McJunkin, K. (2017) International Worm Meeting "The early embryonic mir-35 family of microRNAs regulates sex determination and viability through overlapping target genes."

McJunkin, K.

[International Worm Meeting, 2017]

Gene expression during early embryogenesis is largely controlled by post-transcriptional mechanisms. However, the roles that microRNAs play in controlling biological processes at this stage of development are poorly understood. We have probed the function of the mir-35 family of microRNAs, which is essential for embryonic development. We show that the mir-35 family regulates the sex determination pathway at multiple levels through its target genes encoding suppressor-26 (sup-26) and NHL (NCL-1, HT2A, and LIN-41 repeat) domain-containing-2 (nhl-2), two RNA-binding proteins. Although sex determination is established zygotically after the establishment of dosage compensation, the effect of the mir-35 family on this pathway is partially maternal. This maternal effect on an inherently zygotic process suggests that the mir-35 family may ensure a window of naivete in early development, preventing premature sex-specific gene expression. In this way, the mir-35 family plays a role in developmental timing, analogous to other essential microRNAs families expressed during larval development. In addition to the role this network plays in controlling sex determination, the repression of nhl-2 by mir-35 family members appears to be essential for development. Genome editing events affecting the single mir-35 family seed match in the nhl-2 3'UTR are negatively selected. This is an exciting and rare example of a single microRNA binding site that is essential for development. Here we present our recent progress in elucidating the nature of the deleterious effects of nhl-2 derepression, as well as our efforts to discover other targets whose repression by mir-35 family members is essential.

Yang, B et al. (2019) International Worm Meeting "Identifying essential mir-35 targeting sites in C. elegans."

Yang, B, McJunkin, K

[International Worm Meeting, 2019]

In C. elegans, mir-35 family members have been shown to be essential for the development of embryos [1]. Complete knockout of the mir-35 family (mir-35-41 cluster and mir-42) causes fully penetrant embryonic or L1 larval lethality [1]. Phenotypic characterization of a temperature sensitive deletion strain of mir-35-41 (gk262 or nDf50) at permissive temperature revealed that the mir-35 family plays various roles throughout C. elegans development [2, 3]. The lethal phenotype of mir-35 family mutants is still poorly understood though genetic data suggests that derepression of more than one target gene underlies the lethality. Among the mRNA transcripts that have been identified as direct targets of the mir-35 family, most play roles in non-lethal phenotypes. Interestingly, disruption of the mir-35 seed matching sequence in the nhl-2 3'UTR resulted in a dominant lethal phenotype [5], implying that nhl-2 is one of the essential targets of the mir-35 family. We are taking a CRISPR-Cas9 screening approach to identify other sites at which edited alleles are negatively selected, indicating that they are also essential for mir-35 binding and target repression. Starting with a list of candidate binding sites predicted by Targetscan, we designed a guide RNA to target each of the predicted mir-35 binding sites. To increase the throughput of this large-scale CRISPR-Cas9 screening, we decided to introduce the gRNAs to the animals in a multiplexed way without losing the efficiency of each individual gRNA. After a series of optimizations, multiplexing of 10 gRNAs in one pool proved to be ideal for screening. Transformed animals were selected based on a co-CRISPR marker, and the genomic DNA was collected and subjected to site-specific amplifications. The frequency and position of editing at each site will be analyzed through deep sequencing of these amplicons. Sites in which CRISPR-Cas9 editing of the seed match is not observed will be categorized as potential essential mir-35 binding sites and analyzed in later confirmation studies. This approach will not only allow us to identify essential mir-35 binding sites but also lay the foundation for future multiplexed CRISPR-Cas9 screen for other classes of essential negative regulatory elements in C. elegans. 1. Alvarez-Saavedra, E. and H.R. Horvitz, Curr Biol, 2010. 20(4): p. 367-73. 2. McJunkin, K. and V. Ambros, G3 (Bethesda, Md.), 2014. 4(9): p. 1747-1754. 3. Liu, M., et al., Cell research, 2011. 21(11): p. 1605-1618. 4. Kagias, K. and R. Pocock, Scientific Reports, 2015. 5: p. 11284. 5. McJunkin, K. and V. Ambros, Genes & Development, 2017. 31(4):422-437.

Grimme, A. et al. (2019) International Worm Meeting "Elucidating the role of the miR-100 Family in C. elegans."

McJunkin, K., Grimme, A.

[International Worm Meeting, 2019]

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are important post-transcriptional regulators of gene expression. A miRNA contains a "seed" sequence, typically located in nucleotides 2-7, that has complementarity to a region in a mRNA 3'UTR which allows for target recognition while the miRNA is loaded to the RNA-induced silencing complex (RISC). MiRNA families are comprised of microRNAs that share the same seed sequence, and due to the identical seed sequences, these family members can act redundantly while regulating targets. One of the most highly conserved miRNA families is the miR-100 family, which contains three family members in humans: miR-99a, miR-99b, and miR-100. These miRNAs are implicated in several medical conditions including cancer, obesity, heart disease, and others. In the model organism C. elegans, the miR-100 family microRNAs are known as the mir-51 family members, which includes six microRNAs: mir-51 through mir-56. Current knowledge of the essential functions of this family is relatively limited; one of the most important functions in C. elegans is its role in embryonic development. Given the highly conserved nature of the miR-100 family, understanding the vital functions of the mir-51 family may serve to elucidate how this family is involved in human diseases. While removing all mir-51 family members results in embryonic lethality, this project utilizes a hypomorphic strain with a slow growth phenotype to study the pathways regulated by this family. A portion of this project is dedicated to performing forward genetic screens to isolate suppressors of the slow growth phenotype. Reverse genetic screens using RNAi are performed alongside the forward screens to determine the roles of predicted mir-51 family targets in the slow growth phenotype. Preliminary data from these screens suggest that the slow growth phenotype can be alleviated through isolated suppressors as well as through performing RNAi against certain mir-51 family targets. In the future these results may help to elucidate specific functions of the mir-51 family in C. elegans, which may represent conserved functions of the miR-100 family that can be studied further to determine the connection between this family and human diseases.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Johnson, Christina K. et al. (2019) International Worm Meeting "Requirement of glial Na+/K+-ATPase in touch sensation highlights ionic and metabolic link between glia and touch neurons in C. elegans."

Johnson, Christina K., Bianchi, Laura, Wang, Ying, Fernandez Abascal, Jesus

[International Worm Meeting, 2019]

Isolated microenvironments, such as the tripartite synapse, where the concentration of ions is regulated independently from the surrounding tissues, exist throughout the nervous system, including in mechanoreceptors. Modulation of the ionic composition of these microenvironments has been suggested to be achieved by glia and other accessory cells. However, the molecular mechanisms of ionic regulation and effects on neuronal output and animal behavior are poorly understood. Using the model organism C. elegans, our lab published that Na+ channels of the DEG/ENaC family expressed in glia control neuronal Ca2+ transients and animal behavior in response to sensory stimuli. DEG/ENaC Na+ channels are known to establish a favorable driving force for K+ excretion, which occurs via inward rectifier K+ channels, in epithelial tissues across species. We hypothesized that a similar mechanism exists in the nervous system. Using molecular, genetic, in vivo imaging, and behavioral approaches, we showed that expression in glia of inward rectifier K+ channels and cationic channels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs without disrupting neuronal morphology, supporting our hypothesis. Based on this model, Na+/K+-ATPases are also needed to maintain ionic concentrations following influx of Na+ and excretion of K+. We show here that, in addition to glial Na+ and K+ channels, two specific glial Na+/K+-ATPases, EAT-6 and CATP-1, are needed for touch sensation and that their requirement can be bypassed by a high glucose diet. The effect of glucose is dependent on ATP binding capability of the pump, translation, transcription, and the activity of CATP-2, a third Na+/K+-ATPase ?-subunit. Taken together, our results support metabolic and ionic cooperation between glia and neurons in C. elegans mechanosensors, a mechanism that is essential to regulating neuronal output and may be conserved across species.

Sherrard, R. et al. (2017) International Worm Meeting "Regulation of programmed cell death by miRNAs and RNA-binding proteins."

Holzkamp, H., Conradt, B., Luehr, S., McJunkin, K., Memar, N., Sherrard, R.

[International Worm Meeting, 2017]

Programmed cell death is an important process in animal development and requires strict control to avoid the unwanted death of cells. In C. elegans, the pro-apoptotic BH3-only gene egl-1 is the most upstream factor of the core apoptotic pathway, and its activity is thought to determine the life-versus-death decision during development. Previous studies have identified regulatory modules that control the transcription of egl-1 in a lineage-specific manner; however, investigations into the post-transcriptional regulation of egl-1 are largely absent. We find that the mir-35 family and mir-58 bantam family of miRNAs directly target the 3'UTR of egl-1 mRNA and act cooperatively to suppress its expression. This suppression affects both mRNA copy number and translation efficiency, and is crucial to prevent the precocious and collateral death of mothers and sisters of cells programmed to die. Using single-molecule RNA FISH, we show that egl-1 is already transcribed in mothers of cells programmed to die and that mir-35 family and mir-58 bantam family miRNAs prevent their precocious death by keeping the copy number of egl-1 mRNA below a critical threshold. These miRNAs also act to dampen the transcriptional boost of egl-1 that occurs specifically in the daughter that is programmed to die, but they are generally not required for the degradation of egl-1 mRNA in the daughter that is programmed to survive. We propose that these miRNAs function to adjust lineage-specific differences in egl-1 transcriptional activation and thereby ensure that egl-1 expression reaches a level sufficient to trigger death only in the daughters that are programmed to die. Finally, we investigate the role of PUF (Pumilio/FBF) RNA-binding proteins (RBPs) in the post-transcriptional regulation of egl-1, whose 3'UTR contains two FBF binding elements. Mutations in these elements mediate increased reporter expression in both the germline and early embryos, suggesting that egl-1 is directly regulated by FBF during development. Although miRNAs and RBPs can function independently, recent studies have found these two pathways can interact in the regulation of shared targets, and we speculate that egl-1 may be one such target.

Maelle Jospin et al. (2001) International C. elegans Meeting "Basic electrophysiological properties of body wall muscle from the Nematode C elegans."

Maelle Jospin, Laurent Segalat, Bruno Allard

[International C. elegans Meeting, 2001]

Electrophysiological properties of striated muscle cells were investigated with the patch clamp technique in the Nematode C elegans . Worms were immobilised with cyanoacrylate glue and longitudinally incised using a tungsten rod sharpened by electrolysis. Recording pipettes were sealed on GFP-expressing body wall muscle cells. In the whole cell configuration, under current clamp conditions, in the presence of Ascaris medium in the bath and K-rich solution in the pipette, no action potential could be induced in response to current injection. Under voltage clamp control and in the same ionic conditions, depolarizations above -30 mV from a holding potential of -70 mV gave rise to outward K currents. Outward K currents resulted from two components, one fast inactivating component blocked by 4-aminopyridine, one delayed sustained component blocked by tetraethylammonium. In the presence of both blockers, an inward Ca current was revealed and inhibited by cadmium. Single channel recording using the inside-out configuration revealed the existence of a Ca-activated Cl channel and a Ca-activated K channel. Single channel experiments are currently performed to characterise voltage-gated conductances at the unitary level.

K Omata et al. (1999) International C. elegans Meeting "Habituation of C. elegans for the touch sensitivity"

K Kawamura, F Yonezawa, K Omata

[International C. elegans Meeting, 1999]

In order to study the habituation of C. elegans for the touch sensitivity, we carry out computer simulations, in which the neural circuit is formed by making use of the data table constructed recently by Oshio et al [1]. The i -th neuron is connected with the neighboring j -th neuron through the coupling strength K ij , which is varied dynamically by the Hebb rule. Note that K ij is not necessarily equal to K ji because there are one-way connections between the neurons by chemical synapses. As a reference state, we first deal with the neural circuit consisting only of the neurons ALM, AVM, PLM, PVM, AVA, AVB, PVC, AVD, A and B, that are related to the forward and backward movement directly. We give periodic stimuli to the sensory neurons PLM, PVM, and monitor the response of the motor neuron A. We find that the frequency of the response decreases with time, which indicates that the habituation to the touch sensitivity actually takes place. As one deviation from the reference state, we kill the inter-neuron AVD, and perform the same analysis described in the above. There is a tendency that the decay of the response curve becomes faster, and the habituation is enhanced. As the other deviations, there are several possibilities of killing the inter-neurons AVA, AVB, PVC and/or AVD. We discuss the enhancement of the habituation in relation with the recent experimental results by Hosono. [1.] K. Oshio, S. Morita, Y. Osana and K. Oka; C. elegans synaptic connectivity data'', Technical Report, CCEP, Keio Future No.1 (1998)

Jamie White et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Neurons necessary for sex-specific social behaviors"

Thomas Nicholas, Eliott Davidson, Jamie White, Erik Jorgensen

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

C. elegans nematodes sense and respond to other individuals in their environment via signals released by hermaphrodites. The response is sex-specific: males are attracted to the signals and hermaphrodites avoid them. This is interesting because the male and hermaphrodite nervous systems have highly similar anatomy, and the stimulus detected by each is the same--hermaphrodite-derived signals--but the response is opposite. This could arise from (1) sex-specific neurons, (2) sex-specific connectivity of anatomically similar neurons, (3) sex-specific physiology of similar neurons, or (4) a combination of these mechanisms. We are therefore interested in which neurons are necessary and sufficient for male-attraction and hermaphrodite avoidance behaviors. To determine this, we are using a combination of mutant analysis, neuron-specific rescue, and laser ablation. Avoidance requires tax-4 and osm-3. These genes overlap in the ASE, ASG, ASI, ASJ, and ASK pairs. Avoidance behavior is normal in unc-130 (required for the ASG fate) and in che-1 (required for proper ASE fate) mutants, indicating that ASI/J/K are most likely necessary for avoidance behavior. Avoidance can be rescued in a tax-4(p678) background by tax-4(+) expression from an odr-4 promoter, which includes expression in ASG and ASI/J/K but not ASE, indicating that ASI/J/K are sufficient for avoidance. ASI/J/K will be laser-ablated in the rescued strain to show which are necessary. Attraction behavior requires osm-9. Attraction behavior can be partially rescued in an osm-9(ky10) background by osm-9(+) expression in either male-specific neurons (pkd-2 promoter), or by expression in the AWB, AWC, and ASI/J/K pairs (odr-1 or daf-11 promoters). This indicates these two sets of neurons are each partially sufficient for attraction behavior. Of the second set, attraction is normal in ceh-36 mutants (required for AWC fate), indicating the AWB and/or ASI/J/K pairs are sufficient for partial rescue. We predict full rescue will be obtained by a combination of expression from the pkd-2 and odr-1/daf-11 promoters. Laser ablation will be carried out in the fully rescued strain to show which neurons of the sufficient set are necessary. It appears, then, that the different sexes may use the same neurons--one or more of the ASI/J/K pair--to generate sex-specific behaviors, but males need at least some input from sex-specific neurons.