Carmela Bergamasco et al. (2006) European Worm Meeting "Signalling in quinine avoidance"

Paolo Bazzicalupo, Giovanni Esposito, Carmela Bergamasco, Salvatore Arbucci

[European Worm Meeting, 2006]

Chemical sensitivity allows animals to identify and respond appropriately to the chemical composition of the environment. Noxious water-soluble compounds that are avoided by C. elegans are generally sensed as bitter by humans and are discarded in double choice test by mice. We have used C. elegans to focus on the molecular mechanisms involved in primary sensing of quinine, a molecule detected as bitter by humans. ASH is the main sensory neuron involved in sensing quinine. Two G? subunits, GPA-3 and ODR-3 are necessary for the response of ASH to repellent stimuli (Hilliard et al., 2004 and 2005). In addition the TRPV channel proteins, OSM-9 and OCR-2, are also necessary for the ASH avoidance responses (Colbert et al 1997, Tobin et al 2002). Finally we identified a novel protein, QUI-1, as an essential components of the response to quinine (Hilliard et al., 2004). With regard to the molecular function of QUI-1, we demonstrate that QUI-1 function is required in ASH for the response to quinine and, using specific antibodies, that the protein is localized to the sensory cilia. These results, together with the discovery that QUI-1 contains an RGS (Regulator of G protein Signaling) domain, strongly suggest that this novel protein might be involved in quinine signaling.. Are there other components of the quinine signal transduction pathway?. We are using a best candidate approach and a variety of behavioral assays to identify new molecules involved in sensing repellent chemicals and in particular quinine. We analyzed behaviorally loss of function and overexpression mutants in several molecules known to act in the G protein signaling pathways (G? subunits, G? subunits, RGS proteins, etc.). The results obtained will be discussed.. Colbert, H. A., Smith, T. L. and Bargmann, C. I. (1997). OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci 17, 8259-69.. Hilliard, M. A., Apicella, A. J., Kerr, R., Suzuki, H., Bazzicalupo, P. and Schafer, W. R. (2005). In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. Embo J 24, 63-72.. Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H. and Bazzicalupo, P. (2004). Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans. Embo J 23, 1101-11.. Tobin, D., Madsen, D., Kahn-Kirby, A., Peckol, E., Moulder, G., Barstead, R., Maricq, A. and Bargmann, C. (2002). Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron 35, 307-18.

Giordano-Santini, R. et al. (2013) International Worm Meeting "Studying membrane fusion during development and maintenance of C. elegans neurons."

Hilliard, M. A., Giordano-Santini, R.

[International Worm Meeting, 2013]

EFF-1 and AFF-1 are highly efficient fusogens known to mediate cell-cell fusion events during the development of different C. elegans tissues. Both fusogens are also expressed in neurons, and EFF-1 has recently been shown to play a role in the development and remodelling of neurites1,2, indicating that cell membrane fusion is an important neuronal event in C. elegans. However, very little is known about the molecular mechanisms underpinning membrane fusion within these cells; importantly, it is also unclear how this process is highly restricted to the individual cell, with fusion between neurites of adjacent neurons almost never observed. In non-neuronal tissues where cell-cell fusion occurs, numerous transcription factors and regulators have been identified as being critical for proper cell-cell fusion3. Our hypothesis is that similar mechanisms are in place in neurons, controlling fusion of neurites within individual cells and preventing neurites of adjacent neurons from fusing. We used different pairs of tightly associated neurons, AWCR/AWCL (head) as well as PLM/PLN (tail), to test this hypothesis and to study EFF-1 and AFF-1-mediated fusion in neurons. Using transgenic strains and microscopy techniques, we have found that overexpression of EFF-1 or AFF-1 under neuron-specific promoters leads to mixing of cytoplasms between individual neurons. We confirmed that the cytoplasms are indeed connected by using the photoconvertible protein Kaede. We also determined the temporal requirement of the fusogen by using a construct where EFF-1 is under a heat-shock promoter. In addition, using a fluorescent-tagged version of EFF-1, we are characterising the site of fusion between adjacent neurons when the fusogen is overexpressed. Finally, we have started a genetic screen using neuronal-specific RNAi, where genes known as cell-cell fusion regulators are investigated for their capacity to induce mixing of cytoplasms between associated neurons. The results presented here will give us new insights into the molecular mechanisms that control membrane fusion during development and remodelling of C. elegans neurons. 1Oren-Suissa et al., Science, 2010, 2Gosh-Roy et al., J. Neurosci. 2010, 3Podbilewicz, Wormbook, 2006.

Ghorashian N et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "A Parallelized Microfluidic Chamber Device for High-Throughput Nerve Regeneration Studies in C. elegans"

Ghorashian N, Hilliard M A, Ben-Yakar A

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

We fabricated and tested a high-throughput microfluidic platform to study nerve regeneration in C. elegans. The device consists of an array of small chambers in a parallel fluidic circuit allowing for simultaneous trapping of dozens of C. elegans worms in individual visualization chambers for in-vivo imaging and laser ablation of fluorescently labeled axons. With proper liquid nutrients, the animals can easily survive in the microfluidic chambers for three days or more for monitoring nerve regeneration. This device could serve as the optical and fluidic interface for automated genome-wide nerve regeneration studies using femtosecond laser nano-axotomy and fluorescence microscopy. Using our device and conventional methods, we investigated the regenerative capacity of the oxygen sensory neuron, PQR. This neuron is located in the left lumbar ganglion on the posterior-lateral side of the worm's body, and has only two processes emerging from the cell body – a dendrite extending posterior toward the tip of the tail and an axon extending anterior joining the ventral nerve cord. We looked at regeneration rates in animals in which either only one or both neurites were severed. We observed that the dendrite process regenerated with a higher frequency when the axon was simultaneously severed. This result suggests that the molecular machinery responsible for regeneration is more efficiently recruited in a given process when there is additional damage to other parts of the neuron.

Kirszenblat L et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Wnt signals and Frizzled receptors regulate dendrite formation in C. elegans"

Neumann B, Hilliard M A, Pattabiraman D, Kirszenblat L

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

Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. While many molecules involved in axon development have been discovered, there is little known about the ligands and receptors that regulate dendrite development. To understand how dendrites develop in C. elegans we focused on the PQR oxygen sensory neuron. PQR has its cell body positioned in the left lumbar ganglion on the posterior-lateral side of the body. A single dendrite extends posterior with sensory cilia at its tip, while the axon extends anterior along the ventral nerve cord. PQR is born post-embryonically allowing easy visualization of dendrite development using the gcy-36::GFP transgene. In a genetic screen for dendrite defective mutants we isolated a previously uncharacterized mutation in lin-17, a C. elegans Frizzled receptor gene. We found that in lin-17(vd002), the PQR dendrite was absent, shortened or misrouted anterior. Similar dendrite defects were also observed in other known alleles of lin-17. Cell-specific expression of wild-type LIN-17 in PQR indicated a non-cell-autonomous role of this molecule in regulating dendrite development. LIN-44 is a Wnt ligand known to bind the Frizzled receptor LIN-17 and is expressed by four hypodermal cells in the tip of the tail. We found that lin-44 mutants presented PQR dendrite defects similar to those observed in lin-17 mutants. We expressed LIN-44 ectopically from more anterior regions of the body and found that it rescued the PQR dendrite defects of lin-44 mutants, indicating LIN-44 functions as a permissive cue. Using a heat-shock promoter to drive LIN-44 we determined that the presence of this ligand at the time of PQR dendrite formation was sufficient to rescue the dendrite defects of the lin-44 mutant. Analysis of the lin-17 lin-44 double mutant indicated a genetic interaction between these molecules. Our studies provide the first direct evidence that specific Wnt signals and Frizzled receptors regulate dendrite formation in vivo. We propose a model in which PQR dendrite formation is achieved by the interaction of LIN-44 and LIN-17 acting on PQR through its neighbouring cells.

Bonacossa-Pereira, I. et al. (2019) International Worm Meeting "Neuron-glia interaction: remodelling axonal attachment following axonal injury."

Hilliard, M. A., Bonacossa-Pereira, I., Coakley, S.

[International Worm Meeting, 2019]

Distinct cell types adhere to each other to form functional systems in an organism. Specifically, in vertebrates, ensheathing glia attach to axons forming a functional unit that responds to injury. In C. elegans, the posterior lateral mechanosensory (PLM) neurons have an intimate relationship with their surrounding epidermis. During development, the PLM axon becomes ensheathed within the overlying epidermis and is mechanically coupled to this tissue via specialized trans-epidermal attachment structures. The transmembrane protein LET-805/Myotactin is a component of these attachment structures, and is proposed to be required for correct attachment of the mechanosensory neuron axon to the epidermis as well as for the attachment of the epidermis to the body wall muscles. We visualized the localisation of LET-805, and attachment sites, using a CRISPR/Cas9 engineered C-terminal wrmScarlet tag in a wild-type background, together with a PLM neuron-specific cytosolic GFP marker. To characterize the role of neuronal attachment in axonal maintenance and repair after axonal injury, we axotomized the PLM neuron using UV-laser and visualized LET-805::wrmScarlet before, during, and after injury. In uninjured animals at the L4 stage, LET-805::wrmScarlet localized to periodic puncta over the PLM axon. After injury, we observed that LET-805::wrmScarlet was slowly lost in regions corresponding to the disconnected distal axon fragment of PLM, often occurring after the loss of any visible cytosolic neuronal marker. On the regrowing proximal axon, we observed that LET-805::wrmScarlet did not localize to the newly regrowing axons for at least 48 hours, after which it reassembled into puncta following the path traced by the regrowing axon. Taken together, our data suggests that following injury the attachment of the PLM neuron to its surrounding tissue is maintained and is not highly dynamic. We propose that the regrowing axonal fragment induces re-attachment to the epidermis. We are currently testing whether modulation of axonal attachment impacts axonal degeneration or regeneration in this system, and how injuries on PLM axon are detected by the surrounding epidermis.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

[Development & Evolution Meeting, 2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Neumann B et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Axonal Regeneration Proceeds Through Axonal Fusion in C. elegans Neurons"

Hilliard M A, Neumann B, Ben-Yakar A, Hall D H, Nguyen K CQ

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

Invertebrate axons and those of the mammalian peripheral nervous system are able to regenerate in the adult. Functional recovery takes place when a damaged axon regains connection with its target tissues. In C. elegans, following laser axotomy, the regrowing axon still attached to cell body (proximal) is able to reconnect with its separated distal segment through unknown mechanisms. Using the mechanosensory neurons ALM and PLM as a model system, we have found that during axonal regeneration reconnection between the proximal and distal axonal fragments occurs through a mechanism of axonal fusion, with reestablishment of cytoplasmic and membrane continuity. We found that when axonal fusion does not occur the distal fragment inevitably undergoes Wallerian degeneration and the original axonal tract cannot be restored. Through the use of dual colour labeling of adjacent axonal pairs, we found a high level of specific recognition occurring between a proximal re-growing axon and its own separated distal fragment, revealing possible cross talk between the two processes. Finally, from a candidate mutant approach, we have identified a molecule with homology to a human protein implicated in axonal degeneration, as being necessary for successful regeneration and specifically involved in the process of axonal fusion. We anticipate that a similar mechanism of axonal regeneration could be exploited to improve the outcome of axonal regeneration following injury in mammalian systems.

Carmela Bergamasco et al. (2005) International Worm Meeting "Quinine avoidance: cells and molecules involved"

Carmela Bergamasco, Giovanni Esposito, Paolo Bazzicalupo

[International Worm Meeting, 2005]

We are interested in elucidating the cellular and molecular basis of the avoidance behavior generated by repellents in C. elegans. Among the repellents we particularly focused on quinine which is poisonous for cells and organisms and is perceived as bitter by humans. ASH is the main sensory neuron mediating quinine avoidance. To identify the molecules that, inside ASH, are necessary for sensing quinine we are following two approaches, the identification of new genes with forward screens and a best candidate approach. In the first one, to obtain new quinine-non-avoider mutants we conducted a clonal screen after EMS mutagenesis. We screened single F2 worms using the drop test (Hilliard et al., 2002). This screen allows one to identify non-avoider mutants largely independently of other influences (adaptation, defective movement, social behavior, etc.). We identified 17 new mutants. Two of them do not complement grk-2 (rt97) which has been shown to mediate also octanol avoidance (Fukuto et al., 2004) and three of the mutants, do not complement qui-1 (gb404), a gene identified in a previous screen and directly involved in quinine avoidance (Hilliard et al., 2004). QUI-1 is a novel protein containing in N-terminal region a Nacht Domain, (a predicted nucleoside triphosphatase (NTPase) domain) and in C-terminal region 12 WD repeats. Expression studies based on reporters indicate that it is expressed in the ASH and ADL sensory neurons, which we know are involved in sensing quinine and other repellents. Using cell specific promoter, we showed that expression of QUI-1 in ASH is sufficient to rescue the quinine avoidance phenotype. To begin to understand the function of qui-1 we are carrying out genetic experiments to explore its role in quinine signalling; we have obtained specific antibodies and are using them to identify QUI-1 cellular localization; finally we are performing two hybrid experiments to identify some of its partners. In the second approach we have begun to test if mutants in genes involved in sensory signal transduction are involved in quinine signalling. We had already determined that two Ga proteins, GPA-3 and ODR-3, (Hilliard et al. 2004.) are involved in quinine avoidance. With different approaches we have also studied the role in the avoidance response of osm-9, egl-19 and gpc-1 (Hilliard et al., 2005). Thus, together with QUI-1 and GRK-2 we have identified several players acting within ASH to mediate avoidance and are trying to come up with a reasonable and testable model to explain their interaction and functioning.

Donato, A. et al. (2015) International Worm Meeting "Molecular and cellular mechanisms regulating axonal compartmentalization in C. elegans RIA interneurons."

Ren, J., Donato, A., Zhang, Y., Hilliard, M. A.

[International Worm Meeting, 2015]

Neurons are highly compartmentalized cells consisting of three main structural and functional domains, cell body (soma), axon, and dendrites. In the last decade, increasing experimental evidence has revealed that additional levels of compartmentalization are in place within the processes, providing higher computational potential than previously thought. However, little is known on the molecular and cellular mechanisms that regulate the establishment of these subcellular compartments during development and their maintenance during the lifetime of the animals. A recent study from Yun Zhang's laboratory has shown that the unipolar RIA interneurons display distinct compartmentalized activity in different axonal domains. Remarkably, calcium activity in specific axonal domains is correlated with the direction of the dorsal-ventral head bending and the compartmental activity regulates the amplitude of the head bending (Hendricks et al., 2012). These results reveal the properties and function of RIA axonal compartmentalization. Here, we propose to elucidate the underlying molecular and cellular mechanisms. First, we tested the hypothesis that the functional compartmentalization of RIA results from compartmentalized localization of the muscarinic acetylcholine receptor GAR-3. Previously, it has been shown that the axonal compartmentalized activity of RIA requires the synaptic output of the cholinergic head motor neurons SMDD/V and the function of GAR-3 in RIA (Hendricks et al., 2012). Thus, we analysed the distribution of the GAR-3 receptor in RIA, and found that its localisation is diffused and does not correlate with compartmentalized calcium activity, suggesting different underlying mechanisms. Second, we tested the hypothesis that specific localization of mitochondria in RIA may regulate RIA compartmental activity by buffering calcium waves in certain axonal domains. We found that mitochondria distribution in RIA is not consistent with a potential role in generating calcium compartmentalisation in this cell. Finally, using the GFP reconstruction across synaptic partners (GRASP) (Feinberg et al., 2008), we have specifically labelled the synaptic contacts between SMDs and RIAs, which will allow us to investigate if the calcium compartmentalisation is governed by the precise synaptic organisation in the RIA axon and how changing in the synaptic organisation can affect neuronal activity and therefore the animal behaviour. .

Nirav M Amin et al. (2005) International Worm Meeting "fozi-1, a gene required for proper development of the postembryonic mesoderm."

Jun Liu, Nirav M Amin, Kejin Hu, Dino Terzic

[International Worm Meeting, 2005]

Our lab is studying mesodermal patterning and cell fate specification by using the C. elegans M lineage as a model system. The M lineage arises from a single precursor cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic divisions to form 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). The SMs then migrate to the vulval region and further divide to give rise to uterine and vulval muscles. In a screen for mutants affecting the proper development of the M lineage (mesodermal lineage specification or mls mutants), we isolated four mutations of the same complementation group on chromosome III, cc607, cc608, cc609, and cc610. All four mutant alleles resulted in a terminal phenotype of missing M-derived CCs and extra SMs. Preliminary lineage analysis using M lineage specific GFP markers on the strongest allele cc609 revealed patterning defects after the third division of M. We are currently further characterizing these defects. Using a combination of recombination mapping, cosmid rescue, and RNAi, we identified the corresponding wild-type gene as K01B6.1. All four of our mutant alleles contained either point mutations or a deletion in the coding region of K01B6.1, further confirming this identity. K01B6.1 encodes a putative transcription factor with a glutamine rich region, a C2H2 zinc finger, and a large C-terminal FH2 (Formin Homology 2) domain. Thus we have named this gene fozi-1, standing for formin zinc finger protein-1. We are currently in the process of analyzing the expression pattern of fozi-1 by using both antibodies and GFP fusion constructs. In addition to its function in the M lineage, FOZI-1 also plays a role in other processes during C. elegans development, as a fozi-1 allele was identified in a screen by the Hobert lab for mutants that affect ASEL/ASER specification (personal communication).