Watson JD et al. (2008) BMC Genomics "Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system."

Dexheimer PJ, Wang S, Miller DM, Kurn N, Watson JD, Spencer WC, Von Stetina SE, Heath JD, Levy S

[BMC Genomics, 2008]

ABSTRACT: BACKGROUND: DNA microarrays provide a powerful method for global analysis of gene expression. The application of this technology to specific cell types and tissues, however, is typically limited by small amounts of available mRNA, thereby necessitating amplification. Here we compare microarray results obtained with two different methods of RNA amplification to profile gene expression in the C. elegans larval nervous system. RESULTS: We used the mRNA-tagging strategy to isolate transcripts specifically from C. elegans larval neurons. The WT-Ovation Pico System (WT-Pico) was used to amplify 2 ng of pan-neural RNA to produce labeled cDNA for microarray analysis. These WT-Pico-derived data were compared to microarray results obtained with a labeled aRNA target generated by two rounds of In Vitro Transcription (IVT) of 25 ng of pan-neural RNA. WT-Pico results in a higher fraction of present calls than IVT, a finding consistent with the proposal that DNA-DNA hybridization results in lower mismatch signals than the RNA-DNA heteroduplexes produced by IVT amplification. Microarray data sets from these samples were compared to a Reference profile of all larval cells to identify transcripts with elevated expression in neurons. These results were validated by the high proportion of known neuron-expressed genes detected in these profiles and by promoter-GFP constructs for previously uncharacterized genes in these data sets. Together, the IVT and WT-Pico methods identified 2,173 unique neuron-enriched transcripts. Only about half of these transcripts (1,044), however, are detected as enriched by both IVT and WT-Pico amplification. CONCLUSION: We show that two different methods of RNA amplification, IVT and WT-Pico, produce valid microarray profiles of gene expression in the C. elegans larval nervous system with a low rate of false positives. However, our results also show that each method of RNA amplification detects a unique subset of bona fide neural-enriched transcripts and thus a wider array of authentic neural genes are identified by the combination of these data sets than by the microarray profiles obtained with either method of RNA amplification alone. With its relative ease of implementation and greater sensitivity, WT-Pico is the preferred method of amplification for cases in which sample RNA is limiting.

Earls LR et al. (2010) Proc Natl Acad Sci U S A "Coenzyme Q protects Caenorhabditis elegans GABA neurons from calcium-dependent degeneration."

Miller DM, Hacker ML, Earls LR, Watson JD

[Proc Natl Acad Sci U S A, 2010]

Mitochondria are key regulators of cell viability and provide essential functions that protect against neurodegenerative disease. To develop a model for mitochondrial-dependent neurodegeneration in Caenorhabditis elegans, we used RNA interference (RNAi) and genetic ablation to knock down expression of enzymes in the Coenzyme Q (CoQ) biosynthetic pathway. CoQ is a required component of the ATP-producing electron transport chain in mitochondria. We found that reduced levels of CoQ result in a progressive uncoordinated (Unc) phenotype that is correlated with the appearance of degenerating GABA neurons. Both the Unc and degenerative phenotypes emerge during late larval development and progress in adults. Neuron classes in motor and sensory circuits that use other neurotransmitters (dopamine, acetylcholine, glutamate, serotonin) and body muscle cells were less sensitive to CoQ depletion. Our results indicate that the mechanism of GABA neuron degeneration is calcium-dependent and requires activation of the apoptotic gene, ced-4 (Apaf-1). A molecular cascade involving mitochondrial-initiated cell death is also consistent with our finding that GABA neuron degeneration requires the mitochondrial fission gene, drp-1. We conclude that the cell selectivity and developmental progression of CoQ deficiency in C. elegans indicate that this model may be useful for delineating the role of mitochondrial dysfunction in neurodegenerative disease.

Visentin C et al. (2017) Hum Mol Genet "Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model."

Natalello A, Regonesi ME, Gatta E, Pellistri F, Bonanomi M, Penco A, Relini A, Visentin C, Tortora P, Vertemara J, Airoldi C, De Gioia L

[Hum Mol Genet, 2017]

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e., (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analyzed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compound prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.

Smith CJ et al. (2012) Nat Neurosci "Netrin (UNC-6) mediates dendritic self-avoidance."

Miller DM, Colon-Ramos DA, Watson JD, Smith CJ, VanHoven MK

[Nat Neurosci, 2012]

Dendrites from a single neuron may be highly branched but typically do not overlap. Self-avoidance behavior has been shown to depend on cell-specific membrane proteins that trigger mutual repulsion. Here we report the unexpected discovery that a diffusible cue, the axon guidance protein UNC-6 (Netrin), is required for self-avoidance of sister dendrites from the PVD nociceptive neuron in Caenorhabditis elegans. We used time-lapse imaging to show that dendrites fail to withdraw upon mutual contact in the absence of UNC-6 signaling. We propose a model in which the UNC-40 (Deleted in Colorectal Cancer; DCC) receptor captures UNC-6 at the tips of growing dendrites for interaction with UNC-5 on the apposing branch to induce mutual repulsion. UNC-40 also responds to dendritic contact through another pathway that is independent of UNC-6. Our findings offer a new model for how an evolutionarily conserved morphogenic cue and its cognate receptors can pattern a fundamental feature of dendritic architecture.

Petersen SC et al. (2011) J Neurosci "A transcriptional program promotes remodeling of GABAergic synapses in Caenorhabditis elegans."

Miller DM, Sarov M, Watson JD, Petersen SC, Walthall WW, Richmond JE

[J Neurosci, 2011]

Although transcription factors are known to regulate synaptic plasticity, downstream genes that contribute to neural circuit remodeling are largely undefined. In Caenorhabditis elegans, GABAergic Dorsal D (DD) motor neuron synapses are relocated to new sites during larval development. This remodeling program is blocked in Ventral D (VD) GABAergic motor neurons by the COUP-TF (chicken ovalbumin upstream promoter transcription factor) homolog, UNC-55. We exploited this UNC-55 function to identify downstream synaptic remodeling genes that encode a diverse array of protein types including ion channels, cytoskeletal components, and transcription factors. We show that one of these targets, the Iroquois-like homeodomain protein, IRX-1, functions as a key regulator of remodeling in DD neurons. Our discovery of irx-1 as an unc-55-regulated target defines a transcriptional pathway that orchestrates an intricate synaptic remodeling program. Moreover, the well established roles of these conserved transcription factors in mammalian neural development suggest that a similar cascade may also control synaptic plasticity in more complex nervous systems.

Sundararajan L et al. (2019) PLoS Genet "Actin assembly and non-muscle myosin activity drive dendrite retraction in an UNC-6/Netrin dependent self-avoidance response."

Smith CJ, Miller DM, Tyska MJ, Millis BA, Sundararajan L, Watson JD

[PLoS Genet, 2019]

Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.

Von Stetina SE et al. (2007) Genome Biol "Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system."

Von Stetina SE, Fox RM, Olszewski KL, Miller DM, Roy PJ, Watson JD, Spencer WC

[Genome Biol, 2007]

ABSTRACT: BACKGROUND: With its fully sequenced genome and simple, well-defined nervous system, the nematode C. elegans offers a unique opportunity to correlate gene expression with neuronal differentiation. The lineal origin, cellular morphology and synaptic connectivity of each of the 302 neurons are known. In many instances, specific behaviors can be attributed to particular neurons or circuits. Here we describe microarray-based methods that monitor gene expression in C. elegans neurons and thereby link comprehensive profiles of neuronal transcription to key developmental and functional properties of the nervous system. RESULTS: We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Microarray Profiling C. elegans cells) method, we used Fluorescence Activated Cell Sorting (FACS) to isolate GFP-tagged embryonic neurons for microarray analysis. To profile the larval nervous system, we used the mRNA-tagging technique in which an epitope-labeled mRNA binding protein (FLAG-PAB-1) was transgenically expressed in neurons for immunoprecipitation of cell-specific transcripts. These combined approaches identified approximately 2,500 mRNAs that are highly enriched in either the embryonic or larval C. elegans nervous system. These data are validated in part by the detection of gene classes (e.g. transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. Of particular interest are 19 conserved transcripts of unknown function that are also expressed in the mammalian brain. In addition to utilizing these profiling approaches to define stage-specific gene expression, we also applied the mRNA-tagging method to fingerprint a specific neuron type, the A-class group of cholinergic motor neurons, during early larval development. A comparison of these data to a MAPCeL profile of Embryonic A-class motor neurons identified genes with common functions in both types of A-class motor neurons as well as transcripts with roles specific to each motor neuron type. Conclusion: We describe microarray-based strategies for generating expression profiles of embryonic and larval C. elegans neurons. These methods can be applied to particular neurons at specific developmental stages and therefore provide an unprecedented opportunity to obtain spatially and temporally defined snapshots of gene expression in a simple model nervous system.

Smith CJ et al. (2010) Dev Biol "Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans."

Watson JD, Albeg A, Spencer WC, Smith CJ, Miller DM, O'Brien T, Cha B, Treinin M

[Dev Biol, 2010]

Nociceptive neurons innervate the skin with complex dendritic arbors that respond to pain-evoking stimuli such as harsh mechanical force or extreme temperatures. Here we describe the structure and development of a model nociceptor, the PVD neuron of C. elegans, and identify transcription factors that control morphogenesis of the PVD dendritic arbor. The two PVD neuron cell bodies occupy positions on either the right (PVDR) or left (PVDL) sides of the animal in posterior-lateral locations. Imaging with a GFP reporter revealed a single axon projecting from the PVD soma to the ventral cord and an elaborate, highly branched arbor of dendritic processes that envelop the animal with a web-like array directly beneath the skin. Dendritic branches emerge in a step-wise fashion during larval development and may use an existing network of peripheral nerve cords as guideposts for key branching decisions. Time-lapse imaging revealed that branching is highly dynamic with active extension and withdrawal and that PVD branch overlap is prevented by a contact-dependent self-avoidance, a mechanism that is also employed by sensory neurons in other organisms. With the goal of identifying genes that regulate dendritic morphogenesis, we used the mRNA-tagging method to produce a gene expression profile of PVD during late larval development. This microarray experiment identified>2,000 genes that are 1.5X elevated relative to all larval cells. The enriched transcripts encode a wide range of proteins with potential roles in PVD function (e.g., DEG/ENaC and Trp channels) or development (e.g., UNC-5 and LIN-17/frizzled receptors). We used RNAi and genetic tests to screen 86 transcription factors from this list and identified eleven genes that specify PVD dendritic structure. These transcription factors appear to control discrete steps in PVD morphogenesis and may either promote or limit PVD branching at specific developmental stages. For example, time-lapse imaging revealed that MEC-3 (LIM homeodomain) is required for branch initiation in early larval development whereas EGL-44 (TEAD domain) prevents ectopic PVD branching in the adult. A comparison of PVD-enriched transcripts to a microarray profile of mammalian nociceptors revealed homologous genes with potentially shared nociceptive functions. We conclude that PVD neurons display striking structural, functional and molecular similarities to nociceptive neurons from more complex organisms and can thus provide a useful model system in which to identify evolutionarily conserved determinants of nociceptor fate.

Amigoni, L. et al. (2019) International Worm Meeting "Caenorhabditis elegans as a model for searching compounds capable of preventing Spinocerebellar Ataxia Type 3."

Bonanomi, M., Visentin, C., Sciandrone, B., Airoldi, C., Amigoni, L., Tortora, P., Regonesi, M.E., Natalello, A.

[International Worm Meeting, 2019]

Ataxin-3 (ATX3) is the protein that triggers the neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3) when its polyglutamine stretch exceeds the critical length of 55 residues. This expansion results in misfolding and structural rearrangements, leading to aberrant interactions and the consequent formation of fibrillar amyloid-like aggregates. Currently, no effective treatment is available for SCA3 disease. In order to find natural compounds able to prevent ATX3 aggregation, we studied the effect of tetracycline (TET) and epigallocatechin-3-gallate (EGCG) on ATX3 fibrillogenesis. We displayed that TET increased the solubility of the different aggregated species, without affecting the secondary structures of the final aggregates. Otherwise, EGCG interfered with the early steps of aggregation, accelerating the Josephin Domain (JD) misfolding, and leading to the formation of off-pathway, non-amyloid, final aggregates. By NMR analyses, we proved that the different behavior could be related to the capability of the sole EGCG to bind the monomeric JD. Successively, among a small library of tetracycline, we identified methacycline (MET) as the most effective compound, which exerts the same mechanism of action of TET. The pharmacological efficacy of EGCG, TET and MET has been evaluated on a SCA3 C. elegans model and we demonstrated that all compounds induced a significant increase in mobility without extending the lifespan. MET was proven to be the most effective in relieving the locomotion defects. Even more remarkably, MET treatment also resulted in an ameliorated motility even in wild-type worms suggesting that MET fulfills its action via diverse mechanisms, besides interfering with amyloid aggregation. Furthermore, the drug did not significantly modify the life span in any of the strains investigated, which supports the expectation that it may be devoid of toxic effects in long-term administration.

JD Kormish et al. (1999) International C. elegans Meeting "A genetic screen for genes involved in gut development and differentiation"

JD McGhee, JD Kormish

[International C. elegans Meeting, 1999]

To investigate genes potentially involved in gut development and differentiation, we are developing a genetic screen for gut obstructed (gob) mutants. The gut specific elt-2 GATA factor appears necessary for correct gut cell development. elt-2 null mutants arrest as L1s with a blocked intestinal lumen and abnormal brush border (1). When elt-2 null mutants are fed on a mixture of fluorescent beads and Escherichia coli , the beads collect as a large "gob" in the posterior pharynx and anterior gut that can be easily detected under the dissecting microscope. We are now using this gut obstructed phenotype to screen for mutants that should potentially identify genes involved in gut development. Preliminary results of this screen are promising, as several candidates have already been isolated. 1) Fukushige T, Hawkins MG and McGhee JD (1998) Dev Biol 198(2):286-302