Calarco, John et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Detection and analysis of extensive spatio-temporal regulation of alternative splicing during C. elegans development"

Calarco, John, Blencowe, Benjamin, Pan, Qun, Mavandadi, Sepand, Ramani, Arun, Fraser, Andrew, Zhen, Mei, Lee, Leo, Morris, Quaid

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

Recent studies suggest that spatio-temporal regulation of alternative splicing (AS) plays a critical role in metazoan development. However, little is currently known about the factors and mechanisms regulating AS during development in vivo. Moreover, assigning functional roles to regulated alternative splicing events and understanding their contribution to the development of an organism remains a long-standing goal in biology. C. elegans provides an attractive model system to study AS during development in a living organism. Using AS microarray profiling and RNA-Seq, we have profiled annotated cassette exons and novel splice variants throughout C. elegans developme nt. Our analysis has uncovered many novel AS events and suggests that hundreds of exons are differentially spliced during development. To complement these experiments, bichromatic fluorescent protein-based reporters have been developed that are capable of monitoring spatio-temporal splicing patterns at single cell resolution in vivo. These reporters reveal a remarkable degree of regulatory complexity that exquisitely modulates isoform expression among individual cell types during C. elegans development, particularly in the nervous system. In order to assess the functions of such isoforms during development, a fosmid recombineering-based gene rescue approach has been used to selectively re-introduce individual splice variants into strong loss-of-function mutant strains for a number of genes of interest. These experiments have identified an AS event in the collagen-like gene cle-1 that plays an important role in proper synapse development. The functions of additional AS even ts that display striking cell type-specific regulatory patterns are currently being investigated. Collectively, the resources described above reveal novel patterns of AS regulation at single cell resolution during C. elegans development, and further facilitate the elucidation of important in vivo functions for such splice variants.

Gracida, Xicotencatl et al. (2017) International Worm Meeting "Cell type-specific transcriptome profiling using the Translating Ribosome Affinity Purification technique."

Gracida, Xicotencatl, Calarco, John A., Zhang, Yun

[International Worm Meeting, 2017]

Tissues and defined cell types execute specialized functions in multicellular organisms, largely through tailored gene expression programs. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Classical methods to detect gene expression differences have utilized samples from whole-animals, dissected tissues, or sorted cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods. We adapted the Translating-Ribosome Affinity Purification (TRAP) method for C. elegans to detect cell type-specific gene expression signatures. In TRAP, the ribosomal protein RPL-1 is fused to GFP and is expressed under cell-type specific promoters to mark genetically defined cell types in vivo. Affinity purification of GFP in lysates of animals expressing the tag enriches for ribosome-associated mRNAs of the targeted tissue. The purified mRNA is then used for making cDNA libraries subjected to high-throughput sequencing to obtain genome-wide snapshots of the translating mRNAs in a given cell type. We obtained translational profiles from L4 larvae across three major cell types: neurons, intestinal cells, and body wall muscle cells, and validated the reproducibility and specificity of our procedure. Furthermore, we adapted TRAP and obtained translational profiles of a smaller subset of cells corresponding to two major neuromodulatory cell types, the dopaminergic and serotonergic neurons. We have identified hundreds of tissue-enriched mRNAs in these tissues and cell types, and found a strong agreement between our TRAP data and previously validated patterns. Additionally, we have found genes that were not known to be expressed these tissues or cell-types. We validated and functionally characterized the case of a subunit of an E3 ubiquitin ligase complex, the Elongin C ortholog elc-2 that we found specifically expressed in a subset of serotonergic sensory neurons. In addition to detection of relative transcript levels across samples, with TRAP we have also obtained global patterns of alternative mRNA splicing in a tissue and cell-type specific manner. Taken together, the ease of exposing C. elegans to diverse stimuli, coupled with available cell type specific promoters, makes TRAP a useful approach to be adapted in other laboratories to enable the discovery of molecular components in response to external or genetic perturbations.

Almeida CA et al. (1994) Worm Breeder's Gazette "Function of a Domain of the Myosin Heavy Chain Implicated in Familial Hypertrophic Cardiomyopathy"

Collins JJ, Almeida CA, Swift KE

[Worm Breeder's Gazette, 1994]

Function of a Domain of the Myosin Heavy Chain Implicated in Familial Hypertrophic Cardiomyopathy Craig A. Almeida, Kerry E. Swift and John J. Collins Department of Biochemistry and Molecular, University of New Hampshire, Durham, NH 03820

Gracida, Xicotencatl et al. (2015) International Worm Meeting "Genome-wide analyses of actively translating mRNAs in neurons identify a heat-sensitive transcription elongation factor in a pair of serotonergic sensory neurons."

Dion, Mike, Gracida, Xicotencatl, Calarco, John A.

[International Worm Meeting, 2015]

Specific cell types or tissues adjust their gene expression programs in response to sensory stimuli to help an organism cope with its environment. However, transcriptome-wide analyses are often limited by a lack of spatial resolution when studying whole-animal samples. This difficulty makes it challenging to identify the extent of broader gene expression differences in individual tissues or in specific cell types in vivo. To tackle this problem, we adapted the Translating Ribosome Affinity Purification method (TRAP) (Heiman et al. 2008) in C. elegans. In TRAP, cell-type specific promoters drive expression of eGFP-tagged Ribosomal Protein L10a of the Large subunit to tag sets of genetically defined cell types in vivo. These GFP-tagged ribosomes can be subsequently affinity purified to obtain enriched populations of ribosome-associated mRNAs from cells of interest. We have recently coupled TRAP to deep sequencing and computational analyses to identify tissue- and neuron-specific patterns of gene and isoform abundance. By using TRAP, we globally surveyed the repertoire of mRNAs in the nervous system and specific neuronal classes expressing the neurotransmitters dopamine and serotonin. Comparative analyses between these samples and non-neuronal tissues revealed hundreds of tissue- and neuron-biased mRNA isoforms, and neuronal-enriched mRNAs encoding conserved proteins whose functions have not been characterized. Among these we found elc-2/ElonginC/TCEB1, a regulatory subunit of the transcription elongation factor complex Elongin/SIII. Using a GFP-tagged fosmid we found that ELC-2 protein is selectively expressed in a pair of sensory neurons that we confirmed to be the serotonergic ADFs. ADF neurons are a hub for integrating several stress responses that ultimately lead to the release of serotonin to alter animal physiology. We unexpectedly found that upon heat shock ELC-2 redistributes in the ADF soma, becoming more nuclear enriched. Concomitantly, a backcrossed natural variant containing an elc-2 deletion fails to increase pharyngeal pumping rates upon heat shock, a phenotype of increased serotonin release. We are investigating whether other stresses also induce ELC-2 redistribution, and what the role of its nuclear accumulation is for coordinating heat shock-dependent gene regulation in ADF. We hypothesize that redistribution of this transcription elongation factor subunit ELC-2 may act as a modulator of sensory-dependent transcriptional programs that induce longer lasting neural effects that help the organism successfully respond and adapt to its environment. .

Gracida, Xicotencatl et al. (2017) International Worm Meeting "Cell-specific protein degradation regulates"

Zhang, Yun, Calarco, John A., Gracida, Xicotencatl, Harris, Gareth

[International Worm Meeting, 2017]

Neuromodulatory cells transduce environmental information into lasting modulatory effects on physiological and behavioral processes. However, our understanding of how specific neuronal cell types regulate these modulations is limited by the complexity of the nervous system. To overcome this challenge, we adapted the Translating Ribosome Affinity Purification (TRAP) approach in C. elegans to profile ribosome-associated mRNAs from the neuromodulatory dopaminergic and serotonergic cells. From these profiles, we find that elc-2, an ortholog of the mammalian Elongin C, is specifically expressed in the ADF serotonergic sensory neurons, a neuronal class involved in stress response. We show that elc-2 plays a critical role in mediating a heat-induced long-lasting change in the feeding behavior. We demonstrate a role for ELC-2 and the von Hippel-Lindau VHL-1 protein, both of which are components of a conserved Elongin-Cullin-SOCS-box (ECS) E3 ligase involved in ubiquitin-mediated protein degradation, in modulating feeding behavior after experiencing the heat stress. Disrupting serotonin production in the ADF neurons, mutating a metabotropic serotonin receptor, or inhibiting neuromodulation recapitulates behavioral defects found in the ECS mutants. At the molecular level, the heat stress induces a transient redistribution of ELC-2 within the nucleus. Because the ECS components are expressed in many mammalian tissues, including the brain, our results raise the intriguing possibility that these factors play a similar role in the mammalian nervous system. Together, our results characterize the subcellular and dynamic regulation of an E3 ubiquitin ligase in specific neuromodulatory neurons and identify a novel function for an ECS-mediated protein degradation in integrating stress signals to regulate serotonergic neuromodulation of lasting behavioral states.

Norris, Adam D. et al. (2013) International Worm Meeting "A pair of RNA binding proteins shape alternative splicing regulatory networks in distinct neuronal subtypes."

Norris, Adam D., Calarco, John A., Zhen, Mei

[International Worm Meeting, 2013]

Alternative splicing provides an important mechanism for increasing transcriptomic diversity in metazoans. This increased complexity has likely played a particularly important role in the cellular diversification of the nervous system, where different classes of neurons have evolved to perform distinct functions. However, it has remained a challenge to study the mechanisms and physiological impact of alternative splicing regulation at the level of individual neuronal subtypes. In order to study neuronal subtype-specific regulation of alternative splicing, we created fluorescent two-color reporters to observe alternative splicing in vivo and at single neuron resolution. Our results reveal a remarkable diversity of alternative splicing patterns among individual neuron types. One striking example involved differential inclusion of an alternative exon between GABAergic and cholinergic motor neurons. We conducted a forward genetic screen for regulators of this neuron-type specific splicing pattern and identified two conserved RNA binding proteins, UNC-75/CELF and EXC-7/ELAV. Analysis of splicing patterns in mutant animals showed that UNC-75 and EXC-7 act combinatorially to achieve neuron subtype specificity through partially non-overlapping expression. mRNA-Seq and CLIP-Seq experiments found hundreds of differentially regulated alternative splicing events when either or both factors are absent, and targeted alternative splicing events are enriched in genes associated with synaptic transmission and locomotion. Initial results indicate that the splicing regulatory network can be utilized to implicate both known and previously uncharacterized genes in locomotory behavior and synaptic transmission, and that the impact of individual targeted isoforms on neuronal phenotypes can be teased apart in vivo. Taken together, our findings suggest that partially overlapping expression patterns of multiple alternative splicing factors can create specialized post-transcriptional regulatory networks that contribute to the identity and function of distinct cell types in the nervous system.

Gracida, Xicotencatl et al. (2015) International Worm Meeting "Genome-wide analyses of actively translating mRNAs across tissues and specific neuronal cell-types."

Dion, Mike, Calarco, John A., Gracida, Xicotencatl

[International Worm Meeting, 2015]

Useful approaches for surveying spatiotemporal gene expression patterns at cellular resolution are crucial for a systems-level understanding of cell type-specific gene expression programs during development and in response to stimuli. However, it currently remains challenging to identify the extent of broader expression differences in individual tissues or in specific cell types in vivo. To address this challenge, we have adapted the Translating Ribosome Affinity Purification method (TRAP) (Heiman et al. 2008) in C. elegans. In TRAP, cell-type specific promoters drive expression of eGFP-tagged Ribosomal Protein L10a of the Large subunit to tag sets of genetically defined cell types in vivo. These GFP-labeled ribosomes can then be immunoprecipitated to obtain enriched populations of ribosome-associated mRNAs from cells of interest. We coupled TRAP to deep sequencing to identify tissue-biased patterns of gene expression. In addition, our computational analyses have focused on detecting tissue-biased isoform usage arising from alternative mRNA splicing. By using TRAP, we globally surveyed the repertoire of neuronal, muscle and intestinal mRNAs and measured the abundance of tissue-biased gene isoforms. We also pursued TRAP from specific neuronal classes expressing the neurotransmitters dopamine and serotonin. Our approach unraveled hundreds of tissue- and neuron type-specific isoforms, and tissue-biased evolutionary conserved mRNAs whose functions remain to be characterized. We also used TRAP with a neuronal splicing factor mutant to detect global mRNA isoform changes in the nervous system, and demonstrate that TRAP increases tissue-specific detection sensitivity over whole animal RNA-Seq samples. We are currently coupling this technology with stimulus-triggered neuronal activity in a model of thermal memory by tagging the two major thermosensory neurons. With this technology we are now taking advantage of being able to detect broader gene expression changes in individual tissues or in specific cell types in vivo. .

Plenefisch JD et al. (1994) Worm Breeder's Gazette "Cloning mua-3: Some Observations on the New Molecular Era"

Plenefisch JD, Hedgecock EM

[Worm Breeder's Gazette, 1994]

Cloning mua-3: some observations on the new Molecular Era John Plenefisch and Edward Hedgecock, Dept. of Biology, Johns Hopkins University, Baltimore MD 21218

Chalfie M (1993) J Neurobiol "Touch receptor development and function in Caenorhabditis elegans."

Chalfie M

[J Neurobiol, 1993]

Mutations causing a touch-insensitive phenotype in the nematode Caenorhabditis elegans have been the basis of studies on the specification of neuronal cell fate, inherited neurodegeneration, and the molecular nature of mechanosensory transduction. (C) 1993 John Wiley & sons, Inc.

Parham C et al. (1994) Worm Breeder's Gazette "Tc4 and Tc5: What Makes Them Move and Why It Matters"

Beinhorn J, Collins JJ, Butze K, Parham C

[Worm Breeder's Gazette, 1994]

Tc4 and Tc5: what makes them move and why it matters Christi Parham, Kristie Butze, Joanna Beinhorn and John Collins. Dept. of Biochemistry and Molecular Biology, University of New Hampshire. Durham, NH 03824