Simske JS et al. (1994) Worm Breeder's Gazette "Specification of Vulval Cell Fates By Sequential Signalling Pathways."

Simske JS, Kim SK

[Worm Breeder's Gazette, 1994]

Specification of vulval cell fates by sequential signaling pathways Jeffrey S. Simske and Stuart K. Kim, Dept. of Developmental Biology, Stanford University Medical Center, Stanford CA 94305

Li WQ et al. (1992) Worm Breeder's Gazette "Characterization of the Axonal Guidance and Outgrowth gene Unc-33"

Li WQ, Shaw JE, Herman RK

[Worm Breeder's Gazette, 1992]

Characterization of the axonal guidance and outgrowth gene unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108

Uekusa T et al. (1994) Worm Breeder's Gazette "Molecular Analysis of unc-103"

Nishiwaki K, Miwa J, Uekusa T, Uchida MK

[Worm Breeder's Gazette, 1994]

Molecular analysis of unc-103 Tomoyuki Uekusa (uekusa@exp.cl.nec.cojp)l 2, Kiyoji Nishiwakil, Masaatsu K. Uchida2, and Johji Miwa1 1Fundamental Research Laboratories, NEC Corporation, Miyukigaoka, Tsukllba 305, Japan 2Department of Molecular Pharmacology, Meiji College of Pharmacy; 1-35-23, Setagaya- ku, Tokyo 154, Japan.

Hecht RM et al. (1980) Worm Breeder's Gazette "enzymatic activity in sperm and a sperm specific isozyme."

Hecht RM, Fairlie S, Husta-Butler M, Krass MR

[Worm Breeder's Gazette, 1980]

As part of our biochemical analysis of spermatogenesis we have been assaying enzyme activities in purified sperm preparations (isolated by the technique described by M. K. in WBG3(1):9-12, and detailed in a manuscript submitted to Devel. Biol.). The table below shows the enzymes tested so far. There was no activity for arginine kinase and there was a sperm specific variant form of malate dehydrogenase. All the other enzyme activites have been detected in adult worms also. [See Figure 1]

Lisa Girard et al. (2001) Worm Breeder's Gazette "egl-5 promoter analysis provides clues toward understanding anterior/posterior addressing at the cis-regulatory level"

Lisa Girard, Paul Sternberg

[Worm Breeder's Gazette, 2001]

The Hox genes encode a conserved set of transcription factors that are essential for anterior/posterior patterning in many animals and are expressed in a series of consecutive domains along this axis. These genes are almost always expressed in cells that are related by position rather than lineage and mutations in these genes often result in the transformation of one region to an adjacent one. Hox genes are typically found in clusters, with the order of the genes in the cluster colinear with their order of expression along the anterior/posterior axis, except for in C. elegans in which the order of two of the genes is inverted. In C. elegans, the Hox cluster contains a homolog of the anterior group genes, ceh-13, which is most similar to labial in the Drosophila HOM-C complex; two genes homologous to medial-group genes; lin-39 (sex-combs reduced, deformed, proboscipedia); mab-5 (Antennapedia) and a homolog of the posterior group, egl-5 (Abdominal-B)(1). Additionally, two more posterior homologs not contained within the quasi-cluster, nob-1 and php-3, have been characterized (2). The expression of Hox genes in positionally-related groups of cells raises interesting questions at the cis-regulatory level. Functional analysis of Hox gene promoters will help us understand how positional cues are read and interpreted resulting in correct anterior/posterior addressing along the body-length. C. elegans is ideal for such an analysis because fate-specification changes consequential to small perturbations in these promoters can be observed in individual cells, rather than the fields of cells as in many other organisms. To understand how cell-fate is specified in the posterior region of the hermaphrodite by the egl-5 Hox gene, we have undertaken, in a joint project with Yingqi Teng and Scott Emmons, a promoter analysis of this gene. egl-5 is expressed in the hermaphrodite specific neuron (HSN), body wall muscle, posterior lateral microtubule neuron, PVC interneuron, M, V6, the rectal epithelial cells K, F, B, U and the P12 neuroectoblast cell (3). The experiments described here focus on our progress delineating cis-regulatory elements which direct expression in K, F, B, U and P12. egl-5 and its adjacent Hox gene mab-5 are divergently transcribed with approximately 30 kb between them. A gfp translational fusion containing approximately 13 kb of promoter was sufficient to direct expression in K, F, B, U, P12 while a second construct, containing the most proximal 7 kb of promoter was not, suggesting that the 6 kb differential fragment between the two may be sufficient to direct expression in these cell-types. We tested this 6 kb region fused to a heterologous pes-10 basal promoter driving gfp, and found that indeed it was sufficient. We then examined a series of egl-5 promoter truncations, with this 6 kb piece as a starting point and identified a 1.3 kb region within the 6 kb that is sufficient for expression. Within this 1.3 kb fragment we identified a 469 bp sub-fragment sufficient to drive gfp expression in B, but not K, F, U, P12 and a 446 bp piece sufficient to drive expression in K, but not F, B, U or P12. F, U and P12 elements have proven more difficult to define. The 1.3 kb region sufficient to drive gfp expression in K, F, B, U, and P12 contains six sites (denoted here in distal to proximal order as 1-6) in the range of 20-30 bp each which are conserved between C. elegans and the closely related species C. briggsae. There are several notable consensus sites for trans-acting factors within these conserved regions including a binding site for SOX5 and FORKHEAD in site 3. The six sites are arranged in two quasi-clusters, with approximately 300 bp between the clusters. Specific deletions of either 1, 2 and 3 or 4, 5 and 6 abolishes expression in K, F, B, U and P12 suggesting that sequences in both clusters may be important for expression in these regions. One caveat of these types of experiments is that these deletions could alter spacing in the promoter, disturbing enhancer-promoter interactions. However, constructs which specifically deleted 3, 4 and 5, removing a similar number of base pairs as the 1,2,3 and 4,5,6 constructs, and found that that expression in K, F, B, U and P12 was not affected, suggesting that the spacing in this region is not affected by deletions of this magnitude. These conserved regions are being further studied to assess their relative contributions to egl-5 expression in the rectal epithelial cells and P12. The study of the cis-regulation of egl-5 will reveal the natural taxonomy of differential anterior/posterior addressing within C. elegans. 1. Salser, S.J. and Kenyon, C. (1994) Trends in Genetics 10, 159-164 2. Van Auken, K., Weaver, D.C., Edgar, L.G. and Wood, W.B. (2000) Proc. Natl. Acad. Sci. 97(9), 4499-4503 3. Ferreira, H.B., Zhang, Y., Zhao, C. and Emmons, S.W. (1999) Dev. Biol. 207, 215-228

Fleischhauer R et al. (1999) Worm Breeder's Gazette "Expression of the EXP-2 K+ channel in Xenopus oocytes produces a current with properties similar to those of the negative spike channel"

Avery L, Joho RH, Davis MW, Fleischhauer R

[Worm Breeder's Gazette, 1999]

The original, semi-dominant exp-2(sa26) allele was isolated in a screen for worms with defective defecation behavior (Thomas, 1990). This gain-of-function allele also causes hyperactive head movements, defective egg laying, and brief, shallow pharyngeal contractions. Animals homozygous for this allele arrest as L1 larvae, a phenotype that can be caused by a complete inability to feed. We have isolated loss-of-function alleles which have prolonged pharyngeal muscle contraction. Current clamp recording shows the action potentials from exp-2 loss-of-function pharyngeal muscles stay depolarized for several seconds and repolarize slowly (Figure 1A) instead of the fast repolarization after ~200 ms that is seen in wild-type. We have cloned exp-2 and found that it encodes a voltage activated K+ channel. WBG 14(4): 72 Byerly and Masuda (1979) characterized a K+ current in Ascaris pharyngeal muscles that is responsible for the fast repolarization at the end of the action potential. They called the channel that carries this current the negative spike channel (NSC). Because the electrophysiological defects seen in the exp-2 loss-of-function pharynxes could be explained by loss of NSC function, we hypothesized that exp-2 encodes a subunit of the C. elegans NSC. In order to characterize the properties of EXP-2 and see if they were indeed similar to those of the NSC, we have expressed exp-2 cRNA in Xenopus oocytes. We found that the EXP-2 channel, like the NSC and the HERG channel from the vertebrate heart, produces an inward rectifying K+ current that requires depolarization for activation (Figure 1B). Rectification occurs in all of these channels because inactivation is faster than activation. We find that the EXP-2 channel has an activation time constant of 56 ms at +20 mV (figure 1D) and an inactivation time constant that is too fast to measure. The voltage for half maximal steady state activation was -16 mV (figure 1C), similar to the NSC (-13 mV) and HERG (-21 mV). These data demonstrate that exp-2 does indeed encode a K+ channel subunit whose kinetics resemble those of the Ascaris NSC. These properties would be well suited to repolarize the muscle quickly at the end of the action potential. The channel should be nearly fully open and inactivated at the end of a normal 150 ms action potential, and would then begin to pass current when the potential dropped enough to de-inactivate the channel. Once de-inactivation begins, K+ efflux through the channel drives the membrane potential further negative, causing even more de-inactivation and faster repolarization in a feed-forward manner. We have not yet observed substantial outward K+ current through the EXP-2 channel, and so we are expressing cRNA from a different splice variant of the channel in the hope that this form might allow us to see such current. In addition, experiments are underway to characterize the effect of the gain-of-function mutation on channel kinetics. Byerly, L., and Masuda, M. O. (1979). Voltage-clamp analysis of the potassium current that produces a negative-going action potential in Ascaris muscle. J Physiol (Lond) 288, 263-84. Thomas, J. H. (1990). Genetic analysis of defecation in Caenorhabditis elegans. Genetics 124, 855-72.

Davis MW et al. (1994) Worm Breeder's Gazette "eat-6 Rescue: Probably a Na/K ATPase alpha Subunit"

Davis MW, Avery L

[Worm Breeder's Gazette, 1994]

Mutations in eat-6 cause a distinctive pharyngeal phenotype consisting of incomplete or delayed relaxations of the corpus and terminal bulb and a reduction or lack of negative transients in the electropharyngeogram (EPG)(fig. 1). We have mapped eat-6 to the right of lin-25 by three point mapping and left of arP3 by its failure to complement arDf1 . spa-1 ,which encodes a Na/K ATPase, had been cloned by Doug Fambrough and Delores Sommerville based on its homology to vertebrate ATPases and was mapped to this region of the physical map. This caused us to wonder whether the defect in eat-6 could be a failure of the ATPase to maintain a proper resting potential and thereby to repolarize properly at the end of an action potential. To determine what effect a defect in Na/K ATPase function might have on pumping, we applied the inhibitor ouabain to dissected N2 pharynxes and recorded EPG's. This drug phenocopied the eat-6 phenotype fairly well (fig.1). Based on these results, we concentrated on injecting the cosmid R07B8 ,which should contain the entire spa-1 gene. We obtained two stably rescued lines. We have obtained several lambda clones from a genomic library using probes obtained from D. Sommerville and D. Fambrough and plan to inject these soon. Although not yet proven, it seems likely based on the phenotypes, pharmacology and molecular data that eat-6 is spa-1 and that the failure of the pharynx to repolarize following a pump is due to a reduction of the Na+ and K+ gradients across the muscle membranes.

Gengyo-Ando K et al. (1989) Worm Breeder's Gazette "sequence analysis of unc-15 mutants and its intragenic revertants."

Kagawa H, Gengyo-Ando K

[Worm Breeder's Gazette, 1989]

To understand the molecular mechanism of thick filament assembly, we are investigating the mutation site of EMS induced unc-15 mutants at the DNA level. These mutant alleles are distinguished two categories. One is e1214, has no detectable paramyosin on SDS-PAGE gels and apparently null phenotype. This mutant had amber nonsense mutation (Q to Amber) near the N-terminus. The others are missense alleles which accumulate normal level's paramyosin, nevertheless result in the disruption of thick filament assembly. The latter alleles are of variable phenotype, the most severely paralyzed allele, e73 caused the reversion of electric charge (E- to K+) by the point mutation. Further sequence analysis of two intragenic e73 revertants revealed that second mutations are exactly in the unc-15 gene, not in a closely linked gene, and also caused the reversion of electric charge (E- to K+). These mutation might not disrupt the secondary structure of paramyosin, but only change the charge on the surface of alpha-helical coiled coil structure. This might mean the assembly of paramyosin itself and/or paramyosin-myosin heavy chain result from charge interactions between the molecules. We hope to calculate what kind of changes are induced by each of substituted amino acid with the computer program exploited by Dr. McLachlan (McLachlan, A. D. and Karn, J,1982, Nature 299, 226-231; McLachlan, A. D. and Karn, J, 1983, J. Mol. Biol. 164, 605-626; Kagawa, H., Gengyo, K., McLachlan, A. D., Brenner, S. and Karn, J., 1989, J. Mol. Biol., 207, 311-333). Molecular organization of unc-15 alleles summarized. More detailed epitope mapping of paramyosin is in progress. [See Figure 1]

Cassada RC et al. (1980) Worm Breeder's Gazette "initial characterization of ts embryonic defective (emb) mutants (Goettingen set)."

Culotti MR, Von Ehrenstein G, Cassada RC, Isnenghi E

[Worm Breeder's Gazette, 1980]

[See Figure 1] Already published allelism: Wood et al., 1980: b117=b189; b246 = let- 2.Miwa et al., 1980: hc57 = hc62; hc61 = hc67. Our global complementation results: g1 = g4; g16 = g65 = hc61 = hc67; g36 = hc65; g23 = g34 = hc70 = b117 = b189; g37 = b246 = let-2; b84 = hc66; g14 = g43; g57 = b1O. From frequency of multiple alleles we estimate 200-400 genes essential for embryogenesis. Mapping is in progress. We are also doing tsp's, R + H Tests, and cellular defects ( Isnenghi, Cassada, Radnia, Schierenberg, K. Smith, v. Ehrenstein).

Byerly L et al. (1978) Worm Breeder's Gazette "voltage-clamp studies of the electrical currents of the Ascaris pharyngeal muscle."

Masuda M, Byerly L

[Worm Breeder's Gazette, 1978]

It has proven difficult to study the currents that generate the electrical activity in the somatic muscle cells of Ascaris due to the inability to control the voltage across the excitable membrane. Therefore, we have directed our attention to the pharyngeal muscle, where it is possible to directly measure the voltage and pass large currents across the excitable membrane. We have developed a system which allows us to do current-clamp and voltage-clamp experiments on an isolated segment of the pharyngeal membrane. We find that this membrane has no pacemaker activity. In the absence of nervous input the membrane potential is flat at a level near -40 mV. Two types of spontaneous postsynaptic potentials are frequently seen; one type has a reversal potential near -40 mV and the other has a reversal potential near -10 mV. When the membrane is at the resting level, this second type PSP triggers a positive-going action potential, which reaches a level between +30 and +50 mV. The membrane potential then falls to a plateau near O mV, where it remains until a negative-going PSP triggers a negative-going action potential that reaches about -50 mV (the potassium reversal potential). The membrane potential remains at the plateau level for periods ranging from 100 msec to several minutes. The positive-going action potential is produced by an inward current that appears to be carried by both Na+ and Ca++. This current is prolonged, showing little inactivation by 200 msec after a positive voltage step. Clamping the membrane to positive potentials elicits essentially no delayed-rectification K current, the current that normally repolarizes active membranes. However, stepping the membrane potential back to the resting level after a large positive pulse elicits a strong, transient outward K+ current; this is the current that produces the negative-going action potential. We have done a detailed analysis of this current and have shown that it is a voltage- inverted analogue of the Hodgkin-Huxley Na+ current. It is activated by negative steps in potential. It shows inactivation, being completely inactivated at the resting potential. Conditioning pulses to levels more positive than -10 mV are necessary to remove the inactivation from the channel. This is a new K+ conductance that has not been found in any other animal. This demonstration of unique mechanisms in nematode physiology should serve as a caution in trying to interpret the function of the nematode nervous system. The pharyngeal nervous system must not only generate the signal that triggers the pharynx to contract (open the lumen), but also the signal to relax (close the lumen). Since the relaxation of the pharynx is the 'power stroke' of this muscle, it is not surprising that the membrane has developed a special K+ current to produce a fast, separately-triggerable repolarization.