Narbonne, Patrick et al. (2013) International Worm Meeting "Regulation of self-renewal and differentiation capacities of germline stem cells during ageing."

Labbe, Jean-Claude, Maddox, Paul S., Narbonne, Patrick

[International Worm Meeting, 2013]

Stem cells have the amazing capacity to proliferate and expand in numbers almost indefinitely in vitro. In vivo however, the efficiency of stem cell-dependent processes, such as regeneration/repair and reproduction, declines with age in most organisms. It is thus likely that stem cell capacities decay during ageing in vivo, but the mechanisms remain unclear. Yet, understanding this phenomenon could lead to the identification of genes or pathways that could be targeted by drugs to restore stem cell function in ageing humans. This may be useful to treat many degenerative disorders, but also to elucidate how cancer stem cells somehow escape from this regulation and acquire/maintain the ability to expand in numbers within an ageing organism. I use the C. elegans germline stem cells (GSCs) as a model to study stem cell activity in vivo during ageing. By staining the germ line of unmated hermaphrodites fixed at different ages with an M-phase marker (anti-phospho-H3ser10) and a differentiation marker (anti-HIM-3), I have determined their mitotic index until 10 days of adulthood at 25 deg C. The mitotic index of GSCs progressively decreases as worms age, leading to a decreased GSC pool size. At a population level, the mitotic index reaches a basal level (below 1%) after 4 days, and then remains more or less stable until day 10. GSC population size follows the same trend, reaching a basal size (~100) after 5 days and remaining stable until day 10. Similarly, mitosis-to-meiosis transition of the GSCs is slower in 4 day-old adults compared to 1 day-old. In continuously mated hermaphrodites, the mitotic index remains above 1% for a longer time, but eventually also declines in older animals, and a similar trend is observed in males. This suggests that GSC division rate is coupled to oocyte demand until a certain age, after which the capacity of GSCs to expand in numbers ceases to respond properly to the demand. This is consistent with stem cell capacities progressively deteriorating with age in vivo. I am currently generating mitotic index curves relative to age for a variety of aging, metabolic, epigenetic and developmental mutant animals to find out which genes and genetic pathways are involved in this process.

Narbonne, Patrick et al. (2015) International Worm Meeting "Localized insulin signaling inhibition couples germline stem cell activity to oocyte needs in aging C. elegans adults."

Narbonne, Patrick, Maddox, Paul S., Labbe, Jean-Claude

[International Worm Meeting, 2015]

The efficiency of stem cell-dependent processes declines with age, largely owing to a decline in stem cell capacities, but the mechanisms remain unclear. Using the C. elegans germline stem cells (GSCs) as model, we found that two general mechanisms underlie stem cell activity decline during aging: a passive decline that results from aging, and an active induction of quiescence to meet stem cell progeny needs. The adult C. elegans germline can be viewed as a biological assembly line, with stem cells as the raw material that is turned, in a stepwise manner, into mature oocytes. Under replete growth conditions, the two gonad arms produce about 300 oocytes over three days in self-fertilizing hermaphrodites, but can produce more if they mate with males. We found that the accumulation of unfertilized oocytes, which occurs after sperm depletion, triggers a feedback signal leading to a reversible complete germline arrest, including the induction of stem cell quiescence. Accumulation of intermediate differentiation products or sperm does not trigger the feedback, which once induced, is able to strongly suppress stem cell proliferation throughout the gonad. The active induction of stem cell quiescence in otherwise healthy and well-fed adults only occurs when there is an accumulation of terminally-differentiated stem cell progeny (oocytes) and requires a localized, daf-16-independent down-regulation of insulin signaling in the germline. This in turns locally activates the energy-sensing kinase AMPK, which can then phosphorylate regulators of the ERK/MAPK pathway, including MPK-1 itself, to inhibit MPK-1 activity and shut down GSC proliferation and differentiation. Our results demonstrate that stem cell assembly units (from the stem cells to their terminally-differentiated progeny) can function as self-regulated, independent entities within multi-cellular organisms. This allows different stem cell populations, across and within tissues, to differentially respond to systemic information according to the local needs.

Patrick Narbonne et al. (2008) European Worm Meeting "Hypodermal LKB1/AMPK signalling is critical to dauer longevity"

Richard Roy, Patrick Narbonne

[European Worm Meeting, 2008]

The alteration of genes regulating dauer formation often has a dramatic. effect on adult lifespan. Dauer is a developmentally-arrested, non-feeding. stage that is long-lived and very resistant to harsh conditions. It is. therefore proposed that what confers adult lifespan extension in dauer-. constitutive mutants is the inappropriate expression of a plethora of dauer-. specific genes during adulthood. If the alteration of signalling downstream. of the same genes during dauer is responsible for the extreme longevity of. this stage, dauer-constitutive mutations should not extend dauer lifespan.. We therefore compared the lifespan of naturally-induced (crowding +. starvation) N2 dauers to that of daf-2 dauers and indeed found that reduced. insulin signalling does not extend dauer lifespan.. Mutations in the insulin pathway largely require aak-2 (an AMPK catalytic. subunit) activity to extend adult lifespan. Interestingly, aak-2 mutants. form morphologically normal dauer larvae, which however have a very short. lifespan. We observed similar effects in strd-1; par-4 mutants (AMPK. activators), as well as in aakb-1; aakb-2 mutants (AMPK regulatory. subunits), but not in aak-1 (the second AMPK catalytic subunit) animals. We. therefore believe that an aak-2-specific LKB1/AMPK cascade is necessary for. the extreme dauer longevity, likely through potentiating the effects of the. naturally reduced level of insulin signalling during this stage. To. localize the defect in LKB1/AMPK deficient dauer larvae, we attempted to. rescue the dauer lifespan of aak-2 mutants using tissue-specific promoters. that drive a wild type aak-2 cDNA. We tested intestinal (elt-2), neuronal. (unc-119), muscle (unc-54) and hypodermal (dpy-7) specific promoters to. restore aak-2 function in these tissues. Only hypodermal expression of the. aak-2 cDNA significantly rescued dauer lifespan. A major function of the. hypodermis is to act in energy storage and we found that aak-2 mutant. dauers rapidly deplete their stored lipids while these are maintained in. wild-type dauers. We are currently testing potential AMPK targets for their. ability to suppress energy storage defects and lethality of aak-2 mutant. dauers.

Patrick Narbonne et al. (2007) International Worm Meeting "The LKB1/AMPK cascade functions in the hypodermis to regulate dauer lifespan."

Patrick Narbonne, Richard Roy

[International Worm Meeting, 2007]

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 adult lifespan. Dauer is a developmentally-arrested, non-feeding stage that is long-lived and very resistant to harsh conditions. It is proposed that what confers lifespan extension in insulin signaling pathway mutants results from the inappropriate expression of a plethora of dauer-specific genes that are regulated by DAF-16, the downstream transcription factor of the insulin signaling cascade, during adulthood. Although largely accepted, the theory that daf-16 indeed mediates the extreme longevity of the dauer larva remains difficult to test because daf-16 mutants are dauer defective. In addition to daf-16 activity, mutations in the insulin pathway largely require AMPK activity to extend adult lifespan. Interestingly, aak-2 (an AMPK catalytic subunit) mutants form morphologically normal dauer larvae, which however have a very short lifespan. While aak-1 (the second AMPK catalytic subunit) null mutants do not show such a phenotype, we find that double mutant combinations between par-4/LKB1 (an AMPK activator) and strd-1 (an LKB1 co-factor), or between both of the AMPK beta regulatory subunit isoforms (aakb-1 and aakb-2) also severely reduce dauer lifespan. We therefore believe that an aak-2 specific LKB1/AMPK cascade is necessary for the extreme dauer longevity, likely through potentiating the effects of the naturally reduced level of insulin signaling during this stage. To localize the defect in LKB1/AMPK deficient dauer larvae, we have attempted to rescue the dauer lifespan of aak-2 mutants using tissue-specific promoters that drive a wild type aak-2 cDNA. We used the sur-5 promoter as a positive control for ubiquitous somatic aak-2 cDNA expression, which largely rescued dauer lifespan. We used intestinal (elt-2), neuronal (unc-119), muscle (unc-54) and hypodermal (dpy-7) specific promoters to restore aak-2 function in these tissues. Only hypodermal expression of the aak-2 cDNA significantly rescued dauer lifespan, albeit only partially. Since the co-injection of these four constructs does not enhance the rescue above that observed with the injection of the hypodermal construct alone, we believe that aak-2 must be required in additional tissues/cells. While we are confident that the somatic gonad or germline are not necessary for dauer lifespan since their ablation does not phenocopy aak-2 mutants, we are currently testing alternative tissues/cells, such as the excretory cell and coelomocytes for potential complementary rescue.

Patrick Narbonne et al. (2005) International Worm Meeting "Regulation of germline proliferation and lifespan during dauer development"

Patrick Narbonne, Richard Roy

[International Worm Meeting, 2005]

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, presumably as a result of profound metabolic changes, as well as the establishment and maintenance of cell cycle quiescence throughout the animal, including the cells of the germline. Surprisingly, the Notch ligand (LAG-2) is strongly expressed in the distal tip cells throughout the dauer stage, suggesting that the germ cells presumably receive signals to proliferate, yet they nevertheless arrest without entering the default meiotic program. We reasoned therefore that dauer-inducing signals repress mitosis downstream of the Notch ligand/receptor interaction, while the latter may continue to block entry into meiosis.From a genetic screen designed to identify these potential regulatory genes, we have characterized one mutant (aak-2) in which the dauer larvae show pronounced germline hyperplasia when induced by either of the three signalling pathways mentioned above. In glp-1 (Notch receptor) mutants, germ cells enter meiosis prematurely, but arrest in pachytene during dauer. However, in glp-1; aak-2 dauer larvae, germ cells enter meiosis and complete spermatogenesis during dauer development, indicating that aak-2 not only suppresses germ cell mitosis, but also blocks progression through meiosis during dauer development. From our genetic analysis, we found that daf-18/PTEN and par-4/LKB1, the loss of which causes cancer in humans (Cowden and Peutz-Jeghers syndromes, respectively), are also required to suppress germline proliferation during dauer formation triggered by either reduced insulin-like or TGF- signalling.A striking, independent phenotype of aak-2 dauer larvae is that they are short-lived. aak-2 encodes a C. elegans homolog of the mammalian AMP-activated kinase alpha-2 catalytic subunit, a metabolic "master switch", suggesting that the longevity of the dauer larva may require a substantial metabolic change that is largely triggered by aak-2. LKB1, is a major activator of AMPK and reduced par-4 activity partially phenocopies and enhances aak-mediated defects, suggesting that both proteins may cooperate downstream of daf-18 in insulin-like and TGF- signalling-dependent germline regulation, providing genetic evidence that defective AMPK activation may participate in tumorigenesis in patients affected with Cowden or Peutz-Jeghers syndromes.

Patrick Narbonne et al. (2003) International Worm Meeting "Characterisation of a mutant with compromised germ line cell cycle arrest during dauer."

Jimmy Ouellet, Richard Roy, Patrick Narbonne

[International Worm Meeting, 2003]

In C. elegans, the execution of dauer development results in a complete developmentally-regulated cell cycle arrest. In the germ line, even though the distal tip cells continue to strongly express the lag-2 ligand, as displayed by the lag-2::GFP reporter, no divisions occur in the germ cell nuclei. The cyclin-dependent kinase inhibitor CKI-1 was shown to be a mediator of the dauer-associated germ line cell cycle arrest as cki-1(RNAi) caused proliferation of germ cell nuclei in the dauer gonad. We have found that in dauer larvae, germ cell nuclei are arrested at mitotic pro-metaphase. We have sought to elucidate how this germ line cell cycle arrest is accomplished at the genetic level by screening a population of dauer-arrested worms to find mutants that have more germ cell nuclei in their gonad than wild-type dauer larvae, which bear a mean of 33.48 germ cell nuclei (n=25). Among the 3800 haploid genomes screened, we isolated a mutant (rr48) in which the dauer larvae have an enlarged gonad containing, on average, about twice the wild-type number of germ cell nuclei (mean of 70.28; n=25). This mutation is zygotic, semi-dominant, and fully penetrant. It causes partial dauer-dependent sterility when worms spend 24 hours in dauer (28.57%; n=28). In addition, rr48 worms which spend 96 hours in dauer show poor recovery (26.67%; n=60), while most of those which spend longer durations in dauer die (98.41%; n=251). Moreover, mutant worms that do not enter the dauer stage do not seem to be affected, suggesting that the mutation occurs in a gene involved in a dauer-specific pathway. Finally, we demonstrate that rr48 is required for the proper dauer-induced germ line cell cycle arrest, since many nuclei are visibly beyond pro-metaphase as evidenced by DAPI staining. We observed germ cell nuclei undergoing radical morphological and cytological changes in 6 day-old rr48 dauer larvae (18.75%; n=16), suggesting that this mutation may affect downstream developmental pathways independent of mitotic regulation. The mapping and characterisation of this mutation are currently underway.

Patrick Narbonne et al. (2004) East Coast Worm Meeting "A germline-specific cell cycle inhibitor involved in dauer and adult lifespan"

Richard Roy, Patrick Narbonne

[East Coast Worm Meeting, 2004]

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 morphological changes that take place during the formation of the dauer larva. One feature associated with this stage is the progressive establishment of cell cycle quiescence throughout the animal, including the cells of the germline. Surprisingly, we noticed that a lag-2::GFP transgene is strongly expressed in the DTCs throughout the dauer stage, suggesting that the germ cell nuclei presumably receive signals to proliferate and/or to block entry into meiosis, and should be free to divide mitotically in the dauer larva, yet they do not. We reasoned therefore that genes downstream of the dauer-inducing signals may be required to repress mitosis in the germline by affecting lag-2 or genes downstream. To identify these potential regulatory genes, we performed a pilot screen from which we have isolated and cloned one mutant ( rr48 ) in which the dauer larvae show pronounced germline hyperplasia when induced by reduced TGF-beta or insulin signalling. Our genetic analysis suggests that rr48 acts in a dominant negative manner in the germline, and a time-course examination of germline proliferation revealed that rr48 is required to progressively block germline proliferation during dauer formation. Moreover, we observed that daf-2;rr48 dauer larvae die after 9-11 days in this stage, suggesting that rr48 is required to enhance survival of the dauer larva over extended periods. Consistent with this, daf-2;rr48 adults have a considerably reduced lifespan relative to daf-2 animals, indicating that rr48 is required for the full lifespan extension that is induced by low insulin-like signalling. The reduced dauer lifespan is not suppressed by germline ablation, indicating that germline overproliferation is not responsible for the observed rr48 -mediated dauer lethality. To investigate what downstream genes rr48 may affect to repress the cell cycle specifically in the germline, we characterised its relationship with components of the Notch signalling pathway, which are known to be important in regulating cell divisions in this tissue. A hypomorphic mutation in lag-2 that significantly represses proliferation (without allowing germ cells to execute the meiotic programme) prior to dauer formation in daf-2 animals, partially suppressed the rr48 -induced germline hyperplasia, arguing that a lag-2 signal is required for the mitotic proliferation observed in the rr48 mutant. We also found that rr48 is epistatic to glp-1 with respect to germline meiotic cell cycle progression. Germ cell nuclei are never seen beyond meiotic pachytene in daf-2 glp-1 dauers. However, they are often seen to have completed meiosis in daf-2 glp-1;rr48 dauers as they resemble sperm nuclei after DAPI staining. We believe that rr48 is acting in a daf-16 independent branch of the insulin-like pathway downstream of daf-2 to repress cell cycle progression in the germline. This may occur through altering the effect of Notch signalling at a step downstream of glp-1 . How this crosstalk between the insulin-like pathway and germline cell cycle regulation may occur is currently under investigation and should be clarified when rr48 is characterised molecularly.

Patrick Narbonne et al. (2007) International Worm Meeting "An insulin-dependent neuro-endocrine signal targets PTEN, LKB1 and AMPK to regulate germline stem cell quiescence."

Patrick Narbonne, Roy Richard

[International Worm Meeting, 2007]

During adverse environmental conditions, the signaling level of any of three parallel pathways (cGMP, TGF-<font face=symbol>b</font>, or insulin) can induce C. elegans larvae to enter a diapause-like stage called dauer. All three pathways converge to regulate the production of a sterol-related molecule that binds the nuclear hormone receptor DAF-12 and thereby stimulates reproductive development. When any one of these pathways is down-regulated, the hormone is no longer secreted, and DAF-12 induces dauer formation. Dauer development is tightly associated with the progressive establishment and maintenance of cell cycle quiescence throughout the animal during the extended pre-dauer (L2d) stage. In the germline stem cell population, dauer-dependent quiescence cell autonomously requires the activity of the two AMPK catalytic subunit isoforms, aak-1 and aak-2. To fulfill this role, aak-1/2 further require, at least in part, the activity of par-4/LKB1 (an AMPK-activating kinase), strd-1/STRAD (an LKB1 co-factor) and of aakb-2, which is the primary AMPK <font face=symbol>b</font> regulatory subunit used to establish germline quiescence. The insulin signaling cascade, from the receptor daf-2 down to the transcription factor daf-16, functions in neurons to regulate this developmental switch. We observe that neuronal daf-16 activity is also sufficient to down-regulate germline proliferation during dauer development. This is unlikely to occur solely through DAF-12 regulation because daf-2; daf-12 mutants, despite the fact that they do not form dauers, grow into sterile adults with underdeveloped germlines typical of daf-2 animals. Interestingly, neuronal daf-18/PTEN (an intermediate player in the insulin cascade) is not sufficient to rescue germline quiescence in daf-2; daf-18 or daf-7; daf-18 mutants, and in fact functions cell autonomously in the germline to block proliferation, in addition to its critical function in the neuronal cascade involved in dauer formation. We therefore propose a model where a daf-12 independent insulin-regulated signal is produced in the nervous system and targets daf-18, par-4 and aak-1/2 in the germline to regulate proliferation of the stem cell population during dauer development. Our epistasis analysis suggests that aak-2 requires daf-18 for function, while par-4 works, at least in part independently of daf-18, through aak-1 and yet unidentified gene(s).

Valet, Matthieu et al. (2021) International Worm Meeting "Combining engineering with machine learning to automatically and reliably measure the C. elegans brood size."

Valet, Matthieu, Narbonne, Patrick

[International Worm Meeting, 2021]

Brood size evaluations are routinely used in C. elegans to characterize new alleles, the number of which has exploded following the implementation of efficient CRISPR/Cas9 technology. Manual brood size measurements (number of eggs laid, hatched larvae, adult progeny) are labour intensive, error-prone, and may also be disruptive for animals. Indeed, the experimenter must take individuals outside of the incubator for the long and tedious microscopic analysis of their progeny, during which larvae move constantly and L1 are especially hard to visualize. This creates variability between independent assessments. Manipulations also inevitably cause thermal1,2, mechanical3 as well as photooxidative4 stresses to the sample. Even though such classical evaluations of brood size are somewhat imprecise, that method has been used for the past few decades because no alternative has yet been proposed. Here, we present a novel machine learning-based method that fully automates C. elegans brood size measurements. All that the experimenter will have to do is to pick 1 animal per plate into a 24-well plate, place it in the incubator, and wait that the numbers come out. Although still in development, this project has passed the proof-of-concept stage. We are thus confident that it will be able to acquire, and subsequently analyze, images of entire 15.6mm wells up to once every minute. Our system will allow to measure embryonic lethality, as well as larval lethality at each larval stage. Variations in the environmental parameters will be minimal since the device fits on an incubator shelf, while light exposure, temperature, pressure and vibrations will be recorded. Beyond these basic features, we are working on expanding our device to record other parameters. For example, the neural network used in machine learning could be trained to recognize notable phenotypes (e.g. Dpy, Unc, Rol) which could be useful for segregation analyses. We also aim to couple this system to an additional high-magnification microscopic lense, in order to characterize features (e.g. length, width, turns, and velocity) of the parent during the first days of brooding. As such, we may be able to define adult features that would be predictive of an animal's brood size. Using both macroscopic and microscopic lenses along with a controllable moving platform, we expect to characterize brood size and features of hundreds of strains, with replicas easily exceeding a sample size of 50. We estimate that the cost to build the entire system will be under 500 USD.

Deng, Jichao et al. (2021) International Worm Meeting "DAF-18/PTEN functions in the muscles and proximal somatic gonad to couple to promote oocyte arrest in the absence of sperm"

Gagne, Olivier, Narbonne, Patrick, Deng, Jichao

[International Worm Meeting, 2021]

DAF-18/PTEN is an important tumour suppressor, and loss of PTEN activity is associated with various types of cancers. In C. elegans hermaphrodites that lack sperm, such as fog-1 mutants, DAF-18 is required for oocytes to arrest and accumulate in the proximal gonad. As such, fog-1; daf-18 double mutants lay unfertilised oocytes, and fail to downregulate germline stem cell (GSC) proliferation. However, the tissue(s) in which DAF-18 acts to permit oocyte arrest and accumulation in the absence of sperm, and to eventually suppress GSC proliferation, is still unknown. Here, we transgenically rescued daf-18, specifically in the gut, neurons, hypodermis, muscles, proximal somatic gonad, and germline, of fog-1; daf-18 null mutants. Interestingly, we found that daf-18 expression in the muscles, or in the proximal somatic gonad, was sufficient to allow unfertilized oocytes to arrest and accumulate in the proximal gonad of fog-1;daf-18 double mutants. DAF-18 is also required to suppress the proliferation of germ and somatic gonadal cells during dauer formation, wherein DAF-18 acts in the somatic gonadal cells. Using our germline-specific DAF-18 transgene, we further show that maternally-provided DAF-18 is sufficient to rescue the dauer phenotype of daf-18(-) mutants, but not the arrest of unfertilized oocytes in spermless adults. Overall, our results demonstrate that DAF-18 can act from at least two separate tissues to non-autonomously mediate the arrest of oocytes in the absence of sperm, and allow for their accumulation in the proximal gonad. These results may provide new insights into the how PTEN prevents tumour formation.