Chen, Sway et al. (2015) International Worm Meeting "Functional and anatomical analysis of the L1 motor circuit."

Schalek, Richard, Laskova, Valeriya, Chen, Sway, Cook, Steven, Suriyalaksh, Manusnan, Lichtman, Jeff, Guan, Asuka, Mitchell, James, Neubauer, Marianna, Witvliet, Daniel, Wen, Quan, Zhen, Mei, Samuel, Aravinthan, Lu, Yangning, Mulcahy, Ben

[International Worm Meeting, 2015]

At all developmental stages, C. elegans navigates its environment by generating and propagating bending waves along its body. The neuronal circuit controlling locomotory behavior, however, changes significantly at the end of the L1 larval stage. During the L1 stage, B- and A-type cholinergic motor neurons innervate dorsal body wall muscles but not ventral muscles. D-type GABAergic motor neurons innervate ventral muscles but not dorsal muscles. Calcium imaging and optogenetic stimulation suggest that D-type neurons are inhibitory at the L1 stage, causing ventral muscles to relax. At the L2 stage and beyond, both ventral and dorsal muscles are symmetrically innervated by cholinergic and GABAergic motor neurons, and phasic excitation coupled to contralateral inhibition is thought to be responsible for the propagation of undulatory waves. So how does the worm produce undulatory behavior at the L1 stage given the profound ventral/dorsal asymmetry in excitatory cholinergic and inhibitory GABAergic inputs? To answer this question, we have reconstructed the L1 motor circuit using serial section electron microscopy. We are now using targeted cellular ablation, calcium imaging, and optogenetics to pinpoint the mechanism for ventral muscle contraction, and understand how alternating waves of excitation and relaxation drive undulation during the L1 stage. Our goal is a detailed mechanistic model of L1 locomotion. Comparing the circuit basis of L1 with adult worm locomotion should shed light on conserved principles of rhythmic locomotion and development of motor circuits.

Berger, Daniel et al. (2015) International Worm Meeting "The mind of an L1 worm - how do neuronal circuits function and remodel throughout development?."

Samuel, Aravinthan, Emmons, Scott, Neubauer, Marianna, Lichtman, Jeff, Witvliet, Daniel, Chisholm, Andrew, Laskova, Valeriya, Mitchell, James, Mulcahy, Ben, Cook, Steven, Zhen, Mei, Schalek, Richard, Berger, Daniel, Hall, David, Holmyard, Douglas, Kersen, David, Qu, Angie

[International Worm Meeting, 2015]

When humans and worms are born, they have neuronal circuits in place to integrate sensory cues and effect appropriate behavioural or homeostatic responses. During the course of development, a substantial amount of remodelling occurs in the nervous system. New neurons are born and integrated into existing circuits, and existing neurons and circuits are remodelled. The rules of this remodelling are poorly characterised, because in order to investigate neuronal circuit development one needs a connectome-level analysis of the circuit across multiple time points, in multiple animals. This strategy requires serial-section electron microscopy, which has been very low-throughput and labour-intensive.C. elegans represents an ideal organism to perform these studies on, because of their small size and stereotypical development - some of the reasons they were chosen for the original adult serial-section EM reconstruction that culminated in the acquisition of the adult connectome, dubbed 'The mind of a worm'. Since the publication of this landmark paper, new technologies have been developed that improve the fidelity and throughput of the connectome reconstruction, including better fixation methods, increased computational power and more advanced microscopes.We are reconstructing the entire nervous system of multiple L1 worms using serial-section transmission and scanning electron microscopy. The L1 worm is of special interest because of the major remodelling that occurs at the end of L1, including the birth of new major classes of motor neurons. We are finding both conserved and unreported structure and connectivity from the published adult wiring diagram. This dataset will be used as a framework to functionally interrogate the interplay between developing circuits and sensorimotor behaviours, and the mechanisms that allow this dialogue to take place.

Angela K Spartz et al. (2003) International Worm Meeting "SMU-2, the C. elegans homologue of a mammalian spliceosome associated protein, RED, regulates alternative splicing of unc-52 and interacts with the SMU-1 protein"

Angela K Spartz, Jocelyn E Shaw, Robert K Herman

[International Worm Meeting, 2003]

We are using a genetic approach to identify new genes that function in vivo to regulate alternative splicing in the nematode Caenorhabditis elegans. Mutations in the genes smu-1 and smu-2 were isolated as extragenic suppressors of the synthetic lethal phenotype of certain mec-8 unc-52 double mutants. mec-8 encodes a putative RNA binding protein that affects the accumulation of alternatively spliced transcripts of unc-52 (Lundquist et al. Development 1996 122:1601). We have found that smu-2 also regulates the alternative splicing of unc-52. smu-2 mutants suppress the uncoordination of viable unc-52 mutants that contain premature stops in exon 17 but not in exon 18. Accordingly, we have observed by RT-PCR and RNase protection experiments that mutation in smu-2 leads to enhanced skipping of exon 17 but not exon 18 of unc-52 pre-mRNA. We have cloned smu-2 and find that it encodes a highly conserved protein. Its mammalian homologue, named RED, was identified by Neubauer et al. (Nat. Genet. 1998 20:46) as a protein tightly associated with human spliceosomes. A rescuing smu-2::gfp reporter indicates that SMU-2 is a ubiquitously expressed nuclear protein. We have previously reported similar modulation of unc-52 transcript splicing by SMU-1, a WD repeat protein that is also ubiquitously present in the nucleus (Spike et al. Mol. Cell. Bio. 2001 21: 4985). Genetic evidence suggests that these proteins function in the same pathway: smu-1 and smu-2 mutants have very similar phenotypes, and smu-1 smu-2 double mutants are no better at suppressing unc-52 mutations than is either single mutant. Indeed, we believe that SMU-1 and SMU-2 physically interact. In vitro co-immunoprecipitation experiments indicate that myc tagged SMU-2 can pull down SMU-1 but not control nuclear proteins. In vivo, we have observed that expression of rescuing SMU-1::GFP and SMU-1::HA fusion constructs is dramatically reduced in smu-2 mutants. Western analysis also shows a reduction of SMU-1::HA in a smu-2 mutant background. We hypothesize that formation of a SMU-1/SMU-2 complex in vivo stabilizes SMU-1 protein.

Herman RK et al. (2000) Midwest Worm Meeting "Hypomorphic mutations in smu-2, the homologue of a mammalian spliceosome associated protein, RED, suppress mec-8 and unc-52"

Spartz AK, Herman RK, Shaw JE

[Midwest Worm Meeting, 2000]

Recessive, loss-of-function mutations in smu-2 were previously isolated and characterized as extragenic suppressors of mec-8(ts); unc-52(ts) synthetic lethality. In addition, these mutants suppress several phenotypes associated with mec-8 alleles and the Unc phenotype of unc-52(viable) alleles that have nonsense mutations in exon 17. MEC-8 is an RNA binding protein that regulates the production of certain alternatively spliced unc-52 transcripts. Our preliminary evidence suggests that smu-2 also regulates splicing of unc-52. RT-PCR experiments show that in smu-2 mutants, the levels of some alternatively spliced unc-52 transcripts that skip exon 17 increase relative to internal controls. Increased levels of these transcripts are thus able to compensate for the loss of unc-52 transcripts resulting from mutation in exon 17. The genetic interactions among mec-8, unc-52 and smu-2 suggest that smu-2 may be directly involved in the splicing process. We have cloned the smu-2 gene and find evidence consistent with this hypothesis. smu-2 is the homologue of a ubiquitously expressed nuclear protein known as RED (or IK factor). Although the function of RED is unknown, it has been identified in purified human spliceosomes by mass spectrometry (Neubauer et al., Nature Genetics 20:46). SMU-2 contains a domain rich in arginine (R), serine (S), and aspartic acid (D) residues. These types of domains (RS and RD/RED domains) have been identified in several splicing factors and are proposed to mediate protein-protein interactions. Using a rescuing in-frame fusion construct, we find that SMU-2::GFP is present in the nucleus and is often concentrated in sub-nuclear structures. Similar nuclear "speckles" are observed for many mammalian splicing factors including SR proteins and snRNPs. Additionally, smu-2::gfp is expressed at many stages of development and in many cell types. Interestingly, when we sequenced the smu-2 alleles obtained in the suppressor screen, we found that none is a candidate null. In fact, we are able to rescue smu-2 mutant phenotypes by transformation with smu-2 mutant DNA, presumably by overexpression. Transformation with a smu-2 null made in vitro does not rescue the smu-2 mutant phenotypes. Currently we are addressing the issue of the null phenotype using RNA interference.

Angela K Spartz et al. (2001) International C. elegans Meeting "smu-1 and smu-2 regulate the alternative splicing of unc-52 pre-mRNA"

Robert K Herman, Angela K Spartz, Caroline A Spike, Jocelyn E Shaw

[International C. elegans Meeting, 2001]

Alternative splicing of pre-mRNA is a biologically important but poorly understand process. We are using a genetic approach to identify new genes that function in vivo to regulate alternative splicing. Mutations in smu-1 and smu-2 were isolated as extragenic suppressors of the synthetic lethal phenotype of mec-8 unc-52(viable) double mutants. smu-1 and smu-2 mutations also suppress other phenes of mec-8 mutants, such as mechanosensory and chemosensory defects, apparently by a bypass mechanism; mec-8 encodes a putative RNA binding protein that affects the accumulation of certain alternatively-spliced transcripts of unc-52 and other genes (Lundquist et al. Development 1996 122: 1601). Finally, smu-1 and smu-2 mutations suppress the uncoordination conferred by nonsense mutations in exon 17 but not exon 18 of unc-52 . We hypothesized that smu-1 and smu-2 encode factors that regulate the splicing of various target genes, at least some of which are also targets of splicing control by MEC-8. Indeed, our RT-PCR and RNase protection experiments indicate that mutation in smu-1 or smu-2 leads to enhanced skipping of exon 17 but not exon 18 of unc-52 pre-mRNA. The phenotypes of smu-1 and smu-2 mutants seem to be identical and identical to the phenotype of smu-1 smu-2 double mutants, suggesting that SMU-1 and SMU-2 work together. smu-1 and smu-2 both encode highly conserved proteins that are ubiquitously expressed and nuclearly localized. SMU-1 contains five WD motifs, which are implicated in protein-protein interactions, and is greater than 60% identical in amino acid sequence to a predicted human protein of unknown function; we suggest that this human protein regulates alternative splicing. We have molecularly characterized all six known smu-1 mutations. Two appear to be null and confer a mildly deleterious but viable phenotype. smu-2 encodes a protein that is 37% identical to a mammalian nuclear protein called RED; homologues are also present in Drosophila and Arabidopsis . The similarities between SMU-2 and RED proteins occur throughout their full extents. SMU-2 is the only protein with significant similarity to RED in the C. elegans genomic sequence database. Neubauer et al. (Nat. Genet. 1998 20: 46) identified RED as a protein tightly associated with human spliceosomes. RED was named after the most distinctive feature of the protein, a domain consisting of alternating basic (arginine) and acidic residues (aspartic and glutamic acid); the function of this domain, which seems to be the least well conserved part of the protein, is not known. None of our three smu-2 mutations is a molecular null, but smu-2(RNAi) mimics the smu-2 phenotype: efficient suppression of unc-52 and no inviability. GFP expression of a rescuing smu-1::gfp reporter disappears in a smu-2 mutant background; we suggest that SMU-2 is required to stabilize SMU-1. The first two authors contributed equally to this work.