Bettinger JC et al. (1996) Midwest Worm Meeting "STAGE-SPECIFIC CONTROL OF LIN-29 ACCUMULATION"

Bettinger JC, Rougvie AE

[Midwest Worm Meeting, 1996]

Lateral hypodermal "seam" cells terminally differentiate during the L4-to-adult molt, in a process we term the larval-to-adult (L/A) switch. During the L/A switch the seam cells fuse together and contribute to the synthesis of an adult-type cuticle. lin-29 is required for the execution of the L/A switch. In lin-29 mutant animals, the seam cells fail to terminally differentiate; instead they reiterate their larval program of cell division and larval cuticle synthesis, and the animal undergoes supernumerary molts. We have shown that lin-29 protein accumulates in the nuclei of seam cells, consistent with its predicted role as a zinc finger transcription factor. The earliest detectable LIN-29 accumulation in seam cell nuclei is during the last larval stage (L4), following the final seam cell division, which occurs during the L3-to-L4 molt. We find that LIN-29 accumulates in all hypodermal nuclei during the L4 stage. The time of LIN-29 appearance in the hypodermis is controlled by the heterochronic gene pathway: LIN-29 accumulates in the hypodermis abnormally early, during the third larval stage, in loss-of-function lin-14, lin-28, and lin-42 mutants and fails to accumulate in hypodermis of lin-4 mutants. LIN-29 also accumulates stage-specifically in the nuclei of a variety of non-hypodermal cells during development. For example, during the L3 stage LIN-29 accumulates in many cells of the somatic gonad including the anchor cell, the distal tip cells, the 22 cells that comprise the vulva, and the 16 sex muscle cells. LIN-29 also accumulates in pharyngeal nuclei and a subset of neurons in the head. Intriguingly, LIN-29 accumulation is dependent upon the upstream heterochronic genes in some, but not all, of these non-hypodermal cells. We want to understand the role, if any, of lin-29 in these other cell types. We have begun addressing the role that lin-29 plays in vulval development, and we suspect that its action may not be restricted to the hypodermis. To test this, we are developing the reagents required for mosaic analysis.

Bettinger JC et al. (2000) West Coast Worm Meeting "State-dependent learning in C. elegans."

McIntire SL, Bettinger JC

[West Coast Worm Meeting, 2000]

The execution of learned behaviors may be triggered by contextual information, consisting of environmental cues or the internal state of the organism. State-dependent learning refers to the ability of an organism to more effectively execute learned behaviors if the organism experiences internal contextual influences similar to those experienced when the learning occurred. Such contexts can be pharmacologically manipulated by treating with cholinergic compounds, opiates, cocaine and amphetamines, and with neurodepressants such as ethanol and barbituates1. Worms become intoxicated by ethanol in a manner similar to that of most other organisms tested (see abstracts by A. Davies and H. Kim, this meeting). We have sought to study the mechanisms of ethanol-induced state-dependent learning in worms. Olfactory adaptation in C. elegans is a decrease in the chemotaxis response to an odorant as a result of prior exposure to the odorant2. We demonstrate a form of state-dependent learning in worms by pairing olfactory adaptation and ethanol administration. Ethanol does not interfere with olfactory adaptation, however, worms exposed to an odorant while being treated with ethanol will only show subsequent adaptation to the odorant if ethanol is again administered during chemotaxis testing. If the odorant is presented without ethanol during testing, the animals behave as nave animals and therefore fail to alter their behavior based on their previous experience or prior exposure to the odorant. Further, we demonstrate that the state-dependent effects of ethanol require normal dopaminergic function. The dopamine-defective cat-14 and cat-25 mutants are able to adapt to volatile odorants, however, they do not show state-dependency when they are adapted to volatile odorants while intoxicated by ethanol. These results suggest that C. elegans is capable of a form of learning and indicate a conserved role of dopamine in the modulation of behavioral responses to ethanol. 1 Izquierdo. in Neurobiology of Learning and Memory (eds Lynch, McGaugh, and Wienberger) 333 (Guilford, New York, 1984). 2 Shulz, Sosnik, Ego, Haidarliu and Ahissar (2000) Nature 403, 549, and references therein. 3 Colbert and Bargmann (1995) Neuron 14, 803. 4 Duerr et al. (1999) J. Neuroscience 19, 72. 5 Lints and Emmons (1999) Development 126, 5819.

Bettinger JC et al. (1993) International C. elegans Meeting "lin-29 control of seam cell differentiation."

Bettinger JC, Mansanares K, Rougvie AE

[International C. elegans Meeting, 1993]

Bettinger JC et al. (1995) International C. elegans Meeting "TEMPORAL REGULATION OF LIN-29 IN THE HYPODERMIS AND VULVA"

Rougvie AE, Lee K, Bettinger JC

[International C. elegans Meeting, 1995]

lin-29 activity is required for the terminal differentiation of the lateral hypodermal seam cells at the L4 molt. The seam cells of lin-29 mutants fail to execute this terminal differentiation program; they continue to divide, fail to fuse, and continue to synthesize a larval-type cuticle. Antibody staining has shown that LIN-29 accumulates stage-specifically in the seam cell nuclei and in the nuclei of hyp7 and other hypodermal syncytia of wild-type L4 animals. In addition to hypodermal defects, lin-29 hermaphrodites have a vulval morphogenesis defect, first apparent during the eversion process at the L4 molt. lin-29 adults have ventral protrusions, cannot mate and are 100% Egl. The cause of this defect is unknown. Since lin-29 mutants exhibit no vulval lineage defects but fail to make adult cuticle, it has been suggested that some characteristic of adult cuticle is required for proper vulval morphogenesis [1]. We now have evidence that lin-29 may also play a more direct role in vulval morphogenesis. We have found that LIN-29 accumulates in the anchor cell nucleus very early in the L3 stage, and then in the nuclei of the daughters of P5.p, P6.p, and P7.p, the cells induced to 1o and 2o vulval cell fates. Intriguingly, when the anchor cell is ablated in wild-type worms after vulval induction, eversion usually fails and most of these worms develop ventral protrusions like those seen in lin-29 mutants [2]. Further, we have identified a novel lin-29 allele, ga94, (isolated by D. Eisenmann) that makes normal adult cuticle at the L4 molt, but retains the vulval defect. This allele separates the hypodermal defect from the vulval defect, suggesting that LIN-29 protein is required in the vulva for eversion even in the presence of a wild-type adult cuticle.

Bettinger JC et al. (1997) International C. elegans Meeting "SEAM AND NON-HYPODERMAL REQUIREMENTS FOR lin-29 IN EGG-LAYING"

Bettinger JC, Rougvie AE

[International C. elegans Meeting, 1997]

The lateral hypodermal seam cells terminally differentiate during the L4 molt in a process that requires the activity of the transcription factor LIN-29. We showed previously that LIN-29 accumulates in seam cell nuclei during the L4 stage, and its presence correlates with the timing of seam cell terminal differentiation in various heterochronic mutants1. lin-29 mutants have another phenotype: despite proper vulval cell divisions, the animals develop a large ventral protrusion (a "blip") after the L4 stage and are completely Egl. We showed that LIN-29 accumulates in the nuclei of the anchor cell, distal tip cells, and the sex muscles1. We have now used mosaic analysis to ask whether or not there is a requirement for lin-29 in these cells or in the seam cells for vulval formation and egg-laying. We have found that lin-29 acts cell autonomously in the seam to control terminal differentiation: single seam cells terminally differentiate and synthesize adult alae in an otherwise mutant seam, and single mutant seam cells fail to terminally differentiate in an otherwise wild-type seam. In addition, we have found that the presence of lin-29 (+) in a seam cell is insufficient to direct its fusion to a mutant neighbor. We have found an absolute requirement for lin-29 in at least 3 of the 4 seam cells directly overlying the vulva (decendents of V3L and V3R) for the vulva to form properly. These seam cells are the sites of attachment of the somatic gonad uterine seam cell (utse)2, which are visible on lin-29(+) but not lin-29(-) seam cells by DIC microscopy. Interestingly, we have uncovered a role for lin-29 in egg-laying in animals with wild-type seams. We traced this requirement to MS.a descendants, and of these, we have eliminated the anchor cell and distal tip cell. We are investigating a possible role for lin-29 in the sex muscles. 1Bettinger, Lee, and Rougvie (1996). Development 122, 2517-2527 2Newman, White, and Sternberg (1996). Development 122, 3617-3626

JC Bettinger et al. (1999) International C. elegans Meeting "Behavioral Effects of Exposure to Ethanol"

SL McIntire, JC Bettinger

[International C. elegans Meeting, 1999]

Exposure to ethanol interferes with complex behaviors in many model systems, but it has been difficult to correlate effects of ethanol on behavior with observations of its effects on specific molecular targets. Recently, studies of Drosophila demonstrated a link between ethanol sensitivity and a learning pathway 1 : A screen for mutations that cause flies to be hypersensitive to the effects of ethanol on postural control yielded an allele of amnesiac , a putative neuropeptide known to be involved in learning 2 . After exposure to ethanol, C. elegans display uncoordinated movement (characterized by a decreased amplitude of the sine waveform and lethargy), and decreased rate of pumping and egg-laying (SLM, unpublished observations). After several hours of exposure, worms develop an acute tolerance to ethanol, and recover to resemble untreated controls. We are interested in determining whether or not exposure to and development of tolerance to ethanol alter any of the more complex behaviors exhibited by worms, including chemotaxis. Our preliminary experiments on the effect of ethanol on chemotaxis suggest that brief or prolonged exposure to moderate concentrations of ethanol (100-200 mM) does not prevent chemotaxis to the volatile odorant benzaldehyde. With extended exposure, worms become insensitive to chemoattractants in a process termed adaptation. Worms that have adapted to a particular chemoattractant will not climb a gradient of that chemoattractant 3 . Given that worms are able to chemotax in the presence of ethanol, we can test the effect of ethanol on adaptation. We are determining whether or not exposure to low-to-moderate concentrations of ethanol interferes with the process of adaptation to benzaldehyde and other odorants. 1 Moore, M.S.; DeZazzo, J.; Luk, A.Y.; Tully, T.; Singh, C.M. and Heberlein, U. (1998). Cell 93: 997-1007. 2 Feany, M.B. and Quinn, W.G. (1995). Science 268: 869-873. 3 Colbert, H.A. and Bargmann, C.I. (1995). Neuron 14: 803-812.

Raabe, R et al. (2013) International Worm Meeting "A Potential Role for Palmitoylation in the Acute Response to Ethanol."

Bettinger, J.C., Davies, A.G., Raabe, R

[International Worm Meeting, 2013]

An individual's naive level of response (LR) to ethanol is predictive of their lifetime likelihood to abuse alcohol, and this initial LR is genetically influenced. This suggests that the genes that are responsible for LR may also be central to the development of abuse disorders, making them particularly attractive targets for research. Our laboratory uses C. elegans to investigate the genetic influences on responses to acute ethanol exposure. We have focused on two components of ethanol response: initial sensitivity, measured as the change in mean velocity over the first ten minutes of treatment, and the development of acute functional tolerance (AFT), measured as an increase in the mean velocity at thirty versus ten minutes of exposure. We have previously found that SLO-1, the large-conductance potassium (BK) channel, is likely to be a direct target of ethanol. slo-1 mutants are profoundly resistant to ethanol, and are defective in AFT. Recently, we used a forward genetic screen for mutations that disrupt the ability of worms to develop AFT to identify ctbp-1, a transcriptional repressor that affects AFT through its negative regulation of a triacylglyceride lipase, lips-7. lips-7 mutants have the opposite phenotype to ctbp-1; enhanced development of AFT. Preliminary analysis of the lipid profile of the lips-7 mutant versus wild type has suggested that lips-7 mutants may have an increase in palmitic acid. This led us to ask if and how palmitic acid may be involved in AFT. Palmitoylation, the reversible addition of a lipid to a protein, can alter ion channel activity directly and indirectly. Mouse mSLO-1 has multiple palmitoylation sites that affect localization, internalization, activity, and trafficking. This suggests one model for AFT in which the effects of ethanol on targets such as SLO-1 are modulated by palmitoylation. We have tested the ethanol responses of a combination of RNAi and loss-of-function mutations in various genes related to the synthesis, degradation, or use of palmitic acid. We have identified several suspected palmitoylation-related genes that alter both initial sensitivity and AFT. Our goal is to better understand how the palmitoylation machinery modifies acute responses to ethanol.

Mathies, L.D. et al. (2015) International Worm Meeting "SWI/SNF chromatin remodeling regulates alcohol-response behaviors."

Blackwell, G.G., Davies, A.G., Bettinger, J.C., Mathies, L.D.

[International Worm Meeting, 2015]

Alcohol abuse is a significant social problem for which there are few treatments. One reason for the difficulty in generating effective pharmacological interventions is that we have only a limited understanding of the direct and indirect physiological targets of ethanol. We use C. elegans to study the mechanisms underlying acute responses to ethanol. Here we use locomotion as our behavioral readout and we measure two different responses to ethanol: initial sensitivity is the reduction in locomotion speed upon initial exposure to ethanol and acute functional tolerance (AFT) is the increase in locomotion speed that develops during the course of an exposure. Together, initial sensitivity and development of AFT contribute to the initial "level of response" that in humans predicts susceptibility to alcohol use disorders.We have identified roles for the SWI/SNF chromatin remodeling complex in regulating the behavioral response to alcohol in C. elegans. We found that SWI/SNF components are required in adults for the normal behavioral response to ethanol, and that different SWI/SNF complexes regulate different aspects of the acute response to ethanol (1). Specifically, BAF (Brahma-associated factors) subunits are required for wild-type levels of initial sensitivity and PBAF (Polybromo BAF) subunits are required for wild-type rates of AFT development. We further showed that the PBAF subunits SWSN-9 and SWSN-7 are required in neurons and muscle for the development of AFT. SWI/SNF is a multi-protein complex that alters the interaction between nucleosomes and DNA and thereby influences gene expression. We predict that the SWI/SNF complex regulates the expression of genes that determine the acute level of sensitivity and rate of AFT development. Further, as a potential epigenetic regulator, SWI/SNF is in a good position to modulate gene expression changes that occur as a response to ethanol exposure. We are currently testing SWI/SNF genes for roles in the behavioral response to extended or repeated ethanol exposure. Our ultimate goal is to determine how SWI/SNF regulates chromatin structure to modify behavioral responses to ethanol.(1) Mathies et al., (2015) Proc Natl Acad Sci U S A. 112(10):3032-7.

Mathies, L.D. et al. (2019) International Worm Meeting "Towards identifying genes regulating multipotency and differentiation in the SGP/hmc cell fate decision."

Lopez-Alvillar, K.J., Kim, A.C., Davies, A.G., Mathies, L.D., Bettinger, J.C.

[International Worm Meeting, 2019]

We use progenitors of the C. elegans reproductive system as a model for defining the genetic determinants of multipotency. The two somatic gonadal precursors (SGPs) are multipotent progenitors that generate all somatic tissues of the reproductive system. Each SGP is the product of a cell division that produces one SGP and one differentiated cell, the head mesodermal cell (hmc). Therefore, in this single cell division the potential to generate all of the somatic gonadal types is differentially segregated into one daughter cell. We have used fluorescence-activated cell sorting (FACS) to isolate SGPs and hmcs from the same worms and performed RNA sequencing to identify genes that are differentially expressed in the two cell types. We found a surprisingly large number of differentially expressed genes (~6000) when comparing these two sister cells. GO term enrichment analysis of our dataset provided interesting insights. Genes with higher expression in SGPs are enriched for GO terms related to transcription and translation, as might be expected for a multipotent progenitor cell preparing to undergo multiple cell divisions; among these are 175 of the 934 C. elegans transcription factors. Interestingly, genes with higher expression in the hmc are enriched for GO terms related to neuronal function, such as "synaptic vesicle exocytosis" and "calcium mediated signaling". To date, the function of the hmc has not been determined, and our data strongly suggest that it may have neuronal signaling properties, an idea that is consistent with its neuron-like cellular morphology. Our ultimate goal is to identify determinants of multipotency in SGPs, as well as genes that promote the terminal differentiation of the hmcs. To this end, we have begun to screen the SGP-biased transcription factors for roles in the SGP/hmc cell fate decision using RNA-mediated interference to knock down gene function. We score animals at the first larval stage and assess cell fates using a strain containing markers for SGPs (red fluorescence) and hmcs (green fluorescence). We will report on our progress at the meeting.

Hawkins, E.G. et al. (2015) International Worm Meeting "Tolerance to a cholinergic activating effect of alcohol requires a specific function of the EAT-6 Na+/K+-ATPase."

Kondo, L.M., Davies, A.G., Hawkins, E.G., Martin, I., Bettinger, J.C.

[International Worm Meeting, 2015]

Alcohol (ethanol) produces activating and depressing behavioral effects in humans. We have investigated the basis for an activating effect of ethanol in C. elegans that produces body hypercontraction. Acute ethanol exposure induces a shortening of the animal that requires normal acetylcholine levels and a functional UNC-63 acetylcholine receptor (AChR) subunit. Elimination of the two receptors currently known to contain the UNC-63 subunit, the ACR-2R and the levamisole-sensitive AChR, do not alter sensitivity to ethanol. This suggests that UNC-63 may be acting in an unidentified receptor to mediate this effect of ethanol. AChRs in mammals have been implicated in mediating activating effects of ethanol, and genetic variation in AChR subunit-encoding genes has been associated with the development of alcoholism in humans. We are pursuing the tissue-specific requirements for unc-63 expression for its role in this ethanol effect.The hypercontracted animals show a recovery of pre-treatment body length despite continuous exposure to ethanol (and a gradual increase in internal ethanol concentration). This observation suggests that a physiological tolerance is developing to the drug. We identified the eat-6(eg200) mutation as a mutant that fails to develop tolerance to this ethanol effect. An examination of several existing alleles of eat-6 found that mutants with strong effects on pharyngeal pumping did not alter the response to ethanol-induced hypercontraction, whereas the ad601 allele, which resembles the eg200 mutant in having weak effects on pumping but strong effects on aldicarb sensitivity, does alter the maintenance of tolerance to ethanol. These data suggest that eg200 and ad601 alter a specific function of the Na+/K+-ATPase that is important for the development and/or maintenance of tolerance to ethanol for this cholinergic activating effect of ethanol. One possibility that we are testing, is that EAT-6 has a structural relationship with an ethanol-sensitive AChR, and that the mutations eg200 and ad601 prevent desensitization or compensatory trafficking of that receptor.