Richard Roy | McGill University (original) (raw)

Papers by Richard Roy

Developmental Biology

Developmental plasticity refers the ability of an organism to adapt to various environmental stre... more Developmental plasticity refers the ability of an organism to adapt to various environmental stressors, one of which is nutritional stress. Caenorhabditis elegans require various nutrients to successfully progress through all the larval stages to become a reproductive adult. If nutritional criteria are not satisfied, development can slow or completely arrest. In poor growth conditions, the animal can enter various diapause stages, depending on its developmental progress. In C. elegans, there are three well-characterized diapauses: the L1 arrest, the dauer diapause, and adult reproductive diapause, each associated with drastic changes in metabolism and germline development. At the centre of these changes is AMP-activated protein kinase (AMPK). AMPK is a metabolic regulator that maintains energy homeostasis, particularly during times of nutrient stress. Without AMPK, metabolism is disrupted during dauer, leading to the rapid consumption of lipid stores as well as misregulation of metabolic enzymes, leading to reduced survival. During the L1 arrest and dauer diapause, AMPK is responsible for ensuring germline quiescence by modifying the germline chromatin landscape to maintain germ cell integrity until conditions improve. Similar to classic hormonal signalling, small RNAs also play a critical role in regulating development and behaviour in a cell non-autonomous fashion. Thus, during the challenges associated with developmental plasticity, AMPK summons an army of signalling pathways to work collectively to preserve reproductive fitness during these periods of unprecedented uncertainty.

During periods of energetic stress, Caenorhabditis elegans can undergo a global quiescent stage k... more During periods of energetic stress, Caenorhabditis elegans can undergo a global quiescent stage known as “dauer”. During this stage, all germline stem cells undergo G2 cell cycle arrest through an AMPK-dependent mechanism. In animals that lack AMPK signalling, the germ cells fail to arrest, undergo uncontrolled proliferation and lose their reproductive capacity. These germline defects are accompanied by an altered chromatin landscape and gene expression program. We identified an allele of tbc-7, a RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is regulated by tbc-7 and show tha...

Methods in molecular biology (Clifton, N.J.), 2018

Environmental variation experienced early in life can result in long-term reproductive consequenc... more Environmental variation experienced early in life can result in long-term reproductive consequences. We have recently identified an important role for AMPK in the prevention of transgenerational defects following starvation of L1 stage larvae in C. elegans. Here we describe a means of analyzing these transgenerational defects following a single exposure to energy stress during early larval development. We also provide methods to quantify the histone modifications that are affected by this stress, along with the resulting reproductive defects that arise in later generations.

Experimental Biology and Medicine, 2008

5&amp;#39;-AMP-activated protein kinase (AMPK) has been called &amp;quot;the meta... more 5&amp;#39;-AMP-activated protein kinase (AMPK) has been called &amp;quot;the metabolic master switch&amp;quot; because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5&amp;#39;-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

Development, 1998

C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, an... more C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage.

Nucleic Acids Research

During starvation, organisms modify both gene expression and metabolism to adjust to the energy s... more During starvation, organisms modify both gene expression and metabolism to adjust to the energy stress. We previously reported that Caenorhabditis elegans lacing AMP-activated protein kinase (AMPK) exhibit transgenerational reproductive defects associated with abnormally elevated trimethylated histone H3 at lysine 4 (H3K4me3) levels in the germ line following recovery from acute starvation. Here, we show that these H3K4me3 marks are significantly increased at promoters, driving aberrant transcription elongation resulting in the accumulation of R-loops in starved AMPK mutants. DNA-RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq) analysis demonstrated that a significant proportion of the genome was affected by R-loop formation. This was most pronounced in the promoter–transcription start site regions of genes, in which the chromatin was modified by H3K4me3. Like H3K4me3, the R-loops were also found to be heritable, likely contributing to the transgenerational ...

(A)-(B) Signal overlap between ATGL-1::GFP (Green) and C1&... more (A)-(B) Signal overlap between ATGL-1::GFP (Green) and C1-BODIPY-C12-stained lipid droplets (Red) was compared in control daf-2 and daf-2; aak(0) mutant animals at 32 (A) and 48 hours (B) after shifting to restricted temperature. ATGL-1::GFP signal was closely associated with the labeled lipid droplets in daf-2; aak(0) mutant animals (white arrowheads in the insets) while the signals are clearly distinguishable from each other in control daf-2 animals. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified by the same magnification. (C) Western blot analysis of the endogenous ATGL-1 levels in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 (48 hours after shifting to restricted temperature) dauer extracts of control daf-2 and daf-2; aak(0) mutant animals. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level according to the recent proteomic study on C. elegans lipid droplets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130480#pone.0130480.ref039" target="_blank">39</a>]. (D) Lipid droplet isolation method verified by significant C1-BODIPY-C12 staining and triglyceride enrichment in the isolated lipid droplets portion comparing to the cytoplasm (remaining portion of the total lysate) from daf-2 day 0 dauer larvae.</p

(A) CGI-58::GFP was expressed in the hypodermis and the intestine at ... more (A) CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control daf-2 and daf-2; aak(0) mutant dauer larvae. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005284#sec010" target="_blank">Materials and Methods</a>). Scale bar = 20 μm. (B) CGI-58::GFP localizes to the surface of the lipid droplets at dauer day 0 (48 hours after being shifted to 25°C at the L1 stage) in both daf-2 and daf-2; aak(0) mutant dauers. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene. Scale bar = 10μm. Insets (B' and B") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same magnification. (C) Optimal ATGL-1 lipase activity requires cgi-58. ATGL-1-dependent lipase activity was determined in daf-2; aak(0) mutant dauer larvae with wild type or compromised cgi-58/atgl-1 function. ** indicates statistical significance (P<0.01) compared to all three of the other genotypes. Error bars indicate SD of three independent experiments. (D) Elimination of cgi-58 resulted in the dissociation of ATGL-1 from the lipid droplets. Both strains carry the same hjIs67[Patgl-1::atgl-1::GFP] transgene. Scale bar = 10μm. Insets (D' and D") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same final magnification. (E) ATGL-1 association with the lipid droplets is dependent on appropriate CGI-58 levels. Immunoblot analysis was used to determine the levels of ATGL-1 in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 dauer extracts of each genotype. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level. (F) CGI-58 does not contribute to ATGL-1 stability in AMPK mutant dauers. ATGL-1 levels were determined by immunobloting using anti-ATGL-1 antisera in lysates obtained from AMPK mutants with or without cgi-58. (G) CGI-58 physically interacts with ATGL-1 in vivo in both control and AMPK mutant dauers. Co-immunoprecipitations were performed with daf-2 and daf-2; aak(0) day 0 dauer larvae carrying the same ex[Pcgi-58::cgi-58::GFP;rol-6(gf)] transgene using anti-ATGL-1 serum for pull down and blotted with anti-GFP serum.</p

(A) Comparison of ATGL-1::GFP levels between control daf-2&l... more (A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to restrictive temperature (25°C) at the L1 stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130480#sec002" target="_blank">materials and methods</a>). Scale bar = 10μm. (B) Western blot analysis of GFP levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (C) Western blot analysis of endogenous ATGL-1 levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (D) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 4 animals using semi-quantitative RT-PCR. act-1 was used as loading control.</p

During periods of starvation organisms must modify both gene expression and metabolic pathways to... more During periods of starvation organisms must modify both gene expression and metabolic pathways to adjust to the energy stress. We previously reported that C. elegans that lack AMPK have transgenerational reproductive defects that result from abnormally elevated H3K4me3 levels in the germ line following recovery from acute starvation1. Here we show that H3K4me3 is dramatically increased at promoters, driving aberrant transcription elongation that results in the accumulation of R-loops in the starved AMPK mutants. DRIP-seq analysis demonstrated that a significant proportion of the genome was affected by R-loop formation with a dramatic expansion in the number of R-loops at numerous loci, most pronounced at the promoter-TSS regions of genes in the starved AMPK mutants. The R-loops are transmissible into subsequent generations, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK null germ lines show considerab...

Regulation of germline stem cell proliferation downstream of nutrient sensing

Genetics, Aug 1, 2016

When faced with suboptimal growth conditions, Caenorhabditis elegans larvae can enter a diapause-... more When faced with suboptimal growth conditions, Caenorhabditis elegans larvae can enter a diapause-like stage called "dauer" that is specialized for dispersal and survival. The decision to form a dauer larva is controlled by three parallel signaling pathways, whereby a compromise of TGFβ, cyclic guanosine monophosphate, or insulin/IGF-like signaling (ILS) results in dauer formation. Signals from these pathways converge on DAF-12, a nuclear hormone receptor that triggers the changes required to initiate dauer formation. DAF-12 is related to the vitamin D, liver-X, and androstane receptors, and like these human receptors, it responds to lipophilic hormone ligands. When bound to its ligand, DAF-12 acquires transcriptional activity that directs reproductive development, while unliganded DAF-12 forms a dauer-specifying complex with its interacting protein DIN-1S to regulate the transcription of genes required for dauer development. We report here that din-1S is required in parall...

Cell division, Jan 6, 2006

Stem cells have recently attracted significant attention largely due to their potential therapeut... more Stem cells have recently attracted significant attention largely due to their potential therapeutic properties, but also because of their role in tumorigenesis and their resemblance, in many aspects, to cancerous cells. Understanding how stem cells are regulated, namely with respect to the control of their proliferation and differentiation within a functional organism, is thus primordial to safely profit from their therapeutic benefits. Here, we review recent advances in the understanding of germline stem cell proliferation control by factors that respond to the nutritional status and/or insulin signaling, through studies performed in C. elegans and Drosophila. Together, these data uncover some shared fundamental features that underlie the central control of cellular proliferation within a target stem cell population in an organism. These features may indeed be conserved in higher organisms and may apply to various other stem cell populations.

East Coast Worm Meeting, 2004

Genetic analysis has demonstrated that the decision to execute dauer development is controlled by... more Genetic analysis has demonstrated that the decision to execute dauer development is controlled by three parallel pathways. Little is known however concerning the downstream effectors common to these three pathways that mediate the important metabolic and ...

International Worm Meeting, 2007

The genetic basis underlying C. elegans adult lifespan regulation is emerging from studies carrie... more The genetic basis underlying C. elegans adult lifespan regulation is emerging from studies carried out in several laboratories. It appears that, among others, genes regulating dauer formation such as those involved in insulin signaling, have a dramatic effect on ...

International Worm Meeting, 2005

During adverse environmental conditions, C. elegans larvae can enter a diapause-like stage called... more During adverse environmental conditions, C. elegans larvae can enter a diapause-like stage called" dauer", and three parallel signalling pathways are implicated in this decision (insulin-like, TGF-, or cGMP). The dauer larva is specialized for long-term survival ...

Frontiers in Bioscience, 2008

Introduction 3. Niche signaling and stem cell fate 4. Cytoskeletal changes 4.1. Asymmetric divisi... more Introduction 3. Niche signaling and stem cell fate 4. Cytoskeletal changes 4.1. Asymmetric division 4.2. Symmetric division 5. Germline stem cell quiescence 5.1. Nutrient/insulin regulation 5.2. Downstream effectors 5.3. Quiescence in haematopoietic stem cells-a link? 6. Downstream effectors regulate both growth and polarity 6.1. The LKB1 network in neuronal and epithelial development 6.2. PTEN and Akt in neuronal and epithelial development 7. Summary and perspectives 8. Acknowledgements 9. References

Development, 2006

In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive del... more In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. We show here that the C. elegans orthologues of LKB1 and AMPK cooperate during conditions of reduced insulin-like signalling to establish cell cycle quiescence in the germline stem cell population, in addition to prolonging lifespan. The inactivation of either protein causes aberrant germline proliferation during diapause-like `dauer' development, whereas the loss of AMPK uncouples developmental arrest from lifespan extension. Reduced TGF-β activity also triggers developmental quiescence independent of the insulin-like pathway. Our data suggest that these two signalling pathways converge on the C. elegans PTEN orthologue to coordinate germline proliferation with somatic development during dauer formation, via the regulation of AMPK and its upstream activator LKB1, rather than through the canonical insulin-like signalling cascade. In humans, germline mutatio...