Pamela Opitz et al. (2002) European Worm Meeting "Identification of factors specifically involved in the regulation of physiological germ cell death"

Nicole Wittenburg, BARBARA CONRADT, Pamela Opitz

[European Worm Meeting, 2002]

In the germ line of C. elegans hermaphrodites, programmed cell death (PCD) is the fate of approximately 50% of all germ cells. This physiological germ cell death requires the same central cell death machinery (consisting of ced-9, ced-4, and ced-3) as somatic cell death during development and DNA-damage induced non-physiological germ cell death. The factors leading to the activation of the central cell death machinery in 50% of all germ cells, however, appear to be different. Somatic cell death, as well as non-physiological germ cell death, are dependent on the BH3-only cell death activators EGL-1 and CED-13 (see N. Wittenburg et al., EWM 2002 abstract), which induce PCD by antagonizing the function of the anti-apoptotic Bcl-2-like CED- 9. In contrast, loss-offunction mutations in either egl-1 or ced-13 do not affect physiological germ cell death. Therefore, it is likely that factors other than BH3-only proteins negatively regulate the function of CED-9 to induce germ cell death. We try to elucidate how physiological germ cell death is activated in the germ line by using several approaches. We performed clonal F2 screens for mutants with reduced germ cell death. So far about 6,000 haploid genomes have been screened and 11 mutants were isolated. Two of these mutants also have reduced somatic cell death and were subsequently shown to carry lossof-function mutations in ced-3 and ced-4. The other nine mutations specifically block PCD in the germ line. Complementation analysis showed that five of these nine candidates might define one complementation group. Another cause of non-physiological germ cell death, besides DNA damage, is the infection of C. elegans with Salmonella typhimurium (Aballay and Ausubel, 2001). We wondered whether the level of so called "physiological" germ cell death could be due to the exposure of the animals to E. coli (also gram negative, potentially pathogenic). We therefore raised animals in different bacteria-free/axenic media. When we looked at the progeny of animals that were raised in commonly used liquid Hb medium, we found that a large proportion of the animals formed dauer larvae. This could be due to a high concentration of pheromone, but we think it was due to the lack of nutrients, which did not allow proper development into adults. This is supported by the observation that L4 larvae that were transferred into liquid Hb medium had small underdeveloped gonads later on as adults, indicating a reduction in germ cell proliferation, similar to what is found in starved animals. We also raised animals on plates seeded with a drop of milk. When we transferred L4 larvae onto these plates, we found that the adults had fully developed gonads. The level of germ cell death of engulfment defective ced-6(n2095) animals raised on either milk or on E. coli was the same, indicating that the cultivation of C. elegans on E. coli does not induce germ cell death. We also studied the effect of various mutations and environmental stress factors on the level of germ cell death. Taken together, our results so far indicate that the physiological death of 50% of all germ cells is in fact due to a germ line intrinsic program rather than being influenced by environmental factors.

Claus Schertel et al. (2003) International Worm Meeting "A genetic screen for genes involved in the programmed death of germ cells"

Claus Schertel, Nicole Wittenburg, Pamela Opitz, BARBARA CONRADT

[International Worm Meeting, 2003]

During the development of the soma of a C. elegans hermaphrodite, 131 cells are eliminated by programmed cell death (referred to as developmental cell death). These cell death events are determined by the essentially invariant cell lineage and appear to occur in a cell-autonomous manner. Programmed cell death also occurs in the gonad of adult hermaphrodites: about 50% of all germ cells are eliminated by programmed cell death (physiological germ cell death). In contrast to developmental cell death, physiological germ cell death is not determined by lineage and might be induced by cell-autonomous as well as cell non-autonomous factors. Three genes define the central cell death pathway in C. elegans: ced-9, ced-4 and ced-3 (ced, cell death abnormal). Their inactivation results either in ectopic programmed cell death (ced-9) or a block in programmed cell death (ced-3, ced-4) in both soma and germ line. However, the factors that act upstream of the central pathway to regulate its activation appear to differ in soma and germ line. For example, loss-of-function (lf) mutations in the cell-death activator gene egl-1 (egl, egg-laying defective), which acts upstream of ced-9, block developmental cell death but not physiological germ cell death. To identify genes involved in the control of physiological germ cell death, we performed a genetic screen for mutants with reduced levels of physiological germ cell death. The screen was performed in the background of a ced-6(lf) mutation, which causes a defect in engulfment and an accumulation of germ cell corpses. To visualize and quantify the number of germ cell corpses, the reporter Plim-7CED-1::GFP (lim, lim domain family) was used, which drives the expression of a CED-1::GFP fusion protein in the sheath cells. A total number of 8,600 haploid genomes were screened and 41 independent mutants identified. Of the 41 mutations, three suppress the engulfment defect of ced-6(lf) animals, five block the proliferation of germ cells, and two block programmed cell death in the germ line as well as the soma. Of the remaining 31 mutations, eight reduce the level of germ cell death to less than 20% of the wild-type level and are analyzed further. These eight mutations fall into two classes: Class A mutations reduce specifically physiological germ cell death whereas Class B mutations affect physiological as well as DNA damage-induced, non-physiological germ cell death. The genetic and molecular characterization of the genes defined by Class A and B mutations are ongoing.

Suzuki N et al. (1992) Worm Breeder's Gazette "Cloning of Manganese Superoxide Dismutase in C. elegans"

Ishii N, Suzuki K, Suzuki N

[Worm Breeder's Gazette, 1992]

The reduction of molecular oxygen in all aerobic cells results in intermediates (superoxide radicals, hydrogen peroxide and hydroxy ions) which are highly toxic. Superoxide radicals may play an important role in aging. Superoxide dismutase (SOD) are metalloenzyme which eliminate, through a dismutation reaction, superoxide radicals produced within cells. There are two distinct types of SOD in eukaryotic cells. The copper- and zinc-containing enzyme (CuZn SOD) is found principally in the cytosole and the manganese-containing enzyme (Mn SOD) is found exclusively in the mitochondorial matrix. Pamela Larsen has cloned the Cu-Zn SOD in C. elegans and sequenced (Gazetti, Vol. 11 No. 5, p. 27). Now we cloned and sequenced the Mn SOD in C. elegans. One oligonucleotide was designed from the highly conserved regions that be found in Mn SOD gene which has cloned from several animals. The oligonucleotide was used as probe to hybridize with Barstead cDNA library. [See Figure 1]

Hao JC et al. (2010) Dev Cell "The tripartite motif protein MADD-2 functions with the receptor UNC-40 (DCC) in Netrin-mediated axon attraction and branching."

Mebane L, Bargmann CI, Adler CE, Hao JC, Tessier-Lavigne M, Gertler FB

[Dev Cell, 2010]

Neurons innervate multiple targets by sprouting axon branches from a primary axon shaft. We show here that the ventral guidance factor unc-6 (Netrin), its receptor unc-40 (DCC), and the gene madd-2 stimulate ventral axon branching in C. elegans chemosensory and mechanosensory neurons. madd-2 also promotes attractive axon guidance to UNC-6 and assists unc-6- and unc-40-dependent ventral recruitment of the actin regulator MIG-10 in nascent axons. MADD-2 is a tripartite motif protein related to MID-1, the causative gene for the human developmental disorder Opitz syndrome. MADD-2 and UNC-40 proteins preferentially localize to a ventral axon branch that requires their function; genetic results indicate that MADD-2 potentiates UNC-40 activity. Our results identify MADD-2 as an UNC-40 cofactor in axon attraction and branching, paralleling the role of UNC-5 in repulsion, and provide evidence that targeting of a guidance factor to specific axonal branches can confer differential responsiveness to guidance cues.

Mariam Alexander et al. (2008) C.elegans Neuronal Development Meeting "MADD-2, A Homolog of the Opitz Syndrome Gene Mid1, Regulates Muscle Arm and Axon Extension to the Midline through an UNC-40 Pathway"

Peter Roy, Serena D'Souza, Guillermo Selman, Mariam Alexander

[C.elegans Neuronal Development Meeting, 2008]

The post-synaptic membrane of the neuromuscular junction elaborates extensively during animal development through mechanisms that are poorly understood. In C.elegans, the postsynaptic membrane of the body wall muscles (BWMs) resides at the termini of plasma membrane extensions called ''muscle arms''. Muscle arms extend from the BWMs to the motor neurons at the midline and double in number during larval development. To better understand muscle arm extension, we screened for muscle arm development defective (Madd) mutants and isolated six alleles of madd-2, a previously uncharacterized member of the RBCC (Ring-Bbox-Coiled Coil) superfamily of proteins. Tagging MADD-2 coding sequence with GFP within the context of a genomic clone revealed expression in BWMs, sex muscles, the anchor cell, developing vulval epithelium, seam cells, HSNs, SDQR, SDQL, AVM and PVM neurons. We demonstrate that MADD-2 functions autonomously in the BWMs, where it is subcellularly localized to dense bodies and muscle arm termini. Recreation of select missense mutations in a MADD-2 fusion protein rendered the transgene non-functional and eliminated MADD-2::CFP localization to the leading edge, but not the dense bodies, demonstrating that MADD-2 functions at the leading edge to regulate muscle arm extension. In addition to muscle arm extension, we found that madd-2 plays a role in HSN, AVM, and PVM axon guidance, as well as the extension of the sex muscles in both males and hermaphrodites to the ventral midline. Genetic analyses of the muscle arm, sex muscle, and HSN axon guidance defects reveal that madd-2 functions in an unc-40/DCC pathway. In the AVM and PVM, madd-2 acts in parallel to a slt-1/SLIT pathway to guide the axons to the ventral midline. We are currently investigating the role of MADD-2 in the UNC-40/DCC pathway. In a striking analogy, mutations in one of six human MADD-2 homologs, called mid1, give rise to a congenital disease called Opitz syndrome that is characterized by the errant development of ventral midline structures, most of which express either DCC, and/or it''s paralog Neogenin. We therefore speculate that Mid1 may be mediating DCC/Neogenin function at the ventral midline of humans, the disruption of which leads to Opitz syndrome.

Alexander M et al. (2010) Dev Cell "MADD-2, a homolog of the Opitz syndrome protein MID1, regulates guidance to the midline through UNC-40 in Caenorhabditis elegans."

Selman G, Seetharaman A, Byrne AB, Chan KK, Roy PJ, Alexander M, D'Souza SA

[Dev Cell, 2010]

The body muscles of Caenorhabditis elegans extend plasma membrane extensions called muscle arms to the midline motor axons to form the postsynaptic membrane of the neuromuscular junction. Through a screen for muscle arm development defective (Madd) mutants, we previously discovered that the UNC-40/DCC guidance receptor directs muscle arm extension through the Rho-GEF UNC-73. Here, we describe a gene identified through our mutant screen called madd-2, and show that it functions in an UNC-40 pathway. MADD-2 is a C1-TRIM protein and a homolog of human MID1, mutations in which cause Opitz Syndrome. We demonstrate that MADD-2 functions cell autonomously to direct muscle and axon extensions to the ventral midline of worms. Our results suggest that MADD-2 may enhance UNC-40 pathway activity by facilitating an interaction between UNC-40 and UNC-73. The analogous phenotypes that result from MADD-2 and MID1 mutations suggest that C1-TRIM proteins may have a conserved biological role in midline-oriented developmental events.

Morf MK et al. (2013) Dev Biol "The Caenorhabditis elegans homolog of the Opitz syndrome gene, madd-2/Mid1, regulates anchor cell invasion during vulval development."

Hajnal A, Rimann I, Alexander M, Morf MK, Roy P

[Dev Biol, 2013]

Mutations in the human Mid1 gene cause Opitz G/BBB syndrome, which is characterized by various midline closure defects. The Caenorhabditis elegans homolog of Mid1, madd-2, positively regulates signaling by the unc-40 Netrin receptor during the extension of muscle arms to the midline and in axon guidance and branching. During uterine development, a specialized cell called anchor cell (AC) breaches the basal laminae separating the uterus from the epidermis and invades the underlying vulval tissue. AC invasion is guided by an UNC-6 Netrin signal from the ventral nerve cord and an unknown guidance signal from the vulval cells. Using genetic epistasis analysis, we show that madd-2 regulates AC invasion downstream of or in parallel with the Netrin signaling pathway. Measurements of AC shape, polarity and dynamics indicate that MADD-2 prevents the formation of ectopic AC protrusions in the absence of guidance signals. We propose that MADD-2 represses the intrinsic invasive capacity of the AC, while the Netrin and vulval guidance cues locally overcome this inhibitory activity of MADD-2 to guide the AC ventrally into the vulval tissue. Therefore, developmental cell invasion depends on a precise balance between pro- and anti-invasive factors.

Valenzuela MRL et al. (1991) International C. elegans Meeting "STRUCTURES AND FUNCTIONS OF THE REPEATING MOTIFS IN TWITCHIN."

Valenzuela MRL, Benian GM

[International C. elegans Meeting, 1991]

Twitchin, the 753,570 Da polypeptide encoded by the unc-22 gene, was the first member of a growing family of intracellular and mostly muscle proteins, found in diverse animals, composed of multiple copies of motif I (fibronectin type III domain-like) and motif II ( immunoglobulin C2 domain-like). To date, this family includes, smooth muscle and non-muscle myosin light chain kinases, titin, C-protein, 86 kDa protein, skelemin and probably insect projectin. The similarities to motifs found in extracellular and cell surface proteins engaged in recognition or adhesion suggests that motifs I and II of muscle proteins are also involved in binding--probably to myosin, other thick filament components, and also binding of these proteins to themselves and other family members. We hope to be able to demonstrate binding of small numbers of these motifs to thick filament components both in vitro and in vivo. Crystallographic structure determination will be done in collaboration with Pamela Bjorkman (Cal Tech). Using the pGEX- 2T vector, we have been able to express and purify large quantities of motif I, motif II and the predominant triplet I,I,II as glutathione-S- transferase (GST) fusion proteins in E. coli. We have succeeded with the thrombin cleavage of the motifs from GST and are trying ELISA-type binding assays with nematode and rabbit myosin. For in vivo 'binding' studies, we have cloned motif I, motif II, the trio I,I,II and the C- terminal 5 motif IIs into Andy Fire's pPD30.38 vector which directs body wall muscle expression. These constructs will be microinjected into unc-22 nulls and transgenics examined by immunofluorescence for co-localization to myosin.

Ruiz, Manuel (2021) International Worm Meeting "Glucose impacts HSN morphology and induces an egg-laying defective phenotype dependent of the serotonin-signaling pathway"

Ruiz, Manuel

[International Worm Meeting, 2021]

Glucose impacts HSN morphology and induces an egg-laying defective phenotype dependent of the serotonin-signaling pathway Manuel Axel Ruiz and Pamela Padilla Peripheral neuropathy is a common and often painful complication that results from a chronic state of hyperglycemia. In recent years, research has shown that in humans altered levels of serotonin is highly associated to a chronic state of hyperglycemia. Additionally, altered levels of serotonin are associated with various neurological disorders. However, the mechanism by which hyperglycemia impacts serotonin processes is not understood. Here we are using the genetic model system Caenorhabditis elegans as a hyperglycemia model to further understand the impact of a glucose-supplemented diet on the animal's egg-laying behavior. C. elegans possess a nervous system that is composed of 302 neurons. In C. elegans the Hermaphrodite-Specific serotonergic motor Neurons (HSN) play an essential role in regulating egg-laying. Using this model organism, we conducted genetic and cell biological analysis to examine the role a glucose-supplemented diet has on the serotonergic HSN motor neurons. Our preliminary data shows that a glucose-supplemented diet impacts egg-laying and HSN morphology. A significant proportion of animals raised in a glucose-supplemented diet have a higher incidence of intra-uterine egg-hatching, HSN axonal degeneration, and abnormal HSN morphology. We used genetic mutants to dissect out the role the serotonin pathway has on the response to a glucose diet. Furthermore, through RNA-sequencing, we examined how a glucose-supplemented diet alters the expression of genes responsible of serotonergic functions. This work will lead to the further understanding of the molecular mechanisms that are involved on altered serotonergic neuronal functions as a result of a chronic hyperglycemic state.

Morf, Matthias et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "madd-2 regulates anchor cell invasion"

Alexander, Mariam, Hajnal, Alex, Morf, Matthias, Roy, Peter, Nusser, Stefanie, Rimann, Ivo

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Invasion, the process during which cells cross tissue borders, needs to be tightly regulated since uncontrolled invasion can lead to metastatic cancer growth. During larval development, a specialized cell in the somatic gonad called anchor cell (AC) breaks two basal laminae and then invades the adjacent vulval tissue in order to form a connection between the developing vulva and uterus. Multiple signals from the vulval cells and the ventral nerve cord regulate AC invasion, however, how these signals are integrated by the AC is largely unknown. In a screen for new LIN-12 NOTCH targets, we have identified madd-2 , a conserved RING finger gene, as a putative LIN-12 target in the somatic gonad. madd-2 has independently been identified by the P. Ro y lab as a gene acting upstream of the UNC-40 netrin receptor in controlling muscle arm extensions. The human madd-2 homolog, MID1 , is mutated in most cases of Opitz syndrome, a disease characterized by ventral midline defects. By examining markers for the invasive membrane, we find that AC morphology is altered in madd-2 mutants. Also, the invasion of the AC is delayed in madd-2 mutants judged by examining the removal of the extracellular matrix protein HIM-4. Epistasis experiments with the Netrin receptor UNC-40 suggest that MADD-2 plays a dual role during AC invasion, on the one hand promoting UNC-40 signaling and on the other hand repressing another positive regulator of invasion. Our work adds lin-12 Notch and madd-2 as new factors controlling AC invasion. We postulate that AC invasion is not only activated at the late L3 stage, when invasion normally begins, but also repressed at earlier stages. We further propose that invasion is controlled by multiple, o pposing pathways such as LIN-12 NOTCH and UNC-6 Netrin.