Elvis Huarcaya Najarro et al. (2006) Development & Evolution Meeting "Mutations in tcl-1 cause defects in T cell polarity"

Elvis Huarcaya Najarro, Krista Williams, Michael Herman, Jennifer Griffiths

[Development & Evolution Meeting, 2006]

In C. elegans, the anteroposterior polarities of a pair of cells in the tail, called TL and TR, are controlled by a WNT signal encoded by lin-44. Mutations in lin-44 cause the reversal of T cell polarity. Mutations in lin-17, which encodes a Frizzled-like protein, cause the loss of T cell polarity, suggesting that LIN-17 is a receptor of the LIN-44 WNT signal. Other Wnt pathway components that have been shown to function in the control of T cell polarity include LIT-1/NLK, WRM-1/beta-catenin, SYS-1/beta-catenin and POP-1/TCF. As in many other anterior-posterior asymmetric cell divisions, the nuclear level of POP-1 is higher in the anterior T cell daughter, T.a, than it is in the posterior daughter, T.p. We have identified several mutations that define new genes involved in the control of T cell polarity, including tcl-1. The tcl-1 mutation causes the loss of neural cell fates in the T cell lineage and is incompletely penetrant. Genetic tests suggest that our tcl-1 allele might cause a weak novel function. Analysis of T cell lineages revealed that T.p often fails to divide in tcl-1 hermaphrodites. We also have determined that tcl-1 affects distribution of GFP::POP-1 to the T cell daughters, thus TCL-1 functions early in the control of T cell polarity. tcl-1 mutants also appear to have an additional defect affecting either the neurons or the supporting cells of the sensory phasmid in the tail. We have rescued tcl-1 with cosmid F55A12, on which there are 10 candidate predicted genes. A genomic fragment that contained both F55A12.2 and ppk-1 rescued tcl-1 mutants, whereas a fragment containing only ppk-1 did not. Furthermore, F55A12.2 is only 712 bp downstream of ppk-1 and is trans-spliced to SL2, suggesting that these two genes are in an operon. Although, it is not yet clear which corresponds to tcl-1 and how it might function in T cell asymmetry, ppk-1 encodes a type I phosphatidylinosital phosphate kinase. These enzymes phosphorylate phosphatidylinositol 4-phosphate to generate phosphatidylinositol 4,5 bisphosphate (PI4, 5P2) that can modulate many cellular behaviors, including cell proliferation. Establishing a role for ppk-1 in cellular asymmetry would be an important finding and could link cell signaling to cell polarity.

Elvis Huarcaya et al. (2007) International Worm Meeting "Mutations in tcl-1/bro-1 cause defects in T cell polarity."

Elvis Huarcaya, Michael A Herman, Jennifer Griffiths, Krista Williams

[International Worm Meeting, 2007]

In C. elegans, the anteroposterior polarities of a pair of cells in the tail, called TL and TR, are controlled by a WNT signal encoded by lin-44. Mutations in lin-44/Wnt cause the reversal of T cell polarity and mutations in lin-17/Fz, cause the loss of T cell polarity, suggesting that LIN-17 is a receptor of the LIN-44 WNT signal. Other Wnt pathway components that have been shown to function in the control of T cell polarity include LIT-1/NLK, WRM-1/<font face=symbol>b</font>-catenin, SYS-1/<font face=symbol>b</font>-catenin and POP-1/TCF. As in many other anterior-posterior asymmetric cell divisions, the nuclear level of POP-1 is higher in the anterior T cell daughter, T.a, than it is in the posterior daughter, T.p. The tcl-1(mn593) mutation causes the loss of neural cell fates in the T cell lineage and is incompletely penetrant. We also have determined that tcl-1 affects distribution of GFP::POP-1 to the T cell daughters, thus TCL-1 functions early in the control of T cell polarity. We have rescued tcl-1 with a 4.8 kb fragment of cosmid F56A3 that only contains the bro-1 gene and have identified the lesion associated with the mn593 allele. Thus, tcl-1 corresponds to bro-1, a homolog of CBF<font face=symbol>b</font>/brother that have been shown to function as cofactors for the RUNX family of transcription factors. RUNX transcription factors play conserved roles in controlling proliferation and differentiation during metazoan development. Mutations in rnt-1, the C. elegans RUNX homolog, cause defects in the divisions of the lateral seam cells, including the T cell. The seam cell lineage defects appears to be caused by defects in proliferation in that loss of rnt-1 function caused upregulation of CKI-1, a CDK inhibitor (1). The T cell lineage defect appears to be caused by defects in cell polarity. RNT-1 functions downstream of LIN-44, downstream or in parallel to POP-1 and upstream of TLP-1. Specifically, rnt-1 did not have a large effect on GFP::POP-1 asymmetric distribution, but did affect TLP-1::GFP asymmetric distribution (2). It is not yet clear how to reconcile these two views or whether both might be correct at different times, with the polarity defect taking precedence during the L1 stage and the proliferation defects in later stages. The phenotype of tcl-1/bro-1 mutants are similar to that of rnt-1 mutants. However, rnt-1 has little effect on GFP::POP-1 asymmetric distribution whereas we observed that tcl-1/bro-1 has a large effect. This might suggest that tcl-1/bro-1 has additional roles in addition to its interaction with RNT-1 or that TCL-1/BRO-1 might more directly interact with POP-1/Tcf in the control of T cell polarity. 1. Nimmo et al., 2005; 2. Kagoshima et al., 2005.

X Lin et al. (1999) International C. elegans Meeting "Isolation of the pat-6and pat-12genes"

BD Williams, X Lin

[International C. elegans Meeting, 1999]

Caenorhabditis elegans body-wall muscle cell development is an advantageous system for the study of integrin signal transduction in vivo. Mutations in the pat-2 and pat-3 genes, which code for integrin alpha and beta subunits, respectively, severely disrupt early cytoskeletal assembly events that occur at the muscle cell membrane. The mutant embryos arrest development with the Pat phenotype ( P aralyzed, A rrested-elongation at T wo-fold), characterized by non-functional body wall muscle cells. The pat-2 and pat-3 genes were identified in a previously reported genome-wide screen for mutants with the Pat phenotype (Williams and Waterston, 1994). We have recently shown that a third gene identified in this screen, pat-4, codes for the C. elegans homologue of Integrin-Linked Kinase, a serine-threonine kinase implicated in integrin-mediated signal transduction (see abstract by Mackinnon and Williams). With the goal of identifying other proteins involved in the integrin-mediated events of muscle cell development, we are pursuing the positional cloning of the pat-6 and pat-12 genes, which were also defined in our original screen for Pat mutants. pat-6 maps genetically to the right end of chromosome IV. We are attempting to molecularly isolate pat-6 by transformation rescue and are currently injecting pools of cosmid DNA from the corresponding region of the physical map. We are taking a similar approach with pat-12. We will present our progress at the meeting. We will also present additional characterization of the muscle assembly defects in pat-6 and pat-12 mutants as revealed by immunostaining experiments with an existing panel of antibodies to many different muscle cell proteins.

Sundararajan, Lakshmi et al. (2011) International Worm Meeting "UNC-40/DCC, PTP-3/LAR, and MIG-21 regulate anterior-posterior polarization and migration of the Q neuroblasts."

Lundquist, Erik, Sundararajan, Lakshmi

[International Worm Meeting, 2011]

Polarization and migration of neurons is essential during nervous system development. We use Q neuroblasts as a system to study neuronal cell migration. The QR and QL neuroblasts, born in the posterior lateral region of the worm, undergo initial polarizations at about one hour after hatching in the anterior and posterior directions respectively. They then migrate in the direction of protrusion and divide to produce three neurons of which AQR (from QR) migrates anteriorly to near the anterior deirid, and PQR (from QL) posteriorly to near the phasmid ganglia. Consistent with previous studies (Honigberg and Kenyon, 2000; Williams et al., 2003), we show that mutations affecting the transmembrane receptors UNC-40/DCC and PTP-3, a LAR type receptor tyrosine phosphatase, cause defects in direction of QR and QL polarization and migration. We found that UNC-40 and PTP-3 might act redundantly in directing posterior migration of QL, as defects were significantly more severe for QL in ptp-3(mu256); unc-40(RNAi) than the single mutants alone. Furthermore, we show that loss of MIG-21, a small transmembrane protein with thrombospondin repeats (Williams 2003), caused near randomization of QL and QR protrusion and migration as previously observed. In an attempt to understand how these genes might be acting together in controlling Q cell migrations, we constructed double mutants of mig-21 with unc-40 and ptp-3. Alone, mig-21 mutants displayed many defects in QR anterior migration (QR often migrated posteriorly). Surprisingly, in mig-21; unc-40 double mutants, the defects in QR anterior migration were suppressed (QR migrated to the posterior), suggesting that posterior migration of QR in mig-21 mutants required functional UNC-40. In contrast, mig-21; ptp-3 resembled mig-21 alone for both QR and QL, suggesting that MIG-21 and PTP-3 might be working in the same pathway or in an independent pathway. Taken together, our data suggest that UNC-40 and PTP-3 might redundantly control posterior migration of QL. Furthermore, MIG-21 might normally inhibit UNC-40 in QR, such that in a mig-21 mutant, UNC-40 drives posterior migration of QR. Current studies are directed at determining the focus of action of these genes. unc-40 and mig-21 have been reported to be expressed in QL and QR (Chan et al., 1996; Williams et al. 2003), and we have found that the ptp-3B promoter is active in cells that are consistent with QL and QR, suggesting cell autonomous function. Furthermore, unc-40(RNAi) driven in QL, QR, and the seam cells induces Q cell defects. We are using mosaic analysis to pinpoint where these genes are required.

Yao Li (2002) Mid-west Worm Meeting "Cloning Characterization of pat-9: A Gene Required For The Assembly Of Thin Filaments Into The Myofilament Lattice"

Yao Li

[Mid-west Worm Meeting, 2002]

The related dense body and M-line structures that are found in C. elegans body wall muscle cells are integrin-based transmembrane attachments that couple the basal lamina to the muscle cell cytoskeleton. In an effort to genetically dissect the assembly of dense body and M-lines, we have been studying mutants with the Pat (Paralyzed, Arrested elongation at Two-fold) phenotype. To date all of the molecularly isolated genes associated with the Pat phenotype are known to encode dense body components, includingunc-52 (UNC-52/perlecan) (1), pat-2 (integrin alpha subunit) (Williams and Waterston, unpubl.), pat-3 (integrin beta subunit) (2), unc-112 (UNC-112) (3), deb-1 (DEB-1/vinculin) (4), unc-97 (UNC-97/PINCH) (5), pat-4 (PAT-4/integrinl-linked kinase) (6), and pat-6 (PAT-6/actopaxin) (Lin and Williams, unpubl.). These mutants, analyzed with antibodies to specific attachment proteins and/or functional Green Fluorescent Protein fusions, have produced an assembly dependence pathway for dense bodies and M-lines (6, 7). Here we focus on the pat-9 gene, which has not yet been molecularly cloned. pat-9 mutants have defects in body wall muscle assembly that most closely resemble those found in deb-1vinculin mutants. In particular, the recruitment of actin thin filaments to the muscle cell membrane appears to be completely disrupted, while deficits in the recruitment of myosin thick filaments are less severe. We predict that pat-9 encodes a dense body protein. To positionally clone pat-9, we are currently refining its location on the genetic map. Previously we mapped pat-9 to the interval defined by the right end points of deficiencies mnDf8 and mnDf1 on the right end of the X linkage group. We are now using single nucleotide polymorphisms between N2 and the wild-type Hawaiian C. elegans strain CB4856 to further refine the pat-9 map location. We are currently selecting Unc recombinants from unc-3 pat-9/ + + heterozygotes, and then assaying them for several different SNPs that span the right end of X.

Norman KR et al. (1995) International C. elegans Meeting "ISOLATION AND CHARACTERIZATION OF MUTANTS AFFECTING EARLY STAGES IN MUSCLE DEVELOPMENT."

Moerman DG, Norman KR

[International C. elegans Meeting, 1995]

Myogenesis in C. elegans is composed of several stages: the generation of presumptive myoblasts; the migration of these cells dorsally and ventrally from their lateral position to form the muscle quadrants; the accumulation of muscle specific proteins in these cells; the polarization of the cells; and, finally, the organization of sarcomeres and attachment structures within the cells (see Hresko et al., JCB 124:491-506). We are interested in identifying genes involved in this process. To identify such genes, we are using a screen similar to one described by Williams and Waterston(JCB 124: 475-490). One modification to the protocol involved widening the time interval for isolating embryonic lethals, from approx. 290 min.(prior to the last round of division generating muscle cells) to approx. 550 min.(late 3-fold). Arrested embryos are then stained with muscle specific antibodies and those with abnormal staining patterns are retained. In an initial EMS screen of approx. 500 F1s, 9 embryonic lethal mutants were isolated that had abnormal staining patterns, and these fall into 4 classes: (I) 2 spheroid shaped embryonic lethal mutants with severely disorganized myofilaments; (II) 3 Pat mutants, all with severely disorganized myofilaments (one is an unc-52 allele); (III) 3 mutants that arrest at, or slightly before, 3-fold and have discontinuities in their muscle quadrants; (IV) one mutant that arrests between 2 and 3-fold and has some disorganization of the myofilaments. Strikingly, this mutant has extra wide muscle quadrants (reflected in a wider ECM as well). Unlike the four "A-bands" per quadrant found in wild type embryo's at this stage, this mutant appears to have as many as six "A-bands" per quadrant. The mutants in class II are similar to the mutants described by Williams and Waterston, but the other three classes are different. In particular, the class IV mutant appears to identify a novel muscle affecting phenotype. Further phenotypic characterization of mutants from this screen will be presented as well as genetic map data.

Sundararajan L et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "UNC-40/DCC And PTP-3/LAR Control Posterior Protrusion and Migration Of The Q Neuroblasts"

Sundararajan L, Lundquist E

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Polarization and migration of neurons to their appropriate locations is essential during nervous system development. The QR and QL neuroblasts, born in the posterior lateral region of the worm, undergo initial polarizations at about 1 hour after hatching in the anterior and posterior directions respectively. They then migrate in the direction of protrusion and divide to produce three neurons of which AQR (from QR) migrates anterior to the pharyngeal bulb and PQR (from QL) posterior to the phasmid ganglion. Consistent with previous studies (Williams et al., 2003), we show that mutations affecting the transmembrane receptors UNC-40/DCC and PTP-3, a LAR type receptor tyrosine phosphatase, cause defects in direction of Q cell polarization and migration (Williams 2003). Mutations in these genes have also been shown to cause defects in direction and extent of migration of Q cell descendents. We show that UNC-40 and PTP-3 might act redundantly in directing posterior migration of QL because the direction of migration defects were significantly more severe for QL and PQR in ptp-3 mutants constructed with unc-40(RNAi) than the single mutants alone. Furthermore, we show that loss of MIG-21, a small transmembrane protein with thrombospondin repeats, caused near randomization of QL and QR protrusion and migration, with QR often migrating posteriorly and QL often migrating anteriorly. In an attempt to understand how these genes might be acting together in controlling Q cell migrations, we constructed double mutants of mig-21 with unc-40 and ptp-3. Surprisingly, in mig-21; unc-40 double mutants, QR migrated anteriorly most of the time, suggesting that posterior migration of QR in mig-21 mutants requires UNC-40 and that MIG-21 might normally repress UNC-40 in QR. In contrast, mig-21; ptp-3 double mutants resembled mig-21 alone, suggesting that MIG-21 and PTP-3 might be working in the same pathway, possibly in parallel to UNC-40. Our data indicate that MIG-21 might be a negative regulator of UNC-40 in QR, and that PTP-3 and UNC-40 act redundantly in posterior Q neuroblast protrusion and migration.

Korswagen HC et al. (1995) International C. elegans Meeting "PHYSICAL MAPPING OF Tc1 SEQUENCE-TAGGED SITES IN THREE Tc1-POLYMORPHIC STRAINS"

Plasterk RHA, Shmookler Reis RJ, Korswagen HC, Thaden JJ

[International C. elegans Meeting, 1995]

Williams et al. [(1992) Genetics 131, 609] have described a genetic mapping system based on polymorphic sequence-tagged sites (STS). It is an alternative to classical two- or three-factor crosses, and can also be used to map polygenic traits [Ebert et al. (1993) Genetics 135,1003; Ayyudevara et al. poster]. One weakness has been that fewer than 80 C. elegans polymorphisms had been sequenced and mapped. This has changed. By shotgun sequencing, Korswagen et al. [WBG 13(5),90] have generated over 800 sequences adjoining Tc1 insertions in strains RW7000, KR1787, and CB4000. At this writing, 35 Tc1 alleles map to sequenced genome regions. This number will slowly increase as sequencing proceeds. We are mapping Tc1 alleles by YAC grid annealing to pooled DNA probes. Pooling is via the "matrix addressing" strategy [Zwaal et al. (1993) PNAS 90, 7431]; DNAs are arranged in 3-D arrays and pooled along each plane. An allele thus contributes to three orthogonal probe pools. A YAC or adjacent YACs labeled by only those three pools will in principle map the allele. In preliminary work, we identified and set aside alleles that align with repetitive DNA elements or anneal to total genomic DNA. We are evaluating probe mixtures of increasing complexity, to optimize 3-D array size.

Gilchrist EJ et al. (1991) International C. elegans Meeting "GENETICS INTERACTIONS BETWEEN CLASS I AND CLASS II ALLELES OF unc-52."

Moerman DG, Gilchrist EJ

[International C. elegans Meeting, 1991]

Two classes of mutations in the unc-52 locus have been identified. The first type of mutation was initially described by S. Brenner ( Genetics 77: 71-94, 1974). This class of mutants exhibit a progressive paralysis of the body wall muscles of the worm, and the degree of paralysis and the timing of its onset vary depending upon the allele. We have also noted gonadal defects when mutants of this class are observed using Nomarski optics. A second class of mutations in this gene has been identified by B. Williams (personal communication), and results in lethality at the two-fold stage of embryogenisis. We have done complementation testing of the various class I alleles of unc-52 against one another and against the lethal allele st549. In all cases the heteroallelic class I animals we have constructed have a phenotype intermediate to either of the parental alleles. Animals heteroallelic for a class I and a class II allele are viable, but show more severe defects in the muscle and gonad than any of the class I alleles on their own. These results suggest that class I alleles may be hypomorphs. One allele, el421, shows a curious interaction with the lethal allele, st549. In crosses where el421 is maternally inherited the heteroallelic animals are similar in phenotype to other class I/class II hermaphrodites, but when the reciprocal cross is done the heteroallelic animals are larval lethals.

Ebert RHA et al. (1995) International C. elegans Meeting "ANTIOXIDANT ENZYME ACTIVITIES AND MAPPING OF H2O2 RESISTANCE IN THREE STRAINS OF C. ELEGANS"

Ebert RHA, Shammas MA, Sohal BH, Sohal RS, Shmookler Reis RJ

[International C. elegans Meeting, 1995]

One factor determining the rate of senescence of an organism may be the level of oxidative stress it experiences throughout its lifespan. Recent studies of the long-lived mutant age-1 (Larsen, PNAS 90, 8905, 1993; Vanfleteren, Biochem J 292, 605, 1993) reveal that activity levels of both catalase and Cu-Zn SOD are elevated in old worms compared to controls. Our work with N2 BO (Ebert et al., Genetics 135, 1003, 1993) and N2 DH424 (submitted) recombinant-inbred (RI) populations indicates that there are genes with allelic differences between the parental strains, which have differential effects on longevity. Here we have tested whether SOD and catalase activities vary in an age- or strain- dependent manner. Middle-aged worms (~70-85% of the cohort still alive) were compared with old worms (~4-6% alive). SOD activity levels decreased with age for cohorts of strains DH424 and RW7000 (BO) by ~50%, but remained at the same level for an N2 population. Catalase activity levels dropped with increasing age for populations of all three strains tested and the decrease was largest for N2 (to 38% of the earlier value). Currently, we are determining the sensitivity to acute hydrogen peroxide (H2O2) exposure for these strains and for a cohort of N2 BO RI progeny. We are also analyzing RI survivors of acute H2O2 exposure by single-worm PCR (Williams et al., Genetics 131, 609, 1992) to map any polymorphic genes responsible for their resistance to this pro-oxidant.