Rankin, Aysha C. et al. (2015) International Worm Meeting "Genes involved in embryonic motor neuron positioning along the AP axis."

Tanner, Matthew, Slatculescu, Cristina, Robinson, Claire, Rankin, Aysha C., Colavita, Antonio, Perkins, Theodore

[International Worm Meeting, 2015]

The six embryonically-derived DD motor neurons are born evenly spaced along the AP axis in newly hatched larvae and maintain this spacing into adulthood. We have found that components of a planar cell polarity (PCP)-like pathway that includes prkl-1/Prickle and vang-1/Van Gogh are important for properly positioning DD motor neurons in the ventral nerve cord. In prkl-1, and to a lesser extent, vang-1 mutants, DD neurons are shifted anteriorly and display distinctive cell spacing defects. These defects can be rescued by prkl-1 or vang-1 expression from a pan-neuronal promoter suggesting cell-autonomous roles. DA and DB neurons are also mispositioned in prkl-1 and vang-1 mutants. Neuronal positioning defects are present in newly hatched larvae suggesting an embryonic origin for the defect. DD1,3,5 and DD2,4,5, derived from left and right sides of the embryo, undergo cell intercalation during the comma stage to form a single line of DD neurons at the midline. prkl-1 and vang-1 mutants display DD intercalation defects that are suggestive of a delay in cell intercalation or improper cell-cell neighbor interactions during intercalation. In order to identify new genes involved in cell intercalation and the proper positioning of motor neurons along the AP axis as well as new insight into PCP-like signaling we are in the process of performing a forward genetic screen for DD neuron spacing defects. PCP signaling is known to play an important role in cell intercalation during neural tube and organ morphogenesis in vertebrates. Our findings show for the first time that a PCP-like pathway involving the core components Prickle and Van Gogh is involved in some aspects of cell intercalation during C. elegans development.

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

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

WB Derry, S van Kreeveld, 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.

Claire Lecroisey et al. (2006) European Worm Meeting "Implication of the Dense Body Protein Dyc-1 in the Dystrophin Dependent Muscle Degeneration"

Kathrin Gieseler, Laurent Segalat, Claire Lecroisey

[European Worm Meeting, 2006]

Claire Lecroisey, Kathrin Gieseler and Laurent Sgalat. The molecular mechanism underlying muscle necrosis in dystrophinopathies remains elusive. Our group addresses this question by using the genetically amenable animal model Caenorhabditis elegans, which has the same overall sarcomere composition and architecture as those of vertebrates.. In C. elegans, mutation of the dystrophin homologue, dys-1(cx18), produces a peculiar behavioural phenotype (hyperactivity, a tendency to hypercontract). In a sensitized hlh-1(cc561ts) background, which is a mild mutation of the myogenic factor MyoD, the dys-1(cx18) mutation also leads to a progressive muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to a phenotype similar to that of dys-1(cx18) mutation, which suggests that the two genes are functionally linked. Like dys-1(cx18); hlh-1(cc561ts), the double mutant dyc-1(cx32); hlh-1(cc561ts) also shows a progressive muscle degeneration. Moreover, Dyc-1 overexpression partially suppresses the dys-1(cx18); hlh-1(cc561ts) phenotype.. In the sarcomere, Dyc-1 is localized at the edge of the dense body, the nematode muscle adhesion structure functionally equivalent to vertebrate Z disc, where actin filaments are anchored. Two hybrid and immunocytochemistry experiments indicate that Dyc-1, Deb-1 (the vinculin homologue and main component of dense bodies), Zyx-1 (a focal adhesion protein), and Atn-1 (alpha-actinin homologue) may form a complex at the dense body. Our results suggest that the dense body might be the site of early pathological events occurring in the absence of dystrophin.. We are currently trying to demonstrate that dystrophin impairs the function of dense bodies proteins.

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

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

[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.

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."

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

[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.

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.

KL Williams et al. (1999) International C. elegans Meeting "Identification and analysis of a new mutation affecting T cell polarity"

KL Williams, MA Herman

[International C. elegans Meeting, 1999]

We are interested in how cell polarity is controlled during C. elegans development. Our approach is to identify and study genes involved in the control of cell polarity by identifying mutations that disrupt the polarities of individual cells. Mutations in lin-44 cause the polarities of certain cells in the tail, called TL and TR, to be reversed with respect to the body axis. LIN-44 is a WNT signaling protein that is made by the skin cells at the tip of the developing tail and functions to specify the polarity of more anterior tail cells. Mutations in lin-17 and egl-27 cause the loss of TL and TR cell polarities. LIN-17 is a Frizzled-like protein, suggesting that it is a receptor of LIN-44 WNT signal. EGL-27 contains a domain similar to MTA1, recently shown to be a subunit of a complex with chromatin-remodeling and histone deacetylase activities, and is required in the T cells for proper cell polarity. We want to learn how LIN-44 WNT signal from the tail skin cells influences the polarities of the receiving cells. The defects in T cell polarity observed in lin-44, lin-17 and egl-27 mutants cause the two neurons of the phasmid, a sensory structure in the tail, to fail to fill with fluorescent dyes. New genes that may interact with lin-44, lin-17 and egl-27 were identified by screening for additional mutations that result in a phasmid dye-filling defect caused by defects in T cell polarity. We are currently studying mn593, a mutation isolated by Claire Kari in a preliminary screen of this type. Subsequent screens have identified three other new mutations affecting T cell polarity (see Abstract by X. Zhao, et al. ). mn593 complements mutations in all other known genes that affect T cell polarity. The mn593 mutation causes an incompletely penetrant phasmid dye-filling defect. Furthermore, one or both pairs of phasmid socket cells are missing in most mn593 animals, indicating a T cell lineage defect. Analysis of mn593 hermaphrodite T cell lineages is underway and will determine the exact nature of the defect. In addition, mn593 males have abnormal tail morphologies. We have mapped mn593 to the interval between dpy-5 and unc-63 on LGI. We are currently in the process of cloning the gene affected by mn593 by microinjection of the relevant cosmid and YAC clones into mn593 hermaphrodites.

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

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

[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.