Janet Duerr et al. (2005) International Worm Meeting "Dopamine synthesis, transport, and behavior in C. elegans"

Janet Duerr, Nanda Filkin

[International Worm Meeting, 2005]

We are studying the contribution of dopamine transporters to dopamine dependent neurotransmission and behavior in C. elegans. Dopamine is synthesized by tyrosine hydroxylase, TH (encoded by cat-2; Lints and Emmons 1999) and packaged into synaptic vesicles by the vesicular monoamine transporter, VMAT (encoded by cat-1, Duerr et al. 1999). Dopamine stimulation is at least partially terminated by re-uptake by the dopamine plasma membrane transporter, DAT (encoded by dat-1, Jayanthi et al. 1998). DAT mutants show hyper-stimulation of some dopamine-dependent behaviors, consistent with the persistence of abnormally high levels of extracellular dopamine. Recently, Sanyal et al. (2004) reported that C. elegans TH (cat-2) or VMAT (cat-1) mutants have low but significant levels of dopamine at the whole organism level. It is not known whether the dopamine is released by the appropriate neurons. We are examining the function of this residual dopamine with a combination of genetics and behavior. If the residual dopamine in TH mutants is totally without extracellular function, then lack of DAT should have no effect on TH mutants behavior. However, preliminary results suggest that the behavior of the double mutant cat-2;dat-1 is not identical to that of either single mutant. Similarly, if the dopamine in VMAT mutants is totally without extracellular function, then lack of DAT should have no effect on VMAT mutants behavior. Again, preliminary results suggest that the behavior of the double mutant cat-2;cat-1 is not identical to that of either single mutant. We are continuing to characterize these double mutants in order to understand the significance of the behavioral changes and their relationship to neuronal and vesicular levels of dopamine in this organism. Since serotonin is involved in modulating many of the same behaviors as dopamine, we are also examining the roles of serotonin, thorough the use of additional mutants in serotonin synthesis (tph-1; Sze et al. 2000) and serotonin re-uptake (mod-5; Ranganathan et al. 2001).

Nanda Filkin et al. (2007) International Worm Meeting "Monoamine Re-uptake Transporters."

Janet Duerr, Nanda Filkin

[International Worm Meeting, 2007]

We are using mutant analysis to probe the relative contributions of different transporters to dopamine (DA) levels and DA-dependent behaviors in C. elegans. DA is synthesized by the action of two enzymes, tyrosine hydroxylase (TH; Lints and Emmons 1999), and amino acid decarboxylase (Hare and Loer 2004). DA is packaged into synaptic vesicles by the vesicular monoamine transporter, VMAT. After vesicular release of DA, DA signaling is at least partially terminated by re-uptake of DA by the DA plasma membrane transporter, DAT (Jayanthi et al. 1998). DAT- mutants, like VMAT- (cat-1) or TH- (dopamine-deficient cat-2) mutants, are healthy and show normal thrashing in liquid. However, they show subtle locomotion defects that are consistent with DAT- mutants having dopamine over-stimulation of some dopamine-dependent behaviors (e.g. egg-laying) and under-stimulation for other behaviors (e.g., movement in food). We have used glyoxylic acid-induced fluorescence (IF) to characterize monoamine levels in the dopamine trafficking mutants. As expected, the TH- or TH-DAT- double mutants have serotonin-like IF in the serotonin neurons but no detectable DA-like IF. Surprisingly, after soaking in DA, we observe faint DA-like IF in the serotonergic NSM neurons, but not in the normally DA neurons. This suggests that mod-5, the serotonin re-uptake transporter, may transport DA into serotonergic cells in these mutants. This contrasts with the findings of Ranganathan et al. (2001), who observed that the converse was not true, i.e., there was no uptake of serotonin by DAT in mod-5. We have tested our hypothesis by examining DA-like IF after soaking the triple mutant cat-2; dat-1; mod-5 in DA; as predicted, DA-like IF is no longer found in the serotonergic NSMs. We have also found that soaking in L-dopa leads to faint DA-like IF in the NSMs in cat-2;dat-1, but not cat-2;dat-1;mod-5, suggesting that mod-5 can also transport L-dopa. We are currently determining whether dopamine uptake by the serotonin transporter is normally detectable or in only induced in specific mutant backgrounds.

Arvinbayar B et al. (2000) European Worm Meeting "Species identification based on Bachrest analysis of the sequences of the ITS region of the 18S-5.8S-28S operon of C. elegans and Heterorhbditis spp"

Fodor A, Arvinbayar B, Pamjav H

[European Worm Meeting, 2000]

There are 55 copies of the 18S - ITS1 -5.8S - ITS2 - 28S rRNS opreon in th gnom of C. elegans. The whole operon is rather conserved but the sequence of the 18S rDNA is even more coinserved: Blaxter et al. (1998) used it to identify different genera within Phylum Nematoda. The comparative sequence analyses of C. elegansand 18 different strains belonging to six phylogenetic species of genus Heterorhabitis were carried out. Previously on the basis of PCR PAGAE RFLP studies the phylogenetic Heterorhabditis species could be unambigously identified and separated. The results of the compartive sequence analyses are presented in the poster.

Yukinobu Arata et al. (2007) International Worm Meeting "Quantitative measurement of the timing of cell division in C. elegans embryo."

Hitoshi Sawa, Yukinobu ARATA

[International Worm Meeting, 2007]

During animal development, cell cycle progression seems to be regulated tightly. In this study, we quantitatively measured cell volume and cell cycle duration. We found that cell cycle duration was in inverse proportion to the 0.26 (~1/4) th power of cell volume in the AB and MS lineages and the 0.34 (~1/3) th power of cell volume in the C lineage, but was independent of cell volume in the E and D lineages. These findings indicate that cells divide in size-dependent and -independent manners during the development, and suggest that cells possess unidentified mechanisms to regulate cell cycle progression, depending on cell volume. In addition, we also observed a relationship between cell division timing and cell position in the embryo, and showed that most cell divisions occur semi-synchronously throughout the embryo; anteriorly-positioned cells tended to divide slightly earlier than those in the posterior. This phenomenon seems to correspond to the mitotic wave that has been reported in Drosophila</I>, Xenopus</I> and zebrafish development. Is cell division timing affected by cell division in adjacent cells? We changed the relative positions of cells in embryos cultured in vitro</I>. Although most cells divide in the same order as in the embryo in egg shell, the division order of E cell daughters was earlier. This result suggests that cell division timing is affected by the embryonic microenvironment. Given these findings, we propose that cell cycle progression is regulated by size- and position-dependent mechanisms in the C. elegans</I> embryo.

K Omata et al. (1999) International C. elegans Meeting "Habituation of C. elegans for the touch sensitivity"

K Kawamura, F Yonezawa, K Omata

[International C. elegans Meeting, 1999]

In order to study the habituation of C. elegans for the touch sensitivity, we carry out computer simulations, in which the neural circuit is formed by making use of the data table constructed recently by Oshio et al [1]. The i -th neuron is connected with the neighboring j -th neuron through the coupling strength K ij , which is varied dynamically by the Hebb rule. Note that K ij is not necessarily equal to K ji because there are one-way connections between the neurons by chemical synapses. As a reference state, we first deal with the neural circuit consisting only of the neurons ALM, AVM, PLM, PVM, AVA, AVB, PVC, AVD, A and B, that are related to the forward and backward movement directly. We give periodic stimuli to the sensory neurons PLM, PVM, and monitor the response of the motor neuron A. We find that the frequency of the response decreases with time, which indicates that the habituation to the touch sensitivity actually takes place. As one deviation from the reference state, we kill the inter-neuron AVD, and perform the same analysis described in the above. There is a tendency that the decay of the response curve becomes faster, and the habituation is enhanced. As the other deviations, there are several possibilities of killing the inter-neurons AVA, AVB, PVC and/or AVD. We discuss the enhancement of the habituation in relation with the recent experimental results by Hosono. [1.] K. Oshio, S. Morita, Y. Osana and K. Oka; C. elegans synaptic connectivity data'', Technical Report, CCEP, Keio Future No.1 (1998)

Gidi Shemer et al. (2001) International C. elegans Meeting "FROM BIRTH TO"

Gidi Shemer, Clari Valansi, William A Mohler, Benjamin Podbilewicz

[International C. elegans Meeting, 2001]

Although cell fusion plays an important role in the development of organisms, no fusogens directly involved in cell-cell fusion have been found. In order to find new genes that participate in this complex process, we have concentrated on epithelial cell fusion in C. elegans , which involves almost one third of all the somatic cells in this organism [1]. Since vulva formation in C. elegans involves twenty cell fusion events, we have started with primary screening for vulval defects. Pvl (protruded vulva) and Muv (multivulva) mutants were subjected to a secondary fluorescence-based screening leading to the isolation of eff-1(hy21) [ e pithelial f usion f ailed]. eff-1(hy21) was discovered to be defective not only in vulval fusion, but also in general epithelial cell fusion, as all the epithelial cells in this mutant remained unfused [3]. This cell fusion failure led to gross morphological defects. Organogenesis of the vulva and the male tail was abnormal, resulting in low fertility (Egl phenotype). Defective hypodermal cell fusion also led to severe elongation problems during embryogenesis and larval development (expressed as the Dpy phenotype). The mutant animals exhibited irregular body form with lumpiness during larval stages and bulged tail throughout development. The postembryonic defects in eff-1(hy21) were more severe at 25 o C than at 15 o C. We have found that the temperature sensitive periods (TSP) of the elongation and fertility defects were almost identical to the TSP of the fusion defects (at 12h-33h post fertilization), thus correlating the resulting phenotypes directly to lack of cell fusion. Our results, along with preliminary data on the gene structure [2] and its regulation [3] lead us to propose that eff-1 is the first potential fusion effector, directly involved in the actual process of cell fusion. 1. Shemer and Podbilewicz (2000) Dev. Dyn. 2. Mohler et al. (2001) 13 th International C. elegans meeting 3. Shemer and Podbilewicz (2001) 13 th International C. elegans meeting

William A Mohler et al. (2001) International C. elegans Meeting "THE GENE eff-1 IS REQUIRED FOR CELL FUSION IN EPITHELIAL SYNCYTIA"

Victoria Scranton, Gidi Shemer, Benjamin Podbilewicz, John G White, William A Mohler, Jacob del Campo

[International C. elegans Meeting, 2001]

Cell fusion is a major factor in the development of the C. elegans body plan. As the molecules and physical mechanisms responsible for cell membrane fusion are unknown in any developmental system, we have focused on C. elegans as a uniquely suited model for genetic and structural studies of the process of cell fusion. Some of the advantages of studying cell fusion in the worm are: A) the predictable postion and timing of cell fusion events based on the lineage of precursor cells [1]; B) the amenability of embryos to live-imaging and EM studies of the intermediate structural stages in a cell fusion event [2]; C) the surprising viability of worms with genetic defects in cell fusion [3,4]. In two distinct screens - one for embryonic hypodermal fusion defects and another for vulval morphogenesis/fusion defects - we have isolated recessive mutant alleles of a gene that is required for cell fusion among the precursors of epithelial syncytia [3,4]. Live-imaging experiments confirmed that correctly patterned syncytial precursors are blocked at the earliest stages of cell fusion in these mutants, before formation of an initial cytoplasmic bridge between partner cells. Mutations oj55 and hy21 both lie on Chromosome II and fail to complement in the trans-heterozygote. Allele oj55 was further mapped to postion +0.79, and was rescued by injection of cosmid C26D10. This cosmid also rescues hy21 , suggesting that both mutations lie within the same gene, which we have named eff-1 (Epithelial Fusion Failure). Preliminary results from dsRNAi and a single rescue injection experiment appear to confine the functional eff-1 gene to a 12.5 kb region of C26D10. Confirmation of the identity of the gene and characterization of the molecular lesions underlying eff-1(oj55) and eff-1(hy21) will be presented at the 13 th International C. elegans meeting. The two eff-1 alleles vary in the numbers of unfused cells per animal and in the overall effects on body morphology: hy21 has a stronger loss of function than oj55. Yet it appears that all morphogenetic defects in both mutants can be attributed to failed cell fusions [3,4]. Other aspects of hypodermal and vulval patterning and differentiation appear normal. We therefore believe that the Eff-1 gene product functions specifically within the mechanism controlling the fusion of plasma membranes on neighboring cells. To test this hypothesis we will compare the developmental expression pattern of eff-1 with the known temporospatial pattern of embryonic and larval cell fusions [1]. Furthermore, we will examine the dynamic sub-cellular localization of Eff-1 protein to assess whether it appears at the known sites for initiation and widening of the opening between actively fusing cells [2]. Podbilewicz and White (1994). Developmental Biology 161:406-424. Mohler et al. (1998). Current Biology 8: 1087-1090. Mohler and White (1999) 12 th International C. elegans meeting: 595. Shemer et al. (2001) 13 th International C. elegans meeting.

Konop C et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Cellular Localization of AF19 and Related Peptides in the Cephalic Region of the Nematode Ascaris suum."

Reinitz C, Konop C, Jarecki J, Knickelbine J, Stretton A OW

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Over fortyAscaris FMRFamide-like (AF) peptides have been sequenced in the parasiticnematode Ascaris suum by the Strettonlab. Many of these peptides have beenshown to have potent effects on the A.suum locomotory nervous system (Davis &amp; Stretton, 1996). Locomotion isvital to the survival of the parasite in its host so an in-depth knowledge ofpeptide localization and behavioral effects is essential to develop neweffective anthelmintics. The FMRFamide-like peptide AF19 (AEGLSSPLIRFamide)has been shown to have inhibitory effects on locomotion (Davis &amp; Stretton,2001). Injection of the peptide in the head-restricted behavioral assayabolished all locomotory waveforms and their propagations (Reinitz &amp;Stretton, 2000). Ananti-peptide antibody specific to AF19 reproducibly stains a subset of neuronsin the cephalic region of A. suum. ALA , one of the two cells in the dorsal ganglion , shows AF19-immunoreactivity. This isdata is corroborated by single cell mass spectrometric (MS) and MS/MS data. Cloning, based on th sequence ofa novel peptide in ALA, yieldedthe transcript that encodes AF19 (afp-13)and two other amidated peptides. In situ hybridizationexperiments are planned to further corroborate the cellular localization ofAF19. Supported by NIH grant AI15429.

Sze JY et al. (1997) International C. elegans Meeting "VP16-activation of the neural specific transcription factor UNC-86 alters the egg-laying and chemotaxis behaviours"

Ruvkun GB, Sze JY

[International C. elegans Meeting, 1997]

unc-86 encodes a POU-domain transcription factor. In wild-type animals, UNC-86 accumulates in the nuclei of 57 neurons, including all the touch receptor neurons, interneuron AIZ, and the motor neuron HSN. We are interested in identifying the genetic cascades downstream of unc-86. We reasoned that if unc-86 gene activity could be hyperactivated, it may increase or constitutively activate the expression of the genes normally regulated by it, and that such elevated expression of downstream genes might confer phenotypes in wild type or suppress weak mutations in downstream genes. To activate UNC-86, we inserted the potent VP16 transactivation domain into an unc-86 genomic clone. We find that UNC-86/VP16 is expressed in the correct neurons and can replace wild type UNC-86 for the formation and function of the touch receptor neurons. UNC-86/VP16 also rescues the chemotaxis defects of unc-86 null-mutants, but displays novel genetic activities: it enhances the animal's sensitivity to volatile odorants. Since UNC-86 is not expressed in the odorsensory neurons AWA and AWC, UNC-86/VP16 may increase the expression of components that integrate the volatile odorant response in, for example, the connected interneuron AIZ. UNC-86/VP16 does not rescue the egg-laying defects of unc-86 null-mutants, and causes dominant Egl in wild-type animals. We find that unc-86 is necessary for the expression of a putative tryptophan hydroxylase (TH) (located on cosmid ZK1290) in several serotonergic neurons, including HSN. Tryptophan hydroxylase catalyzes the rate limiting step of serotonin synthesis. We find unc-86/VP16 transgenic animals express TH/GFP, but fail to accumulate serotonin in HSN and cause HSN degeneration in adults. unc-86/VP16 induced HSN defects are suppressed by unc-2 (e55), which encodes an a-subunit of a calcium channel in HSN, but not by snt-1 or unc-104, which may regulate serotonin secretion. Thus, the lack of HSN serotonin in unc-86/VP16 animals is not due to excess release of serotonin. UNC-86/VP16 may upregulate the expression or the activity of HSN signaling components to cause excitotoxicity triggered by excessive Ca2+ accumulation. We have isolated 11 suppressors of unc-86/VP16 induced Egl phenotype from a genetic screen. These suppressors could be mutations in unc-86 downstream genes that have roles in the maintenance of HSN function.

Kohtaro Kawamata et al. (2006) East Asia C. elegans Meeting "C. elegans CREB functions in XXX cells to regulate dauer formation"

Sachiko Yoshimura, Masatoshi Hagiwara, Yoshishige Kimura, Kohtaro Kawamata, Hidehito KUROYANAGI

[East Asia C. elegans Meeting, 2006]

CREB (cAMP-response element binding protein) is ubiquitously expressed and is involved in multiple biological phenomena. We have reported that disruption of CREB/crh-1 gene in C.elegans caused transient formation of dauer-like larvae at 27&deg;C but not at 25&deg;C, and that crh-1 functioned upstream of daf-12 and daf-16 (15^th biennial international C.elegans conference 2005, 277). In this report, we show that crh-1 functions in XXX cells, which are considered to prevent dauer-like larva formation by releasing steroid hormone(s), dafachronic acid(s). XXX-specific expression of CRH-1 cDNA rescued the crh-1 mutant phenotype, while expression in other tissues did not. XXX-specific expression of dominant negative CREB mimicked dauer-like constitutive phenotype of crh-1 mutant. These resulsts indicate that expression in XXX cells is required and sufficient for the crh-1 gene in regulating dauer-like larva formation. Furthermore, crh-1 dauer-like constitutive phenotype was rescued by addition of lathosterol or 4-cholestene-3-one, but not by cholesterol or 7-dehydrocholesterol, which are considered to be intermediate compounds in the biosynthesis of dafachronic acids (Veerle et. al. 2006). These results suggest that CREB/crh-1 functions in XXX cells by cell-autonomously regulating steroid hormone biosynthesis.