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

Muimo R et al. (1993) Worm Breeder's Gazette "5-HT (Serotonin) Metabolism: Identification of a Developmentally Regulated 5-HT N-acetyltransferase"

Muimo R, Isaac RE

[Worm Breeder's Gazette, 1993]

5-HT is found in a number of neurons in C elegans including the Hermaphrodite Specific Neurons (HSN) which innervate the sex muscles of hermaphrodites and control egg-laying (Desai et al., 1988, Nature 336, 638-646). The addition of 5-HT to worms in M9 results in stimulation of egg-laying and mutants (e.g.. egl-l, egl-5 and egl-10 )with HSN abnormalities become bloated with eggs but respond to exogenously applied 5-HT by laying eggs, demonstrating an important regulatory role for 5-HT (Trent et al.,1983, Genetics 104, 619-647). We are interested in the inactivation of biogenic amines in nematodes and have studied the metabolism of 5-HT in C. elegans. Initially, we incubated cut worms with [14C]5-HT and after 2h at 20 C, the radiolabelled metabolites were extracted and analyzed .together with marker compounds on two dimensional silica TLC. Autoradiograms of the developed plates identified the major metabolite as N-acetyl 5-HT and the identity was confirmed by chromatography on two different reversed phase HPLC systems. There were no metabolites corresponding to products of oxidative deamination. N-acetylation of [14C]5-HT by C. elegans homogenate was dependent upon the addition of acetyl CoA to the incubation mixture. The N-acetyltransferase (NAT) was localized in a cytosol fraction and displayed Michaelis-Menten kinetics with a high affinity for 5HT (apparent Km, 30 +/- 8 mM; +/- SEM, n=3). Soluble enzyme was prepared from staged worms (L1, L2 ,L3 ,L4 and adults) and the specific activity of the NAT in each preparation determined from linear reaction rates (fig.). The specific activity was very low (< 8 nmol NA-5HT/h/mg of protein) in L1 and L2 stages but increased in an exponential manner with development, reaching the highest activity ( 144 nmol NA-5HT/h/mg of protein) in adult worms. [See Figure] The increase in the specific activity of the 5-HT NAT occurring at around the L4 /adultstage correlates with the appearance of 5-HT in the HSN cells and the appearance of the sex muscles that are innervated by these neurons. This correlation and the high affinity for 5-HT is consistent with a physiological role for the NAT in the metabolism of this amine but it is also possible that other biogenic amines, such as dopamine and octopamine, can also serve as substrates for this enzyme. We are also considering the possibility that N-acetylated amines are not necessarily end-products of metabolism but may have a signaling role in their own right. Future work will focus on defining the precise function of this enzyme in amine metabolism in adult nematodes.

Weinshenker DZ et al. (1997) Worm Breeder's Gazette "Serotonin and acetylcholine act in parallel to induce egg laying."

Weinshenker DZ, Thomas JH

[Worm Breeder's Gazette, 1997]

We have been investigating the role of ACh (acetylcholine) in egg laying. 5-HT (serotonin) is a strong inducer of egg laying in both the wild type and in animals that lack HSN neurons (Trent et al., Genetics 104, pp.619-647, 1983), suggesting that it acts directly on the egg-laying muscles. Levamisole, a nicotinic ACh agonist, is also a strong inducer of egg laying in the wild type, but on its own fails to induce egg laying in animals that lack HSN neurons (Weinshenker et al., J Neurosci 15(10), pp. 6975-6985, 1995). Based on this evidence, it has been proposed that ACh acts upstream of the HSNs. However, we showed that mutants defective in ACh transmission, such as cha-1 and unc-17, do not lay eggs in response to high levels of 5-HT (Weinshenker et al., ibid.). Based on this experiment, we proposed a model in which 5-HT and ACh act in parallel on the egg-laying muscles to induce egg laying. To further address this model, we tested whether levamisole could potentiate 5-HT-induced egg laying in egl-1 mutants, which lack HSN neurons. We found that levamisole significantly increased the number of eggs laid by 5-HT-treated egl-1(n487) animals in a well assay (Figure 1). Other combinations of drug dosages showed similar though less robust results. This demonstrates that levamisole does not require HSN neurons to enhance egg-laying. The egg-laying muscles are innervated by two classes of neurons: the HSNs, which contain both 5-HT and Ach vesicle transporters (VMAT and VAChT, respectively), and the VCs, all of which contain VAChT and a subset (VC4 and VC5) which contain VAChT and VMAT (Duerr et al., 1997 IWM abstract). The localization of both transmitter systems in neurons that innervate the egg-laying muscles, in conjunction with our pharmacological data, support the model that 5-HT and ACh act in parallel to induce egg laying. [The original article contains a figure.] Figure 1. Pharmacology of egl-1(n487) egg-laying. Each data point represents 48 animals. Error bars represent SEM. * indicates a P value calculated by the Mann-Whitney nonparametric test of 5-HT 5 mg/ml and 5-HT 5 mg/ml + levamisole 10 ug/ml.

Otsuka AJ (1990) Worm Breeder's Gazette "ISOLATION AND CHARACTERIZATION OF UNC-104 CDNA CLONES"

Otsuka AJ

[Worm Breeder's Gazette, 1990]

Although the unc-104 fragments isolated from an Arhinger-Kimble library were instrumental in obtaining approximately 4 kb of the 6 kb mRNA sequence, it was desirable to obtain full length cDNA clones to both complete the cDNA sequence and to produce the recombinant protein product in bacteria. The discrepancy between the abundance of unc-104 cDNA clones in the Arhinger-Kimble library (approximately 1 per 10,000) and in the Kim-Horvitz (none in 200,000) and Thacker-Capecci libraries (none in 100,000), led us to screen the Barstead-Waterston and Schauer-Wood libraries. The abundance of cDNA clones in these additional libraries suggests that an insufficient number of clones was screened in the cases of the Kim and Thacker libraries. cDNA clones were isolated from 2.6 million phage (1.4 million phage screened from the Barstead-Waterston bank, 0.6 million the Schauer- Wood early embryonic and him-8 embryonic libraries). The probe was the 5'-most cDNA fragment obtained from the library. The results are shown below: [See Figure 1] The two cDNA clones from the Barstead library overlap and cover a region of 4.7 kb. DNA sequencing of the cDNA clones is currently in progress.

Choy RKM et al. (1996) Worm Breeder's Gazette "Serotonin-Independent Neuromuscular Targets of Fluoxetine (Prozac)"

Thomas JH, Choy RKM

[Worm Breeder's Gazette, 1996]

Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and tricyclics such as imipramine have been shown to block serotonin (5-HT) reuptake in vitro and to be efficacious antidepressants in vivo. However, little direct evidence exists that demonstrates a causal relationship between these two effects. In spite of this, fluoxetine has been prescribed for over 20 million people worldwide and generates over $2 billion in sales each year. Our lab has screened a wide variety of drugs for neuromuscular effects on C. elegans. We typically assayed 8-10 adult worms in drug dissolved in 25-50 ul of M9 in microtiter wells. Previous work in our lab found that SSRIs and tricyclics rapidly induce nose and body muscle contraction1. Two pieces of evidence suggest these effects are not mediated by block of 5-HT reuptake: 1) cat-4(e1141) animals, which lack immunocytochemically detectable 5-HT, are still fully sensitive to fluoxetine-induced nose and body muscle contraction, and 2) exogenous 5-HT does not mimic the effects of fluoxetine on nose and body muscles. Previous work in our lab also suggests that similar doses of SSRIs are capable of blocking 5-HT reuptake in the egg-laying system of C. elegans1. In order to investigate the biochemical actions of fluoxetine in worms, we screened for mutants that were nose resistant to fluoxetine (Nrf). After incubating adult F2 progeny of EMS mutagenized N2 in 1 mg/ml fluoxetine for 20 minutes, we picked animals with noses that failed to contract. From 11,000 mutagenized genomes, we isolated 16 alleles that define eight complementation groups. All Nrf mutants tested so far are cross-resistant to nose contraction by a second SSRI, (paroxetine) as well as the tricyclic clomipramine. This finding suggests that SSRIs and tricyclics share a target other than 5-HT reuptake. Since we screened for mutants defective in muscle contraction, one concern was whether the Nrfs were specifically defective in fluoxetine response or their nose muscles had a non-specific excitability defect. We found that all Nrfs are sensitive to nose contraction by the cholinergic agonist levamisole, even at low doses, demonstrating that their nose muscles are still competent to contract. In addition, levamisole-resistant unc-29(e1072) animals are still sensitive to fluoxetine induced muscle contraction. Three Nrf alleles fail to complement ndg-4(lb108). This mutant was isolated in a screen for resistance to lethal exposure to the lipoxygenase inhibitor nordihydroguaiaretic acid (NDG)2. NDG has been shown to block an arachidonic acid metabolite signaling pathway that regulates S-type K+ channels in Aplysia. In worms, NDG acutely induces nose and muscle contraction in a manner similar to SSRIs and tricyclics. ndg-4 mutants have pale eggs (Peg) and we found that Nrf mutants in three other complementation groups, including one with four alleles, also have pale eggs. These Peg mutants produce roughly 50-80% dead eggs, including some that are abnormally shaped or arrest in development. Adult Peg animals accumulate large globules of what appears to be yolk in the pseudocoelomic space (W. Shreffler, pers. comm. and our observations) and the pale eggs contain less yolk than wild-type embryos. Yolk proteins are synthesized in the intestine and then transported through the pseudocoelomic space into the gonad where they are absorbed by oocytes. Peg mutants may be defective in some stage of this transport process. One speculation is that the Peg mutations disrupt the establishment of an ionic gradient that is used to transport yolk proteins across cell membranes and that a similar ionic gradient regulates the excitability of motorneurons or muscle cells. We are currently mapping the Nrf mutations and further pharmacologically analyzing the mutants. Many of the mutations have been localized to regions already sequenced by the genome project. We plan to identify molecularly several of the genes involved in this fluoxetine response. References: 1. Weinshenker et al. J. Neuroscience 15:6975 (1995) 2. Shreffler et al. Genetics 139:1261 (1995)

Loer CM et al. (1990) Worm Breeder's Gazette "Neurotransmitter Synthesis in Worms: An Aromatic Amino Acid Hydroxylase Gene from C."

Kenyon CJ, Loer CM

[Worm Breeder's Gazette, 1990]

Specification of neurotransmitter phenotype is an important requirement for generating a functional nervous system. A specific neurotransmitter phenotype requires the coordinate expression of biosynthetic enzymes, a degradation or re-uptake system, and perhaps a specific packaging apparatus. The Drosophila dopa decarboxylase gene ( Ddc), the last enzyme required for serotonin (5-HT) and dopamine (DA) synthesis is differentially regulated and spliced in the nervous system and the epidermis, where is required for cuticle synthesis ( Morgan et al., 1986, EMBO J. 5: 3335; Bray et al., 1988, EMBO J. 7:177). A number of neuron-specific enhancers in the 5' region of Ddc have been identified, including one that binds a POU-type homeodomain protein. Mutation in this enhancer region eliminate DA expression in a subset of dopaminergic CNS neurons (Johnson and Hirsh, 1990, Nature 343: 467). The biogenic amine neurotransmitters 5-HT and DA are used by a number of neurons in C. elegans, including the HSN, NSM, and some CPs (5-HT) and ADE, PDE, CEPs, and Ray neurons 5A, 7A, and 9A (DA) ( Sulston et al., 1975, J. Comp. Neurol., 163: 215; Horvitz et al., 1982, Science 216:1012). 5-HT and DA are derived by two enzymatic steps from the aromatic amino acids tryptophan and tyrosine, respectively (see below). In vertebrates and flies, specific enzymes catalyze the first step in the reaction (i.e., tryptophan hydroxylase and tyrosine hydroxylase), whereas the decarboxylation step for both 5- HT and DA is catalyzed by a single enzyme with broader specificity (i. e., dopa decarboxylase). [See Figure 1] As a first step towards analyzing the control of neurotransmitter phenotype, we are seeking to clone genes encoding biosynthetic enzymes for these neurotransmitters. We used the polymerase chain reaction ( PCR) with degenerate oligonucleotides encoding highly conserved regions of aromatic amino acid hydroxylases to amplify a genomic fragment encoding a C. elegans AAA Hydroxylase. Of 5 clones sequenced, 3 were garbage and 2 encoded the AA sequence shown below. As indicated, the sequence included a typical small worm intron with good consensus splice donor and acceptor sequences at an evolutionarily conserved splice site. Typical C. elegans codon usage was also apparent in the coding region sequenced. [See Figure 2] We used this 185 bp fragment (TH1,2) to isolate a lambda genomic clone and also to probe a genomic southern blot. At high stringency, the TH1,2 fragment showed a single band in all lanes except for a SacI digest (TH1,2 contains a SacI site); thus, the gene seems to be found in a single copy in the genome. We are using the lambda genomic clone to isolate cDNAs for sequencing. We plan to use in situ hybridization to define sites of expression of the gene. Since we may not be able to determine what kind of AAA hydroxlase this gene encodes by sequence alone, such information may clearly indicate the probable specificity of the enzyme (e.g., if it is found only in dopaminergic cells, it should be a tyrosine hydroxylase!). We also plan to fuse -gal or another reporter gene into the AAAH gene to use for identifying mutations that alter its expression and to examine expression in certain known mutants. Such a screen should yield genes that are required for the regulation of neurotransmitter synthesis (the cat mutations may be alleles of such genes) as well as genes that determine which neurons produce a specific neurotransmitter.

Rosenbluth RE et al. (1985) Worm Breeder's Gazette "update on linkage group V (left)."

Johnsen RC, McKim KS, Rosenbluth RE, Baillie DL, Turner LM

[Worm Breeder's Gazette, 1985]

Our analysis of LGV(left) is continuing. At present 10 deletions, providing 13 different breakpoints, divide the region into 14 zones ( figure). The table shows recessive lethal mutations that have been mapped to the zones. [See Figure 1] At Cold Spring Harbor (1985) we noted that deletion mapping placed unc-34 to the left of unc-60. This has been additionally confirmed by 3-factor mapping. The two genes are 0.8 - 3.4 m.u. apart. Another gene, let-336 has now been deletion-mapped to the left of unc-34 and therefore becomes the current left-end gene of LGV. One of us (Kim McKim) has begun an intragenic mapping study of unc- 60. We are in possession of the alleles, e677, e723, e890 and m35 and would greatly appreciate receiving others. We have noticed that at least e677 and m-35 differ greatly regarding their viability as heterozygotes over sDf28: e677/sDf28 barely survives while m35/sDf28 survives well. This relationship correlates with their respective homozygous phenotypes, - m35 being more mobile than e677.

Seydoux GC et al. (1995) Worm Breeder's Gazette "Evidence for a maternally-provided dowry of SL1 precursor RNA."

Seydoux GC, Fire A

[Worm Breeder's Gazette, 1995]

Thanks to Kim Ferguson and Joel Rothman for gifts of SL1 plasmid and zen-1 strains. We have previously described the embryonic distribution of RNAs containing the splice leader SL1 (Seydoux and Fire, 1994, Development 120, p. 2823). Using an oligo complementary to the 22 nucleotides of SL1 that are transpliced, we had detected SL1-containing RNAs in the cytoplasm of all cells, and in P granules, from the one-cell stage to the pretzel stage. We have now examined the distribution of the full-length SL1 precursor RNA. To do this, we used a probe complementary to the SL1 precursor RNA, under conditions where hybridization to the 22bp trans-spliced leader would not occur. We detected SL1 precursor RNA in the germline syncytium, in oocyte nuclei, and in interphase and prophase nuclei of early embryos (1-28/50 cell stage). No SL1 precursor RNA was detected past the 50-cell stage, except for occasional staining in 1 or 2 nuclei, which we tentatively identified as the nuclei of P4, and Z2/Z3. To test whether the SL1 precursor RNA detected in embryos was maternal or zygotic, we repeated our in situ hybridization using embryos derived from unc-76(e911) zen-1(e2482); wEx127 (pRF4, SL1/5SrRNA) hermaphrodites. These hermaphrodites segregate 65% viable embryos (which carry the SL1 gene on an extrachromosomal array) and 35% dead embryos (which presumably have lost the array and have no copy of SL1). If the embryonic distribution of SL1 was due to zygotic transcription, we would have observed 35% of embryos with no staining. Instead, we found that 95% of 1-28-cell stage embryos were positive for the SL1 precursor, indicating that this early RNA is independent of zygotic transcription. This result suggests that SL1 precursor RNA synthesized in the germline is inherited by the embryo and repeatedly localized to the nucleus following each mitosis. What about zygotic transcription of SL1? Kim Ferguson and Joel Rothman have demonstrated a zygotic requirement for SL1 as early as the 28-cell stage (wm95, p4). It appears, therefore, that SL1 is transcribed in the early embryo, in addition to being maternally inherited. We have not detected SL1 precursor RNA in somatic nuclei past the 50-cell stage: one possibility is that, in these later stages, SL1 precursor RNA no longer accumulates to detectable levels as increased numbers of zygotic transcripts are available for trans-splicing.

Pilgrim DB et al. (1992) Worm Breeder's Gazette "Preliminary Sequence Analysis of the daf-7 Gene"

Johnsen RC, Riddle DL, Pilgrim DB

[Worm Breeder's Gazette, 1992]

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tc1 tagging. On a chromosomal walk toward fem-2 ,one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9 was used to probe a cDNA library constructed by Stuart Kim. Twenty cDNA clones of various sizes up to 2.3kb were isolated from the library. Hybridization results indicate that all these clones share common sequences. Probing restriction digested genomic DNA isolated from the Tc1 -insertionmutant DR1039 ,daf-7 (m434),revealed differences consistent with a 1.6kb Tc1 insertion in DR1039 DNA when compared with non-mutant DNA. For example, when probed with cDNA a 11kb BamHI fragment found in wild-type genomic DNA corresponds to a 12.6kb fragment in mutant DNA. We sequenced both ends of the largest (2.3kb) cDNA clone, and have orfs totaling 460 amino acids. Unfortunately, no appropriate initiator codon or poly A addition site are evident in the cDNA. Therefore, we are screening Bob Barstad's cDNA library to identify a more complete clone. The sequence so far shows no strong homology to anything in the NBRF-PIR data base, although it does have weak homology to some calcium dependent ATPases.

Huang LS et al. (1990) Worm Breeder's Gazette "Molecular cloning of lin-15 by the candidate gene approach"

Sternberg PW, Huang LS, Tzou P, Mendel JE

[Worm Breeder's Gazette, 1990]

We have rescued the lin-15 multivulva.(Muv; visible or AB) and synthetic (syn; class A and class B) Muv phenotypes with cosmid PS#74B3. This cosmid maps to the far left (formerly, far right) end of the clb-1 contig on the X chromosome and was originally isolated because it weakly hybridizes to the G-protein PCR clone 1, as reported in WBG11(2):32. As we had been trying to clone lin-15 (see WBG11(3) :60 for last update), we knew from the map data that lin-15 should lie either at the right edge of the clb-1 contig or in the gap off the end. Since PS#74B3 appeared to map closely to lin-15 on the physical map and because the putative G-protein was a candidate gene for a genetic locus presumably involved in signal transduction, we decided to microinject the cosmid to test whether it rescued lin-15. As we had hoped, PS#74B3 rescued lin-15, although the G-protein hybridizing region did not rescue. A 'reverse northern' analysis identified a 6.8 kb BamHI fragment which lay within the region defined as necessary for lin-15 function by injection. When the 6.8 kb fragment was used to screen Stuart Kim and Chris Martin's cDNA libraries, we identified two classes of non-cross hybridizing cDNAs which mapped to opposite sides of our 6.8 kb fragment, with cDNA2 apparently more abundant than cDNA1.