Tao Cai et al. (2008) C.elegans Neuronal Development Meeting "Loss of the Transcriptional Repressor PAG-3 Results in Enhanced Neurosecretion that is Dependent on the Dense-Core Vesicle Membrane Protein IA-2"

Ping Yu, Guofeng Zhang, Tao Cai, Michael Krause, Hiroki Hirai, Tetsunari Fukushige, Abner Notkins

[C.elegans Neuronal Development Meeting, 2008]

It is generally accepted that neuroendocrine cells regulate dense core vesicle (DCV) biogenesis and cargo packaging in response to secretory demands, although the molecular mechanisms of this process are poorly understood. One factor that has previously been implicated in DCV regulation is IA-2, a catalytically inactive protein phosphatase present in DCV membranes. Our ability to visualize a functional GFP-tagged version of IA-2 directly in live C. elegans animals has allowed us to capitalize on the genetics of the system to screen for mutations that disrupt DCV regulation. We found that loss of activity in the transcription factor PAG-3, that functions as a repressor in many systems, results in a dramatic up-regulation of IA-2 and other DCV proteins. The up-regulation of DCV components was accompanied by an increase in presynaptic DCV numbers and resulted in phenotypes consistent with increased neuroendocrine secretion. Double mutant combinations revealed that these PAG-3 mutant phenotypes were dependent on wild type IA-2 function. Our results support a model in which IA-2 is a critical element in DCV regulation and reveal a novel genetic link to PAG-3-mediated transcriptional regulation.

Tao Cai et al. (2003) International Worm Meeting "IA-2, a Vesicle Membrane-Associated Protein, Regulates Synaptic Transmission in C. elegans"

Michael Krause, Abner L Notkins, Tao Cai, Tetsunari Fukushige

[International Worm Meeting, 2003]

IA-2, a major autoantigen in type 1 diabetes, is a receptor-tyrosine phosphatase-like protein associated with the membrane of secretory granules of neural and endocrine-specific cells. IA-2 lacks phosphatase activity due to an amino acid substitution within the catalytic domain and the function of IA-2 remains unclear. In order to gain insight into the cellular functions of IA-2, we have studied the C. elegans homolog, CeIA-2 encoded by the ida-1 gene. Using two independent, putative null, deletion alleles of ida-1 we show that animals lacking CeIA-2 are viable with only subtle visible phenotypes. Pharmacological studies show loss of CeIA-2 results in presynaptic neuronal defects involving neurotransmitter release. Furthermore, ida-1 interacts genetically with unc-31 and unc-64, two genes encoding factors required for neuronal vesicle function. These results suggest that CeIA-2 is a novel regulator of synaptic transmission that regulates vesicle release.

Hiroshi Ichijo et al. (2006) East Asia C. elegans Meeting "Analysis of mutants defective in olfactory learning induced by AWC-sensed odorants."

Isao Katsura, Hiroshi Ichijo, Ichiro Torayama, Kotaro Kimura

[East Asia C. elegans Meeting, 2006]

C.elegans changes its behavior based on its experience, which can be regarded as learning. For example, animals decrease the efficiency of taxis to a stimulus after they have received the same stimulus under starving conditions: Animals conditioned with starvation and NaCl no longer migrate toward NaCl, and the animals that had been starved at certain temperature avoid the starvation-temperature upon temperature gradient (Saeki et al., 2001; Mohri et al., 2005). In contrast, we discovered that conditioning with food and the AWC-sensed odorant butanone (Bu) enhances chemotaxis to this odorant, which we call butanone enhancement (Torayama et al., submitted).Recently we found that such olfactory enhancement was not specific to butanone: animals showed olfactory enhancement after conditioning with food and AWC-sensed odorant Isoamyl alcohol (Ia) or 2,3-pentanedione (Pd), although animals that conditioned with food and the AWC-sensed odorant benzaldehyde or the AWA-sensed odorant diacetyl exhibited almost no change or decrease (i.e., adaptation) rather than increase in the response to the same odorants.To elucidate the molecular mechanism of olfactory enhancement, we used a forward genetic approach. We screened 7000 genomes and isolated seven candidate mutants for the Ia olfactory enhancement (ut321 - ut327). We are analyzing the phenotypes of these mutants and other mutants that were previously isolated for the Bu olfactory enhancement defective (ut301-ut315). One of the Ia olfactory enhancement mutants (ut324) and some of the Bu olfactory enhancement mutants were also defective in the Pd olfactory enhancement. In this meeting, we will also report on the progress in the genetic mapping of ut324 and some other mutants.

Makoto Tsunozaki et al. (2002) West Coast Worm Meeting "Mechanisms of odor discrimination"

Makoto Tsunozaki, Cori Bargmann

[West Coast Worm Meeting, 2002]

We are using the chemotactic response to volatile odors by C. elegans to ask how animals discriminate between multiple inputs to produce the appropriate behavior. The AWC amphid neurons of C. elegans sense the attractive odors benzaldehyde (bz), butanone (bu), isoamyl alcohol (iaa), pentanedione (pd), and trimethylthiazole (tmt). The AWC pair alone can discriminate between bu, pd, tmt, and bz/ia - that is, in a uniform background of one odor, animals can still chemotax towards a point source of a different odor. The mechanisms for odor discrimination are not well understood.

Hiroshi Ichijo et al. (2007) International Worm Meeting "Genetic mapping of a novel butanone enhancement mutant."

Isao Katsura, Hiroshi Ichijo, Kotaro Kimura, Ichiro Torayama

[International Worm Meeting, 2007]

We have previously reported that C. elegans enhances its chemotaxis to butanone (bu) after conditioning with food and the odorant, which we call butanone enhancement. This behavioral plasticity is independent to serotonin, which is requires for the modulation of many other food related behavioral plasticity. Analysis of olrn-1(ut305) and olrn-2/bbs-8(ut306) (olrn: olfactory leaning defective) revealed that butanone enhancement is probably conducted in the STR-2-expressing AWC (AWCON) neuron and required the function of Bardet-Biedl syndrome genes, suggesting the importance of sensory cilia in this plasticity (Torayama et al., 2007). Such olfactory enhancement was not specific to butanone: animals also showed olfactory enhancement after conditioning with food and AWC-sensed odorants Isoamyl alcohol (Ia) or 2,3-pentanedione (Pd) (Ichijo et al., EAWM 2006). To elucidate the molecular mechanism of olfactory enhancement, we used a forward genetic approach. We screened 7000 genomes and isolated seven candidate mutants for the Ia olfactory enhancement (ut321 - ut327). We analyzed the phenotypes of these mutants and other mutants that were previously isolated as Bu olfactory enhancement defective mutants (ut301-ut315). Among these mutants, ut311 showed nearly normal chemotaxis to AWC-sensed odorants (Bu, Ia and Pd), and defects in Bu- and Pd-enhancement. To assess whether ut311 has defects in asymmetrical differentiation of AWC neurons and sensory cilia formation, we checked the expression pattern of str-2::gfp and the dye filling of sensory neurons with the fluorescence dye DiI in ut311. ut311 showed neither the 2 AWCOFF phenotype nor dye-filling defects. This suggests that the Bu enhancement defects in ut311 is due to a reason independent of the olrn-1 and olrn-2. To clone the responsible gene for ut311, we mapped ut311 to the 2.5 map unit interval on the chromosome V by snip-SNPs. In this meeting, we will report on the progress of detailed mapping and cloning of ut311.

Diya Banerjee et al. (2004) East Coast Worm Meeting "Molecular connections between developmental timing and circadian timing: The C. elegans homolog of the circadian gene doubletime regulates post-embryonic developmental timing"

Alvin Kwok, Shin-Yi Lin, Diya Banerjee, Frank Slack

[East Coast Worm Meeting, 2004]

Spatial patterning during animal development is genetically controlled by genes such as the Hox cluster. Similarly, the temporal aspect of developmental patterning is under the genetic control of heterochronic genes. The most extensively investigated heterochronic pathway defines a somatic clock that controls the timing of cell fate determination during C. elegans post-embryonic development. Although a number of key heterochronic genes have been identified, the molecular mechanisms that underlie temporal control of cell division and proliferation remain elusive. The heterochronic gene lin-42 is the C. elegans homolog of Drosophila and mammalian period , key regulators of circadian rhythms, which specify behavior and physiology over a 24 hour day/night cycle. lin-42 thus defines a molecular link between two different types of biological clocks: developmetal timing and circadian timing. In addition to lin-42 , the C. elegans genome contains homologs of a number of core circadian clock genes. We have investigated the possible function of these homologs in control of developmental timing by using RNA interference (RNAi) to knock-down gene expression. We describe the identification and characterization of two genes that interact with the developmental clock: kin-20 (casein kinase I e /CKI e ) and kin-19 (casein kinase I a/ CK Ia ), which are C. elegans homologs of Drosophila doubletime . kin-19 / CK Ia and kin-20/CKI e antagonistically regulate cell fate decisions during late larval development. kin-19 is downstream of and negatively regulated by kin-20 , lin-42, lin-41 and hbl-1, which either work together or in parallel. We have also found that the homologs to the Drosophila clock genes timeless , casein kinase II a , protein phosphatase 2A, vrille, and pdp1 appear to function in the developmental clock. Our results, showing function of multiple circadian gene homologs in the developmental clock, suggest that circadian and developmental timing pathways may utilize conserved mechanisms of temporal regulation. Our work also suggests that one or more of the heterochronic genes already identified, including CK Ia and microRNAs, may play a role in circadian timing. Furthermore, our findings point to circadian genes being regulators of cell fate timing, and support reports that circadian genes can function as oncogenes in mammals.

Susan Carlson et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Iowa State University ADVANCE Program's Collaborative Transformation: Advancing Women Faculty in STEM Fields"

Susan Carlson, Jo Anne Powell-Coffman, Lisa Larson, Diane Debinski, Sharon Bird, Kristen Constant, Jan Thompson, Charles Glatz, Bonnie Bowen, Carla Fehr, Fred Janzen, Sandra Gahn, Florence Hamrick

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

The goal of the NSF-funded ISU ADVANCE program is to investigate the effectiveness of a multilevel collaborative effort to produce institutional transformation that results in the full participation of women faculty in science, technology, engineering and math fields in the university. Our approach focuses on transforming departmental cultures, practices, and structures, as well as university policies. Here, we summarize the findings of the Collaborative Transformation Project with the 3 departments that participated in the first round (Departments of Genetics, Development, and Cell Biology; Ecology, Evolution, and Organismal Biology; and Material Science and Engineering) (Bird and Hamrick 2008). A 3-step process for departmental transformation includes (1) focus groups to discuss aspects of department culture, practice and structure, (2) needs assessment meetings tailored to meet the needs of individual departments, and (3) collaborative problem solving sessions involving department faculty and ADVANCE program leaders. The findings include 6 major issues common to all 3 departments. Some issues do not specifically address gender or race, but these issues can have a disproportionate effect on underrepresented groups. Each of the 3 focal departments has developed strategies for change that address the issues identified in their departments. Examples of these strategies will be presented. Bird, Sharon R. and Florence A. Hamrick. 2008. ISU ADVANCE Collaborative Transformation Project: First Round Focal Department Synthesis Report. Ames, IA: Iowa State University ADVANCE Program. http://www.advance.iastate.edu

Mariam Alexander et al. (2005) International Worm Meeting "A Screen For Genes Required for Muscle Arm Development."

Mariam Alexander, Peter J Roy

[International Worm Meeting, 2005]

In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nerve cord to form the neuromuscular junction. Our lab and others have provided evidence that there is likely two distinct phases of muscle arm development; embryonic muscle arms arise by a passive mechanism (CR Norris, IA Bazykina, DH Hall, EM Hedgecock, Worm Breeder's Gazette 14(5): 37), while larval arms actively seek out motor axons (Dixon and Roy, submitted). Although the mechanism by which larval muscle arms reach motor axons is not clear, one model is that a chemotropic cue secreted by motor axons guide muscle arm migration to the nerve cord. To better understand muscle arm development, I performed a forward genetic screen to isolate mutants with muscle arm extension defects. I have screened 36,000 haploid genomes and isolated five mutants with highly penetrant and expressive muscle arm defects. Complementation tests show that tr50 is allelic to unc-60, a gene encoding the C. elegans ADF/cofilin orthologue, while tr51 is allelic to unc-22, which encodes twitchin. The remaining mutants do not map near other genes known to be required for larval muscle arm extension, including lev-11 and unc-54 (Dixon and Roy, submitted), suggesting that these genes may provide novel insight into the development of muscle arms. Surprisingly, one of mutants that has a dramatic reduction in the number of muscle arms (tr64) is not uncoordinated. This suggests that proper muscle arm development is not necessary for wild type movement. I am currently refining the map positions of the genes corresponding to the remaining mutations.

Tobias R Zahn et al. (2001) International C. elegans Meeting "Movement of IDA-1::GFP-tagged vesicles in C. elegans nerve processes"

Margaret A MacMorris, Tobias R Zahn, Joseph K Angleson, John C Hutton

[International C. elegans Meeting, 2001]

In mammals, the closely related protein tyrosine phosphatase-like type 1 transmembrane proteins ICA512 (IA-2) and phogrin (IA-2beta) are restricted to neuroendocrine tissues including brain, pituitary and pancreatic beta-cells, where they are localized to dense core vesicles (DCVs) of the regulated secretory pathway. Phogrin has been shown to undergo Ca 2+ -dependent phosporylation in response to secretagogues and ICA512 to interact with betaIV spectrin and beta2-syntrophin. These findings together with the restricted expression pattern, intracellular localization and the strong conservation between species suggest a role of these proteins in regulated secretion. They are of additional interest as major targets of autoimmunity in type 1 diabetes. We recently reported the existence of a single ortholog of phogrin and ICA512 in C. elegans , IDA-1 ( i slet cell d iabetic a utoantigen). Using GFP reporter constructs, we localized ida-1 gene expression to a subset of about 30 mostly sensory neurons and the vulval neurosecretory-like uv1 cells. Expression was sex-specific and included the hermaphrodite-specific vulval motoneurons and many male-specific neurons. In contrast to synaptic vesicles (SVs) in C. elegans , very little is known about the larger ‘dark-cored’ vesicles which have been seen in the electron-microscopic analysis of the worm’s nervous system. We hypothesize that IDA-1 in C. elegans like ICA512 and phogrin in mammals is specific to DCVs which are distinct from synaptic vesicles and secrete neuropeptides or hormones in a regulated fashion. SNB-1::GFP-tagged SVs have proven very useful tools, and more recently Scholey and colleagues showed that in live C. elegans animals movement of GFP-tagged molecular rafts in amphid dendrites can be observed with a fluorescent light microscope. We generated transgenic C. elegans strains expressing an IDA-1::GFP fusion protein, showing punctate staining within neuronal cell bodies and their processes. The distribution of IDA-1::GFP in nerve processes is reminiscent of the bouton-like staining of SV markers. In contrast to the reported subcellular distribution of SNB-1::GFP, IDA-1::GFP strongly stains cell bodies though is excluded from the nucleus consistent with DCV biogenesis and recycling at the trans-Golgi network and endosomal compartments. Small IDA-1::GFP punctae shuttle between larger immobile GFP accumulations distributed periodically along axons and dendrites. Our initial analysis of vesicle movement in PHC dendrites reveals rates of transport of ~ 1.2 microm/s in retrograde and more variable ~ 2.6 microm/s in anterograde direction indicative of fast microtubule-based transport. We generated IDA-1::GFP transgenic mutants defective in motor proteins including unc-104 , osm-3 , and che-3 to directly test a requirement of IDA-1::GFP-tagged vesicle transport by those molecular motors. In an attempt to define similarities and differences between C. elegans SVs and the IDA-1::GFP-tagged structures, we introduced the IDA-1::GFP transgene into a variety of mutant backgrounds that are known to severely disrupt the localization of synaptic vesicles and proper formation of synapses, such as unc-11 , syd-2 , and rpm-1 . The observed subcellular localization and movements of IDA-1::GFP are consistent with a vesicular localization of IDA-1 and indicate that the nematode will be a useful model to study the molecular mechanisms involved in DCV transport and recycling.

Vidal-Gadea A G et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Biogenic amines mediate transition between crawling and swimming in C. elegans"

Brauner M, Gottschalk A, Erbguth K, Kressin L, Vidal-Gadea A G, Topper S, Young L, Pierce-Shimomura J T

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

A wide variety of animals must quickly adjust their pattern of locomotion to successfully navigate through different environmental niches. Selection and execution of the appropriate locomotory pattern is therefore paramount to survival. Although C. elegans is capable of performing many adaptive behaviors, it has been controversial whether forward crawling and swimming represent distinct gait-like forms of locomotion or the modulation of a single form of locomotion [1-3]. Biogenic amines have been shown to mediate the transition between gait-like forms of locomotion across taxa as diverse as sea slugs, leeches, lampreys and humans. We previously reported that C. elegans crawls and swims with distinct kinematics and different patterns of muscle activity [2]. We now combine quantitative behavioral analysis, optogenetic tools and neuronal ablation to show that C. elegans uses biogenic amines to switch between crawling and swimming in a gait-like manner. As in other invertebrates, we find that serotonin mediates the smooth transition from crawling to swimming in C. elegans. Serotonin is further required to inhibit motor behaviors (e.g. foraging and pharyngeal pumping) during swimming that normally only accompany crawling. Mirroring the role of dopamine in other invertebrates, C. elegans uses dopamine to successfully initiate and maintain crawling when emerging from liquid. Over 600 million years of separate evolution notwithstanding, the highly conserved role played by biogenic amines such as dopamine and serotonin across taxa attests to how vital their function is to adaptive strategies for locomotion. Korta J, Clark DA, Gabel CV, Mahadevan L, Samuel AD. J. Exp. Bio. 2007 210:2383-9.Pierce-Shimomura JT, Chen BL, Mun JJ, Ho R, Sarkis R, McIntire SL. PNAS. 2008 105:20982-7.Berri S, Boyle JH, Tassieri M, Hope IA, Cohen N. HSFP J. 2009 3:186-93.