Cannataro, Vincent et al. (2009) International Worm Meeting "TMD-1 / Tropomodulin Regulates Intestinal Lumen Diameter in C. elegans."

Silva, Malan, Cannataro, Vincent, Gallagher, Thomas, Cox, Elisabeth

[International Worm Meeting, 2009]

Tubes are a central architectural feature of many human tissues including glands, the urogenital and gastrointestinal tracts, the respiratory tract and the vascular system. Despite their importance, much remains unknown about the molecular mechanisms involved in tube development. This work explores a new role for tropomodulins in regulation of endothelial tube formation. Tropomodulins are one of the few proteins known to regulate the slow growing ends of actin filaments. The C. elegans tmd-1 gene encodes two transcripts (tmd-1a and tmd-1b) that have similar domain structure to mammalian tropomodulins. We have been examining the role of TMD-1 in development of the C. elegans intestine, which is a relatively simple, transparent tube that forms by a cord-hollowing mechanism similar to that used by some capillaries and renal tubules. Antibody staining using a rabbit anti-TMD-1 antibody demonstrates that TMD-1 localizes to the luminal surface of the C. elegans intestine as it undergoes morphogenesis. Interestingly, embryos homozygous for the tmd-1(tm724) allele, which contains a large deletion that affects both transcripts1, exhibit areas of intestinal lumen diameter that are 65% larger than wild-type. tmd-1(sf20) homozygous mutants, which have a premature stop codon affecting both transcripts2, also exhibit luminal expansions (50% larger than wild-type). Currently, investigations are underway to determine the molecular mechanisms by which TMD-1 regulates intestinal lumen diameter. Possibilities include modulation of actomyosin contractility, cooperation with the actin-spectrin cytoskeleton to provide mechanical strength to the luminal surface, and/or regulation of vesicle trafficking. This work is supported by National Institutes of Health grant R15HD059952. 1 Yamashiro et al. (2008) J. Cell Sci. 121: 3867-77. 2 Stevensen et al. (2007) J. Mol. Bio. 374: 936-50.

Portegijs, Vincent et al. (2015) International Worm Meeting "Regulation of spindle positioning through phosphorylation of LIN-5 in asymmetric cell division."

Fielmich, Lars-Eric, Van den Heuvel, Sander, Portegijs, Vincent, Galli, Matilde

[International Worm Meeting, 2015]

Asymmetric cell division is required throughout development to generate cells of unequal size, composition and fate. The correct distribution of cell fate determinants depends on the plane of cell cleavage, which is determined by the position of the mitotic spindle. Spindle positioning is mediated by a conserved Galpha-GPR-1/2­-LIN-5 complex at the cortex, which interacts through dynein with astral microtubules and thereby promotes the generation of pulling forces. We aim to understand how the developmental regulation of cortical pulling forces determines the plane of cell cleavage and contributes to asymmetric cell division.Previous studies of our lab identified four amino acids in the C-terminal domain of LIN-5 that are phosphorylated by the polarity kinase PKC-3. This phosphorylation leads to reduced pulling forces in the anterior, but additional mechanisms contribute to asymmetric spindle positioning. In recent studies, we identified additional phosphorylations of LIN-5 with critical functions in vivo. Moreover, we defined the LIN-5 C-terminal domain that mediates interaction with GPR-1. Hereto, we used reverse yeast two-hybrid screening to identify single amino acid changes in LIN-5 that prevent GPR-1 binding. All of the identified mutations are confined to a 30 amino-acid domain located upstream of the PKC-3 phosphorylated residues of LIN-5. This GPR-1 binding region contains two additional in vivo phosphorylated LIN-5 residues. We used Cas9/CRISPR-mediated genome editing to substitute these amino acids for non-phosphorylatable alanine or phosphomimetic glutamic acid / aspartic acid. Combined with UV-laser spindle severing experiments, we found that phosphorylation of these residues contributes to GPR-1 binding and cortical pulling forces. In a parallel study, we identified residues in the N-terminus of LIN-5 that are phosphorylated by CDK-1. Again, examination of Cas9/CRISPR-mediated knock-in alleles revealed that these N-terminal residues are critical for LIN-5 function. Taken together, our data show that phosphorylation of LIN-5 contributes to the spatiotemporal regulation of the cortical pulling forces that position the mitotic spindle.

Lanier, Vincent et al. (2021) International Worm Meeting "High-throughput EPG recordings reveal the food exploitation-exploration trade-off"

Lanier, Vincent, Lockery, Shawn, Millet, Jonathan, Weeks, Janis, Roberts, William

[International Worm Meeting, 2021]

Whole-animal screens complement traditional screens based on cultured cells and single-celled organisms. In C. elegans, two of the most common whole-animals screens involve locomotion and pharyngeal pumping. Whereas high-throughput locomotion screens are in wide use, high-throughput pharyngeal screens are lacking. Electrophysiological measures of pharyngeal activity - electropharyngeograms (EPGs) - offer higher temporal resolution and signal to noise ratio than manual or optical methods. Recently, the ease and efficiency of EPGs was improved by placing individual worms in tight-fitting microchannels. Electrical resistance formed where the worm contacts the channel walls generates voltage differences sufficient to resolve individual pharyngeal action potentials. However, this approach is currently limited to only eight worms per recording. To address this deficiency, we explored the utility of bulk EPG recordings made by increasing the length and width of the recording channel to accommodate hundreds or even thousands of worms. In bulk recordings, where worms are loosely arrayed in the channel and unrestrained, the voltage signal produced by each worm is insufficient to resolve individual action potentials. However, the composite voltage signal, which can be conceptualized as the sum of many individual, asynchronous EPGs, can be analyzed by computing its power spectrum. We found that EPG power spectra generally have just two peaks: a low frequency peak at 0-2 Hz, and a high frequency peak at 4-6 Hz. Using serotonin dose-response curves, optogenetic inhibition of pharyngeal muscles, and pharyngeal pumping mutants, we found that the high frequency peak reflects neurogenic pumping whereas the low frequency peak reflects myogenic pumping together with locomotion. As serotonin concentration is increased, the high and low frequency peaks are enlarged and diminished, respectively. We propose that the high frequency peak reflects the proportion of worms that are nearly stationary but feeding vigorously (exploitation), whereas the low frequency peak reflects the proportion of worms that are moving rapidly but feeding intermittently (exploration). Thus, the new method quantifies not only neurogenic pharyngeal pumping, but also the distribution of animals across these mutually exclusive behavioral states.

O'Connor, Vincent et al. (2015) International Worm Meeting "Distinct pharyngeal glutamate determinants underpinning the context dependent modulation of feeding behaviour."

Robert, Walker, Nikhil, Bhatla, Robert, Horvitz, Dalliere, Nicolas, O'Connor, Vincent, Lindy, Holden-Dye

[International Worm Meeting, 2015]

Pharyngeal pumping mediates the filter feeding of C.elegans. In the presence of bacteria, worms maintain high pump rates (250 pumps per minute). Although the modalities that control this sensory dependent increase are unknown, removal of bacteria reduces pumping. We show reduced pumping OFF food is not simply loss of an activating ON food cue. Mutants that exhibit high pump rates (>100 pumps per minute) OFF food suggest an important transmitter-mediated reduction of pumping. eat-4 mutants, deficient in glutamate, produce an antonymous regulation in the ON and OFF food contexts. Compared to N2, eat-4 mutants have a reduced pumping ON food while OFF food their pumping is raised. The reduction ON food is consistent with the loss of glutamate release from the pharyngeal neuron M3. Screening glutamate receptor mutants ON food identified the pharyngeal muscle receptors glc-2 and avr-15 have a reduced pumping. In addition, the ceh-2 mutants, disrupted in M3 motor neuron function, phenocopy eat-4 ON food. This reinforces that M3 release of glutamate onto inhibitory glutamate-gated chloride channels that relax muscle after contraction acts as the critical determinants of pumping ON food. In contrast, these determinants of pharyngeal glutamate function do not change the OFF food pumping. Surprisingly, a distinct glutamate gated chloride receptor, avr-14, that has no effect on the pumping ON food, replicate the OFF food phenotype of eat-4. This receptor was not known to be expressed in the pharynx, but re-analysis indicates pharyngeal neuron expression. OFF food signalling has been identified at the level of the extrapharyngeal sensory nervous system but our studies indicate the pharyngeal neuron I2 is a dominating determinant in the OFF food glutamate dependent response. Selective expression of eat-4 in I2 suppresses the elevated pumping of eat-4 mutants, restoring OFF food pumping to wild-type levels. Further, selective ablation of this neuron imparts an eat-4 like phenotype OFF food. This suggests that I2-mediated glutamate release underpins the OFF food imposition of an inhibitory tone. This conclusion is reinforced by the observation that optogenetic activation of I2 mediates a powerful reduction in pharyngeal pumping occluded in the OFF food context. Overall this indicates that major determinants of ON and OFF food behaviour are mediated by discrete pharyngeal microcircuits. Although proprioceptive feedback inhibition from mechnosensory cues in the gut can explain how glutamate transmission is engaged ON food, the upstream modality for the distinct OFF food response is unknown.

O'Connor, Vincent et al. (2015) International Worm Meeting "Neurochip: a microfluidic device for improved throughput to translate agrochemical, pharmacological and biological potential of nematode pharyngeal function.."

Callahorro, Fernando, Hu, Chunxiao, Dillon, James, O'Connor, Vincent, Holden-Dye, Lindy, Ferreiro, Teressa, Morgan, Hywel

[International Worm Meeting, 2015]

The pharyngeal system of C.elegans provides a powerful in vivo route to investigate the biological function of a tractable neuroactive circuit. It consists of a muscular structure made up of the corpus isthmus and terminal bulb. This produces a rhythmic cycle of contraction and relaxation modulated by overriding neural activity. The pharynx expresses the major transmitter pathways and/or their cognate receptors that operate across the phyla. This makes its function sensitive to pharmacological regulation by many classes of neuroactive drugs. This includes those of general bio-medical and agrochemicals relevance and a more selective nematicidal potency. Thus, the pharynx lends it-self to use as a model for drug discovery, better mapping of mode of action and comparative approaches for selective and ecological toxicity. Measurement of pharyngeal function using extracellular measurement provides a powerful tool for the above approaches and supplements visual measurement of pumping. Microelectrode recording from intact C.elegans is a tractable route as it offers the potential to investigate chemically induced changes in the context of whole organism, to scope pharmacokinetics and comparative toxicity. The electropharyngeogram (EPG) is recorded by immobilizing the worm in glass electrode fabricated to make a tight seal around the nose of the worm. By analogy with a grease gap this allows recording of the potential difference between the two sides of the tight seal. We have adapted this principle and embedded it into microfluidic chip we have called neurochip. The central aspect of this device is the delivery of the worm into a PDMS trapping channels in which a double layer design allows the production of a compliant circular aperture that restrains the worm with a tight seal. The device has intrinsic platinum electrodes either side of the seal and when the pharynx contract a waveform with all the characteristic features of the EPG is recorded. We can record EPG in loading buffer or prime them by loading in 5-HT a potent stimulator of pumping. We have optimized a medium through put plate assays where worms are cultivated in drug overnight prior to investigation in the neurochip. This approach is particularly useful in instances where access across the cuticle is slow or mode of action of implies an important role of chronic exposure. Pre-exposure of the inhibitor with known and unknown candidates reveals a dose dependent inhibition of the EPG in which the frequency and pattern of activity are modified. A further iteration of the neurochip design incorporates supporting flow channels designed to direct efficient drug perfusion onto the pre immobilized worms. Restrictions to efficient flow in the immobilizing and recording micro channels is circumvented by incorporating perforated exit ports along the length of the trapped worm. Thus, the device and accompanied optimization of dosing regimes opens up a wide user base for previously technically challenging measurement of EPG. The format and ergonomics of loading are suited to investigation of both the chronic or acute effects of important classes of chemicals on wild type, mutant or genetically modified C.elegans sculpted to express targets of experimentally intractable nematode species. .

Portegijs, Vincent et al. (2021) International Worm Meeting "Multiple levels of regulation ensure robust cell cycle exit during C. elegans vulva formation"

Godfrey, Molly, Portegijs, Vincent, Van den Heuvel, Sander

[International Worm Meeting, 2021]

Regulated cell cycle withdrawal is crucial for cell differentiation and tissue homeostasis, with impaired cell cycle exit leading to hyperplasia, a hallmark of cancer. The reproducible pattern of cell proliferation and differentiation in C. elegans provides an excellent model to study the regulatory pathways that control cell cycle arrest. Using tissue-specific gene knockout combined with lineage tracing, we explored whether known cell cycle inhibitors and tumor suppressor genes regulate cell cycle exit of the epidermal vulva precursor cells (VPCs). Lineage-specific knockout of cki-1, the main C. elegans CIP/KIP cell-cycle inhibitor, resulted in a surprisingly weak extra vulval cell division phenotype. We found that a second CIP/KIP inhibitor, cki-2, becomes transcriptionally upregulated in these cki-1 knockout animals. Simultaneous knockout of both CKIs substantially increased the number of extra vulva cells. In addition to cki-2, we discovered that cep-1 p53 and daf-3 Smad4 contribute to proper cell cycle arrest of cki-1 knockout vulval cells. Genetic analyses support a model in which these transcriptional regulators act upstream of cki-2 to promote cki-2 expression, which is critical only when cki-1 is absent. Furthermore, a forward genetic screen identified the HECT-domain ubiquitin ligase UBR-5 as another factor contributing to the timely VPC cell division arrest. The ubr-5 overproliferation phenotype was markedly enhanced when combined with cki-1 knockout, indicating that UBR-5 may act in parallel to CKI-1. Notably, all tested combinations of negative cell-cycle regulator knockout/knockdown resulted in limited increases in vulval cell numbers. Our data support that multiple redundant levels of regulation ensure remarkably robust control of cell cycle withdrawal of C. elegans vulval cells.

Vincent Hyenne et al. (2007) International Worm Meeting "Defining the role of the brat family in C.elegans embryonic polarity."

Jean-Claude Labbe, Marianne Desrosiers, Vincent Hyenne

[International Worm Meeting, 2007]

We are interested in understanding the molecular mechanisms controlling cell polarity and asymmetric cell division. Asymmetric cell division relies on the cells polarization prior to division and allows the generation of cell diversity. In C.elegans zygote, polarity is established along an antero-posterior axis and leads to the formation of two unequal cells with distinct fates after division. Polarization induces the segregation of cell fate determinants in the anterior or posterior part of the zygote as well as posterior displacement of the mitotic spindle during metaphase/anaphase. The evolutionary conserved PAR proteins (PAR-1 to -6 and PKC-3) are essential for these events: loss of any of them results in a loss of polarity and an abnormal symmetric division. However, the molecular mechanisms involving PAR protein functions remain elusive. Recently, several genes have been identified as new modulators of PAR-protein dependent cell polarity, as their disruption can suppress the embryonic lethality of par-2(it5ts) animals. Two of these genes, nos-3 and fbf-1/2, have Drosophila orthologs that were shown to form a complex with the tumor suppressor Brat and negatively regulate the translation of Hunchback mRNA during embryonic polarization. This suggested that orthologs of Brat could also regulate embryonic polarity in C.elegans. While there are 5 genes encoding predicted orthologs of Brat in C.elegans, we have found that 3 of them, namely ncl-1, nhl-1 and nhl-2 (together referred to as CeBrats), are able to suppress par-2(it5ts) lethality. This suggests that, like in Drosophila, they could function in C.elegans PAR-protein dependent polarity through translational repression. Phenotypic analysis demonstrated that ncl-1, nhl-1 and nhl-2 suppress most of the early polarity phenotypes associated with par-2 depletion. Although disrupting these CeBrats collectively did not result in significant embryonic lethality, embryos individually mutant for these 3 CeBrats displayed distinct polarity phenotypes: ncl-1 and nhl-1 mutants have a more posterior spindle localization whereas nhl-1 and nhl-2 have an enhanced asynchrony of division at the second cell stage. These results suggest that these CeBrats could function through at least partially independent pathways. We are currently establishing whether ncl-1, nhl-1 and nhl-2 function, together or independently, within a complex with nos-3 and fbf-1/2 to regulate translation, and we are searching for putative mRNA targets of such a complex, using a combination of biochemical, bioinformatic and genetic approaches. This study should shed light on a new molecular mechanism controlling cell polarity and asymmetric cell division in a PAR-protein dependent manner.

Vincent Menuz et al. (2005) International Worm Meeting "Identification of Genes Affecting Resistance to Anoxia in Caenorhabditis elegans"

Vincent Menuz, Monique Fornallaz, Michael O Hengartner, Jean-claude Martinou, Marie Gomez

[International Worm Meeting, 2005]

In obligatory aerobe organisms, molecular oxygen is necessary for the production of ATP through the electron transport chain in the mitochondria. Adult C. elegans has evolved the capacity to survive more than 24 hours in the complete absence of oxygen (anoxia). The goal of our study is to identify the mechanisms in C. elegans that confer its resistance to anoxia. During anoxia, C. elegans enters into a reversible state of suspended animation in which cell division, developmental progression and movement cease. Young adults worms (72 hours post L1 stage) were exposed to anoxia for different periods of time. A time course study indicated that 85%, 67%, 30% and 2% of the worms survived 48h, 72h, 96h and 168h of anoxia respectively. We chose to screen for mutants whose survival rate was either higher or lower than the survival rate of wild type worms after 72 h anoxia. A random screen of a mutant library allowed us to identify eight mutants which were unable to survive to anoxia. Among those mutants, the strongest phenotype was displayed by the srf-3 mutant whose survival did not exceed 2% at 72h of anoxia. SRF-3 codes for a nucleotide sugar transporter capable of translocating UDP-galactose and UDP-N-acetylglucosamine into the lumen of the Golgi apparatus. The sensitivity of this mutant to hyperoxia, heat shock, negative pressure, hypertonic and hypotonic shock was not different than that of wild type worms under the same conditions. We are currently investigating the mechanisms by which SRF-3 deficiency sensitizes C. elegans to anoxia.

Hyenne, Vincent et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "RAB-5 contributes to polarity establishment in the C. elegans embryo by localizing anterior PAR proteins independently of actomyosin contractility"

Velmurugan, Ramraj, Hyenne, Vincent, Grant, Barth D, Labbe, Jean-Claude, Loerke, Dinah, Tremblay-Boudreault, Thierry

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In many cell types, cell polarity is controlled by the evolutionary conserved PAR proteins, some of which are asymmetrically localized at the cell cortex where they define specific domains. How asymmetric cortical localization of PAR protein is achieved remains unclear. In the C. elegans zygote, actomyosin contractility participates to the establishment of polarity along an antero-posterior axis by controlling the formation of two exclusive cortical groups of PAR proteins: the PAR-3/PAR-6/PKC-3 complex at the anterior cortex and PAR-1 and PAR- 2 at the posterior cortex. We tested the possibility that vesicular trafficking, which maintains the polarity of epithelial cells, could affect polarity establishment by localizing PAR proteins in the C. elegans embryo. We found that while disrupting several genes involved at various steps of the endocytic pathway could partially suppress par-2 lethality, depleting the early endocytic gene rab-5 could fully suppress all par-2 polarity phenotypes. rab-5 acts positively and upstream of anterior PAR proteins since its disruption resulted in a displacement of cortical PAR-6::GFP toward the anterior, without affecting the velocity of actomyosin movement. We also found that a pool of PAR-3/PAR-6/PKC-3 complex colocalized with RAB-5 positive endosomes. Interestingly, TIRF imaging revealed that PAR-6::GFP cortical association is highly dynamic in wild-type embryos and that this association is perturbed after rab-5 depletion. Finally, depleting rab-5 in mutant embryos with reduced ac tomyosin contractility resulted in an increase in PAR-6 mislocalization. Taken together, these results suggest that a RAB-5-dependent endocytic pathway contributes to polarity establishment in C. elegans by localizing anterior PAR proteins in parallel to actomyosin contractility. Since anterior PAR proteins are themselves positive regulators of endocytosis, our results reflect the possible existence of a feedback mechanism involved in embryo polarization.

Vincent Bertrand et al. (2007) International Worm Meeting "Acquisition of subtype specific features by a single interneuron in C. elegans."

Vincent Bertrand, Oliver Hobert

[International Worm Meeting, 2007]

One characteristic of the nervous system is its high diversity of neuronal subtypes. How this diversity is generated is the object of intense studies in several organisms. In C. elegans, the identity, connectivity and lineage of every neuron is known and thus it constitutes an excellent system to address this question. We are using the well-characterised AIY interneuron to analyse how pan-neuronal and neuronal subtype-specific features are acquired during embryonic development. A battery of at least 40 subtype-specific genes expressed in the AIY interneuron has recently been identified. These genes are directly activated in the post-mitotic AIY interneuron by two homeobox transcription factors TTX-3 and CEH-10 that cooperate to bind to their promoters. A key question to understand the acquisition of the specific identity of AIY is to determine how ttx-3 and ceh-10 are initially co-activated in the AIY interneuron during embryonic development. Via a combination of loss of function and promoter analysis experiments, we have shown that ttx-3 and ceh-10 are activated in the AIY lineage in a sequential manner: ttx-3 is first activated in the AIY precursor before its last cleavage (mid-embryogenesis), while ceh-10 is activated later in the postmitotic AIY interneuron in a ttx-3-dependent manner. We are now conducting a genetic screen in order to identify new genes involved in AIY identity specification. By using a worm sorting machine, we have screened 90 000 genomes so far and recovered 12 mutants affecting the AIY interneuron fate. We have cloned one of those mutants and shown that it affects a zinc finger transcription factor which directly activates ttx-3 expression in the AIY precursor. We are now extending the genetic screen and pursuing the analysis of the new mutants isolated. This study should lead to a better understanding, at a cellular and promoter resolution level, of neuronal subtype identity acquisition during embryonic development.