Starr, D. et al. (2015) International Worm Meeting "UC Davis-University of Maryland Eastern Shore Graduate Admissions Pathway: Collaborating to broaden participation in molecular and cellular biology."

Hom, C., Starr, D.

[International Worm Meeting, 2015]

STEM professionals in the 21st century remain predominantly Caucasian/white, in spite of decades of work by professional societies, colleges and universities, and individual scientists to broaden participation. This multifaceted problem includes concerns among students and faculty at minority-serving institutions about the economics of career choice, family pressure to pursue a career in a biomedical field, and limited exposure to natural history. Further, institutional efforts in recruitment by research universities remain rooted in graduate fairs that target senior undergraduates from groups underrepresented in science, whereas connections made via shared research networks provide a more sure means to admission in molecular and cell biology. The UC Davis-University of Maryland Eastern Shore (UMES) Molecular and Cellular Biology Graduate Admissions Pathways (MCBGAP) program addresses this challenge via collaborations between faculty at the two institutions and a research co-mentoring program that brings UMES undergraduates to UC Davis for summer research. The program is funded by a grant from the University of California Office of the President and the UC Davis College of Biological Sciences.MCBGAP supported two cohorts of five UMES students in the summers of 2014 and 2015. The MCBGAP program consists of reciprocal student-faculty visits, close interactions between key UC Davis and UMES faculty, monthly Skype meetings that involve mentors and students, and research, professional development, and field trips in the summer. MCBGAP has catalyzed change both at UMES, where students are given the opportunity to self-identify as researchers at a tier 1 research university, and at UC Davis, where increased numbers of faculty recognize the need to be proactive in graduate recruiting and admissions, and multiple deans have committed time to mentor students and funds to support additional undergraduates from Historically Black Colleges and Universities for summer research and mentoring. The experience has also inspired us to apply for a Postbaccalaureate Research Education Program (PREP) from the NIH.

Goetting, Desiree et al. (2017) International Worm Meeting "Food deprivation suppresses sleep via AMP kinase and circumvents the lethal effect of sleep loss in C. elegans."

Goetting, Desiree, Van Buskirk, Cheryl, Soto, Rony

[International Worm Meeting, 2017]

A major challenge in understanding the function and evolution of sleep lies in identifying the mechanisms that offset the vulnerabilities that come with it, such as the inability to forage or escape. The nematode C. elegans has recently been identified as a useful model for the dissection of sleep function and evolution, as animals experience a primitive sleep-like state that is triggered by conditions that cause cellular damage. In response to noxious environmental conditions such as extreme heat, animals enter a state of behavioral quiescence characterized by a reduction in sensory responsiveness and a cessation of feeding and locomotion. This recovery sleep, or RS, appears to be beneficial under certain conditions, as sleepless mutant animals are impaired for survival following noxious heat exposure. We wished to investigate how the decision to enter into RS may be influenced by additional environmental inputs that could potentially alter the physiological benefit to be gained from sleep. Here we show that food deprivation suppresses RS, and that this effect is exacerbated as population density increases. In addition to suppressing sleep drive, food deprivation protects against the lethality associated with sleep loss, suggesting that food-deprived animals have a reduced need for sleep. We show that suppression of sleep drive during periods of food deprivation requires AMP kinase. Additionally, we show that competence to engage in RS is dependent on the neuroendocrine signal DAF-7/TGF- beta , activating a previously identified neural circuit that shifts several aspects of development and metabolism from conservation to utilization of energetic resources. These data suggest that recovery sleep in C. elegans is an energetically costly activity that can be suppressed when environmental conditions are unfavorable and animals are required to compete for resources.

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

[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 & 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 & Stretton,2001). Injection of the peptide in the head-restricted behavioral assayabolished all locomotory waveforms and their propagations (Reinitz &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.

Chen LS et al. (1997) International C. elegans Meeting "MOLECULAR AND GENETIC ANALYSIS OF C. ELEGANS NEF-1, A MEMBER OF THE IG/FNIII SUPERFAMILY OF CELL ADHESION MOLECULES"

Chen LS, Bennett V

[International C. elegans Meeting, 1997]

A cell's identity, its interactions with other cells, and its ablity to adapt to its environment are heavily dependent on the proteins associated with its plasma membrane. Ankyrin, an intracellular protein that couples several integral membrane proteins to the spectrin cytoskeleton, is believed to be instrumental in organizing many membrane proteins into specialized domains. Recently, a family of cell adhesion molecules belonging to the Ig/FnIII superfamily was shown to bind ankyrin; these proteins include neurofascin, L1, NrCam and NgCam in vertebrates and neuroglian in Drosophila (1-6). This family of ankyrin binding CAMs is believed to be involved in neurite outgrowth, axonal fasciculation and targeting, cell migration and synaptogenesis during embryonic and postnatal vertebrate development (1-4). A member of the family has been identified in C. elegans. The gene encoding the homologue has been designated nef-1. In addition to the Ig and FnIII domains, it contains a highly conserved ankyrin binding domain. We are interested in elucidating the role of nef-1 in C. elegans development and its interactions with the C. elegans ankyrin homologue, UNC-44. Interestingly, mutations in unc-44 causes defects in axon guidance (7). 1. Sonderegger and Rathgen, 1992. J.Cell Biol. 119:1387-1394. 2. Rathgen and Jessel, 1991. Sem. of Neurosci. 3:297-307. 3. Grumet, 1991. Curr. Opin. Neurobiol. 1:370-379. 4. Hortsch and Goodman, 1991. Ann. Rev. Cell Biol. 7:505-557. 5. Davis et al., 1993. J. Biol. Chem.232:121-133 6. Davis and Bennett, 1994. J. Biol. Chem.267:18955-18972. 7. Otsuka et al., 1995. J.Cell Biol. 129: 1089-1092.

Breimann, Laura et al. (2021) International Worm Meeting "Precise quantification of mRNAs across all C. elegans embryonic stages through a microscopy and machine learning-based approach"

Breimann, Laura, Epstein, Leo, Preibisch, Stephan, Harrington, Kyle I S, Lionnet, Timothee, Bahry, Ella, Kolyvanov, Klim, Ercan, Sevinc

[International Worm Meeting, 2021]

Precise quantification of mRNA transcripts in space and time throughout embryogenesis is essential for understanding gene regulation, a process critical for embryogenesis in all animals, including C. elegans. We developed an imaging approach using 3D widefield microscopy-based single-molecule RNA fluorescence in situ hybridization (smFISH) to quantify mRNA transcripts. To count individual single-molecule mRNA spots, we developed RS-FISH, a fast 3D spot detection method that we implemented in Fiji that combines radial symmetry and RANSAC outlier removal. To assign each fixed, imaged C. elegans embryo to its developmental stage, we used advanced machine learning-based image classification that relies on the concept of auto-encoders. Currently, we are applying our methods to understand the role of condensins in chromosome compaction and transcription regulation. In C. elegans, an X-specific condensin binds to and represses X chromosomes in XX hermaphrodites by 2-fold for dosage compensation. In our study, we want to understand condensin DC's effect on transcript numbers and dynamics in single embryos across development. We obtained thousands of smFISH images for a set of condensin DC-regulated and control genes and extracted mature and nascent RNA counts in 3D, which we use to determine transcription burst characteristics throughout embryonic development. The distribution of total transcripts in wild-type and condensin DC-depleted embryos shows that single genes on the X chromosome are downregulated ~2-fold. Our machine learning approach to separate embryo images by development stage allowed us to observe the timing of condensin DC-mediated transcription repression, which occurs from the 100-cell stage on. RS-FISH is freely available as a Fiji plugin, and details for installation can be found at https://github.com/PreibischLab/RadialSymmetryLocalization and described at https://doi.org/10.1101/2021.03.09.434205

Franks CJ et al. (1997) International C. elegans Meeting "ACTION POTENTIALS RECORDED FROM THE PHARYNX OF CAENORHABDITIS ELEGANS."

Holden-Dye L, Walker RJ, Franks CJ

[International C. elegans Meeting, 1997]

Recently, Davis et al. (1995) have shown that it is possible to demonstrate phenotypic differences between wild-type and mutant C. elegans, using an electrophysiological approach. We have adapted their protocol in order to further characterise the wild-type phenotype, which will act as a basis for future comparative studies. We describe the properties of action potentials recorded from the terminal bulb of the pharyngeal muscle of wild type C. elegans. The anterior region of C. elegans was sectioned from the body and placed in a perfusion chamber on an inverted microscope stage in modified Dent's saline (composition in mM: NaCl 144, MgCl2 10, CaCl2 1, KCl 6, HEPES 5, pH 7.4). Intracellular recordings were made from pharyngeal muscle (60 MW microelectrodes;3M KAcetate), connected to an Axoclamp 2A amplifier. Drugs were applied by perfusion . Data are the mean +- S.E.M. The pharyngeal muscle membrane potential was -68.4 ! 1.5 mV (n=16). Action potentials had an amplitude of 85.5 ! 4.2 mV (n=16) with a duration of 0.26 ! 0.23 s, followed by a transient hyperpolarization of 7.5 ! 1.0 mV (mean of 4 to 10 action potentials from 16 cells; Figure 1). 5-HT (5!M; n=8) increased the frequency of firing in a reversible manner. Application of 4-aminopyridine (250!M) changed the firing pattern to bursting activity with a loss of the after hyperpolarization (n=6). This reversed on washing. We investigated the effect of changing external K+ concentrations (3-10 !M). Increasing [K+]out caused a decrease in the amplitude of the negative going after potential and concomitantly elicited a depolarisation of the membrane potential (n=6). Figure 1. Pharyngeal action potentials recorded from C. elegans. The membrane potential is indicated at the start of the trace. Reference. Davis et al. (1995) Mutations in the Caenorhabditis elegans Na, K-ATPase a-subunit gene, eat-6, disrupt excitable cell function. J. Neurosci. 15(12): 8408-8418.

Carnell, Lucinda (2009) International Worm Meeting "Killing two birds with one stone: Getting your undergraduate class to map your mutants."

Carnell, Lucinda

[International Worm Meeting, 2009]

Science education is moving towards more authentic and inquiry-based approaches to enhance learning. Laboratory courses that provide genuine research experiences are one strategy for generating excitement and enthusiasm. I have used this approach in a one-quarter upper-level majors Molecular Biotechniques course by performing a C. elegans mapping project. During the first third of the class, the students are introduced to and perform the techniques necessary to map the molecular lesion of a mutant to a chromosome. These include isolation of genomic DNA, PCR, and restriction digestion of restriction fragment length polymorphisms (RFLPs) also called snip-SNPs. In addition, the students learn about the broader significance of single nucleotide polymorphisms (SNPs) in phenotypic variation of organisms and disease by identifying examples through literature searches of primary research papers. In the last three weeks of the course, the students apply theses techniques to map a mutant previously isolated in a genetic screen. Each student was given an F2 recombinant from a cross between the mutant (in the N2 background) and the polymorphic mapping strain CB4856, with the goal of collectively mapping the mutation to a chromosome. The students isolated genomic DNA, cloned snip-SNP fragments for each of the six chromosomes, and digested the snip-SNPs using a single restriction enzyme, DraI (Davis et al. 2005). On the last day of the class, the students compiled their results together as a class (the total number of students was 22). The class data was analyzed and suggested that the mutation is on Chromosome V. As an independent research project, two students are going to continue to more finely map the lesion on Chromosome V. Students'' comments indicate that they enjoyed the challenge of such a project, especially when their experiments lead to novel research findings. The novelty of the project can be maintained by using a new mutant each year. Davis et at. 2005 BMC Genomics 2005, 6:118doi:10.1186/1471-2164-6-118.

Wada K et al. (2000) Japanese Worm Meeting "SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development."

Wada K, Kuroyanagi H, Kimura T, Matsumoto K, Hagiwara M, Hisamoto N

[Japanese Worm Meeting, 2000]

SR protein kinases (SRPKs) and their substrates, the serine/arginine-rich pre-mRNA splicing factors are key components of splicing machinery, and well conserved across phyla. These factors in metazoa have been well characterized through biochemical experiments, however, their physiological functions in multicellular organisms are still unclear. Here, we cloned a C. elegans SR protein kinase homologue, SPK-1, and one of its substrate, CeSF2. SPK-1 binds directly to and phosphorylates RS domain of CeSF2 in vitro. In situ hybridization analysis of adult hermaphrodite showed that both spk-1 and CeSF2 are predominantly expressed in gonads. Double-stranded RNA interference (RNAi) revealed that SPK-1 and CeSF2 play essential roles in the embryonic stage and that SPK-1 is also required for germline development of both hermaphrodites and males in C. elegans. We will also show that RNAi by soaking L1 larvae is also feasible for studying function of genes required for germline development.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Soto , Rony et al. (2017) International Worm Meeting "UNC-103/ERG potassium channel contributes to locomotor quiescence during sleep."

Sternberg, Paul W., Soto , Rony, Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In recent years the field of sleep research has been bolstered by studies in non-mammalian model organisms like C. elegans, promising to shed light on the long-standing mystery of the core function of sleep. C. elegans has been found to experience two distinct types of behavioral quiescence that each fulfill all of the behavioral criteria for sleep. The first to be discovered was developmentally-timed sleep (DTS), which occurs at the end of each of the four larval stages (Raizen et al., 2008). The other sleep state can be triggered at any time by exposure to damaging conditions such as noxious heat, tissue damage, and UV exposure, and is referred to as stress-induced sleep (SIS) or recovery sleep (RS) (Hill et al., 2014; Iannacone et al., 2016). We have shown that recovery sleep in C. elegans is dependent on EGF signaling, and that robust sleep can be induced at any time, in an EGFR-dependent manner, via forced expression of the EGF ligand LIN-3 (Van Buskirk and Sternberg, 2007). We have taken advantage of this 'forced-sleep' assay to uncover genes required for sleep regulation. We are particularly interested in determining which potassium channel genes, if any, contribute to C. elegans sleep, as K+ channel mutations produce short sleepers in Drosophila and zebrafish. An RNAi screen of K+ channel genes and their regulators identified UNC-103, an ERG-type potassium channel, as required for EGF-induced sleep. Here we show that UNC-103 is required for locomotor quiescence during recovery sleep (RS) and contributes to locomotor quiescence during lethargus (DTS) as well, indicating that these two types of sleep share common downstream effectors. We present evidence for developmental compensation by other K+ channels in the unc-103 null mutant, as short exposure unc-103 RNAi produces a greater sleep disruption than long exposure. We find that the application of ERG-blockers disrupts sleep in an UNC-103-dependent manner. Last, we present the results of our current site of action analyses aimed at determining in which neurons the widely expressed UNC-103 is required during sleep.