McIntyre, Daniel et al. (2017) International Worm Meeting "Primordial germ cell survival depends on contact with the somatic gonad in the C. elegans embryo."

Nance, Jeremy, McIntyre, Daniel

[International Worm Meeting, 2017]

We are studying the formation of the germline stem cell niche in the C. elegans gonad primordium, and investigating how niche cell contact promotes stem cell survival. Niche-derived cues can control stem cell quiescence, proliferation and differentiation. As such, establishment of niche-stem cell interactions is a critical stage in development. In C. elegans embryos, two primordial germ cells (PGCs, stem cell precursors), and two somatic gonadal precursor cells (SGPs, niche cells) come together to form the gonad primordium. This happens through a fascinating morphogenetic process in which the SGPs migrate to the PGCs and extend their cell membranes, wrapping around these cells. This process is critical for PGC survival. Ablation of the SGPs at this time causes death of the PGCs (Kimble and White, 1981) through an unknown mechanism. We repeated this experiment using fluorescent reporters to follow the fate of the PGCs after SGP ablation. Surprisingly, we observed that unprotected PGCs were often engulfed by neighboring endodermal cells. In a normal embryo, PGCs produce large lobes that are pinched off and digested by endodermal cells, suggesting that endodermal cells may inappropriately cannibalize the PGC cell body when SGPs are absent. Loss of endodermal cells in embryos lacking SGPs prevented PGC death, indicating that this is the case. Endodermal cells remove PGC lobes by assembling a ced-10 and lst-4 dependent scission complex composed of actin and dynamin around PGC lobe necks. We show that this pathway is also needed for PGC death following SGP ablation. We hypothesize SGP wrapping protects the PGC cell body, ensuring only the lobe is eaten by the endoderm. We are testing this hypothesis by specifically disrupting SGP wrapping of PGCs. While signaling between niche and stem cell is important in many systems, our work suggests that close physical contact with the niche also plays an unexpected role in protection of the primordial germ cells from cannibalism by neighboring cells.

McIntyre, Daniel et al. (2015) International Worm Meeting "Primordial germ cell survival requires niche cell conact in the C. elegans gonadal primordium."

McIntyre, Daniel, Nance, Jeremy

[International Worm Meeting, 2015]

Niche-derived signals have been shown to control stem cell quiescence, proliferation and differentiation. Because the niche is needed for stem cells to function, formation of the niche is a critical event in development. Yet, niche formation is poorly understood. This is because in most models, the cells participating in the niche are not well identified prior to niche assembly, and because formation is very difficult to observe in vivo. To understand niche formation, we are studying the gonad primordium in C elegans. The gonad primordium is a simple organ consisting of just four cells - two primordial germ cells (precursors to the germline stem cells), and two somatic gonadal precursor cells (niche cells). The primordium forms via a fascinating morphogenetic process in which the two niche cells migrate posteriorly along the endoderm, turn and recognize the germ cells, and finally wrap almost completely around the germ cells. This process recapitulates many key steps that must occur more generally in niche formation including cell recognition, adhesion with the niche, and activation of the quiescent stem cells. Using transgenic analysis, fluorescent imaging and embryological manipulations including laser ablations we are investigating the early signals exchanged between stem and niche cells, and how niche cell contact promotes stem cell survival. Previously, it was reported that ablation of niche precursors resulted in a loss of the primordial germ cells. We have repeated this experiment using fluorescent reporters to follow cells in the primordium. In animals where niche cells have been ablated, the germ cells are lost through an interesting mechanism in which the germ cells fragment and these pieces are consumed by the neighboring endodermal cells. We are currently investigating the mechanisms used by the niche cells to prevent germ cell death. Ultimately, we hope to identify how the niche cells are able to surround the stem cells, and determine the signals in the niche that protect the germ cells and enable their function in the animal.

McIntyre, Daniel et al. (2019) International Worm Meeting "Somatic gonad precursors assemble a basement membrane that guarantees primordial germ cell quiescence and gonad integrity during embryogenesis."

Nance, Jeremy, McIntyre, Daniel

[International Worm Meeting, 2019]

Somatic niche cells are often critical regulators of germline precursor cells, controlling their survival, proliferation and differentiation. We are investigating how niche cells and germline precursor cells interact with the surrounding basement membrane (BM) in the C. elegans embryo. We find that BM performs two critical roles in development: first, BM helps to establish and maintain niche cell wrapping during embryogenesis; second, BM guarantees primordial germ cell (PGC) quiescence in the embryo. The gonad primordium in C. elegans is a simple, 4-cell structure formed during embryogenesis that will give rise to the entire germ line and somatic gonad in the adult worm. It is comprised of two primordial germ cells (PGC), each of which interacts with a single somatic gonadal precursor cell (SGP). Each SGP extends its cell membrane around the body of a PGC, so as to completely enwrap it. Wrapping establishes a morphology that is carried into larval development, and provides a protective niche for the PGCs in the embryo. The value of this niche is demonstrated by unwrapped PGCs, which die unexpectedly when they are cannibalized by neighboring endodermal cells. We show that the SGPs are required to recruit laminin to the outer surface of the gonad primordium. If the BM is disrupted, SGP wrapping is not maintained and PGCs escape quiescence late in embryogenesis, rather than in fed L1 larva. These failures in development of the gonad primordium lead to the disorganized larval gonad and tumorous, sterile germ line previously reported in BM mutants. Finally, we find that the BM receptor DGN-1/dystroglycan is expressed by the SGPs on surfaces contacting the BM, is required to maintain SGP wrapping, but does not affect PGC quiescence. We conclude that SGPs template a BM that assures the integrity of the niche throughout embryogenesis and that PGCs remain quiescent until after hatching. These results show how a developmentally programed structure, the BM surrounding an organ primordium, plays both structural and signaling roles in the embryonic PGC niche.

(2024) Development "The people behind the papers - Nadia Manzi and Daniel Dickinson."

[Development, 2024]

The mitotic kinase Aurora A has been shown to regulate the anterior-posterior polarity in developing Caenorhabditis elegans embryos. In a new study, Daniel Dickinson and colleagues find that Aurora A has temporally distinct roles in coordinating the localization of Partitioning defective (PAR) proteins to establish cell polarity during development. To find out more about the story behind the paper, we caught up with first author Nadia Manzi and corresponding author Daniel Dickinson, Assistant Professor at the University of Texas at Austin.

(2019) Development "The people behind the papers - Daniel Osorio, Elaine Chan, Joana Saramago and Ana Carvalho."

[Development, 2019]

Animal cytokinesis is driven by an actomyosin ring that assembles at the cell equator and constricts to physically separate the two daughters. Although myosin is known to be essential for cytokinesis in multiple systems, whether this requirement reflects its motor or actin crosslinking activities has recently been a matter of contention. A new paper in Development now addresses this problem using the first divisions of the <i>Caenorhabditis elegans</i> embryo as a model. We caught up with the paper's three first authors Daniel Osorio, Elaine Chan and Joana Saramago, and their supervisor Ana Carvalho, Principal Investigator at the University of Porto's i3S consortium, to find out more about the story.

Morales AR et al. (1990) Worm Breeder's Gazette "Molecular cloning of unc-49"

Mancillas JR, Morales AR

[Worm Breeder's Gazette, 1990]

unc-49 shrinks by the simultaneous contraction of the dorsal and ventral musculature when provided with mechanosensory stimulation of the head region. It also displays severe difficulties in spontaneous backward locomotion and its body length is slightly reduced. Abnormalities in the placement of the axons of the anterior mechanosensory receptors, the ALMs, have been observed (Siddiqui, abstracts of the 1989 C. elegans meeting), but the axons of the cross- inhibitor motorneurons, the DDs and VDs, display normal trajectories ( McIntyre, personal communication). Several lines of evidence suggest that abnormalities in the GABA receptor or regulation of its expression or binding ability may be the primary defect in unc-49 ( McIntyre, personal communication): a) unc-49 specimens fail to exhibit normal behavioral responses to muscimol, a GABA agonist, when both the rate of locomotion or pharyngeal pumping are monitored, b) binding assays reveal abnormal GABA binding kinetics in unc-49 when compared to N2 tissue, and c) immunocytochemical staining with antibodies against GABA suggests normal levels of GABA in ventral cord motorneurons. unc-49 is being cloned by transposon tagging. Eighty two recombinant strains were generated using unc-49 (n1324) and two flanking markers, sma-2 and vab-7. Using Southern blot analysis and probes for the 5 known C. elegans transposons, a Tc1 band that consistently segregates with the unc-49 phenotype was identified. A 4. 5 kb EcoRI fragment containing this Tc1 insertion was isolated and the 2.9 kb of flanking genomic sequences have been cloned and are being used to probe a cDNA library, probe Northern blots for the unc-49 transcript and to probe the cosmid library in Cambridge for the corresponding genomic clones. During a screen using PCR-generated probes sharing sequences with the mammalian GABA receptor, Silver and Way (personal communication) have identified a YAC clone which maps to the unc-49 region in the genetic map, further suggesting that unc-49 may encode the GABA receptor or one of its sub-units.

Vo, An A. et al. (2021) MicroPubl Biol

Ward, Jordan D., Ragle, James Matthew, Levenson, Max T., Vo, An A.

[MicroPubl Biol, 2021]

The AID system has emerged as a powerful tool to conditionally deplete proteins in a wide-range of organisms and cell types (Nishimura et al. 2009; Holland et al. 2012; Zhang et al. 2015; Natsume et al. 2016; Trost et al. 2016; Brown et al. 2017; Daniel et al. 2018; Chen et al. 2018; Camlin and Evans 2019). The system is comprised of two components. A plant F-box protein Transport Inhibitor Response 1 (TIR1) is expressed and forms a complex with endogenous Skp1 and Cul1 proteins to form a functional SCF ubiquitin ligase (Nishimura et al. 2009; Natsume and Kanemaki 2017). TIR1 can either be expressed constitutively or in a tissue-specific manner depending on promoter choice. A degron sequence from the IAA17 protein is fused to the protein of interest (Nishimura et al. 2009; Natsume and Kanemaki 2017). Commonly used auxin-inducible degrons include 44 amino acid (AID*) and 68 amino acid (mAID) fragments of IAA17 (Morawska and Ulrich 2013; Li et al. 2019). Addition of the plant hormone auxin bridges an interaction between TIR1 and the degron and the SCF ligase ubiquitylates the degron-fused protein leading to proteasomal degradation.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Attila L Kovcs et al. (2005) International Worm Meeting "Electron microscopy of autophagy in Caenorhabditis elegans"

Judit Fehr, Tibor Vellai, Krisztina Takcs-Vellai, Zsolt Plfia, Jnos Kovcs, Attila L Kovcs

[International Worm Meeting, 2005]

Cellular autophagy is a process for the degradation of cytoplasmic constituents in eukaryotic cells. Since its discovery in 1957 in the epithelial cells of kidney of the newborn mice (1) electron microscopy has been and still remains an indispensable method for studying autophagy. One of the main reasons of the late start of autophagy research in C. elegans is the relative difficulty of performing transmission electron microscopy with worm samples. Recently we have developed a technique by which autophagic processes of the worm become accessible for systematic morphological and, in three major tissue types, for morphometric analysis by transmission electron microscopy (2). Our poster introduces the method, presents the criteria for the identification and morphological analysis of various types of autophagic vacuoles in all major cell types, and shows the latest morphometric data on autophagy in hypodermal, gut epithelial and body wall muscle cells during postembryonic development including all four larval, as well as the predauer, dauer and postdauer stages. Our results indicate that the cells of continuously feeding worms are practically devoid of autophagic vacuoles. Significant increase in the quantity of autophagic vacuoles can be observed at the end of each larval stage when the lethargus is reactivated. Systematic measurements on Daf-c mutants in the predauer period show that preparation for the dauer stage does not involve constitutive autophagic activity. (1) Clark S.L. (1957) Cellular differentiation in the kidneys of newborn mice studied with the electron microscope, J. Biophysic. Biochem. Cytol. 3, 349 (2) Kovacs AL, Vellai T, Muller F (2004) Autophagy in Caenorhabditis elegans. In: "Autophagy" Ed. Daniel J. Klionsky, Landes Bioscience 2004, Chapter 17, pp 216-223

Astrid Kleinert et al. (2006) European Worm Meeting "Analysis of Insulin-Like Peptides in C. elegans by Mass Spectrometry"

Astrid Kleinert, Daniel Hess, Joy Alcedo, Jan Hofsteenge

[European Worm Meeting, 2006]

Astrid Kleinert, Daniel Hess, Jan Hofsteenge and Joy Alcedo. Genetic analyses in C. elegans have implicated an endocrine signaling pathway, the insulin/IGF-1 pathway, in regulating lifespan. Mutations in the gene daf-2, which encodes an insulin/IGF-1 receptor homologue, can increase worm lifespan by more than 100% (1). In addition, the insulin and IGF-1 pathways have been shown to influence fly and mouse lifespan (2-5).. Although downstream components of the DAF-2 pathway have been studied in detail, far less is known about the putative DAF-2 ligands predicted to be encoded by 38 insulin-like genes in the worm. It remains unknown which of the predicted insulin-like peptides are present in wild type or in longevity mutants. In order to determine this, we have initiated a project to analyze the polypeptide subfractions of the C. elegans proteome by mass spectrometry. Worm lysates will be prepared from mixed stage, wild-type worms, excluding membrane-bound proteins. Acid-ethanol extraction and gel filtration will be tested as methods to obtain a subfraction enriched in small polypeptides. Peptide identification will be done by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Data analyses will be carried out using a MASCOT search database, which contains a subset of the UNIPROT database and a database generated of relevant C. elegans polypeptides. Finally, using the SILAC method for quantification (6), we plan to compare differences in the levels of polypeptides, e.g., insulin-like peptides, between wild-type worms and longevity mutants. . References: (1) Kenyon et al., 1993. Nature 366: 461-4. (2) Clancy et al., 2001. Science 292: 104-6. (3) Tatar et al., 2001. Science 292: 107-110. (4) Holzenberger et al., 2003. Nature 421: 182-7. (5) Blher et al., 2003. Science 299:572-4. (6) Krijgsveld et al., 2003. Nature Biotech. 21: 927-931.