Richard Ikegami et al. (2004) East Coast Worm Meeting "Guidance and cell-matching of a migrating epidermal sheet during ventral enclosure by MAB-20 and PLX-2"

Kristin Simokat, Louise Dixon, Joseph Culotti, Jeff Hardin, Richard Ikegami

[East Coast Worm Meeting, 2004]

During epithelial morphogenesis, epithelial cells undergo dynamic changes in cell shape, cell-cell adhesions and cellular arrangement. Semaphorins, classically identified as repulsive axon guidance cues, play a major role in epithelial morphogenesis in C. elegans. Plexin-2/PLX-2 was identified as one receptor for Semaphorin-2a/MAB-20 to function in a redundant manner with at least one other as yet unidentified receptor during male tail morphogenesis. We present phenotypic and genetic evidence consistent with the role of PLX-2 as one receptor for MAB-20 signaling during ventral enclosure. Mutants of plx-2 including its null share similar but milder ventral enclosure defects compared to mutants of mab-20 . At the cellular level, plx-2 mutants like mab-20 mutants show inappropriate regulation of adhesion between the P cells of the migrating epidermal sheet. In addition, the plx-2 null is unable to enhance the embryonic lethality of the mab-20 null consistent with a genetic role in the same pathway yet suggests that mab-20 must also signal through a pathway parallel to plx-2 . PLX-2 is expressed in a manner that could confer the cell-specific function of ubiquitously expressed MAB-20. PLX-2 is expressed in a discrete set of epidermal P cells (P3/4, P5/6, P9/10, P11/12) that migrate to meet their contralateral partners at the ventral midline. Concomitantly, PLX-2 is expressed in a subset of substrate neuroblasts of the ventral pocket. These neuroblasts undergo short-range migration and arrange into a band of a single cell width. This band bisects the ventral midline of the open pocket counterfacing the leading edge of the migrating PLX-2 non-expressing P7/8 cell. In addition we show that VAB-1 Eph receptor may function to arrange the PLX-2 neuroblasts into a contiguous band just prior to the overlying migration of the epidermis. We present a model for the role of MAB-20/PLX-2 in guiding the migrating epidermal sheet to mediate contralateral cell-matching of opposing cells of the leading edge at the ventral midline during pocket closure.

Mariam Alexander et al. (2005) International Worm Meeting "A Screen For Genes Required for Muscle Arm Development."

Mariam Alexander, Peter J Roy

[International Worm Meeting, 2005]

In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nerve cord to form the neuromuscular junction. Our lab and others have provided evidence that there is likely two distinct phases of muscle arm development; embryonic muscle arms arise by a passive mechanism (CR Norris, IA Bazykina, DH Hall, EM Hedgecock, Worm Breeder's Gazette 14(5): 37), while larval arms actively seek out motor axons (Dixon and Roy, submitted). Although the mechanism by which larval muscle arms reach motor axons is not clear, one model is that a chemotropic cue secreted by motor axons guide muscle arm migration to the nerve cord. To better understand muscle arm development, I performed a forward genetic screen to isolate mutants with muscle arm extension defects. I have screened 36,000 haploid genomes and isolated five mutants with highly penetrant and expressive muscle arm defects. Complementation tests show that tr50 is allelic to unc-60, a gene encoding the C. elegans ADF/cofilin orthologue, while tr51 is allelic to unc-22, which encodes twitchin. The remaining mutants do not map near other genes known to be required for larval muscle arm extension, including lev-11 and unc-54 (Dixon and Roy, submitted), suggesting that these genes may provide novel insight into the development of muscle arms. Surprisingly, one of mutants that has a dramatic reduction in the number of muscle arms (tr64) is not uncoordinated. This suggests that proper muscle arm development is not necessary for wild type movement. I am currently refining the map positions of the genes corresponding to the remaining mutations.

Mariam Alexander et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Search for New Genes Required for Muscle Arm Extension"

Peter Roy, Mariam Alexander

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The guided migration of cells and cell extensions is essential throughout development and is integral to many diseases such as cancer. Although many guidance pathways have been characterized, there remain guidance events that are poorly understood and possibly regulated by uncharacterized guidance systems. For example, in nematode worms, the muscles extend specialized membrane called muscle arms to the nearest nerve cord to form the postsynaptic element of the neuromuscular junctions. These arms are extended in a stereotypical and regulated manner and require conserved cytoskeletal elements such as UNC-60 (ADF/cofilin) and UNC-54 (myosin) ( Dixon and Roy , 2005). Observation suggests that there is a passive embryonic phase of muscle arm development followed by an active phase of muscle arm extension during larval development. How the larval muscle arms reach the nerve cord is unclear. One hypothesis that is supported by multiple lines of evidence is that the muscle arms are guided to the nerve cord by a chemotropic cue that is made by the motor axons within the nerve cord (see Hedgecock et al., 1990; Hall and Hedgecock, 1991; Dixon and Roy , 2005). However, genetic disruption of known guidance pathways, including Netrin, Semaphorin, Ephrin, Slit and Wnt does not result in muscle arm extension defects (MADs). To identify genes required for arm extension to the nerve cords, I performed a forward genetic screen to isolate mutants with a MAD phenotype. I have screened 36,000 haploid genomes and isolated five mutants with highly penetrant and expressive MADs, including unc-60 (tr50), unc-22 (tr51), unc-95 (tr61), and two other mutants named tr63 I and tr64 V. Surprisingly, tr64 is not uncoordinated even though larval muscle arms fail to extend in this mutant. This suggests that either the number of neuromuscular junctions per muscle is not dependent on the number of muscle arms or that a significant reduction in the number of neuromuscular junctions per muscle will not result in any movement defects. I have mapped tr64 to a 150kb region on Chromosome V. All the genes in this region are relatively novel and have no known role in muscle development, membrane extension or guidance. The identification and characterization of this mutant will undoubtedly lead to new insights in guided cell migration and extension.

S Ogg et al. (1999) International C. elegans Meeting "The PTEN homologue DAF-18 acts in the DAF-2 insulin receptor-like metabolic signaling pathway"

S Ogg, G Ruvkun

[International C. elegans Meeting, 1999]

An insulin-like signaling pathway, from the DAF-2 insulin receptor homologue, the AGE-1 phosphoinositide 3-kinase, the AKT-1/AKT-2 serine/threonine protein kinases to the DAF-16 Fork head transcription factor, regulates the metabolism, development and life span of Caenorhabditis elegans. DAF-2 insulin receptor is likely to signal via the AGE-1 PI 3-kinase which generates lipid second messengers (PI-3,4-P 2 and PI-3,4,5-P 3 ) that activate the Akt kinases. The Akt kinases act redundantly to antagonize the activity of DAF-16 FKHR (Paradis and Ruvkun, 1998). AKT-1 can directly phosphorylate DAF-16 in vitro and AKT-1/AKT-2 are likely to directly antagonize DAF-16 FKHR in vivo. Inhibition of daf-18 gene activity bypasses the normal requirement for AGE-1 PI 3-kinase and partially bypasses the need for DAF-2 insulin receptor signaling. Using RNA interference, we have demonstrated that the suppression of age-1 mutations by a daf-18 mutation depends on AKT-1/AKT-2 signaling. This suggests that DAF-18 acts between the AGE-1 PI 3-kinase and the Akt kinases. Our molecular characterization revealed that daf-18 encodes a homologue of the human tumor suppressor PTEN (MMAC1/TEP1). PTEN has both tyrosine phosphatase activity and 3-phosphatase activity towards PI-3,4-P 2 and PI-3,4,5-P 3 (Maehama and Dixon, 1998). DAF-18 PTEN likely antagonizes AGE-1 PI 3-kinase and limits AKT-1 and AKT-2 activation by dephosphorylating AGE-1 PI 3-kinase generated lipid second messengers. DAF-18 PTEN may act to modulate or down regulate DAF-2 insulin receptor/AGE-1 PI 3-kinase signaling and/or isolate distinct PI 3-kinase signaling complexes. The action of daf-18/PTEN in this metabolic control pathway suggests that PTEN may modulate mammalian insulin signaling and may be variant in diabetic pedigrees. These results also suggest a mechanism by which human PTEN suppresses tumor formation by limiting cell growth and survival signals through Akt. Maehama, T. and Dixon, J.E. (1998). J. Biol. Chem. 273:13375-13378. Paradis, S. and Ruvkun, R. (1998). Genes Dev. 12:2488-2498.

Chan, Kevin K et al. (2011) International Worm Meeting "Integrin Cooperates with UNC-40 to Guide Muscle Arm Extension."

Roy, Peter J, Chan, Kevin K

[International Worm Meeting, 2011]

Body muscles of nematodes extend specialized plasma membranes, called muscle arms, to the dorsal and ventral nerve cords to form neuromuscular junctions. The extension of C. elegans muscle arms is stereotypical and likely guided by a chemoattractant that is secreted from motor axon targets. We recently found that an UNC-40/DCC pathway, which includes the UNC-40 receptor, the MADD-2 adaptor protein, and the UNC-73 Rho-GEF, functions cell-autonomously in C. elegans to direct muscle arm extension towards the motor axons (Alexander et al., 2009, 2010). Two lines of evidence suggest that this signalling pathway may be a non-canonical one. First, UNC-6/Netrin, which is the canonical ligand of UNC-40, is dispensable for muscle arm extension to motor axons. Second, the extra-cellular domains of UNC-40 are not required for UNC-40's role in directing muscle arm extension. These data suggest that a second receptor complex may co-operate with UNC-40 to guide muscle arms. To identify other plasma membrane-associated proteins that might cooperate with UNC-40, we screened candidate receptors for their ability to facilitate UNC-40-mediated muscle arm extension. We previously reported that PAT-2/a-integrin, but not INA-1/a-integrin, is required for muscle arm extension to the motor axons (Dixon et al., 2006). Here, we present evidence that the PAT-2/PAT-3 integrin heterodimer is required for UNC-40-dependent muscle arm extension. We are currently investigating the mechanism by which integrin facilitates UNC-40's role in directing muscle arm extension.

Jillian L. Youds et al. (2006) European Worm Meeting "The DOG-1 Helicase, Genomic Instability and Fanconi Anemia"

Ann M. Rose, Nigel J. O'Neil, Simon J. Boulton, Louise J. Barber, Jillian L. Youds

[European Worm Meeting, 2006]

Jillian L. Youds1, Nigel J. O''Neil1, Louise J. Barber2, Simon J. Boulton2 & Ann M. Rose1. In Caenorhabditis elegans, DOG-1 is required for the maintenance of polyG/polyC-tracts (G-tracts). In the absence of DOG-1, it is thought that G-tracts form secondary structures that block replication, leading to deletions that initiate in the G-tracts. Using our assay for deletions forming in the absence of DOG-1, we have assayed the in vivo contribution of various repair genes to the maintenance of these tracts. We show that DOG-1 and the BLM ortholog, HIM-6, act synergistically during replication; simultaneous loss of function of both genes results in replicative stress and an increase in the formation of small deletions that initiate in G-tracts. Similarly, we show that genes implicated in homologous recombinational repair and trans-lesion synthesis are required to prevent G-tract deletions in the dog-1 background. However, genes essential to the non-homologous end-joining and nucleotide excision repair pathways do not appear to be involved in deletion prevention or formation. By investigating the mechanisms that maintain genomic stability in dog-1 mutants, we might better understand the genomic instability associated with Fanconi anemia, as the human gene most similar to dog-1, BRIP1/FANCJ, was recently shown to be mutated in a subset of patients with Fanconi anemia, implicating it in interstrand cross-link (ICL) repair. In collaboration with the Boulton lab, we are currently investigating the possibility that DOG-1 and BRIP1 have functionally conserved roles in DNA repair. Our preliminary data indicate that dog-1 mutants are sensitive ICL-inducing agents and we are assessing the relationship to fcd-2 and other genes involved in ICL repair. . This research is funded by the Natural Sciences and Engineering Council and the Michael Smith Foundation for Health Research.

Scott Dixon et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Insulin Signaling Negatively Regulates Muscle Arm Extension in C. elegans"

Mariam Alexander, Scott Dixon, Peter Roy

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The guided migration of cells and cell extensions is essential for animal development. To better understand the genes required for the initiation and guidance of cell extensions our lab is studying muscle arm extension from the body wall muscles (BWMs) in C. elegans. Muscle arms (MAs) are BWM-membrane extensions that make connections with the motor axons located in the dorsal and ventral nerve cords of the animal (White et al., 1986, Dixon & Roy, 2005). Well-fed L3-stage worms average 3.5 muscle arms per BWM. Interestingly, dauer animals average significantly more muscle arms per BWM than normal, a phenotype we call the supernumerary arm (Sna) phenotype. Unlike other dauer-specific modifications, the Sna phenotype is irreversible: adults recovered from the dauer stage are still Sna. To better understand this phenotype we are examining signaling pathways known to be involved in dauer formation, including the insulin pathway. We find that hypomorphic daf-2/insulin receptor mutants that have not gone through the dauer stage are Sna and that this phenotype is suppressed by null alleles of the downstream transcription factor daf-16/FOXO. Mutations in daf-16 alone have no effect on arm outgrowth. However, expression of DAF-16 in the BWMs and to a lesser extent the intestine, but not the nervous system, of daf-2;daf-16 double mutant worms rescues the Sna phenotype. These data suggest that DAF-16 stimulates the irreversible production of additional (supernumerary) muscle arms under conditions where DAF-2 activity is down-regulated. Studies are underway to see if these supernumerary muscle arms influence animal behavior and to determine if any of the known insulin ligands regulate this phenotype.

Dalley BK et al. (1987) Worm Breeder's Gazette "synchronous young Ascaris embryos are transcribing."

Golomb M, Dalley BK, Bennett KL

[Worm Breeder's Gazette, 1987]

Ascaris embryos offer several advantages with regard to studying early nematode development, including the capability of obtaining large populations of synchronous embryos. The fertilized, but uncleaved eggs remain arrested and viable at 4 C for months. With the intention of producing an oocyte and an embryonic cDNA library to screen with our P granule antibodies, Dr. Bennett's new laboratory has recently isolated polyA+ RNAs from Ascaris oocytes and from Ascaris embryos at two stages, 4-8 cells and 16-32 cells. These RNAs have been tested with various probes, including our Ascaris actin gene. All contain actin message, but it had yet to be established whether young Ascaris embryos carry out new zygotic transcription, or rely entirely on maternal messages. We wanted to establish that the embryonic library will not be redundant. To address this question, we have isolated nuclei from Ascaris embryos using the protocol of Dixon et al.(WBG 9:3,73-74). The embryos used were slightly older than those used in the RNA preparations, in the 5-6th cleavage divisions, our reasoning being with more nuclei we'd have a better chance of success with the first isolations. The nuclei are intact, as assayed by diamidinophenylindole (DAPI) staining. We have used this preparation, along with nuclei from a mixed population of Caenorhabditis eggs, in run-on transcription assays, essentially as described by McKnight and Palmiter (JBC 1979, 254:9050). These Ascaris nuclei incorporate significant amounts of label (~35 fold over background). Preliminary results of testing the alpha- amanitin sensitivity of the run-on transcripts show these young Ascaris nuclei have a striking profile, with the transcripts being over 80% sensitive to 1 microgram/ml alpha-amanitin, implying high levels of Polymerase II transcription. After assuring ourselves that we are optimizing conditions for Ascaris (we're currently using those worked out in the Golomb lab for Caenorhabditis), we will assay transcription with specific probes, including actin. We plan to look at earlier embryos; the synchronous Ascaris populations offer the opportunity to profile transcription throughout nematode embryogenesis. And it's on for the embryonic library!

Golomb M (1988) Worm Breeder's Gazette "mutant RNA polymerase II is super-resistant to alpha-amanitin."

Golomb M

[Worm Breeder's Gazette, 1988]

RNA polymerase II (RpoII) from C. elegans is 50% inhibited by 7 ng/ml of alpha-amanitin, whereas RpoIII is 50% inhibited by 80 g/ml and RNA polymerase is unaffected by 500 g/ml. Dominant mutations have been described in the gene for the large subunit of the enzyme from mammals (Lobban et al. (1976), Cell 8: 65-70), Drosophila ( Greenleaf et al. (1979), Cell 18: 613-622), and the worm (Sanford et al. (1983), J. Biol. Chem. 258: 12804-12809), which render the enzyme partially resistant to alpha-amanitin by decreasing binding of the drug. None of these mutations, however, confer the full resistance characteristic of animal RpoI. Using a selection technique which increases the permeability of worms to alpha-amanitin, Rogalski and Riddle (this issue) have obtained a derivative of a resistant (ama- 1(m118)) strain that is super-resistant to alpha-amanitin. The new mutation (m526) is also at the ama-1 locus and maps very close to m118 (Rogalski and Riddle, op. cit.) RpoII from the ama-1(m118m526) strain is labile to chromatography and cannot be separated in active form from RpoI and III. However, RNA polymerases I, II and III in wild-type nuclei have the same sensitivities to alpha-amanitin as the purified enzymes. These enzymes can, therefore, be distinguished in a nuclear run-off assay using appropriate DNA probes. Nuclei were isolated by the method of Dixon (WBG 9:3, 73-74) and allowed to incorporate alpha-[32P]-CTP in the presence of various concentrations of alpha-amanitin. Nuclear transcripts were hybridized to a Southern blot of restriction fragments from cloned genes. These included genes transcribed by RpoI (Ascaris rDNA obtained from K. Bennett), RpoIII (C. elegans 5S DNA ( Honda et al. (1986), Nucl. Acids Res. 14: 869-881), and RpoII (C. elegans splice leader precursor DNA (Honda et. al., op.cit.; Krause and Hirsh (1987), Cell 49: 753-761)). Splice-leader precursor is transcribed in nuclei by RNA polymerase II (M. Golomb and S. Bektesh, unpublished). In this assay, transcription of splice-leader precursor RNA was unaffected by 500 g/ml alpha-amanitin, a concentration at which 5S RNA transcription was nearly completely inhibited. Thus, the m526 mutation appears to render RpoII fully resistant to alpha-amanitin.

Dalley BK et al. (1987) Worm Breeder's Gazette "are dauer larvae transcriptionally acitve?"

Dalley BK, Golomb M

[Worm Breeder's Gazette, 1987]

Although C. elegans dauer larvae have been reported to lack translatable mRNA, the transcriptional activity of dauer larvae has not been assayed directly. Using the method of Dixon et al. (WBG 9:3, 73-74), we have isolated nuclei from staged cultures, including dauer larvae. For purposes of comparison among developmental stages, the amount of nuclear DNA was measured using a sensitive fluorometric assay. We have optimized conditions for run-on transcription by RNA polymerase II. Under these conditions, approximately 12,000 cpm or 6 pmol of [3H]-UTP were incorporated into total RNA by 10 l of an adult stage nuclear suspension containing 4ng of DNA. The table below shows the rate of nuclear transcription in a 10 min assay (20 C), using nuclei from various developmental stages. [See Figure 1] Relative to nuclei from other stages, dauer nuclei are certainly transcriptionally active, although less so than nuclei from late larval stages and adults. We will next compare transcription of specific mRNAs using appropriate probes. Using an immunological assay, we have measured the quantity of RpoII polypeptides (relative to total protein) in crude extracts from various developmental stages. Not surprisingly, the amounts of RpaII in extracts from all stages, including dauers, is closely similar. RpoII activity is also roughly similar in extracts from different stages. Thus, differences in transcriptional rates among different stages cannot be attributed to changes in enzyme level or activity in a nonspecific assay. In Western blots with anti-RpoII antibody, the polypeptide composition of RpoII from all stages is closely similar, with one exception. The amount of the 30 kd subunit detected by this probe is greatly reduced in whole cell and nuclear extracts of dauers, relative to other stages; this band reappears during dauer recovery. We are currently trying immunoprecipitation experiments to ask whether this change reflects loss or modification of the subunit. The use of nuclear extracts has led us to suspect modification of the large subunit of C. elegans RpoII, which has an Mr of 200,000. Crude extracts, but not purified enzyme, have small amounts of a polymerase-related polypeptide with Mr = 235,000. This is probably the worm equivalent of the IIo variant of the large subunit in other organisms, which is a phosphorylated form of IIA. In contrast to C. elegans whole-cell extracts, nuclear extracts have a high proportion of IIo (approximately 50% by antigenicity). This is useful information because IIo is the form believed to be active in in vitro transcription in other systems. In contrast to the situation with the 30 kDa subunit, modification of the large subunit appears to be similar in all stages, including dauers.