Mathies, LD et al. (2017) International Worm Meeting "SWI/SNF chromatin remodeling complexes regulate the SGP/hmc cell fate decision."

Mathies, LD, Davies, AG, Bettinger, JC

[International Worm Meeting, 2017]

The two somatic gonadal progenitors (SGPs) of Caenorhabditis elegans are multipotent progenitor cells that give rise to all somatic tissues of the reproductive system, including uterus, sheath, spermatheca, distal tip cells and the anchor cell. The SGP sister cells are head mesodermal cells (hmcs); one undergoes programmed cell death and the other migrates anteriorly and terminally differentiates shortly after it is born. We use this cell fate decision, between SGP and hmc, as a simple and genetically tractable model to understand the determinants of multipotency. We have used forward and reverse genetic approaches to identify hnd-1/dHand and three subunits of the SWI/SNF chromatin remodeling complex (swsn-1, swsn-4, and pbrm-1) as regulators of the SGP/hmc cell fate decision. Loss-of-function mutants in any of these genes result in three phenotypes: 1) fewer than the normal number of SGPs, 2) SGPs that sometimes express a marker that is characteristic of the hmc, and 3) the anterior gonadal arm is missing more frequently than the posterior gonadal arm. We suggest that all three of these phenotypes result from SGPs that are partially transformed into hmcs in the mutants. HND-1 is a transcription factor and SWI/SNF chromatin remodeling complexes influence gene expression by altering chromatin structure. Therefore, a simple model is that SWI/SNF acts by facilitating the binding of HND-1 to its target genes to regulate gene expression in SGPs. We are using a combination of fluorescence activated cell sorting and RNA sequencing to identify genes that are differentially expressed in SGPs and hmcs. Among these differentially expressed genes, we expect to find downstream targets of HND-1 and SWI/SNF, including the determinants of multipotency in SGPs. SWI/SNF chromatin remodeling complexes are important for the proliferation of mammalian multipotent progenitors. The identification of downstream targets of SWI/SNF in the SGP/hmc cell fate decision may therefore uncover conserved molecular mechanisms regulating multipotency in diverse species.

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.

Xu, Ningyi et al. (2011) International Worm Meeting "Local triglyceride synthesis by DGAT-2 promotes lipid droplet expansion."

McKinney, Sean, Cole, Ronald, Zhang, Shaobing, Xu, Ningyi, Guo, Fengli, Mak, HoYi

[International Worm Meeting, 2011]

Lipid droplets (LDs) are evolutionarily conserved organelles for cellular fat storage. We have previously shown that a loss of DAF-22 function, which attenuates peroxisomal fatty acid beta-oxidation, causes LD expansion and an increase in triglyceride levels. In order to identify genes that are required for LD expansion, we performed a genetic suppressor screen in the daf-22 mutant background and recovered multiple alleles that fell into at least 5 complementation groups. We report here the molecular cloning of dgat-2, which encodes a conserved diacylglycerol acyltransferase, the terminal enzyme for triglyceride synthesis. Loss of dgat-2 function inhibits LD expansion and reduced triglyceride levels of daf-22 mutant animals while over-expression of DGAT-2 is sufficient to promote LD expansion in wild-type animals. Photo-bleaching demonstrates that DGAT-2 is a LD resident protein. Electron microscopy reveals that DGAT-2 clusters at discreet foci on the LD surface. We engineered a mutant DGAT-2 that was tethered to the ER. This mutant DGAT-2 failed to support LD expansion, indicating that LD localization of DGAT-2 is necessary for its proper function. Our results demonstrate that local synthesis and deposition of triglyceride is critical for LD expansion. We are currently searching for proteins that act in concert with DGAT-2 using genetic and proteomic approaches.

Petcherski AG et al. (1998) Midwest Worm Meeting "Ce-PABP1, A POLY(A)-BINDING PROTEIN FROM C.elegans IS REQUIRED FOR GERM CELL PROLIFERATION AND DIFFERENTIATION."

Kimble JE, Lambie EJ, Mathies LD, Petcherski AG

[Midwest Worm Meeting, 1998]

How are tissues controlled to proliferate and differentiate correctly? In an ongoing project to address this question, we have focused on genes that are required for the regulation of proliferation and differentiation. Our tissue of choice has been the germ line of the nematode Caenorhabditis elegans. Three independently isolated mutations of the glp-2 gene lead to severe defects of both proliferation and differentiation of the germ line. All glp-2 mutants make 4-10 germ cells rather than the wild-type number of about 1500. Furthermore, the glp-2 mutant germ cells fail to differentiate as either sperm or oocytes. By genetic criteria, the glp-2 mutations appear to be severe loss-of-function alleles. No other major defects are observed in glp-2 mutants, although a vulval defect can be observed at low frequency (about 3/100 animals). We have cloned glp-2 by a combination of mutant rescue and RNA interference techniques. The glp-2 locus encodes a C. elegans homologue of poly(A)-binding proteins (PABPs): Ce-PABP1. PABPs bind to poly(A) tails of eukaryotic messages and are thought to be involved in regulation of translational initiation and mRNA turnover. Ce-PABP1 shows 53 % amino-acid identity with human PABP1. The most homologous regions encompass the four RRM domains (65 % aa identity) and a C-terminal region (63 % aa identity). Ce-PABP1 has 49 % overall amino-acid identity with S. cerevisiae Pab1p. Mutations in Ce-PABP1 were identified in two alleles of glp-2. One glp-2 mutation is associated with an 8 bp deletion early in the Ce-PABP1 coding sequence, the other is a nonsense mutation predicted to truncate the protein after the fourth RRM domain. To our knowledge, these are the first mutations in PABP in a genetically tractable metazoan. The C. elegans Genome Sequencing project has identified another PABP homologue, which we called Ce-PABP2. Preliminary RNA interference experiments show that while RNAi against Ce-PABP2 produces no phenotype, double RNAi against both Ce-PABP1 and Ce-PABP2 gives rise to embryonic lethality. We suggest that Ce-PABP1 is required specifically for germline development, and that Ce-PABP1 and Ce-PABP2 act redundantly in other tissues.

Markussen F-H et al. (2000) Midwest Worm Meeting "Establishing the Gonad Primordium"

Kimble JE, Blelloch RH, Henderson ST, Markussen F-H, Mathies LD

[Midwest Worm Meeting, 2000]

The gonad in all organisms contains both somatic and germline tissues that must be united to produce a functional organ. In C. elegans, the somatic and germline precursor cells coalesce during embryogenesis to form the gonad primordium. Specifically, the somatic gonadal precursors (Z1 and Z4) migrate toward and flank the germline precursors (Z2 and Z3). The 4-celled gonad primordium is arranged with two-fold rotational symmetry in both sexes. This symmetry is maintained throughout hermaphrodite development generating a two-armed gonad, but is broken in males to generate a single-armed gonad. We have set out to dissect the genetic controls of gonad primordium formation. Using genetic screens and RNAi, we have identified at least 4 genes that are required for proper assembly of the gonad primordium. In each of these mutants, the symmetry of the gonad primordium is disrupted resulting in aberrantly-shaped adult gonads. Thus, the proper organization of the gonad primordium is essential for establishing organ shape. In the simplest case, the gonad primordium of gnd-2 RNAi animals is frequently missing one of the two somatic precursor cells, giving rise to an adult gonad with a single arm. gnd-1 and gnd-3 mutants can have asymmetric gonad primordia that lead to a more severely disorganized gonad and sterility. We have also examined previously existing mutants and find that mutations in the sex determination gene tra-1 result in an asymmetric gonad primordium. We will report our progress in analyzing these mutants phenotypically and molecularly.

Singh, Varsha et al. (2021) International Worm Meeting "Neuronal control of lipid metabolism by STR-2 G protein-coupled receptor promotes longevity in C. elegans"

Dixit, Anubhuti, Shashikanth, Meghana, Koushika, Sandhya, Singh, Varsha, Modi, Souvik, Sandhu, Anjali, Watts, Jennifer

[International Worm Meeting, 2021]

The G proteincoupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. We find that C. elegans longevity is regulated by a chemosensory GPCR STR2, expressed in AWC and ASI amphid sensory neurons. STR2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR2 regulates expression of delta9 desaturases, fat5, fat6 and fat7, and of diacylglycerol acyltransferase dgat2. Rescue of the STR2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat5, dgat2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wildtype levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.

Friedman LC et al. (2000) Midwest Worm Meeting "Sexual dimorphism of organ polarity in gonad formation"

Markussen F-H, Mathies LD, Kroll-Conner PL, Kimble JE, Siegfried KR, Friedman LC

[Midwest Worm Meeting, 2000]

When the L1 larva hatches, the gonad primordium consists of 4 cells arranged with two-fold rotational symmetry in both sexes. Two of these cells, Z1 and Z4, are somatic and their development determine the shape of the organ. During L1, cell divisions generate 8 somatic cells in hermaphrodite gonads and 6 in males. In the hermaphrodite, the symmetrical arrangement of somatic cells is maintained during L1. The symmetry is continued throughout later development, producing the two-armed gonad of hermaphrodites. In males, symmetry is broken during L1, and most somatic cells coalesce in an asymmetric cluster at the anterior of the developing gonad. This event underlies the asymmetrical one-armed gonad of males. We have set out to dissect the genetic and cellular basis of sexual dimorphism during early gonadogenesis. To this end, we have isolated two mutants with defects primarily in male gonadogenesis. One mutant, tentatively called man-1 for male asymmetric gonad defective, fails to break gonad symmetry in L1 males. Another similar mutant has defects in early male gonadogenesis. We will report our progress in characterizing these mutants and our efforts to describe cellular anatomy of the gonad primordium at a molecular level.

Markussen F-H et al. (1998) Midwest Worm Meeting "A screen for mutations affecting gonad polarity and somatic gonad formation in C. elegans"

Kimble JE, Friedman LC, Siegfried KR, Markussen F-H, Mathies LD, Kroll-Conner PL

[Midwest Worm Meeting, 1998]

We are studying the problem of organogenesis using the C. elegans gonad as a model. The gonad develops postembryonically from a primordium of two somatic and two germ line cells. The four cells are in an arrangement of two-fold rotational symmetry in either sex. In hermaphrodites this polarity is maintained throughout development generating a symmetrical two-armed gonad. In males, the symmetry is broken after the first round of cell division establishing a non-symmetrical single armed gonad. The somatic gonad develops in three phases. An initial round of cell divisions generates 12 somatic cells in hermaphrodites and 10 cells in males. Second, rearrangement of the somatic cells generates the somatic gonadal primordium. This phase consolidates the gonad, laying down a !blueprint! of the final arrangement of tissues. Lastly, a phase of cell division, differentiation, and morphogenesis shapes the final organ. We are doing an F2 screen for mutations disrupting the somatic gonad in either sex. From 6660 mutagenized haploid genomes we have isolated 58 independent mutations specific to somatic gonad development. Based on our current understanding of gonadogenesis, we are classifying the mutants in three broad classes. 1) Gon; gonad-less: absence of the L1 gonad primordium or arrested development at the L1 gonadal primordium stage. 2) Spf; somatic gonadal primordium formation defective. This includes polarity defects, early lineage defects, and defects in primordium consolidation. 3) Sgo; somatic gonad defective: the over-all polarity and organization of the gonad is normal indicating that a somatic gonadal primordium did form. Development fails during the last phase of gonadogenesis often resulting in malformed or absent tissues such as uterus and spermatheca. The number of complementation groups in each class is still unknown. Representative phenotypes of the different classes will be presented.

Tran, Tra My et al. (2021) International Worm Meeting "Influences on fat content, fat density and lipid droplets in C. elegans liquid culture"

Schonfelder, Gilbert, Oelgeschlager, Michael, Vogl, Silvia, Wagner, Aline, Menzel, Ralph, Tran, Tra My, Steinberg, Gianna

[International Worm Meeting, 2021]

C. elegans has become an emerging model system to investigate different toxicological endpoints. In particular, fundamental pathways regulating energy homeostasis are highly conserved between C. elegans and humans and thus, the worm provides an important model for the analysis of mechanisms leading to obesity. For example, the analysis of lipid homeostasis as well as fat accumulation in C. elegans can provide important insights into metabolic diseases and the activity of potential obesogenes. In addition, changes in lipid homeostasis by external factors are suspected to be transmitted to subsequent generations. Thus, lipid metabolism might also be a promising read-out in C. elegans to address inter- or transgenerational effects of xenobiotics. In a first step, we studied the influence of different food regimes on worm development, fat content and changes on the level of lipid droplets (LD), the worm's major form of fat storage, to generate "low body fat" or "high body fat" phenotypes. We exposed C. elegans to different OP50 concentrations in liquid culture for three days (L1 to adulthood) and measured worm sizes, triacylglyceride (TAG) contents, LD sizes and density as well as fatty acid (FA) patterns. After three days, worms fed with higher OP50 concentrations showed an increase in body size, higher TAG levels and significant increases in LD size and volume per microm3 in the anterior part of the intestine. Moreover, GC-MS analysis revealed significant differences in FA pattern of adult worms and their eggs, when comparing the "fat" and "lean" phenotypes. Notably, cyclic FA and polyunsaturated fatty acids (PUFA) were affected in a diet-dependent manner. Interestingly, orlistat, an anti-obesogenic drug, could also influence fat density and FA patterns in adult worms as well as eggs. Currently, we are testing effects of (anti-)obesogenes on fat content and FA composition and if those are transmitted to subsequent generations to assess the applicability of these read-outs for the analysis of inter- or transgenerational (adverse) effects.