Dendrites from a single neuron may be highly branched but typically do not overlap. This self-avoidance behavior has been shown to depend on cell-specific membrane proteins that trigger mutual repulsion. Here we report the surprising 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 C. elegans. We used time lapse imaging to show that dendrites fail to withdraw upon mutual contact in the absence of UNC-6/Netrin signaling. We propose a model in which the UNC-40/DCC receptor captures UNC-6/Netrin at the tips of growing dendrites for interaction with UNC-5 on the apposing branch to induce mutual repulsion. UNC-40/DCC also responds to dendritic contact through an additional pathway that is independent of UNC-6/Netrin. 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.
Although transcription factors are known to regulate synaptic plasticity, downstream genes that contribute to neural circuit remodeling are largely undefined. In C. 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 homologue, 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.
New genotyping scheme facilitates classification of virus sequences.
Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes fatal encephalitis in humans. The initial outbreak of NiV infection occurred in Malaysia and Singapore in 1998–1999; relatively small, sporadic outbreaks among humans have occurred in Bangladesh since 2001. We characterized the complete genomic sequences of identical NiV isolates from 2 patients in 2008 and partial genomic sequences of throat swab samples from 3 patients in 2010, all from Bangladesh. All sequences from patients in Bangladesh comprised a distinct genetic group. However, the detection of 3 genetically distinct sequences from patients in the districts of Faridpur and Gopalganj indicated multiple co-circulating lineages in a localized region over a short time (January–March 2010). Sequence comparisons between the open reading frames of all available NiV genes led us to propose a standardized protocol for genotyping NiV; this protcol provides a simple and accurate way to classify current and future NiV sequences.
Nipah; Nipah virus; outbreak; encephalitis; phylogeny; viruses; Bangladesh
PVD and FLP sensory neurons envelope the body of the C. elegans adult with a highly branched network of thin sensory processes. Both PVD and FLP neurons are mechanosensors. PVD is known to mediate the response to high threshold mechanical stimuli. Thus PVD and FLP neurons are similar in both morphology and function to mammalian nociceptors. To better understand the function of these neurons we generated strains lacking them. Behavioral analysis shows that PVD and FLP regulate movement under normal growth conditions, as animals lacking these neurons demonstrate higher dwelling behavior. In addition, PVD—whose thin branches project across the body-wall muscles—may have a role in proprioception, as ablation of PVD leads to defective posture. Moreover, movement-dependent calcium transients are seen in PVD, a response that requires MEC-10, a subunit of the mechanosensory DEG/ENaC channel that is also required for maintaining wild-type posture. Hence, PVD senses both noxious and innocuous signals to regulate C. elegans behavior, and thus combines the functions of multiple mammalian somatosensory neurons. Finally, strong mechanical stimulation leads to inhibition of egg-laying, and this response also depends on PVD and FLP neurons. Based on all these results we suggest that noxious signals perceived by PVD and FLP promote an escape behavior consisting of increased speed, reduced pauses and reversals, and inhibition of egg-laying.
C. elegans; somatosensory system; nociceptor; proprioceptor; behavior; movement
Transplantation of allogeneic organs has proven to be an effective therapeutic for a large variety of disease states, but the chronic immunosuppression that is required for organ allograft survival increases the risk for infection and neoplasia and has direct organ toxicity. The establishment of transplantation tolerance, which obviates the need for chronic immunosuppression, is the ultimate goal in the field of transplantation. Many experimental approaches have been developed in animal models that permit long-term allograft survival in the absence of chronic immunosuppression. These approaches function by inducing peripheral or central tolerance to the allograft. Emerging as some of the most promising approaches for the induction of tolerance are protocols based on costimulation blockade. However, as these protocols move into the clinic, there is recognition that little is known as to their safety and efficacy when confronted with environmental perturbants such as virus infection. In animal models, it has been reported that virus infection can prevent the induction of tolerance by costimulation blockade and, in at least one experimental protocol, can lead to significant morbidity and mortality. In this review, we discuss how viruses modulate the induction and maintenance of transplantation tolerance.
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 1.5 X 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 the 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.
Dendritic morphogenesis; nociceptor development; transcription factors
Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC50 of ~10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.
“Humanized” mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rγnull mutation; NOD-scid IL2rγnull, NOD-Rag1null IL2rγnull, and BALB/c-Rag1null IL2rγnull mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.
Humanized mice; SCID; hematopoietic stem cells; IL-2R “common” gamma chain
Polymodal nociceptors detect noxious stimuli including harsh touch, toxic chemicals, and extremes of heat and cold. The molecular mechanisms by which nociceptors are able to sense multiple qualitatively distinct stimuli are not well-understood. We show here that the C. elegans PVD neurons are mulitidendritic nociceptors that respond to harsh touch as well as cold temperatures. The harsh touch modality specifically requires the DEG/ENaC proteins MEC-10 and DEGT-1, which represent putative components of a harsh touch mechanotransduction complex. By contrast, responses to cold require the TRPA-1 channel and are MEC-10- and DEGT-1-independent. Heterologous expression of C. elegans TRPA-1 can confer cold responsiveness to other C. elegans neurons or to mammalian cells, indicating that TRPA-1 is itself a cold sensor. These results show that C. elegans nociceptors respond to thermal and mechanical stimuli using distinct sets of molecules, and identify DEG/ENaC channels as potential receptors for mechanical pain.
In an effort to identify genes that specify the mammalian forebrain, we used a comparative approach to identify mouse homologs of transcription factors expressed in developing Caenorhabditis elegans GABAergic neurons. A cell-specific microarray profiling study revealed a set of transcription factors that are highly expressed in embryonic C. elegans GABAergic neurons.
Bioinformatic analyses identified mouse protein homologs of these selected transcripts and their expression pattern was mapped in the mouse embryonic forebrain by in situ hybridization. A review of human homologs indicates several of these genes are potential candidates in neurodevelopmental disorders.
Our comparative approach has revealed several novel candidates that may serve as future targets for studies of mammalian forebrain development.
The rising prevalence of methylmercury (MeHg) in seafood and in the global environment provides an impetus for delineating the mechanism of the toxicity of MeHg. Deleterious effects of MeHg have been widely observed in humans and in other mammals, the most striking of which occur in the nervous system. Here we test the model organism, Caenorhabditis elegans (C. elegans), for MeHg toxicity. The simple, well-defined anatomy of the C. elegans nervous system and its ready visualization with green fluorescent protein (GFP) markers facilitated our study of the effects of methylmercuric chloride (MeHgCl) on neural development. Although MeHgCl was lethal to C. elegans, induced a developmental delay, and decreased pharyngeal pumping, other traits including lifespan, brood size, swimming rate, and nervous system morphology were not obviously perturbed in animals that survived MeHgCl exposure. Despite the limited effects of MeHgCl on C. elegans development and behavior, intracellular mercury (Hg) concentrations (≤ 3 ng Hg/mg protein) in MeHgCl-treated nematodes approached levels that are highly toxic to mammals. If MeHgCl reaches these concentrations throughout the animal, this finding indicates that C. elegans cells, particularly neurons, may be less sensitive to MeHgCl toxicity than mammalian cells. We propose, therefore, that C. elegans should be a useful model for discovering intrinsic mechanisms that confer resistance to MeHgCl exposure.
Caenorhabditis elegans; methylmercury
Activation of TLR4 by administration of LPS shortens the survival of skin allografts in mice treated with costimulation blockade through a CD8 T cell-dependent, MyD88-dependent, and type I IFN receptor-dependent pathway. The effect of TLR activation on the establishment of allogeneic hematopoietic chimerism in mice treated with costimulation blockade is not known. Using a costimulation blockade protocol based on a donor-specific transfusion (DST) and a short course of anti-CD154 mAb, we show that LPS administration at the time of DST matures host alloantigen-presenting dendritic cells, prevents the establishment of mixed allogeneic hematopoietic chimerism, and shortens survival of donor-specific skin allografts. LPS mediates its effects via a mechanism that involves both CD4+ and CD8+ T cells and results from signaling through either the MyD88 or the type I IFN receptor pathways. We also document that timing of LPS administration is critical, as injection of LPS 24 h before treatment with DST and anti-CD154 mAb does not prevent hematopoietic engraftment but administration the day after bone marrow transplantation does. We conclude that TLR4 activation prevents the induction of mixed allogeneic hematopoietic chimerism through type I IFN receptor and MyD88-dependent signaling, which leads to the up-regulation of costimulatory molecules on host APCs and the generation of alloreactive T cells. These data suggest that distinct but overlapping cellular and molecular mechanisms control the ability of TLR agonists to block tolerance induction to hematopoietic and skin allografts in mice treated with costimulation blockade.
We investigated the mechanisms by which toll-like receptor (TLR) agonists affect the induction of mixed chimerism and skin allograft survival in mice treated with costimulation blockade (CB). We report that TLR agonists prevent the generation of mixed chimerism by breaking tolerance in the alloreactive CD4+ and CD8+ T cell compartments, and that type I interferon (IFN) is important in this process. Understanding how environmental perturbations affect CB-induced transplantation tolerance may lead to more effective regimens that can be used as an approach for the treatment of type 1 diabetes, for which the transplantation of pancreatic islets is a promising therapy.
costimulation blockade; Toll-like receptors; hematopoietic chimerism; tolerance
Bacterial quorum sensing is mediated by low molecular-weight signals and plays a critical role in both the pathogenesis of infectious disease and beneficial symbioses. There is significant interest in the development of synthetic ligands that can intercept bacterial quorum sensing signals and modulate these outcomes. Here, we report the design and comparative analysis of the effects of ~ 90 synthetic N-acylated homoserine lactones (AHLs) on quorum sensing in three Gram negative bacterial species and a critical examination of the structural features of these ligands that dictate agonistic and antagonistic activity, and selectivity for different R protein targets. These studies have revealed the most comprehensive set of structure–activity relationships to date that direct AHL-mediated quorum sensing and a new set of chemical probes with which to study this complex signaling process. Furthermore, this work provides a foundation on which to design next-generation quorum sensing modulators with improved activities and selectivities.
combinatorial chemistry; Gram-negative bacteria; homoserine lactones; quorum sensing; structure activity relationships
Bacteria use a language of low molecular weight ligands to assess their population densities in a process called quorum sensing. This chemical signaling process plays a pivotal role both in the pathogenesis of infectious disease and in beneficial symbioses. There is intense interest in the development of synthetic ligands that can intercept quorum-sensing signals and attenuate these divergent outcomes. Both broad-spectrum and species-selective modulators of quorum sensing hold significant value as small-molecule tools for fundamental studies of this complex cell–cell signaling process and for future biomedical and environmental applications. Here, we report the design and synthesis of focused collections of non-native N-acylated homoserine lactones and the systematic evaluation of these ~90 ligands across three Gram-negative bacterial species: the pathogens Agrobacterium tumefaciens and Pseudomonas aeruginosa; the model symbiont Vibrio fischeri. This study is the first to report and compare the activities of a set of ligands across multiple species and has revealed some of the most potent synthetic modulators of quorum sensing to date. Moreover, several of these ligands exhibit agonistic or antagonistic activity in all three species, while other ligands are only active in one or two species. Analysis of the screening data revealed that at least a subset of these ligands modulate quorum sensing via a partial agonism mechanism. We also demonstrate that selected ligands can either inhibit or promote the production of elastase B, a key virulence factor in wild-type P. aeruginosa, depending on their concentrations. Overall, this work provides broad insights into the molecular features required for small-molecule inhibition or activation of quorum sensing in Gram-negative bacteria. In addition, this study has supplied an expansive set of chemical tools for the further investigation of quorum-sensing pathways and responses.
In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.
Marburg virus, similar to its close cousin Ebola virus, can cause large outbreaks of hemorrhagic fever (HF) in rural Africa with case fatalities approaching 90%. For decades, a long-standing enigma has been the identity of the natural reservoir of this deadly virus. In this report, we identify the cave-dwelling Egyptian fruit bat (Rousettus aegyptiacus) as a natural host of Marburg virus based on multiple lines of evidence which include, for the first time ever, the isolation of virus directly from wild-caught and apparently healthy bats. The species R. aegyptiacus is common throughout Africa with distribution into the eastern Mediterranean and Middle East. Our finding of active virus infection in approximately 5% of R. aegyptiacus bats and their population exceeding 100,000 in Kitaka cave in Uganda suggests there are likely over 5,000 Marburg virus–infected bats in this cave, which is only one of many such cave populations throughout Africa. Clearly, these bats could serve as a major source of virus with potential to initiate human epidemics, and the implications for public health are striking. Additionally, we found highly divergent (21%) genome sequences among viruses circulating in these bat populations, a level of diversity that would result from a long-term association with a suitable reservoir host of large population size.
A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.
Muscular diseases affect many people worldwide. While we have learned much about the sarcomere, the basic building block of muscle cells, there are still numerous questions that remain to be answered. We must learn more about proteins expressed in muscle and how they interact so that better treatments for myopathies can be developed. The nematode Caenorhabditis elegans is a valuable model organism for the study of muscle due to similarities between worm body wall muscle and vertebrate muscle, along with its semi-transparent cuticle that allows for visualization of muscle structures in live animals. We have used transcriptional profiling methods to identify the majority of genes that are expressed in the embryonic body wall muscle cells of C. elegans. To gain insight into possible functions performed by these genes and their corresponding proteins, we examined animals and muscle cells for abnormalities after the targeted inactivation of about 3,300 genes. We identified 122 genes necessary for proper myofilament organization, 108 of which had no previously characterized role in muscle. This approach proved to be a rapid and sensitive means to identify genes that affect muscle differentiation and sarcomere assembly.
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.
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.
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.
Fluorescence activated cell sorting and microarray profiling were used to identify 1,312 expressed genes that are enriched in myo-3::GFP-positive muscle cells of Caenorhabditis elegans.
The force generating mechanism of muscle is evolutionarily ancient; the fundamental structural and functional components of the sarcomere are common to motile animals throughout phylogeny. Recent evidence suggests that the transcription factors that regulate muscle development are also conserved. Thus, a comprehensive description of muscle gene expression in a simple model organism should define a basic muscle transcriptome that is also found in animals with more complex body plans. To this end, we applied microarray profiling of Caenorhabtidis elegans cells (MAPCeL) to muscle cell populations extracted from developing C. elegans embryos.
We used fluorescence-activated cell sorting to isolate myo-3::green fluorescent protein (GFP) positive muscle cells, and their cultured derivatives, from dissociated early C. elegans embryos. Microarray analysis identified 7,070 expressed genes, 1,312 of which are enriched in the myo-3::GFP positive cell population relative to the average embryonic cell. The muscle enriched gene set was validated by comparisons with known muscle markers, independently derived expression data, and GFP reporters in transgenic strains. These results confirm the utility of MAPCeL for cell type specific expression profiling and reveal that 60% of these transcripts have human homologs.
This study provides a comprehensive description of gene expression in developing C. elegans embryonic muscle cells. The finding that more than half of these muscle enriched transcripts encode proteins with human homologs suggests that mutant analysis of these genes in C. elegans could reveal evolutionarily conserved models of muscle gene function, with ready application to human muscle pathologies.
A novel strategy for profiling Caenorhabditis elegans cells identifies transcripts highly enriched in either the embryonic or larval C. elegans nervous system, including 19 conserved transcripts of unknown function that are also expressed in the mammalian brain.
With its fully sequenced genome and simple, well-defined nervous system, the nematode Caenorhabditis 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.
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 (for example, 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.
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.
Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo.
Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which ~1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons.
We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system.