Characterization of «par-4-»dependent germ line stem cell quiescence in Caenorhabditis elegans

Upon encountering harsh environmental conditions, Caenorhabditis elegans larvae are able to alter their developmental program and enter the dauer diapause, an alternative developmental stage that enables larvae to endure long periods of stress. During this arrested state, the germ line stem cells, which normally divide during reproductive development, halt their proliferation and are consequently rendered quiescent. Previous work has implicated a role for PAR-4/LKB1 in germ line stem cell quiescence. Inactivating mutations in par-4 result in aberrant germ line stem cell proliferation during the dauer diapause, suggesting that PAR-4 is required for germ cell cycle arrest. LKB1 is a tumor suppressor protein kinase that is implicated in the rare, autosomal dominant disease Peutz-Jeghers syndrome (PJS). In order to better understand its function in tumorigenesis, we characterized its role in regulating cellular quiescence in developmentally arrested larvae using a genome-wide, RNA interference-based screen to identify suppressors of PAR-4-dependent germ line hyperplasia. We identified 50 genes whose loss-of-function was found to rescue the germ line hyperplasia observed in par-4 dauer larvae, suggesting that their expression is misregulated in the absence of PAR-4/LKB1. In addition, we demonstrated the importance of PAR-4-dependent germ line arrest by characterizing the post-dauer, reproductive capacity of par-4 mutants, which was significantly reduced. Future endeavors include the characterization of key candidates—many of which impinge on the cytoskeleton and the extracellular matrix—as well as the post-dauer germ line defects observed in par-4 animals.Lors de conditions environnementales hostiles, la larve Caenorhabditis elegans est capable d'altérer son programme de développement. Elle entre en phase dauer diapause, un stade alternative de développement qui permet d'endurer des longues périodes de stress. Durant ce stade, les cellules souches germinales, qui normalement se divisent durant leur développement reproductif, arrêtent leur prolifération et deviennent quiescentes. Plusieurs études ont impliqués PAR-4/LKB1 comme acteur dans l'arrêt du cycle des cellules germinales. Lorsqu'on inactive par-4 à l'aide d'une mutation, les cellules souches germinales prolifèrent de façon anormales lors de la dauer diapause, ce qui suggère que PAR-4 est nécessaire pour l'arrêt du cycle des cellules germinales. LKB1 est une protéine kinase impliqué dans le syndrome Peutz-Jeghers (PJS). Pour comprendre la fonction de LKB1 dans la tumorogenèse, nous avons caractériser son rôle dans la régulation de la quiescence cellulaire des larves en arrêt développemental, utilisant un crible ARNi global, afin d'identifier les suppresseurs de l'hyperplasie germinale induite par PAR-4. Nous avons identifiés 50 gènes dont la perte de fonction était capable de empêcher l'hyperplasie observée chez les larves dauer par-4, suggérant que leur expression est dérégulé en l'absence de PAR-4/LKB1. De plus nous avons démontré l'importance de PAR-4 dans l'arrêt du cycle des cellules germinales en caractérisant la capacité reproductive nettement diminuée des larves mutantes en post dauer. Dans le future, nous recommandons que des recherches supplémentaires caractérisent les gènes clés qui ont le potentiel de empêcher l'hyperplasie observée chez les larves dauer par-4

Genome-wide surveys reveal polarity and cytoskeletal regulators mediate LKB1-associated germline stem cell quiescence

Abstract Background Caenorhabditis elegans can endure long periods of environmental stress by altering their development to execute a quiescent state called “dauer”. Previous work has implicated LKB1 - the causative gene in the autosomal dominant, cancer pre-disposing disease called Peutz-Jeghers Syndrome (PJS), and its downstream target AMPK, in the establishment of germline stem cell (GSC) quiescence during the dauer stage. Loss of function mutations in both LKB1/par-4 and AMPK/aak(0) result in untimely GSC proliferation during the onset of the dauer stage, although the molecular mechanism through which these factors regulate quiescence remains unclear. Curiously, the hyperplasia observed in par-4 mutants is more severe than AMPK-compromised dauer larvae, suggesting that par-4 has alternative downstream targets in addition to AMPK to regulate germline quiescence. Results We conducted three genome-wide RNAi screens to identify potential downstream targets of the protein kinases PAR-4 and AMPK that mediate dauer-dependent GSC quiescence. First, we screened to identify genes that phenocopy the par-4-dependent hyperplasia when compromised by RNAi. Two additional RNAi screens were performed to identify genes that suppressed the germline hyperplasia in par-4 and aak(0) dauer larvae, respectively. Interestingly, a subset of the candidates we identified are involved in the regulation of cell polarity and cytoskeletal function downstream of par-4, in an AMPK-independent manner. Moreover, we show that par-4 temporally regulates actin cytoskeletal organization within the dauer germ line at the rachis-adjacent membrane, in an AMPK-independent manner. Conclusion Our data suggest that the regulation of the cytoskeleton and cell polarity may contribute significantly to the tumour suppressor function of LKB1/par-4

An Immobilization Technique for Long-Term Time-Lapse Imaging of Explanted Drosophila Tissues

Time-lapse imaging is an essential tool to study dynamic biological processes that cannot be discerned from fixed samples alone. However, imaging cell- and tissue-level processes in intact animals poses numerous challenges if the organism is opaque and/or motile. Explant cultures of intact tissues circumvent some of these challenges, but sample drift remains a considerable obstacle. We employed a simple yet effective technique to immobilize tissues in medium-bathed agarose. We applied this technique to study multiple Drosophila tissues from first-instar larvae to adult stages in various orientations and with no evidence of anisotropic pressure or stress damage. Using this method, we were able to image fine features for up to 18 h and make novel observations. Specifically, we report that fibers characteristic of quiescent neuroblasts are inherited by their basal daughters during reactivation; that the lamina in the developing visual system is assembled roughly 2–3 columns at a time; that lamina glia positions are dynamic during development; and that the nuclear envelopes of adult testis cyst stem cells do not break down completely during mitosis. In all, we demonstrate that our protocol is well-suited for tissue immobilization and long-term live imaging, enabling new insights into tissue and cell dynamics in Drosophila