Sym M et al. (2000) West Coast Worm Meeting "Two new genes regulating neuroblast migration in C. elegans"

Kenyon CJ, Sym M, Ch'ng Q-L, Yang L

[West Coast Worm Meeting, 2000]

In C. elegans, the Q cells are bilaterally symmetric neuroblasts present in the posterior body region of the worm at hatching. During the first larval stage, the Q cells divide and migrate. QR and its descendents migrate anteriorly whereas QL and its descendents migrate posteriorly. Several genes that regulate the anterior migrations of QR and its descendents have been identified. These include: 1) lin-39, a homeobox gene required in QR and its descendents for migration (Wang B. et al, 1993; Clark S. et al, 1993); 2) mig-13, a novel transmembrane protein required outside of QR and its descendents for migration (Sym M et al, 1999); and 3) egl-20, a Wnt homolog expressed in cells in the tail region (Whangbo J.. and Kenyon C, 1999). Two mutant screens (Mary Sym and Queelim Ch'ng) were conducted to identify additional genes that regulate the migration of QR and its descendents. From these screens, mutations in two new genes were identified that cause certain cells in the QR lineage to stop migrating prematurely. These two genes seem likely to be involved in guidance rather than in providing cells with the ability to migrate because, in these mutants, other cells sometimes migrate in the wrong direction. Current efforts are directed toward cloning these two genes and determining how these genes regulate the migration of QR and its descendents together with genes previously known to regulate these migrations. References: Clark SG, Chisholm AD, and Horvitz HR. 1993. Control of Cell Fates in the Central Body Region of C. elegans by the Homeobox Gene lin-39. Cell 74: 43-55. Sym M, Robinson N, Kenyon C. 1999. mig-13 Positions Migrating Cells Along Anteroposterior Body Axis of C. elegans. Cell 98: 26-36. Wang BB, Muller-Immergluck MM, Austin, J, Robinson, NT, Chisholm, A, Kenyon, C. A Homeotic Gene Cluster Patterns the Anteroposterior Body Axis of C.. elegans. Cell 74: 29-42. Whangbo J, Kenyon, C. A Wnt Signaling System that Specifies Two Patterns of Cell Migration in C. elegans. Molecular Cell 4: 851-858.

WB Derry et al. (1999) International C. elegans Meeting "Analysis of the C. elegans homolog of the FHIT tumor suppressor gene"

S van Kreeveld, WB Derry, JH Rothman

[International C. elegans Meeting, 1999]

Alterations in the FHIT gene occur frequently in the development of several human cancers (1). The Fhit protein is a diadenosine P 1 , P 3 -triphosphate hydrolase and is a member of the histidine triad superfamily of nucleotide binding proteins (2). The cellular mechanism of Fhit activity and the relationship between Fhit signaling and tumorigenesis are presently unknown. The C. elegans and Drosophila FHIT genes encode a fusion protein in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases, and are referred to as NitFhit (3). We are interested in understanding the role of NitFhit in development and programmed cell death. RNAi of C. elegans NitFhit causes an embryonic arrest phenotype, suggesting an essential role for this gene in development. We are currently analyzing the loss-of-function phenotype and the effect of ectopic NitFhit expression on viability and programmed cell death in the worm. (1) Huebner, K., Garrison, P.N., Barnes, L.D. & Croce, C.M. (1998). Ann. Rev. Genet ., 32 : 7-31. (2) Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A, Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M. & Huebner, K. (1996). Biochemistry , 35 : 11529-11535. (3) Pekarsky, Y., Campiglio, M., Siprashvili, Z, Druck, T., Sedkov, Y, Tillib, S., Draganescu, A., Wermuth, P., Rothman, J.H., Huebner, K., Buchberg, A.M., Mazo, A., Brenner, C. & Croce, C.M. (1998). Proc. Natl. Acad. Sci. USA , 95 : 8744-8749.

Kenyon CJ et al. (2000) West Coast Worm Meeting "How are anterior cell migrations guided by mig-13?"

Kenyon CJ, Ch'ng Q-L

[West Coast Worm Meeting, 2000]

mig-13 was previously identified as a guidance factor specifically required for the anterior migrations of the QR descendants. mig-13 acts by promoting cell migrations in the anterior direction (Sym et. al., 1999). We are taking several approaches to understand further how the anterior migrations of the QR descendants are guided by mig-13. mig-13 is predicted to encode a novel transmembrane protein containing a CUB domain and LDL-receptor repeat in the extracellular region as well as a proline-rich domain in the intracellular region (Sym et. al., 1999). Our structure/function studies suggest that the extracellular domain of MIG-13 alone can confer partial function in guiding the QR descendants to the anterior. A rescuing mig-13::GFP fusion was previously found to be expressed in the anterior and mid-body ventral cord motor neurons, which cross the migratory track of the QR descendants. Consistent with this expression pattern, mosaic analysis revealed that mig-13 acts non-autonomously to direct the migrations of the QR lineage (Sym et. al., 1999). To determine where mig-13 expression is sufficient to guide the migrating cells, we are expressing mig-13 in broad sets of tissues, as well as in specific subsets of cells that express mig-13::GFP. We also intend to refine previous mosaic analysis to pinpoint the cells in which mig-13 acts. Reference Sym M, Robinson N and Kenyon C. Cell, 1999 Jul 9, 98(1):25-36.

AK Jones et al. (2009) European Worm Neurobiology Meeting "A mutation in the Caenorhabditis elegans nicotinic acetylcholine receptor a subunit, unc-63, provides a model for fast channel congenital myasthenia"

D Rayes, AK Jones, G Hernando, R Jones, SD Buckingham, DB Sattelle, A Al-Diwani, C Bouzat, TPR Maynard

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC and OXION. Abstract: The nematode Caenorhabditis elegans is an established model organism for studying neurobiology since many synaptic molecular components are conserved both in the worm and humans. The unc-63 a subunit of the C. elegans nicotinic acetylcholine receptor (nAChR) plays an important role in fast cholinergic synaptic transmission at the nematode neuromuscular junction (1). Here we show that worms with the unc-63(x26) allele, with its .C151Y mutation that disrupts the Cys-loop, have deficient muscle function as displayed by impaired thrashing locomotion compared to wild-type worms (2). Single-channel recordings from muscle cells of the mutant strain show reduced number of active patches, a 100-fold reduced frequency of opening events, and abnormally reduced duration of channel openings similar to that observed in patients with fast-channel congenital myasthenia syndromes (FCCMS). Interestingly, a mutation in the equivalent position of the e subunit of the human muscle nAChR (.C128S) is associated with a FCCMS due to nAChR deficiency at the endplate (3). Therefore, disruption of the Cys-loop results in qualitatively similar actions on both nematode and human receptors. Therapy for FCCMS is limited in variety and success with 3,4-diaminopyridine (3,4-DAP) and anticholinesterase drugs such as pyridostigmine bromide (PB). We show that PB and 3,4-DAP can partially rescue the motility defect seen in unc-63(x26). Conclusions: We describe the first candidate C. elegans model for screening for drugs aimed at ameliorating the consequences of mutations in nAChRs associated with FCCMS. References: (1) Culetto E et al. J Biol Chem. 2004. 279:42476-83. (2) Jones AK et al. Hum Mol Genet. Submitted 2009. (3) Milone M et al. Ann N Y Acad Sci. 1998. 841:184-8.

Judkins, Joshua C. et al. (2011) International Worm Meeting "Second generation synthesis and bioactivity of novel endogenous ligands of the nuclear hormone receptor DAF-12."

Schroeder, Frank, Mahanti, Parag, Bose, Neelanjan, Hoffman, Jacob, Judkins, Joshua C.

[International Worm Meeting, 2011]

Nuclear hormone receptors (NHR's) are critical components in metazoan signaling cascades, and it is estimated that 284 NHRs exist in C. elegans.1 Although the function of most NHR's may be regulated by small molecule ligands, endogenous ligands for only one C. elegans NHR have been proposed. The two bile-acid-like steroids, D4- and D7-dafachronic acids were predicted as endogenous transactivators of DAF-122, an NHR with high homology to vertebrate Vitamin D and LXR receptors.3 Recent studies by the Schroeder group have revealed the presence of additional ligands of DAF-12. The structural diversity of the newly revealed dafachronic acids highlights the importance of a high yielding and flexible approach to chemically synthesize DAF-12 ligands for biological investigation. Here we present a novel second generation synthesis that is shorter, more efficient, and more versatile than those previously published.4-8 In addition, our synthesis converges on an aldehyde intermediate that allows divergent access to both previously described and novel dafachronic acids. Our synthetic approach provides D4- and D7-dafachronic acids in 6 and 9 steps, respectively. Key features of this synthesis include multiple transformations in a one-pot reaction and a late-stage chiral hydrogenation. The variety of structures attainable through this synthetic method has allowed us to investigate the role of these and related molecules in C. elegans signaling pathways and to examine structure activity relationships (SAR's) of several dafachronic acid variants. We present bioactivity data for several natural and non-natural dafachronic acids. In addition, we compared SAR's obtained from in-vitro experiments with the results from in-vivo studies. [1] Robinson-Techavi et al.; J. Mol. Evol. 2005, 60, 577. [2] Motola et al.; Cell. 2006, 124, 1209. [3] Antebi et al.; Genes & Dev., 2000, 14, 1512. [4] Giroux et al.; Org. Lett., 2008, 16, 3643. [5] Giroux et al.; Org. Lett., 2008, 5, 801. [6] Giroux et al.; JACS, 2007, 129, 9866. [7] Martin et al.; Org. Biomol. Chem., 2010, 8, 739. [8] Gioiello et al.; Tetrahedron, 2011, 67, 1924.

Yung S Lie et al. (2003) International Worm Meeting "Identification of proteins interacting with MIG-13 in Q cell migration"

Cynthia Kenyon, Marc Vidal, Jean-Francois Rual, Mary E Hatten, Yung S Lie, Scott Anderson, John R Yates

[International Worm Meeting, 2003]

We are studying cell migration in the context of anteroposterior migrations of the Q neuroblasts and their descendants. QR and QL are born in right-left symmetrical positions in the animal. QR and its descendants migrate towards the anterior of the body, while QL and its descendants migrate towards the posterior. Proper positioning of QR and its descendants in the anterior requires mig-13. mig-13 encodes a novel transmembrane protein (Sym et al., 1999). We currently know very little about the mechanism by which MIG-13 functions. In order to learn more about how migrating cells determine a final stopping point, we are performing biochemical experiments to isolate proteins that bind to MIG-13. Immunoprecipitations were performed using extracts from worms expressing a rescuing MIG-13-GFP fusion, or as a control, from worms expressing GFP alone. A commercial anti-GFP antibody was used to isolate the fusion protein (or GFP alone in the control) and any associated proteins.Immunoprecipitated samples were analyzed by mass spectrometry to identify proteins present in each sample. We will present data from this mass spectrometry analysis. RNAi was used to disrupt the gene function of candidates identified by mass spectrometry. One such candidate has a QR descendant cell migration phenotype when disrupted by RNAi. We are investigating the interaction of this candidate with MIG-13 by yeast two hybrid and by other biochemical methods. In addition, we plan to complete genetic analysis to determine the functional significance of these protein-protein interactions. We are also currently characterizing a mutant, mu335, which has a phenotype similar to mig-13. QR descendant cell migration is defective in mu335 animals. We have mapped mu335 to the center of LGIII. To identify the gene disrupted by mu335, we are in the process of injecting cosmids for rescue of the migration phenotype. Sym, M., Robinson, N., and Kenyon, C. (1999). MIG-13 positions migrating cells along the anteroposterior body axis of C. elegans. Cell 98, 25-36.

Heestand, Bree et al. (2021) International Worm Meeting "Adult longevity of late-generation Piwi/prg-1 mutants"

Ahmed, Shawn, Lister-Shimauchi, Evan, Simon, Matt, Kennedy, Sophia, Heestand, Bree

[International Worm Meeting, 2021]

The C. elegans Piwi Argonaute protein PRG-1 associated with piRNAs to preventing germ cells from expressing foreign genetic elements such as transposons. PRG-1 promotes germ cell immortality and lines of prg-1 mutants are initially fertile but become sterile if they are grown for many generations. prg-1 mutant sterility is a form of reproductive arrest (1), which may resemble reproductive arrest in response to environmental stresses such as starvation (2). We found that early-generation prg-1 mutants have normal lifespans and fertility, while later generations had reduced fertility yet longer lifespans. Mutation of the stress response transcription factor Daf-16/FOXO, caused the longevity phenotype of late-generation prg-1 mutants to disappear, leading to the conclusion that prg-1 mutant longevity may be a hormetic stress response. Phenotypes that arise from epigenetic defects can be inherited for at least 3 generations (3,4), and we found that longevity of late-generation prg-1 mutants is inherited by F1 but not F2 cross progeny. We are currently asking if the daf-12 germline signaling pathway that regulates aging is required of prg-1 mutant longevity. Overall, our results imply that the longevity of late-generation prg-1 mutant is distinct from the longevity that is transmitted for several generations by germ cells of mutants deficient for the ASH-2/SET-2/trithorax transcriptional activation complex (3,4). 1. Heestand, B., Simon, M., Frenk, S., Titov, D. & Ahmed, S. Transgenerational Sterility of Piwi Mutants Represents a Dynamic Form of Adult Reproductive Diapause. Cell Rep. 23, 156-171 (2018). 2. Angelo, G. & Van Gilst, M. R. Starvation Protects Germline Stem Cells and Extends Reproductive Longevity in C. elegans. Science 326, 954-958 (2009). 3. Greer, E. L. et al. Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans. Nature 479, 365-371 (2011). 4. Lee TW, David HS, Engstrom AK, Carpenter BS, Katz DJ. Repressive H3K9me2 protects lifespan against the transgenerational burden of COMPASS activity in C. elegans. Elife (2019). doi: 10.7554/eLife.48498

QueeLim Ch'ng et al. (2001) International C. elegans Meeting "How does mig-13 guide anterior migrations?"

Cynthia Kenyon, QueeLim Ch'ng

[International C. elegans Meeting, 2001]

mig-13 is a guidance factor that promotes cell migrations in the anterior direction (Sym et. al., 1999). Previous work demonstrated that mig-13 is required for the anterior migrations of the QR descendants and the BDU neurons (Sym et. al., 1999). Consistent with the role of mig-13 in anterior migrations, we have also found that mig-13 also directs the anterior migration of the distal tip cell (DTC) in the posterior gonad arm during late L3. We are taking several approaches to understand how mig-13 can guide many anterior migrations. mig-13 encodes a novel transmembrane protein containing putative protein-protein interaction domains: a CUB domain and a LDL-receptor repeat in the extracellular region as well as a proline-rich domain in the intracellular region (Sym et. al., 1999). We have examined the function of these domains in MIG-13 by deleting them and assaying the in vivo activity of the resulting MIG-13 construct. Our data suggests that a MIG-13 construct lacking the intracellular domain can confer partial function in directing the QR descendants to the anterior. Previous mosaic analysis revealed that mig-13 acts non-autonomously to direct the migrations of the QR lineage (Sym et. al., 1999). To determine where mig-13 expression is sufficient to guide the migrating cells, we have expressed mig-13 in different sets of tissues, as well as in specific subsets of cells. Expression of mig-13 in all neurons but not any of the other tissues we have tested rescues the QR descendant and DTC migrations in mig-13 mutant animals. This suggests that mig-13 might function in neurons. To pinpoint the cells in which mig-13 acts, we are also refining previous mosaic analysis. For the guidance of the QR descendants, we have narrowed the focus of mig-13 activity to the AB.pr (and possibly the AB.pra) lineage. We have also isolated several mosaic animals that have wild-type QR descendant migrations but mutant DTC migration in the posterior gonad arm, raising the possibility that the migrations of the QR descendants and the posterior DTC are guided by different cells. Reference: Sym M, Robinson N and Kenyon C. Cell, 1999 Jul 9, 98(1):25-36.

Janton, Francis et al. (2017) International Worm Meeting "The DVA neuron promotes wakefulness via a cAMP-mediated mechanism."

Brown, Amelia, Nelson, Matthew, Janton, Francis, Vance, Ryan, Szurgot, Mary, Sullivan, Nicole

[International Worm Meeting, 2017]

Caenorhabditis elegans display unique forms of sleep: developmentally timed sleep (DTS) and stress-induced sleep (SIS), which are regulated by overlapping and distinct mechanisms (Trojanowski et al., 2015). The cyclic adenosine monophosphate (cAMP)/Protein Kinase A (PKA) pathway promotes arousal in invertebrates and vertebrates (Crocker and Sehgal, 2010). This signaling pathway is downregulated during DTS in C. elegans (Belfer et al., 2013) and we show the same is true for SIS. To determine where cAMP/PKA functions during sleep we have been expressing and activating the near infrared light-activated adenylyl cyclase called IlaC, which converts ATP into cAMP in the presence of red light, in candidate neurons (Ryu et al., 2014). We have chosen this tool because, unlike blue light, red light does not wake worms up during sleep. We find that pan-neuronal activation of IlaC reduces both DTS and SIS. Induction of cAMP in either the motor neurons, command interneurons or the sleep-regulating interneuron RIA is not sufficient to reduce DTS or SIS. However, activation of IlaC in the DVA interneuron, by expressing it from the full twk-16 promoter or using a DVA-specific enhancer from the twk-16 promoter (Puckett Robinson et al., 2013), significantly reduces SIS, DTS and non-physiological quiescence induced by neuropeptide over-expression (Nelson et al. 2013 and 2014). This suggests that DVA may be a common wake-promoting neuron downstream of distinct sleep promoting circuits. To confirm DVA's place in the known circuitry, we have expressed a cAMP biosensor, Epac1-camps (Shafer et al., 2008) in DVA and are currently measuring cAMP levels during sleep. What lies downstream of the cAMP/PKA pathway during DTS and SIS is still unclear. The cAMP response element binding protein (CREB) is a transcription factor known to play a role in C. elegans sleep and arousal, as well as arousal in mammals (Graves et al., 2003). CRH-1 is the C. elegans CREB homologue; and crh-1 loss-of-function mutants display increased amounts of DTS (Singh et al., 2014), which we have observed as well. We show evidence that CRH-1 functions downstream of cAMP/PKA in the nervous system during DTS, and are currently testing if DVA is the wake-promoting site of action. Surprisingly, we find that crh-1 mutants have reduced SIS, thus cAMP/PKA promotes arousal after SIS in DVA via distinct mechanisms.

Sym M et al. (1996) West Coast Worm Meeting "mig-13 AND CELL MIGRATION"

Kenyon CJ, Sym M

[West Coast Worm Meeting, 1996]

The Q cells are born as left/right homologs (QL and QR) and undergo identical cell lineages. However, QL and QR migrate in opposite directions: QL and its descendants migrate posteriorly; whereas, QR and its descendants migrate anteriorly. The mig-13(mu31) mutation was identified in a screen for mutations affecting Q cell migration (Naomi Robinson, Ph. D. thesis, 1995). The initial migration of QR itself is not affected in mig-13(mu31), but the subsequent anterior migrations of its descendants are significantly shortened. Thus, these cells are found substantially posterior to their wild-type positions. The posterior migration of QL and its descendants is not affected in this mutant, suggesting that migration per se does not require mig-13(+). However, in mutants where the descendants of QL migrate anteriorly instead of posteriorly (e.g., mab-5, egl-20), mig-13(+) is required for anterior migration of both the QR and QL descendants. Molecular characterization of mig-13 has shown that the gene is SL1-spliced and contains 8 exons, which encode a predicted protein of 362 amino acids. Sequence comparisons revealed an N-terminal signal sequence followed by two potential protein interaction domains and a single-pass transmembrane domain. The predicted cytoplasmic C-terminus bears no homology to anything currently in the database. A C-terminal GFP fusion was constructed that exhibits full mig-13-rescuing activity. Worms bearing this mig-13-GFP fusion show reproducible expression in a number of cells, most of which are neurons. The fusion protein is localized to the periphery of each expressing cell, as predicted from the transmembrane domain within MIG-13. During the time of Q cell migration (L1), mig-13-GFP is expressed in the pharygeal-intestinal valve cells and in approximately 10 ventral cord neurons, but is not detected in the Q cell lineage. Most of the ventral cord neurons show staining in the ventral and/or dorsal axon bundles as well as commissural axons, which project into the dorsal cord. Interestingly, the ventral cord neurons that express the fusion protein are located in the anterior two-thirds of the body axis, through which QR and its descendants migrate. Ventral cord neurons in the posterior third of the body do not express mig-13-GFP. Thus, the expression pattern suggests that mig-13 acts non-autonomously to influence Q cell migration. Mosaic analysis is in progress, which should determine whether this is indeed the case. Perhaps MIG-13 acts as an attractive or permissive cue along the migratory pathway to promote anterior Q cell migration.