Zhao, P. et al. (2019) International Worm Meeting "Femtosecond laser microdissection isolates single C. elegans neurons for nerve regeneration RNA-seq studies."

Kreeger, L., Arur, S., ZHAO, P., Ben-Yakar, A., Trimmer, K., Messing, R., Ma, K., Martin, C., Zemelman, B., Jiang, N., Maiya, R.

[International Worm Meeting, 2019]

C. elegans has become a versatile system for studying in vivo nerve regeneration since the advent of precise laser axotomy method for severing specific axons. Through mutant and RNAi screening, a number of regeneration regulator genes have been identified. Nevertheless, their downstream effectors remain elusive. As a complementary approach, we propose to perform single-cell RNA-sequencing on regrowing neurons to capture the genome-wide dynamics underlying nerve regeneration. However, it has been technically unfeasible to isolate regrowing neurons from living C. elegans. The prevalent isolation method uses FACS to sort neurons of interest from chemo-mechanically dissociated animals, thus requires thousands of animals with synchronized nerve injury, which cannot be obtained even with state-of-the-art automated microfluidic systems. We developed a new femtosecond laser microdissection (fs-LM) method to rapidly and precisely isolate single cells directly from living tissue or organisms by leveraging femtosecond laser ablation as a high-precision cutting tool. Compared to traditional laser capture microdissection, our method provides a few crucial advantages. 1) fs-LM yields intact single cells without sample sectioning, freezing, or fixing, thus preventing sample degradation or contamination. 2) compared to the dissociation and sorting method, fs-LM induces less stress response in isolated cells. 3) fs-LM preserves the spatial and phenotypic information of the collected neurons. In addition, by correlating gene expression to the context-dependent regeneration phenotypes, it is possible to further dissect the genetic activities encoding nerve regeneration. 4) fs-LM does can isolate unlabeled cells. We isolated regrowing posterior lateral microtubule (PLM) neurons from larval 4 stage animals. Single cell RNA-sequencing on the isolated neurons identified gene expression patterns underlying axon regeneration. To demonstrate the versatility of our method, we have also dissected and sequenced single C. elegans oocytes and mammalian brain neurons.

Mehmet F Yanik et al. (2005) International Worm Meeting "Nerve injury and regeneration in C. elegans"

Adela Ben-Yakar, Yishi Jin, Andrew CHISHOLM, Mehmet F Yanik, Hulusi Cinar, Hediye N Cinar

[International Worm Meeting, 2005]

Understanding the basics of nerve regeneration can be used to identify novel therapies for neurological diseases. Animal models of nerve regeneration are mostly limited to vertebrate organisms. Establishment of axotomy models in invertebrate organisms such as C. elegans is highly desired because of remarkable research potential promised by genetics and screening methods. We reported a surgical technique based on femtosecond (fs) laser pulses to severe nerve processes (0.2-0.4 ? in radius) in the worm (Nature 432:822 (2004)). In this technique, amplified fs pulses were used in a low-energy processing regime to achieve axotomy with minimal surrounding damage. Anesthetized animals were mounted on a slide and 100 laser pulses with 10-40 nanojoule energy and 200 fs short duration at 1 kHz repetition rate were tightly focused on the fluorescent nerve processes. By cutting circumferential axons of D-type motor neurons, we were able to establish a nerve injury and regeneration model. Operated animals showed uncoordinated backward motion, a manifestation of D neuron deficiency (Nature 364:337-41 (1993)). Lesioned axons recovered in one day and the behavioral defect improved over the same time period. Invertebrate animals have differing capacities for natural recovery after nerve injury, probably regulated by permissive environment and intrinsic qualities of neurons. Since the energy deposited by laser pulses induces structural damage, the assessment of damage extent in the tissue after laser operation becomes important to understand tissue permissivity factors regarding nerve regeneration. To assess surrounding damage by laser pulses, we used a strain where both nerve processes and muscle cell membrane were labeled by GFP. We severed the nerve process and measured ensuing surrounding damage by loss of GFP signal in the lesion periphery. Unwanted damage by laser pulses seemed to be proportional to laser pulse intensity which provides a controllable parameter to curb damage spread, and, thus, facilitate the investigation on the permissive environmental factors. We extended the axotomy model to sensory neurons to determine if intrinsic qualities of neurons confer differential responses to nerve injury. The results, technique, and its potential applications will be discussed.

Ketting RF et al. (1995) International C. elegans Meeting "REGULATION OF TRANSPOSITION IN C.elegans."

Ketting RF, Vos JC, Plasterk RHA, Smits MT

[International C. elegans Meeting, 1995]

We study the process of transposition of the Tc1 and Tc3 elements of C.elegans. The elements encode a protein, a transposase, that is necessary for transposition. These transposases are called Tc1A and Tc3A, respectively. The first 63 and 65 amino acids of the respective transposases are able to bind specifically to the inverted repeats of their elements [Colloms S.D. et al. (1994) Nucl. Acids Res. 22(25), 5548-5554, Vos J.C. et al. (1993) Genes Dev. 7, 1244-1253.]. The binding characteristics of the Tc3A protein have been determined in further detail. A 192 amino acid fragment appears to be more restricted in the DNA it can bind than the first 65 amino acids: both proteins will bind to an oligo of 38 basepairs inverted repeat sequence, but only the first 65 amino acids will bind to a smaller fragment of 22 basepairs, which is similar to what is found for Tc1A, where a GG to CC mutation only abolishes binding of the bigger DNA binding fragment, which is 143 amino acids long [Vos J.C. and Plasterk R.H.A. (1994) EMBO J. 13(24), 6125-6132]. We are currently trying to obtain crystalls of the first 65 amino acids of Tc3A together with its binding site, in order to study the interactions of the transposase with the transposon ends. We are also looking for additional mutator loci in a Bristol background. In wild- type Bristol strains there is no detectable transposition in the germline such as found in Bergerac strains. John Collins has shown that it is possible to increase germline transposition activity in a Bergerac background [Collins J. et al. (1987) Nature 328, 726-728]. One spontaneous Tc1 mutator line has been obtained from a Bistol N2 background [Babity J.M. et al. (1990) Mol. Gen. Genet. 222, 65-70]. Is it also possible to raise the transposition frequency in the Bristol germline to a detectable level by EMS mutagenesis? We are now looking for new mutator lines in Bristol N2 backgrounds, using genetic screens. Progress will be reported.

Nassar, Layla et al. (2015) International Worm Meeting "Understanding how sex modulates the female nervous system to drive distinct reproductive behavior states."

Collins, Kevin, Nassar, Layla, Bode, Addys

[International Worm Meeting, 2015]

We are interested in understanding how mating and reproductive behaviors are coordinated in the female nervous system. Specifically, we are identifying the neural signaling systems that drive two mutually exclusive vulval motor behaviors: mating with males or the release of progeny during egg laying [1]. We hypothesize that: 1. female vulval muscle twitching contractions facilitate male spicule insertion during mating; 2. specific mechanical and chemical signals report successful copulation and insemination; and 3. mating initiates reproductive behaviors including oocyte production and egg release. To investigate these hypotheses, we are using calcium imaging techniques to record vulval signaling events during mating with males, after successful insemination, and during the resumption of normal egg laying behavior. We have found that hermaphrodites with sperm have reduced vulval muscle twitching behaviors resulting in inefficient mating. In contrast, hermaphrodites depleted of sperm have increased vulval muscle twitching that facilitates male spicule insertion and mating. After mating is complete, hermaphrodites have sustained vulval muscle twitching that can result in release of sperm from the uterus. This behavior may act as a mechanism for competition with self-sperm. We are now examining how the other cells in the egg-laying circuit, including the HSN and VC neurons and uv1 neuroendocrine cells, respond during steps of male mating. During egg laying behavior, we found that the uv1 cells are mechanically activated by passage of eggs through the vulva, driving release of tyramine that inhibits egg laying [2]. We predict that mechanical stimulation by male spicules may similarly activate the uv1 cells to inhibit egg release during mating. However, once mating is complete, we expect egg laying to resume or even increase. Together, these results will explain how internal and external signals modulate activity in the same neural circuit to drive distinct behavior states.1. Collins, KM, and Koelle, MR (2013). Postsynaptic ERG Potassium Channels Limit Muscle Excitability to Allow Distinct Egg-Laying Behavior States in Caenorhabditis elegans. J. Neurosci. 33, 761-775.2. Collins KM, Bode A, Fernandez R, Tanis JE, Creamer M, Koelle MR (2015). Unpublished results.

Gilchrist EJ et al. (1995) International C. elegans Meeting "A C. ELEGANS MYOTONIC DYSTROPHY GENE HOMOLOGUE."

Gilchrist EJ, Baillie DL

[International C. elegans Meeting, 1995]

Myotonic Dystrophy is an inherited, autosomal dominant disease that results in a progressive wasting of the skele- tal muscles (and sometimes heart and smooth muscles) in humans. The gene has been cloned and sequenced (R. Korneluk, personal communication) and encodes a large protein that has been localised to neuromuscular and myotendinous junctions. The N-terminus domain bears similarity to cAMP-dependent serine/threonine protein kinases. Yuji Kohara, in his cDNA sequencing project, identified a C. elegans gene that is 75% similar (if conserved amino acids are considered) to the kinase domain of the human myotonic dystrophy gene. He mapped the cDNA to LG I (personal communication), and, through Southern analysis, we have placed the cDNA on cosmid K06G1. This cosmid was injected into wild-type N2 hermaphrodites by Diana Collins, and we are currently trying to rescue some of the genes in this region with the extrachromosomal array. One candidate gene was unc-57. Animals homozygous for mutations in unc-57 show no obvious structural muscle defects. This is consistent with unc-57 being a muscle, or neuro- muscular kinase. However, the Unc-57 phenotype is not rescued in animals carrying the K06G1 extrachromo- somal array. We are now focusing on some of the lethal mutations in this region that have been isolated in the laboratory of Ann Rose at UBC.

Chi-Chang Lee et al. (2007) International Worm Meeting "The Implication of A Nucleolar Protein,CeNopp140, in the Germ-line Development in Caenorhabditis elegans."

Ning-Hsing Yeh, Chi-Chang Lee, Huey-Jen Lai, Szecheng Lo

[International Worm Meeting, 2007]

Nopp140 and TCOF1 are two highly phosphorylated nucleolar proteins and share with a similar arrangement of structural domains. However, Nopp140 has multiple functions, including regulation of rRNA transcription, while mutation of TCOF1 leads to a Treacher Collins Syndrome (TCS), affecting craniofacial development in human. Previously we have identified the human Nopp140 (hNopp140) homologue, dao-5, in C. elegans by BLAST comparison. Although they differ in total amino acid residues (709 vs. 971 residues), they share three structurally conserved domains, N-terminal, central, and C-terminal domain, and two highly identical stretches of amino acids, SSPFRRVREE and GKSFRHEKTKKKRGSYRGG, at the C-terminal conserved domain. We designated Dao-5 as CeNopp140 since it localizes ectopically at nucleolus in HeLa cells as well as expressing at the nucleoli of somatic gonad, intestine and hypodermis in C. elegans. The knockout mutant of CeNopp140 appeared a lower brood size as compared with wild-type N2 strain. After a closer examination on the phenotype and some germ-line events of homozygote mutants of CeNopp140, we found that (1) the gonad of mutants developed well as WT animal until L4 stage, (2) the young adult of mutants possessed a late L4-stage-like gonad, and (3) the gravid adult mutants developed deformed and shriven gonad but occasionally were fertile. Experiments of RNAi-mediated depletion of CeNopp140 also show the phenocopy. Taken these results together, we suggested that the nucleolar protein, CeNopp140, plays a role on the appropriate germ-line development spatially and temporally. This model may be useful in studying the molecular mechanism related to TCS in human.

Chamberlin HM et al. (1995) International C. elegans Meeting "Essential genes required for asymmetric cell divisions in C. elegans"

Chamberlin HM, Baillie DL

[International C. elegans Meeting, 1995]

Many of the initial postembryonic cell divisions in C. elegans are visibly asymmetric: the two daughter cells are morphologically or behaviorly different, and these observable differences correlate with different fates. Although genetic screens have identified viable alleles of some genes that function in these divisions, it is possible that other genes have escaped identification because they have multiple developmental functions and are required for viability. To identify additional genes involved in mediating asymmetric cell divisions, we have begun to screen existing larval lethal mutants for defects in these postembryonic divisions. To date, we have identified five essential genes (out of 168 loci screened) that are required for normal asymmetric cell divisions. Mutations in two of the loci appear to disrupt a wide range of postembryonic initial asymmetric cell divisions, whereas mutations in the other three disrupt some but not other divisions. Although these mutations disrupt the initial cell division, all mutants generally arrest prior to any further development, so it is not clear if the defects in asymmetric cell division correspond to defects in fate specification. To further characterize this group of mutants, we are focusing on one gene, let-765. In let-765 mutants, the asymmetric division of the male B cell is disrupted, but most other initial postembryonic divisions are normal. let-765 maps between sma-4 and sma-3 on LG III. We are using transgenic strains generated by Collins et al. (see abstract) to test rescue of let-765. We are also attempting to identify additional, non-null mutations in let-765.

Stewart HI et al. (1995) International C. elegans Meeting "CAENORHABDITIS ELEGANS: BALANCERS, RECESSIVE LETHAL MUTATIONS AND DEFICIENCIES ON LGIII(LEFT) AND LGV(RIGHT)."

Ha TT, Stewart HI, Baillie DL, Rose AM, Collins D

[International C. elegans Meeting, 1995]

The new balancers sC1 and sDp3 along with the well documented eT1 balancer (Rosenbluth et al, 1981) completely balance LGIII. We have validated sC1 and sDp3 by isolating recessive lethal mutations and have generated a set of overlapping deficiencies spanning these balancer systems. A set of 91 new essential genes were isolated, while validating sDp3, as a balancer. These recessive lethal mutations were also utilized in the initial mapping of the new set of deficiencies. We have 11 confirmed deficiencies and have several other putative deficiencies, balanced by sDp3. Work validating the new balancer sC1 is also well underway. We have isolated over 70 recessive mutations and have also generated a set of deficiencies, in this region, two of which have been confirmed. As the sequencing of LGIII is rapidly being completed by the sequencing consortium, work on the correlation of the genetic and physical maps is also progressing. About 100 transgenic strains, using sequenced cosmids, have been constructed to aid in this work (See poster Diana Collins et al, this session). LGV(Right) is currently balanced by nT1 and sC4. sC4 is a new balancer, unc-76 rol-9/ sC4[dpy-21], which is not homozygous viable. It incompletly balances the right end of LGV. Work is in progress to develop a new balancer for the far right of LGV., using a derivative of nT1. The genetic tools provided by this reasearch are available to the C. elegans genetic community on request. Information on how to access these mutations, through the www site, is available at http: This research is being funded by The National Institutes of Health (NIH) U.S.A.

Rezsohazy R et al. (1995) International C. elegans Meeting "Towards Tc1- and Tc3-mediated enhancer detection assays in Caenorhabditis elegans"

Rezsohazy R, Plasterk RHA

[International C. elegans Meeting, 1995]

We aim to set up an assay to detect tissue- or stage-specific regulatory sequences. A screen of gene expression patterns has been developed by Hope, using promoter-reporter fusions generated by shotgun cloning (1). Our assay will rely on the insertion of the transposable elements Tc1 and Tc3 carrying an appropriate reporter gene. As a preliminary step we developed a selection that allows detection of Tc1, or Tc3 jumping events from an extrachromosomal array to any chromosomal locus in the germ-line. To detect such events the transposable elements have been marked by inserting the pha-1 gene (2) which complements the thermosensitive embryonic lethal allele pha- 1(e2123). The ability of marked elements to jump in somatic cells was confirmed by PCR detection. Transgenes carrying both the rol-6(su1006) gene and the Tc1-pha-1 or Tc3-pha-1 construct have been established in mutator strains (3) that are a homozygous for the pha-1(e2123) allele. This strain is now an obligatory roller at the non-permissive temperature. Among the progeny we look for non-rolling worms able to develop at non- permissive temperature, in which presumably the transposable module has inserted into new chromosomal locations. For use as an enhancer-trapping technique, large scale detection of transposition events will be needed. Therefore it will be necessary to select rather than detect the loss of the transgene. For this purpose the cha-1 gene could be used as a counter-selection marker in homozygous cha- 1(p1152) mutants resistant to acetylcholine esterase inhibitors (4). (1) Hope, I.A. (1991); Development, 113, 399-408. (2) Granato, M., et al. (1994); Nucl Acids Res, 22, 1762-1763. (3) Collins, J., et al. (1987); Nature, 328, 726-728. (4) Rand, J.B. and Russell, R.L. (1984); Genetics, 106, 227-248.

Collins, Kevin M. et al. (2013) International Worm Meeting "Understanding how neurotransmitter signaling drives two-state activity of the C. elegans egg-laying behavior circuit."

Collins, Kevin M., Koelle, Michael R.

[International Worm Meeting, 2013]

We are interested in understanding how neurotransmitter signaling allows a neural circuit to execute distinct behavior states. C. elegans regulates egg laying by alternating between an inactive phase and a ~3 minute "active" state during which clusters of 3-6 eggs are laid. Six VC motor neurons release acetylcholine to excite the vulval muscles, and two HSN motor neurons release serotonin which signals through vulval muscle receptors to promote the active phase. Four uv1 cells release tyramine to inhibit egg laying. We are using GCaMP calcium imaging in behaving animals to understand how the signaling events in the circuit produce its two state behavior. We recently reported that the vulval muscles are rhythmically excited during each locomotor body bend (1). We found the UNC-103 K+ channel depresses muscle excitability below the threshold that drives calcium transients and contraction during the inactive phase, while still allowing signals above threshold during the active phase. To understand how the HSN, VC, and uv1 neurons regulate vulval muscle excitability, we are manipulating neurotransmitter release from these cells and using GCaMP to record changes in neuron and vulval muscle activity. Activation of serotonin release from the HSNs using Channelrhodopsin induces rhythmic vulval muscle twitching and egg-laying behavior-hallmarks of the active phase. We find that VC neuron activity increases during the active phase, and preliminary results show that egg-laying events mechanically distort the uv1 cells and trigger calcium transients. We have previously shown that TRPV channels, which can be mechanically gated, are required for uv1 to inhibit egg laying (2). We propose that successive egg-laying events increase uv1 calcium signaling, promoting release of tyramine and neuropeptides which terminate the active phase of egg laying. (1) Collins and Koelle (2013). J. Neurosci. 33, 761-775. (2) Jose et al. (2007). Genetics 175, 93-105.