimic germline phenotypes found in BPA-treated worms. Here we show that developing germ cells of these mutants have increases in germ cell nuclei apoptosis and reduced fertility when exposed to no or low cholesterol conditions. Protruding vulva, as well as missing or underdeveloped gonads, are also frequently seen in strl-1 (homologous to StAR) worms. To investigate a possible interaction between BPA and mitochondrial cholesterol transport, wild type and strl-1 worms were treated with BPA. Diakinetic analysis of the -1, -2 and -3 oocytes from control-treated worms reveals an increased incidence of abnormal chromatin arrangement in strl-1 mutant worms compared to wild type (29, 49 and 78 versus 0, 5 and 25 percent). Interestingly, the occurrence among wild type and strl-1 worms is similar following BPA treatment (6, 34 and 71 percent in wild type versus 7, 30 and 73 in strl-1). Additionally, BPA treatment reduced the incidence of protruding vulva (4 vs 24% in controls) and other severe gonad defects (6 vs 31% in controls) in strl-1 mutants. Together, the data suggest that BPA can at least partially rescue the sensitive germline phenotype in strl-1 worms. .

Lisa Girard et al. (2001) International C. elegans Meeting "Role of egl-5 in EGF/Wnt signal integration in the specification of P12 fate"

Scott Emmons, Paul Sternberg, Henrique B Ferreira, Lisa Girard

[International C. elegans Meeting, 2001]

The C. elegans Hox gene egl-5 is most similar, based on sequence analysis, to Abdominal-B . Consistent with its assignment into this paralog group, egl-5 is expressed in the posterior region of the worm. Immuno-staining results have shown that in the hermaphrodite, egl-5 is expressed in the hermaphrodite specific neuron, body wall muscle, posterior lateral microtubule neuron, PVC interneuron, the rectal epithelial cells K, F, B, U and the P12 neuroectoblast cell. 1 The two most posterior P cells are P11 and P12. The anterior products of their first division are both neuroblasts. P11.p fuses with the epidermal syncytium and P12.p divides again during late L1. The anterior division products, the epidermal cells P12.pa and P11.p are distinguishable by their distinct nuclear morphologies and positions. Previous genetic analysis indicates that P12 fate specification requires the synergistic action of the EGF and the Wnt signaling pathways. Reduction - or loss of function mutations in components of the EGF or the Wnt pathway result in partially penetrant P12 to P11 or P11 to P12 transformations. Double mutants of EGF and Wnt pathway components significantly enhance the frequency of transformation. P12 is not specified in an egl-5(lf) mutant and overexpression of egl-5 can rescue the loss of P12 specification phenotype of let-23 mutants. 2 In order to understand how information from the EGF and Wnt pathway are integrated at a cis -regulatory level, we have undertaken an analysis of the egl-5 promoter in collaboration with Scott Emmons to determine elements required for egl-5 expression in P12. 3 Do the two pathways converge on the egl-5 promoter or upstream of it? Beginning with a large promoter construct provided by Ferreira and Emmons, we have identified an approximately 1.3 kb. fragment of egl-5 promoter sufficient to drive expression of a heterologous promoter in P12. This 1.3 kb fragment contains six sites of approximately 20-40 bp each which are conserved between C. elegans and C. briggsae . Specific deletion of at least two of these sites in certain combinations eliminates expression in P12, suggesting that these sites might represent P12 enhancers. Complementary to our cis regulatory analysis, we have been using EMS screens to identify, and are currently characterizing, potential trans-acting factors as well as other genes which may participate in signal integration. Ferreira et al .(1999) Dev. Biol. 207:215-228 Jiang and Sternberg (1998) Development 125:2337-2347

Lu C et al. (2000) West Coast Worm Meeting "A b -tubulin gene, tbb-2, functions as an activator of mei-1 and mei-2 in female meiotic spindle formation in Caenorhabditis elegans."

Mains PE, Srayko MA, Lu C

[West Coast Worm Meeting, 2000]

C. elegans female meiosis requires two meiotic-specific genes, mei-1 and mei-2. Loss of mei-1 or mei-2 function blocks female meiotic spindle formation while the subsequent mitotic cleavages are normal. In a mei-1 gain-of-function mutant, however, the mitotic cleavages are disrupted after normal meiotic divisions. Wild-type MEI-1 and MEI-2 localize to the meiotic, but not mitotic spindle.1,2 However, MEI-1(gf) and MEI-2 ectopically localize to mitotic spindles in mei-1(gf) embryos. mei-1 and mei-2 encode homologs of p60 and p80 subunits of the sea urchin microtubule severing protein katanin, respectively, and MEI-1 and MEI-2 together disassemble interphase microtubules in a HeLa cell system.2 We propose that MEI-1 and MEI-2 form a complex in meiosis and regulate meiotic spindle formation by katanin-like activities. In a mei-1(gf) suppressor screen, we recovered three extragenic suppressors. One of them, sb26, was found to be a missense mutation in a b -tubulin gene, tbb-2. tbb-2(sb26) also enhances a weak mei-2(lf) allele. Together, these results suggest that tbb-2 is required for the function of mei-1 and mei-2. Antibody staining shows that TBB-2 is widely expressed during worm development. Neither tbb-2 nor tbb-1 (another b -tubulin highly similar to tbb-2) RNAi has severe effects during early development. However, tbb-2 and tbb-1 double RNAi results in 100% dead eggs indicating that they act redundantly during embryogenesis. We are doing experiments to examine the interactions of MEI-1/MEI-2 with tbb-2 and tbb-1. 1. Clark-Maguire, S. and Mains, P.E. (1994). J. Cell Biol. 126, 199-209. 2. Srayko, M., Buster, D.W., Bazirgan O.A., McNally, F.J., and Mains, P.E. (2000). Genes Dev. 14 (9).

Plasterk RHA et al. (2000) European Worm Meeting "Adaptation to sodium chloride: a behaviour that can be explored genetically"

Plasterk RHA, Weinkove D

[European Worm Meeting, 2000]

Chemotaxis to specific volatile odorants is inhibited by prolonged exposure to those odorants. For example, pre-exposure to benzaldehyde inhibits chemotaxis to a benzaldehyde source. This well characterised effect has been termed adaptation, and, in a screen for worms defective for benzaldehyde adaptation, a single dominant mutant allele, adp-1(ky20), was identified (Colbert and Bargmann Q995). adp-1 worms show normal chemotaxis to attractive odours and soluble compounds but have a selective defect in adaptation to benzaldehyde and butanone (Colbert and Bargmann 1995). We have discovered that the chemotaxis of wild type worms to sodium chloride is significantly reduced by pre-exposure to sodium chloride, particularly when worms are given the choice between sodium chloride and glutamine in a chemotaxis quadrant assay (Wicks et al.). In contrast to benzaldehyde adaptation, which requires a long pre-exposure (60 min) to very high concentrations of odorant, sodium chloride adaptation requires only 10-15 minutes pre-exposure to 50 mM NaCl, the concentration found in NGM agar plates. Furthermore, we have found that adp-1(ky20) worms are also defective in adaptation to sodium chloride. We are using SNP recombination mapping, firstly, to determine whether the same mutation is responsible for both the taste and odorant adaptation defects in the adp-1 strain, and secondly, to identify the adp-1 gene. We are currently testing whether the sodium chloride adaptation behaviour can be also used to identify and characterise further genes involved in adaptation. Colbert, H. A., and Bargmann, C. I. (1995) Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans. Neuron 14, 803-812 Wicks, S. R., de Vries C. J., van Luenen, H. G. A. M. and Plasterk, R. H. A. (2000) Che-3, a cytosolic dynein heavy chain is required for sensory cilia structure and function in C. elegans. Dev. Biol. in press comments: D.W. is supported by the Human Frontiers Science Program

Chenggang Lu et al. (2001) International C. elegans Meeting "The beta-tubulin gene, tbb-2 is an activator of the C. elegans female meiosis specific genes, mei-1 and mei-2"

Paul E Mains, Chenggang Lu

[International C. elegans Meeting, 2001]

mei-1 and mei-2 are specifically required for the C. elegans female meiotic spindle formation. MEI-1 and MEI-2 show homology to the two subunits of the microtubule-severing protein, katanin, and disassemble interphase microtubules when co-expressed in Hela cell. 1 In the wild-type embryo, MEI-1 and MEI-2 localize to meiotic spindle but disappear before the onset of the first mitotic cleavage. However, a mei-1 gain-of-function ( gf ) mutant results in ectopic localization of the proteins into the mitotic spindles and abnormal mitotic spindle formation. This mimics defects observed when embryos are treated with nocodazole, suggesting an ectopic spindle microtubule-destabilizing activity in mitosis. 2 We isolated three extragenic suppressors of mei-1 ( gf ). All genetically behave as activators of mei-1 and mei-2 function but are phenotypically wild-type by themselves. One of the suppressors, sb26 , has a missense mutation in the b -tubulin gene, tbb-2 . The amino acid change is in the carboxyl terminus, which has been shown to be required for the microtubule severing activity of sea urchin katanin. 3 Genetic interactions suggest that tbb-2 ( sb26 ) behaves as a gf suppressor that impairs the microtubule-severing activity of MEI-1 and MEI-2. TBB-2 specific antisera stain microtubule structures through out the worm life cycle. By using RNA interference and indirect immunoflurescence, we showed that tbb-2 and another b -tubulin gene, tbb-1 , function redundantly during early development. The two other extragenic suppressors of mei-1 ( gf ) are possibly allelic and map to the vicinity of an a -tubulin gene. Our work demonstrates genetic evidence that katanin requires specific interaction with spindle microtubules. 1. Srayko, M., Buster, D.W., Bazirgan O.A., McNally, F.J. and Mains, P.E. (2000). Genes Dev. 14, 1072-1084. 2. Strome, S. and Wood, W.B. (1983). Cell 35, 15-25. 3. McNally, F.J. and Vale, R.D. (1993). Cell 75, 419-429.

Chenggang Lu et al. (2003) International Worm Meeting "C. elegans genes tbb-2 and tba-2 are preferred tubulin isotype substrates of the MEI-1/MEI-2 microtubule severing complex during meiotic spindle formation."

Chenggang Lu, Paul Mains

[International Worm Meeting, 2003]

Female meiotic spindle formation requires two meiosis-specific components, MEI-1 and MEI-2, which are similar to the p60 (severing) and p80 (localization) subunits, respectively, of the sea urchin microtubule-severing complex katanin. In addition to their sequence similarities to katanin, MEI-1 and MEI-2 disassemble interphase microtubules when coexpressed in Hela cells.1 In wild-type embryos, MEI-1 and MEI-2 localize exclusively to the female meiotic spindle. MEI-1 and MEI-2 likely regulate the length of meiotic spindle fibres to maintain the unique morphology of the small anteriorly-located, barrel-shaped meiotic spindle, but MEI-1/MEI-2 must be inactivated prior to mitosis. Here we report analysis of three extragenic suppressors of a mei-1(gf) mutant that results in ectopic microtubule-severing activity during mitosis. These suppressors show semi-dominant suppression of the mei-1(gf) defects, but are phenotypically wild type by themselves. One suppressor is a missense allele of the -tubulin gene, tbb-2 (C36E8.5). The other two are missense alleles of an -tubulin gene, tba-2 (C47B2.3). All three suppressors genetically behave as if they generally inhibit microtubule severing: they suppress the mitotic phenotype resulting from ectopic mei-1 activity while enhancing meiotic defects seen when MEI-1 severing activity is compromised during meiosis. Although functional redundancies were seen with and -tubulins during worm development (our data, Philips et al. and Wright et al.), our experiments revealed tubulin isotype preferences of the MEI-1/MEI-2 severing complex. When MEI-1 and MEI-2 are limiting, tbb-2(RNAi) resulted in meiotic defects while tbb-1(RNAi) did not. Thus, MEI-1/MEI-2 prefers the TBB-2 isotype as a substrate. Similarly, TBA-2 is the preferred -tubulin substrate. Therefore, although TBB-1/TBB-2 and TBA-1/TBA-2 isotype pairs are functional redundant, our results reveal differences in their functions. 1. Srayko, M., Buster, D.W., Bazirgan O.A., McNally, F.J. and Mains, P.E. (2000). Genes Dev. 14, 1072-1084.

Wheeler JM et al. (2000) West Coast Worm Meeting "Calcium imaging of the defecation rhythm in C. elegans"

Wheeler JM, Thomas JH

[West Coast Worm Meeting, 2000]

Ultradian rhythms (those with a period shorter than 24 hours) have been shown to regulate a number of biological processes, such as the vertebrate heartbeat and invertebrate swimming behavior. The defecation cycle in C. elegans is an ultradian rhythm regulated by a clock-like mechanism (1). The cycle has a 45 to 50 second periodicity that is insensitive to changes in temperature between 18 and 30C. In addition, the clock mechanism can keep time in the absence of the defecation motor program (when animals leave their food source), indicating that the clock is distinct from the motor program. It has been shown that the inositol trisphosphate (IP3 ) receptor, a calcium channel that regulates cytoplasmic Ca2+ concentration by release from intracellular stores, is required in the intestine for the defecation cycle (2). Furthermore, calcium imaging with fura-2 revealed that periodic calcium spikes occur in the most posterior intestinal cell, just preceding the initiation of each posterior body-wall muscle contraction (pBoc). The pBoc begins at the posterior of the animal and proceeds anteriorly in a wave-like motion. There are two simple possibilities for how the calcium spike signals to the posterior body-wall muscles: 1) Ca2+ may initiate secretion of a muscle activator only in the posterior intestine, which then propagates anteriorly through the pseudocoelomic space or by muscle-to-muscle signaling, or 2) the Ca2+ spike may be propagated anteriorly through the intestinal cells themselves, initiating signaling to adjacent muscle cells along the way. In order to distinguish between these possibilities, I am constructing a strain that expresses fluorescent indicators for Ca2+ ("cameleons") in the intestine. This strain will be used to characterize the defecation cycle in wild type worms, as well as in various mutant strains with cycle defects. 1. D.W. Liu and J.H. Thomas, 1994. J Neurosci 14: 1953-1962. 2. P. DalSanto et al, 1999. Cell 98: 757-767.

Hannes E Buelow et al. (2005) International Worm Meeting "HOX genes define position-specific projection patterns of ventral nerve cord motor neurons"

Oliver Hobert, Hannes E Buelow, Nartono Tjoe

[International Worm Meeting, 2005]

The developing nervous system of all animals consists of a multitude of neuronal cell types with characteristic position-specific projection and innervation patterns. In the developing spinal cord of vertebrates recent work has shown that homeodomain-containing transcription factors of the Hox-c class are sequentially expressed in overlapping domains along the anterior-posterior (ap) axis and thereby determine distinct neuronal cell fates (1). However, little is known about how neuronal fate and cell position is translated into the specific neuronal trajectories that are characteristic for their respective position along the ap axis. The cluster of Hox genes in C. elegans consists of at least four genes ceh-13, lin-39, mab-5, and egl-5 which have been studied widely for their role in different cell fate decisions along the ap axis (2). Furthermore, Hox genes in C.elegans have been shown to define the migratory behavior of neuroblasts along the ap axis, yet few if any axonal phenotypes have been reported for these mutants (3). We show here that in C. elegans mutations in Hox genes lead to homeotic transformations in neuronal cell fates of motor neurons in the ventral nerve cord where specific neurons at specific positions along the ap axis adopt fates of adjacent neurons. These transformations are evidenced by changes in axonal projection patterns of motor neurons along the ap axis and will allow to dissect how cell fate/position is linked to axonal projection patterns. We are currently working on two basic questions relating to the observed axonal phenotypes. Specifically we are asking whether (a) Hox genes act cell-autonomously in neurons to determine position-specific axonal projection patterns and (b) which guidance factors may be under (direct or indirect control) of the Hox genes to determine the axonal trajectories. We are using a combination of expression analyses and cell-specific rescue experiments to address the first question. To answer the second question we are employing a candidate gene approach to identify guidance cues which may be responsible for specific axonal projection patterns. For these experiments we are using both gfp reporters for various guidance cues to determine whether these reporters are under control of Hox genes as well as double mutant analyses. (1) Dasen JS et al., Nature. 2003;425: 926-33. (2) Wang BB et al., Cell. 1993;74(1): 29-42.; Clark, S. et al. Cell. 1993;74(1)43-55. Costa M, et al. Cell. 1988; 55(5):747-56.; Ferreira HB et al., Dev. Biol. 1999; 207: 215-228; Brunschwig K et al., Development. 1999;126(7): 1537-46. (3) Desai C et al. Nature. 1988; 336:638-46.

Yueyi I Liu et al. (2004) West Coast Worm Meeting "Global Identification of C. elegans Regulatory Motifs"

Stuart K Kim, Yueyi I Liu

[West Coast Worm Meeting, 2004]

Regulatory motifs are short sequences of DNA that regulate the level, timing, and location of gene expression. Identifying these motifs and their functions is crucial in our understanding of gene regulation and disease processes. We developed CompareProspector, a motif-finding program that takes advantage of cross-species sequence comparison to identify putative regulatory motifs from sets of co-regulated genes [1] . We applied CompareProspector to 30 sets of genes with very similar patterns of expression, identified from the C. elegans topomap [2] and individual DNA microarray experiments. The statistical significance of each candidate motif identified was evaluated using criteria such as motif enrichment-the ratio of prevalence of the motif in a given set of promoters to its prevalence elsewhere in the genome, and the expression coherence of genes with the motif. We identified twelve significant regulatory motifs, three of which have literature evidence confirming they are true regulatory motifs. Overall, these twelve motifs are found in the upstream regulatory regions of 2970 different genes, and may be involved in gene regulation in 24 clusters of co-expressed genes. The first known motif, with the consensus TGATAA, matches the consensus of known binding sites for GATA factors. As GATA factors are known to be involved in worm intestine development [3] and hyperdermis development, it is not surprising that the GATA motif is identified from a set intestine-specific genes (F. Pauli, unpublished), mount08 of the topomap, which is enriched in genes from the intestine, and several collagen-related datasets (mount14, 17, and 35 of the topomap). We correctly identified GATA sites in the promoters of genes known to be regulatory by GATA factors. Interestingly, the GATA motif is also identified from several data sets involved in the aging process. This result parallels that of Murphy and colleagues, who independently identified this motif from their data set of DAF-16 target genes [4] . Both our result and the result from Murphy suggest that GATA factors may be involved in worm aging. Motif 2, which is identified in the two heat shock-related data sets, matches the consensus of known binding sites for heat shock factors [5] . Motif 3 matches the consensus of heat shock associated sites (HSAS), a motif that was first predicted computationally to be involved in the heat shock process [6] and later experimentally validated to be involved in ethanol stress response (14 th International C. elegans Conference abstract 1113C). We are currently in the process of validating the rest of the motifs and their individual binding sites using mutagenesis studies of promoters with predicted motifs. 1. Liu, Y., Liu, X.S., Wei, L., Altman, R.B. and Batzoglou, S. (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics . Genome Res. 14 , 451-8. 2. Kim, S.K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J.M., Eizinger, A., Wylie, B.N. and Davidson, G.S. (2001) A gene expression map for Caenorhabditis elegans . Science. 293 , 2087-92. 3. Maduro, M.F. and Rothman, J.H. (2002) Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm . Dev Biol. 246 , 68-85. 4. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H. and Kenyon, C. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature. 424 , 277-83. 5. Amin, J., Ananthan, J. and Voellmy, R. (1988) Key features of heat shock regulatory elements . Mol Cell Biol. 8 , 3761-9. 6. GuhaThakurta, D., Palomar, L., Stormo, G.D., Tedesco, P., Johnson, T.E., Walker, D.W., Lithgow, G., Kim, S. and Link, C.D. (2002) Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods . Genome Res. 12 , 701-12 .