Robledo, Jose et al. (2021) International Worm Meeting "Identification of transcriptional regulators impacted by a glucose-supplemented diet in C. elegans"

Burks, David, Robledo, Jose, Padilla, Pamela, Song, Luhua, Azad, Rajeev, Leguizamo, Isai

[International Worm Meeting, 2021]

A high glycemic diet (i.e. high sugar diet) is among the main factors contributing to severe and detrimental human health issues such as type 2 diabetes, cardiovascular diseases, and metabolic abnormalities. Underlying mechanisms involved in gene expression regulation and metabolism as a result of a high sugar diet continue to remain poorly understood. In our study, we use the genetic model system Caenorhabditis elegans to further investigate the impact a glucose-supplemented diet has on gene expression changes/regulation. Previously, we showed that a glucose-supplemented diet increases lipid composition, sensitivity to stress responses, and significantly alters the transcriptome profile (RNA-sequencing studies). We observed differential gene expression chromosomal maps and identified various clusters majorly found in Chromosome IV/V. Given that a link exists between chromosomal clustering and transcriptional regulation we further sought to identify gene regulators. The transcriptomic analysis revealed 140 gene expression regulators among the 2,370 differentially regulated genes as a result of a glucose-supplemented diet. Furthermore, we identified the acs-2 gene is a glucose-responsive gene, as shown by RNA-sequencing analysis, the acs-2::GFP reporter strain, and quantitative RT-PCR. Here, we developed a targeted RNA interference (RNAi) screen to identify the transcriptional regulators that respond to a glucose diet and impact the expression of acs-2::GFP. From our screen, we identified 20 regulators that significantly impact the expression of acs-2 transcript in a glucose diet dependent manner; 18 regulators increase and 2 regulators decrease acs-2::GFP expression. We also identified 13 gene regulators that increase acs-2::GFP expression in standard RNAi diet. Several of the regulators identified also show homology to human gene regulators suggesting conserved gene expression regulators between C. elegans and Humans. Together, these studies add to our understanding of the transcriptional and molecular changes that occur in response to a glucose-supplemented diet.

Nahar, Saifun et al. (2021) International Worm Meeting "Glucose-induced developmental delay is modulated by insulin signaling in C. elegans"

Padilla, Pamela, Song, Luhua, Ruiz, Manuel, Nahar, Saifun, Robledo, Jose

[International Worm Meeting, 2021]

A significant amount of research has shown that type 1 diabetes in children causes a delay in puberty. However, it is unclear how hyperglycemia contributes to this delay in puberty. Hyperglycemia, which is associated with diabetes, refers to elevated blood glucose levels. To model hyperglycemia at a molecular level, C. elegans are fed a glucose-supplemented diet and the physiological and molecular impacts are studied. We found that a glucose-supplemented diet induces a developmental delay in comparison to the control food (OP50 E. coli diet). The glucose induced developmental delay was also observed in animals fed the deltaPTS E. coli OP50 bacteria strain, which lacks a glucose transporter, indicating the phenotype is not due to metabolites produced by OP50. Developmental delay can be induced by environmental stress or genetic mutations. For example, mutations that impact ceramide biosynthesis, electron transport chain activity, ubiquinone biosynthesis, or insulin signaling (hyl-2(tm2031), isp-1(qm150), clk-1(e2519) and daf-2(e1370), respectively) lead to developmental delay. We tested if a glucose diet prolonged developmental progression in these mutants. A glucose-supplemented diet further exacerbates the developmental delay of the hyl-2(tm2031), isp-1(qm150) and clk-1(e2519) mutants but did not induce a developmental delay in the daf-2(e1370) animal. The resistance to glucose-induced developmental delay observed in the daf-2(e1370) animal is suppressed by daf-16(mu86). To further understand how glucose impacts insulin signaling and developmental progression we are using RNA sequencing and genetic analysis to identify the DAF-16 regulated genes that have a role in developmental progression. This could provide insight into how hyperglycemia and altered insulin signaling is linked to delayed puberty in children with type 1 diabetes.

King, Skylar D. et al. (2017) International Worm Meeting "Ceramides localize to granules within the P cell of C. elegans embryos."

Schmidt, Kevin, Padilla, Pamela A., King, Skylar D., Robledo, Jose

[International Worm Meeting, 2017]

Disruption of the ceramide biosynthesis impacts many biological processes including stress responses (e.g. O2 deprivation, apoptotic signals in the germline, mitochondrial surveillance system), lifespan, and fecundity. Ceramides, which are composed of sphingosine and a fatty acid, have a role in membrane structure (mitochondria, ER) and are important signaling molecules. In C. elegans the hyl-1, hyl-2, and lagr-1 genes code for ceramide biosynthetic enzymes. Simultaneous disruption of hyl-1 and hyl-2 results in lethality indicating that ceramide biosynthesis is an essential process. We used an anti-ceramide antibody (mAb15B4) to examine the localization pattern of ceramides in embryos. Surprisingly, the mAb15B4 recognized centrosomes (marked by anti-gamma tubulin) and granule-like perinuclear structures within the P cell. Ceramides, which impact programmed cell death in the germline and fecundity, are thought to colocalize with a mitochondrial marker (anti coxIV) in oocytes. However, to our knowledge there are no reports of the localization of ceramides to granule-like structures in the P cell. Thus, we tested the hypothesis that mAb15B4 recognizes ceramides that colocalize to the P granules. We used the MEX-3::GFP and PGL-1::GFP strains and determined there is overlap between the anti-GFP and mAb15B4 localization pattern. Additionally, the hyl-2 mutant shows a loss of mAb15B4 localization to the granule-like structures in the P cell, indicating that the mAb15B4 antibody is recognizing an antigen that is dependent on ceramide biosynthesis. The mAb15B4 localization to the granules in the P cell is restored in the fat-7(wa36);hyl-2(tm2031) double mutant demonstrating that alteration of lipid biosynthesis genes do impact granules recognized by the antibody. However, the P granules, as recognized by the K76 antibody (anti PGL-1) or MPMP2 antibody, remained intact in the hyl-2 mutant. These data suggest that ceramides localize to P granules but the alteration of ceramides do not alter PGL-1 localization. Given that ceramides are not only mitochondrial and ER structural membrane components but are also important signaling molecules we are investigating whether their presence in the P cell is for signaling.

Gallotta I et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Cell Specific Knock-down of Gene Function in Targeted C. elegans Neurons"

D'Orta M, Vitale M, Castro S, Esposito G, Hilliard M A, Di Schiavi E, Gallotta I

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

An important aspect in the study of gene function is the possibility to dissect the role exerted by a gene of interest selectively in specific cells or groups of cells. In C. elegans the available approaches are time consuming and largely restricted to non-essential genes for which mutants are available. We have developed a simple reverse genetics approach to reduce the function of specific genes in chosen C. elegans neurons (Esposito et al., 2007). By expressing sense and antisense RNA corresponding to a gene of interest under cell-specific promoters, we obtain an efficient, heritable and cell autonomous knock-down of the targeted gene function in the specific neurons. This strategy has now been used by several other labs, with a variety of genes and on different neurons (Harris et al. 2009; Yamada et al. 2009; Jose et al. 2009; Ezak et al. 2010). To optimize the efficiency of this approach we have tested the influence of genetic backgrounds that affect RNA interference. We show that in a rrf-3(pk1426) background silencing is more efficient. Although this result indicates the involvement of RNAi pathways in the silencing mechanism we have shown that there is no spreading of the silencing effect at least among amphidial chemosensory neurons and within the motorneurons of the ventral cord. We are now targeting chemosensory and mechanosensory neurons and two classes of motorneurons (GABAergic and HSN) using a variety of different promoters. We are testing the effect of silencing several essential genes on the development, morphology and survival of these neurons as well as on the behavior that they control.

Collins, Kevin M. et al. (2013) International Worm Meeting "Understanding how neurotransmitter signaling drives two-state activity of the C. elegans egg-laying behavior circuit."

Collins, Kevin M., Koelle, Michael R.

[International Worm Meeting, 2013]

We are interested in understanding how neurotransmitter signaling allows a neural circuit to execute distinct behavior states. C. elegans regulates egg laying by alternating between an inactive phase and a ~3 minute "active" state during which clusters of 3-6 eggs are laid. Six VC motor neurons release acetylcholine to excite the vulval muscles, and two HSN motor neurons release serotonin which signals through vulval muscle receptors to promote the active phase. Four uv1 cells release tyramine to inhibit egg laying. We are using GCaMP calcium imaging in behaving animals to understand how the signaling events in the circuit produce its two state behavior. We recently reported that the vulval muscles are rhythmically excited during each locomotor body bend (1). We found the UNC-103 K+ channel depresses muscle excitability below the threshold that drives calcium transients and contraction during the inactive phase, while still allowing signals above threshold during the active phase. To understand how the HSN, VC, and uv1 neurons regulate vulval muscle excitability, we are manipulating neurotransmitter release from these cells and using GCaMP to record changes in neuron and vulval muscle activity. Activation of serotonin release from the HSNs using Channelrhodopsin induces rhythmic vulval muscle twitching and egg-laying behavior-hallmarks of the active phase. We find that VC neuron activity increases during the active phase, and preliminary results show that egg-laying events mechanically distort the uv1 cells and trigger calcium transients. We have previously shown that TRPV channels, which can be mechanically gated, are required for uv1 to inhibit egg laying (2). We propose that successive egg-laying events increase uv1 calcium signaling, promoting release of tyramine and neuropeptides which terminate the active phase of egg laying. (1) Collins and Koelle (2013). J. Neurosci. 33, 761-775. (2) Jose et al. (2007). Genetics 175, 93-105.

Shu Liu et al. (2008) European Worm Meeting "Towards the search for a thermonociceptor"

Shu Liu, Maren Hertweck, Ralf Baumeister

[European Worm Meeting, 2008]

Upon exposure to noxious temperature, Caenorhabditis elegans executes an. escape reflex (Tav response) similar to the response to body touch. We had. previously shown that sensory neurons in the head and tail, but not in the. midbody region, are involved in the preception of heat. In vertebrates, the. capsaicin-sensitive vanilloid receptor VR1 of the TRP family has been shown. to be involved in the perception to heat, capsaicin, and low pH, and. several attempts have been made in other labs to relate TRP channels to the. sensation of external stimuli. We have begun a systematic phenotypic. analysis of available TRP channel mutants. Of 11 TRP mutants tested, only. osm-9 ocr-2 double mutants exhibited a severe Tav response defect. OSM-9. and OCR-2 have previously been demonstrated to function coordinately in C.. elegans olfaction and nose-touch responses (Tobin et al., 2002), and our. data support a similar model of coordinated sensory transduction in. thermonociception. OCR-2 and OSM-9 expression overlaps only in a few head. and tail neurons (Jose et al., 2007), suggesting that these could in. principle include candidates for thermonociceptors. We next tested a. variety of mutants whose genes have been reported to be expressed in. subsets of these neurons. sem-4 and unc-86 indeed displayed a reduced Tav. response in the tail, similar to the osm-9 ocr-2 double mutants'' defect. In. addition, one receptor mutant expressed in several classes of head neurons. also resulted in a mild yet significant anterior Tav defect, similar to. that seen in eat-4 and osm-9ocr-2 (79% Tav response). Taken together, we. are narrowing down our search for (a) sensory neuron(s) involved in the. perception of thermonoxious stimuli. We plan to carry out neuron ablations. to verify our findings.

Johnson, Matthew et al. (2013) International Worm Meeting "Sexually dimorphic synaptic connectivity in the C. elegans tail."

Ryan, Deborah, Portman, Douglas, Johnson, Matthew

[International Worm Meeting, 2013]

Sex-specific wiring of neural circuits is likely to have important roles in behavior. However, little is known about how sexual cues might influence synapse formation or stability. Interestingly, the recently described connectome of the C. elegans male tail (Jarrell et al., 2012, Science 337:437) has revealed several instances of sex-specific synaptic connections between non-sex-specific ("shared") neurons. In particular, the architecture of phasmid and posterior touch sensory circuitry appears to to be significantly different in the adult male compared to the adult hermaphrodite, perhaps to enable efficient male copulatory behavior. Using GRASP and other fluorescent markers, we are asking several questions about these sexually dimorphic connections. (1) Are sex differences in connectivity established in the embryo, or do these arise later, as a re-wiring process that occurs in parallel with male-specific tail neurogenesis and circuit formation? (2) What determines the sex-specificity of these connections? Is the genetic sex of the pre- and/or post-synaptic cells important, or do non-autonomous sexual cues have a role? (3) How do these sex differences in connectivity alter neural circuit function and behavior? Our preliminary results indicate that one putative hermaphrodite-specific connection, PHB-AVA, is indeed present in larval males. Unexpectedly, we can also often detect strong PHB-AVA GRASP signal in adult males. One possibility is that PHB-AVA synapses are removed as part of male tail re-wiring in L4, but that GRASP is unable to detect (or perhaps even disrupts) this removal. We are currently using GRASP to label other putative sex-specific connections to explore this further. The unique tools available in C. elegans should offer the opportunity to understand how genetic sex regulates modulators of synapse specification and maintenance to bring about sex differences in circuit connectivity. We are grateful to S. Emmons (Albert Einstein) for sharing unpublished data on male tail connectivity and to M. VanHoven (San Jose State) for sharing the wyIs157 (PHB-AVA GRASP) transgene.

Sean M O'Rourke et al. (2003) International Worm Meeting "Exploring the centrosome with proteomics and functional genomics."

Sean M O'Rourke, Bruce Bowerman

[International Worm Meeting, 2003]

The centrosome is a large eukaryotic organelle that organizes microtubules for cellular functions including setting up the mitotic spindle. Despite the important role of the centrosome, relatively little is known about its composition and regulatory mechanisms in metazoans. SPD-5 is a component of C. elegans centrosomes and contains multiple coiled-coil domains that may interact with other proteins thereby governing centrosomal structure (1). Because SPD-5 is thought to be a central component of centrosomes, it represents an excellent handle to pursue other genes and proteins that are also involved in centrosome function. We are taking two experimental routes to uncover additional centrosomal components and regulators.First, SPD-5 antibodies are being used to isolate protein complexes from embryonic extracts with the goal of purifying SPD-5-interacting proteins. Such proteins will be identified by mass spectrometry. To date, we have prepared embryonic extracts from N2 animals and we have successfully immunoprecipitated SPD-5 protein. Interestingly, two SPD-5 isoforms are apparent in our extracts, the significance of which remains to be determined. We are currently optimizing the purification procedure to obtain larger quantities of SPD-5 and interacting proteins. The interacting proteins we identify will be initially characterized by localization and RNAi inhibition studies.In the second route to understanding centrosome function, we are employing genome interaction screens to identify genes which, when knocked-down by feeding RNAi, alter the phenotypes of temperature-sensitive mutants. Building on the work of Jose Eduardo Gomes (see Gomes and Bowerman abstract), we have begun to use multiple-well plates for culturing worms and a robot to easily and accurately dispense the dsRNA-producing E. coli. We have performed a pilot semi-automated screen using the chromosome I library (2). Current results will be presented. In addition to examining spd-5, we are planning to perform genome-wide interaction RNAi feeding screens with 20 different temperature-sensitive mutants defective in several different processes. We envision generating a genetic interaction map that will be useful for determining how many different processes require any given gene. We anticipate that our use of many different sensitized genetic backgrounds will enable us to identify gene requirements missed in previous classical and genomic screens.(1) Hamill, D. R. et al. Developmental Cell 3, 673-684 (2002).(2) Fraser, A. G. et al. Nature 408, 325-330 (2000).

Chalfie M (2010) J Vis Exp "The 2009 Lindau Nobel Laureate meeting: Martin Chalfie, Chemistry 2008."

Chalfie M

[J Vis Exp, 2010]

American Biologist Martin Chalfie shared the 2008 Nobel Prize in Chemistry with Roger Tsien and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Martin Chalfie was born in Chicago in 1947 and grew up in Skokie Illinois. Although he had an interest in science from a young age--learning the names of the planets and reading books about dinosaurs--his journey to a career in biological science was circuitous. In high school, Chalfie enjoyed his AP Chemistry course, but his other science courses did not make much of an impression on him, and he began his undergraduate studies at Harvard uncertain of what he wanted to study. Eventually he did choose to major in Biochemistry, and during the summer between his sophomore and junior years, he joined Klaus Weber's lab and began his first real research project, studying the active site of the enzyme aspartate transcarbamylase. Unfortunately, none of the experiments he performed in Weber's lab worked, and Chalfie came to the conclusion that research was not for him. Following graduation in 1969, he was hired as a teacher Hamden Hall Country Day School in Connecticut where he taught high school chemistry, algebra, and social sciences for 2 years. After his first year of teaching, he decided to give research another try. He took a summer job in Jose Zadunaisky's lab at Yale, studying chloride transport in the frog retina. Chalfie enjoyed this experience a great deal, and having gained confidence in his own scientific abilities, he applied to graduate school at Harvard, where he joined the Physiology department in 1972 and studied norepinephrine synthesis and secretion under Bob Pearlman. His interest in working on C. elegans led him to post doc with Sydney Brenner, at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. In 1982 he was offered position at Columbia University. When Chalfie first heard about GFP at a research seminar given by Paul Brehm in 1989, his lab was studying genes involved in the development and function of touch-sensitive cells in C. elegans. He immediately became very excited about the idea of expressing the fluorescent protein in the nematode, hoping to figure out where the genes were expressed in the live organism. At the time, all methods of examining localization, such as antibody staining or in situ hybridization, required fixation of the tissue or cells, revealing the location of proteins only at fixed points in time. In September 1992, after obtaining GFP DNA from Douglas Prasher, Chalfie asked his rotation student, Ghia Euskirchen to express GFP in E. coli, unaware that several other labs were also trying to express the protein, without success. Chalfie and Euskirchen used PCR to amplify only the coding sequence of GFP, which they placed in an expression vector and expressed in E.coli. Because of her engineering background, Euskirchen knew that the microscope in the Chalfie lab was not good enough to use for this type of experiment, so she captured images of green bacteria using the microscope from her former engineering lab. This work demonstrated that GFP fluorescence requires no component other than GFP itself. In fact, the difficulty that other labs had encountered stemmed from their use of restriction enzyme digestions for subcloning, which brought along an extra sequence that prevented GFP's fluorescent expression. Following Euskirchen's successful expression in E. coli, Chalfie's technician Yuan Tu went on to express GFP in C. elegans, and Chalfie published the findings in Science in 1994. Through the study of C. elegans and GFP, Chalfie feels there is an important lesson to be learned about the importance basic research. Though there has been a recent push for clinically-relevant or patent-producing (translational) research, Chalfie warns that taking this approach alone is a mistake, given how "woefully little" we know about biology. He points out the vast expanse of the unknowns in biology, noting that important discoveries such as GFP are very frequently made through basic research using a diverse set of model organisms. Indeed, the study of GFP bioluminescence did not originally have a direct application to human health. Our understanding of it, however, has led to a wide array of clinically-relevant discoveries and developments. Chalfie believes we should not limit ourselves: "We should be a little freer and investigate things in different directions, and be a little bit awed by what we're going to find."