Blelloch RH et al. (1996) Midwest Worm Meeting "GON-1 IS REQUIRED FOR GONADOGENESIS IN C. ELEGANS"

Hodgkin JA, Anna-Arriola SS, Kimble JE, Blelloch RH

[Midwest Worm Meeting, 1996]

The two somatic gonadal precursor cells, Z1 and Z4, generate 12 (hermaphrodite) or 10 (male) cells during L1 and L2. In each sex, two cells become distal cells, and the remaining ones form a somatic gonadal primordium from which the various specific structures of the somatic gonad arise. We will describe the genetic and phenotypic characterization of a gene required for development of the somatic gonad: gon-1. We will also report progress on cloning of this gene.

Blelloch RH et al. (1997) International C. elegans Meeting "gon-1, DTC MIGRATION AND MORPHOGENESIS OF THE C. elegans GONAD"

Blelloch RH, Hodgkin JA, Kimble JE, Anna-Arriola SS

[International C. elegans Meeting, 1997]

Development of the C. elegans gonad follows a stereotypical pattern of cell divisions, migrations, differentiations, fusions, and deaths. In hermaphrodites, the gonadal distal tip cells (DTCs) play at least two critical roles during this process. First, the DTCs "lead" the germ line during extension and growth of the two gonadal arms and, second, the DTCs provide a proliferative signal to the germline. The leader function is responsible for generating the two U-shaped germline tubes. In gon-1 mutants, the hermaphrodite DTCs fail to migrate disrupting the morphogenesis of the U-shaped tubes. However, the DTCs of gon-1 mutants express lag-2 and signal the germline to proliferate. Therefore, the gon-1 locus is essential for extension of germline arms without affecting specification of the DTCs. In wild-type males, the linker cell provides leader function, while the DTCs signal germline proliferation. Consistent with the hermaphrodite gon-1 phenotype, the linker cell does not migrate in gon-1 mutant males, but the DTCs signal germline proliferation. By Nomarski, the gon-1 hermaphrodite DTCs are enlarged and ruffled, and by EM, they are rounded up and have massive infoldings of plasma membrane relative to their wild-type counterparts. We speculate that the gon-1 locus is essential for polarizing the DTCs for their migrations.

Blelloch RH et al. (1998) Midwest Worm Meeting "A predicted metalloproteinase with thrombospondin repeats is required for organ morphogenesis in C. elegans"

Anna-Arriola SS, Hodgkin JA, Blelloch RH, Kimble JE

[Midwest Worm Meeting, 1998]

During organ development, the basement membrane must be remodeled and expanded to accomodate and perhaps to influence both size and shape changes. We are working on the regulation of organogenesis in the nematode C. elegans, and have discovered a gene that may play a key role in basement membrane remodelling during cell migration and organ growth. Our work focuses on gonadogenesis, where growth of the organ is paralleled by growth of the surrounding basement membrane. This growth is led by distal tip cells, DTCs, in hermaphrodites and linker cell, LC, in males. These specialized gonadal cells lead the gonadal tube and direct morphogenesis of that tube during its extension. We have identified a gene, gon-1, that is essential for the leader function of these cells. In the absence of gon-1 activity, neither DTCs nor LCs migrate and morphogenesis of the growing tubes is abnormal. However, gon-1 is not required for a second function of hermaphrodite DTCs, its role in signaling the germline to proliferate. Therefore, the gon-1 locus is essential for gonadal tube extension per se rather than cell fate specification. We have recently cloned the gon-1 gene. The predicted GON-1 protein is well conserved from C. elegans to humans and contains a metalloproteinase domain followed by multiple thrombospondin domains. Molecular analysis of gon-1 mutants suggests that a truncated protein lacking the thrombospondin domains is not active. GON-1 is predicted to be secreted and, therefore, the thrombospondin domains may localize GON-1 within the basement membrane. We propose that DTC/LC migrations may differ from other migrations in C. elegans. First, gon-1 function appears to be specific to DTC/LC migration. Consistent with this assertion, gon-1 mutants do not have the terminal phenotypes seen in mutants with defects in other cell migrations or in axon guidance. Furthermore, several specific migrations that have been examined in gon-1 mutants are not affected. Second, while most migrations occur along pre-existing basement membranes, the DTC/LC migration occurs within a basement membrane that normally delimits the edge of the organ. Therefore, DTC/LC migration depends on a constant remodeling of the basement membrane surrounding the gonad. In conclusion, the specificity of the Gon-1 phenotype for gonadal migration together with its molecular identity suggests that the GON-1 protein is involved in the remodeling of the basement membrane at the leading edge of the DTC/LC migration. We envision a constant and rapid turnover of basement membrane at this leading edge to allow gonadal tube extension. We also suggest that understanding DTC/LC migration at the molecular level will provide an excellent paradigm for migration of cells confined by a basement membrane.

Singh, Akanksha (2021) International Worm Meeting "CRISPR- Nanobodies from C. elegans as an therapeutic approach for Erythroblastosis Fetalis"

Singh, Akanksha

[International Worm Meeting, 2021]

Erythroblastosis fetalis is a consequence of incompatibility amongst Rh-negative antigen from mother and Rh-positive antigen of foetus thus resulting in hemolysis. The hemolysis usually results in respiratory problems, neurological disorders or even heart failure. C.elegans is known to be homologous to some extent with human genome and produces monoclonal antibodies. The nanobodies from C. elegans are engineered with CRISPR Cas 9 technology where Cas 9 protein has been incorporated into nanobodies that will accelerate its efficiency. The engineered nanobodies will target genes like RHD and RhCE that are prominent in RBC membranous environment. The nanobodies with Cas-9 might block the interaction of Rh-negative antibodies with Rh-positive antibodies of foetus as by refusing the recruitment of Rh-negative antibodies in placenta. Antibodies from the C. elegans are extracted and engineered to produce nanobodies with encoded Cas 9 protein. The haemoglobin obtained from foetus are mixed with haemoglobin from mother with Rh-antigens in vitro. After a period of 24-48 hours, when engineered nanobodies are treated with blood by ELISA technique indicated reduced count of Rh-positive antibodies thus blocking RHD gene not allowing the interaction with Rh-negative antibodies. The aim of the study was to study molecular interaction occurring after exposure of nanobodies to Rh- antigens present in foetal blood. Keywords: Erythroblastosis fetalis, Rh-negative, hemolysis, C. elegans, CRISPR Cas 9, RHD , RHCE, haemoglobin, nanobodies.

Flavia I Pellerone et al. (2003) International Worm Meeting "Investigation of the functional role of Rh proteins in the nematode Caenorhabditis elegans."

Susan M Howitt, Flavia I Pellerone, Carolyn A Behm, Inge J Stewart

[International Worm Meeting, 2003]

Rhesus (Rh) factors have been extensively studied as blood group antigens. Recently it has become evident that Rh proteins share ~20% homology with methylamine permeases (mep) and ammonium transporters (amt) of yeast, bacteria and plants. Non-erythroid Rh proteins, expressed in kidney and liver, have also recently been identified in mammals. This implies that Rh proteins may function as the elusive ammonium transporter in animals. Putative Rh homologues also occur in lower organisms (green algae, slime mold) and invertebrates such as Caenorhabditis elegans and Drosophila melanogaster. Thus Rh proteins have functions not restricted to red blood cells. The model nematode C. elegans expresses four amt genes, plus two Rh genes (rhr-1 and rhr-2) that share significant identity with human RhAG genes. The considerable biological resources of C. elegans provide an excellent tool for studying the functions of these genes. To test whether the Rh proteins transport ammonium we have commenced complementation studies in a yeast strain lacking all three native ammonium transporters. In initial experiments, rhr-2 cDNA failed to complement but further work is under way. We will also undertake functional characterization of the Rh proteins by expressing them in Xenopus oocytes. We have used RNA interference to investigate the function of these proteins. Silencing expression of rhr-1 and rhr-2, individually or together, reduced mRNA expression by ~80% but induced no obvious phenotype in C. elegans. However, two dimensional gel electrophoresis of protein extracts from these worms has revealed differentially expressed proteins, which are undergoing further analysis.

Dan Hesselson et al. (2002) Mid-west Worm Meeting "Suppressors of gon-1"

Dan Hesselson, Judith Kimble

[Mid-west Worm Meeting, 2002]

The gon-1 gene is required for distal tip cell (DTC) migration and gonadal morphogenesis. gon-1encodes a secreted metalloprotease of the ADAMTS family (Blelloch and Kimble, 1999). Previous work identified an allelic series of gon-1 alleles, although all alleles cause sterility (Blelloch et al., 1999). We are concentrating on the null allele, gon-1(q518), and a weaker allele, gon-1(e1254). To identify targets or regulators of gon-1, we have conducted suppressor screens for mutations permitting DTC migration in the absence of GON-1. As a pilot screen, GFP was employed under control of the lag-2promoter as a DTC marker. Specifically, we mutagenized a balancedgon-1(q518); lag-2::GFP strain and scored F2/F3 progeny for DTC migration. From 660 F1 animals, two mutations were found that suppressed a gon-1(0) mutant to fertility. Linkage tests are in progress. Given the finding that fertile suppressors could be obtained, we have used the COPAS Biosort (Union Biometrica) to isolate 25,000 mutagenized F1 gon-1(e1254) animals and 50,000 F1/F2/F3 gon-1(q518) animals. These were plated in pools of 100-400 animals and screened for fertility. So far, 140 individual suppressors have been isolated. Progress with these screens will be reported. Blelloch and Kimble (1999) Control of organ shape by a secreted metalloprotease in the nematode Caenorhabditis elegans. Nature 399(6736):586-90. Blelloch et al (1999) The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans. Dev. Biol. 216(1):382-93.

Markussen F-H et al. (2000) Midwest Worm Meeting "Establishing the Gonad Primordium"

Kimble JE, Blelloch RH, Henderson ST, Markussen F-H, Mathies LD

[Midwest Worm Meeting, 2000]

The gonad in all organisms contains both somatic and germline tissues that must be united to produce a functional organ. In C. elegans, the somatic and germline precursor cells coalesce during embryogenesis to form the gonad primordium. Specifically, the somatic gonadal precursors (Z1 and Z4) migrate toward and flank the germline precursors (Z2 and Z3). The 4-celled gonad primordium is arranged with two-fold rotational symmetry in both sexes. This symmetry is maintained throughout hermaphrodite development generating a two-armed gonad, but is broken in males to generate a single-armed gonad. We have set out to dissect the genetic controls of gonad primordium formation. Using genetic screens and RNAi, we have identified at least 4 genes that are required for proper assembly of the gonad primordium. In each of these mutants, the symmetry of the gonad primordium is disrupted resulting in aberrantly-shaped adult gonads. Thus, the proper organization of the gonad primordium is essential for establishing organ shape. In the simplest case, the gonad primordium of gnd-2 RNAi animals is frequently missing one of the two somatic precursor cells, giving rise to an adult gonad with a single arm. gnd-1 and gnd-3 mutants can have asymmetric gonad primordia that lead to a more severely disorganized gonad and sterility. We have also examined previously existing mutants and find that mutations in the sex determination gene tra-1 result in an asymmetric gonad primordium. We will report our progress in analyzing these mutants phenotypically and molecularly.

Tanaka, R. et al. (2013) International Worm Meeting "Quiescence of entomo-phoretic nematode Caenorhabditis japonica."

Kanzaki, N., Hirooka, Y., Kikuchi, T., Tanaka, R.

[International Worm Meeting, 2013]

Entomo-phoretic nematodes are not parasitic to insects but use insects for their transportation. Caenorhabditis japonica, a bacteria-feeding nematode, has a species-specific phoresy with a shield bug, Parastrachia japonensis, and its lifecycle is synchronized to the bug's life. The active dauer larvae (DL) show intensive host-seeking, high sensitivity to oxidative stress and less than 15 days of longevity without attaching the bug. On the other hand, quiescent DL on the bug survive more than 11 months. Thus, the quiescence associated with the bug seems an essential factor for nematode survivability. In the present study, survivabilities of quiescent and active DL were compared under several different conditions to examine the involvement of the bugs in nematode's longevity. Then transcripts and proteins were analyzed to compare gene expression between quiescent and active DL. The quiescent DL on the bug and active DL were kept in a container with 85% (dehydrated condition) or 97% (lightly dehydrated condition which immobilize the DL) of relative humidity (RH). The most active DL died in one week because of desiccation (85% RH) or fungal infection (97% RH). On the other hands, quiescent DL on the bug showed significantly higher survival rate under the same conditions. Further, the survivability of surface-sterilized active DL kept in 97% RH was almost the same as quiescent DL. Therefore, the bugs are likely to work as the shelter from dehydration, and are also providing anti-microbe activity to DL. The expressed genes and proteins also differed between quiescent and active DL. Expression of genes and proteins involved in several stress resistance, metabolic regulation and cuticle formation were significantly higher in quiescent DL. On the other hand, expression of genes and proteins involved in several metabolic related (activity regulation) were significantly higher in active DL. Our results suggest C. japonica DL use their host bug not only for transportation, but also as shelter from environmental stresses and microbe infection. Further, the quiescent stage-specific gene expression allows the extraordinary longevity of this stage.

Nettell JJ et al. (1998) European Worm Meeting "EXPRESSION PATTERN OF C. ELEGANS RH (RHESUS) HOMOLOGUES -AN ATTEMPT TO ASSIGN FUNCTION TO HUMAN RH PROTEINS"

Nettell JJ, Tanner MJ, Kuwabara PE

[European Worm Meeting, 1998]

Rh antigens are highly immunogenic in man and are important in clinical haematology and blood transfusions. They form the major cause of haemolytic disease of the newborn in caucasian populations. The antigens are expressed on two 30kDa non-glycosylated membrane proteins (Rh30) and complex with a glycoprotein (Rhgp) of very close homology. The function of these erythroid specific proteins is unknown. In an attempt to elucidate the function of Rh we have been studying two recently discovered Rhgp homologues in the nematode C. elegans (CERH1/2). We have begun to localise the expression of both genes by constructing GFP-tagged promoter vectors containing a nuclear localisation signal (NLS). The gonad of N2 hermaphrodite C. elegans was injected and double rol-6/GFP transformed worms were visualised by laser confocal microscopy. GFP fluorescence, resulting from CERH promoter activity, indicated that expression predominates in seam cells and in some of the large polyploid nuclei of the intestine. The results also suggest CERH1 and CERH2 co-localise in these cell types. Further work should confirm this co-localisation and determine protein targeting by protein-GFP fusion constructs. Immunocytochemistry, using prepared anti-CERH peptide antibodies, will further investigate such expression patterns.

Aisa Nakashima et al. (2008) European Worm Meeting "Two new germ cell immortality mutants"

Jan Laroque, Theresa M Zucchero, Julie Hall, Aisa Nakashima, Shawn Ahmed, Lauren Garwood

[European Worm Meeting, 2008]

Germ cells represent canonical stem cells that possess the remarkable. quality of being able to proliferate from one generation to the next,. indefinitely, free of replicative damage. mortal germline mutants initially. display normal levels of fertility, but become progressively sterile when. grown for multiple generations. Of 16 original mortal germline mutants (1),. most were temperature-sensitive and only became sterile when grown at 25oC.. Of the ts mutants, two were resistant to RNAi feeding constructs targeting. embryonic lethal genes at all temperatures: 1c and 10i. The 10i mutation. mapped to the right arm of chromosome IV, between unc-22 and dpy-4, and. fails to complement rsd-2 for its RNAi resistance phenotype. rsd-2 has been. reported to be is deficient for spreading of dsRNA-mediated RNAi from. somatic cells to germ cells (2), suggesting that this function may be. relevant to maintaining germ cells over multiple generations during growth. at high temperatures. The mechanism by which this occurs is being studied.. (1) Ahmed S, Hodgkin J (2000). MRT-2 checkpoint protein is required for. germline immortality and telomere replication in C. elegans. Nature.. (2) Tijsterman M, May RC, Simmer F, Okihara KL, Plasterk RH (2004). Genes. required for systemic RNA interference in Caenorhabditis elegans. Current. Biology.