Characterization of hyl-2(gnv1) animals

We observed that at all developmental stages, except for embryos (figure 38 a and b) and starved L1 larvae (figure 38 c), the hyl-2(gnv1) animals were highly sensitive to anoxia. Moreover, as N2 animals, 10 day-old hyl-2(gnv1) showed a very good adaptation to anoxia (figure 38 d). Hence, the hyl-2 gene is required neither for

unfed larvae nor for 10 day-old adults, suggesting that the molecular mechanims are responsible for adaptation to anoxia differ with age.

Figure 38: The resistance to anoxia of hyl-2gnv1) depends on the stage of development and on the age. a. The majority of hyl-2(gnv1) embryos are resistant to 72 h of anoxia. White arrowheads show dead embryos (propidium iodide, 100µg ml^-1). b. The majority of hyl-2(gnv1) embryos developed normally to L1 stage larvae within the 24 h of recovery period following 72 h of anoxia. White head arrows show dead embryos (propidium iodide, 100µg ml^-1). c. Survival of different larval stages of hyl-1(gnv1) to 48 h of anoxia compared to N2 (mean ± SD, n=3 for each stage; L1: unfed L1 larvae, 24 h post-L1 (24L1):L2-L3, 48 h post-L1 (48L1):L4 and 72 h post-L1 (72L1):young adult). d. Survival of 10 d old hyl-2(gnv1) animals compared to N2 (mean ± SD, n=3).

We used confocal microscopy to monitor propidium iodide (PI) uptake over time following a short-term exposure to anoxia (16 h). PI positive animals showed either necrotic cells in the head or a massive necrosis through the gut. This necrosis was accompanied with multiple vacuoles and retractile corpses throughout the entire body (figure 38).

Figure 39: Propidium iodide (PI) uptake immediately after 16 h of anoxia. N2 animals displayed a non-specific coloration in the mouth (a, white arrow). Alive hyl-2(gnv1) animals displayed an identical non-specific staining in the mouth (b, white arrow). Different patterns in dying animals were observed:

distinct nuclei in the head (c, we did not identified these specific cells), a general necrosis throughout the body (d, white arrowhead) or a massive necrosis (e). Black arrow shows vacuoles commonly observed after anoxia of hyl-2(gnv1) animal (panel c). Pictures were taken using a Leica SP2 confocal microscope.

We checked the resistance of hyl-2(gnv1) animals to other stress stimuli, including hyperoxia (100% oxygen), heat shock and hypotonic shock.

We found that hyl-2(gnv1) animals, as N2, reproduced normally in hyperoxia (100% O₂) and had a normal development (16). We showed that, compared to N2, young adults hyl-2(gnv1) animals were not sensitive to heat-shock at 30°C (figure 40 a) but did not tolerate incubation at 36°C (figure 40 b).

Figure 40: Survival of hyl-2(gnv1) animals to mild (30°C) and high (36°C) heat-shock (HS) incubation. a. Aging of hyl-2(gnv1) animals incubated at 30°C compared to N2 (mean ± SD, n=3). b.

Survival of hyl-2(gnv1) to 36°C compared to N2 (mean ± SD, n=3, adapted from S. Gentina).

Interestingly, it has been previously shown that hif-1 was necessary to HS resistance (258) and that the up-regulation of heat-shock proteins (hsp) during hypoxia is part of the low oxygen stress response in D.melanogaster (259), C.elegans (249) as well as in mammalian tissues (260). Moreover, the

implication of HS proteins in ischemic brain injury has been known for long (261). Finally, Mendenhall showed that the hyper resistance to anoxia displayed by the daf-2(e1370ts) mutation was abolished by knocking down the gene hsp-12.6 (personal communication, International Worm Meeting 2006, poster n°420B).

Altogether, these observations suggested that the mechanisms implicated in the response to low oxygen and heat adaptation, share common mechanisms. Hence, we wondered if animals with mutations in heat-shock genes (hyp-12.6, hsp-3, hsp-16.48, hsp-16.2 and hsf-1) would be sensitive to anoxia. None of these mutants showed sensitivity to anoxia (figure 41).

Figure 41: Survival of several animals mutated in heat-shock predicted gene to 48 h of anoxia.

Long term incubation in hypotonic medium can be deleterious for C.elegans.

Animals with a mutation in the gene srp-6 were found to by hypersensitive to this stress as they die within minutes when soaked into double distilled water (262). hyl-2(gnv1) animals did not show any sensitivity to hypotonic shock (double distilled sterile water, figure 42 b and c). However, we observed that once soaked into water, half of hyl-2(gnv1) animals stopped rapidly their thrashing behaviour, became rod-like, sunk down the plate and moved only when stimulated by a platinum wire (figure 42 b), while N2 animals did not stop swimming (figure 42 a). The hypotonic solution was not responsible for this behaviour since the animals behaved similarly in M9 (figure 42 b). After > 10 h of soaking either in M9 or in double distilled sterile water, the animals totally recovered indicating that the suspended animation following the soaking was transient and did not lead to death (figure 42 c). If these experiments exclude a sensitivity of hyl-2(gnv1) to hypotonic shock, they raise the question of

their ‘paralysis’ when placed in liquid. We wondered whether an energy impairment could explain this behaviour. Using a standard luciferase-based assay for ATP measurement (263), Molecular Probes kit, see material and methods), we found that hyl-2(gnv1) worms contained increased levels of ATP compared to N2 (figure 42 d).

The relevance of these results have not been explored further, but will deserve a particular attention in the future.

Figure 42: Survival of hyl-2(gnv1) animals to hypotonic shock and hyl-2(gnv1) ATP measurement. a. Survival of of hyl-2(gnv1) animals soaked into 4ml of M9 for 4.5 h at room temperature (RT, mean ± SD, n=3). b. Survival of hyl-2(gnv1) animals soaked in 4ml of double distilled water for 4.5 h at RT (mean ± SD, n=3). For panel a and b: spont. mov.: spontaneous movement; stimul. mov.: stimulated movement; dead: animals non responding to platinum wire stimulation were scored as dead. c. Black bars represent N2 animals and white bars represent hyl-2(gnv1) animals. After 10 h of incubation either in H2O or in M9, moving and non moving animals were picked out in fresh plate and allowed to recover for 24 h at 20°C. Animals crawling through the plate were scored as alive (n > 100 animals). d. ATP relative contents of N2 compared to hyl-2(gnv1) animals (mean ± SD, n=3).

IV.7. hyl-1 and hyl-2 can both functionally replace ceramide synthase activity in S. cerevisiae

hyl-2 gene (homolog of yeast longevity assurance gene 2) belongs to a large eukaryotic gene family known as longevity assurance genes (Lass genes) that are all related to the yeast LAG1 gene. LAG1 was identified as a gene whose expression decreases with aging of yeast (264). Moreover, a deletion of this gene results in a 50%

increase in the lifespan of the mutated yeast cells (264). Several members of the Lass gene family have been shown to encode a ceramide synthase for the de novo

sphingolipid pathway (159, 265, 266). The similarity of the Lass genes resides in a domain called “Lag motif” that is found among all Lass gene family members (figure 43) and that is essential for enzyme activity (266). C.elegans possesses three Lass genes: hyl-1 (C09G4.1), hyl-2 (K02G10.6) and lagr-1 (Y6B3B.10) (267) (figure 43).

hyl-1 and hyl-2 are most closely related compared to lagr-1 (267).

Figure 43: Alignment of amino acid sequences of the Lag motif of different Lass genes. The CLUSTALW algorithm was used to create the alignment. The black boxes are the two conserved histidines, essential for the correct activity of the protein. The grey boxes represent the two modified amino acid, arginine and tyrosine, in the hyl-2(gnv1) mutant (modified from 266).

The S.cerevisiae Lass genes LAG1 and LAC1 are required for de novo ceramide synthase activity and a double knock-out ΔLAG1ΔLAC1 is lethal (265). It has already been shown that hyl-1 is functionally homologue to LAG1 and LAC1 (265). In order to test whether hyl-2 is also functionally homolog to the yeast Lass genes, we transformed ΔLAG1ΔLAC1 yeast with an expression vector carrying the cDNA of either LAG1, hyl-1 or hyl-2 (see material and methods). All expression vectors were able to rescue the lethal phenotype of ΔLAG1ΔLAC1 strain (figure 44 a and b) indicating that, as hyl-1, hyl-2 is functionally related to LAG1. Importantly, expression of hyl-2(gnv1) cDNA in ΔLAG1ΔLAC1 yeast failed to rescue the lethal phenotype demonstrating that His¹⁶⁸ and His ¹⁶⁹ are required for HYL-2 function (figure 44 b, bottom panel). This strongly supports previous studies reporting that these two evolutionary conserved His residues in the Lag motif are essential for enzymatic function (266, 268) (figure 44 b). Thus the inability of C.elegans to adapt to oxygen deprivation is due to a loss of function of the HYL-2 ceramide synthase.

Figure 44: hyl-2 is functionally homologue to LAG1 and LAC1. a. Schematic representation of the complementation. Basically, yeast strains RH6602 (double deletion ΔLAG1ΔLAC1 transformed with pRS416-LAG1, a Ura-based plasmid) were transformed by pRS424 (Trp-based plasmid) containing either LAG1, hyl-1 or hyl-2. Transformed yeasts were spotted on SD (-Trp) and SD (-Trp) + 5-FOA plates. The plates were incubated at 30°C for 3 days. Vector pRS424 without insert was used as negative control. b. functional complementation of ΔLAG1ΔLAC1 by hyl-1 and hyl-2. Replacing the His¹⁶⁸His¹⁶⁹ by Gln¹⁶⁸Tyr¹⁶⁹ affected the protein activity (bottom line). From Kate Howell.

IV.8. hyl-1 mutation confers hyper-resistance to anoxia and HYL-1 is functionally different from HYL-2 in C.elegans

In order to determine the expression pattern of hyl-1 and hyl-2, we engineered two different transgenic lines: a GFP::hyl-2 fusion gene under the control of the promoter of hyl-2 (figure 45 a) and a GFP::hyl-1 fusion gene under the control of the hyl-1 promoter (figure 45 e, supplementary figure 2).

Each transgene was injected using a standard procedure into hyl-2(gnv1) animals and the fluorescence was observed using a confocal microscope. Both transgenes were expressed in embryos and in all larval stages (data not shown).

In adulthood, while the promoter of hyl-1 drove the expression of GFP in the gut and in the whole pharynx (figure 45 f and g), the promoter of hyl-2 was shown to be active in the gut (figure 45 b), the hypoderm, the posterior bulb of the pharynx and in different unidentified cells of the tail and of the head (figure 45 c and d).

Figure 45: Expression pattern of hyl-1 and hyl-2 genes in young adult C.elegans. a. A GFP::hyl-2 fusion gene on the control of the promoter of hyl-2 (gnvEx16) was injected into hyl-2(gnv1) animal. b.

expression pattern of hyl-2 gene and detail of the head (c) and the tail (d). e. A GFP::hyl-1 fusion gene on the control of the promoter of hyl-1 (gnvEx19) was injected into hyl-2(gnv1) animal. f. expression pattern of the hyl-1 gene and detail of the head (g). Pictures were taken using a SP2 Leica confocal microscope.

In contrast to hyl-1;hyl-2 double knockouts that are not viable, animals with inactivation of only hyl-1 or hyl-2 was indistinguishable from N2 animals in terms of development, fertility, brood size or pharyngeal pumping. Moreover, in contrast to ΔLAG1 or ΔLAC1 that both resulted in a 50% increase in the lifespan in yeast (264), none of the mutated alleles hyl-1(ok976), hyl-1(gk203), hyl-2(gnv1) or hyl-2(tm2031) resulted in increased lifespan when compared to N2 (figure 46 a) (267).

Since both hyl-1 and hyl-2 were found to be functionally homologous to the ceramide synthase LAG1 (figure 44 b), we wondered whether a loss of function of hyl-1 would also impair resistance of C.elegans to anoxia. Interestingly, the alleles hyl-1(ok976) and hyl-1(gk203), two deletions in the hyl-1 gene (see supplementary figure 2), were found to be resistant to a 30°C heat-shock (figure 46 b) and hyper-resistant to anoxia compared to N2 (figure 46 e and f). Hence, HYL-2 and LAGR-1 are sufficient to promote normal survival of C.elegans during anoxia.

Figure 46: Aging, heat-shock (HS, 36°C) and anoxia of hyl-1 and hyl-2 mutants. a. aging of N2, hyl-1(gk203), hyl-1(ok976), hyl-2(gnv1) and hyl-2(tm2031) at 20°C. Life span is defined as the day animals were at the L1 larval stage (time t=0) until the day they were scored as dead. Results are cumulative from 3 independent experiments with ≥ 40 animals per trial. Adapted from S. Gentina b.

Survival of young adults incubated 2.5 h at 36°C (mean ± SD, n=3). Adapted from S. Gentina c-f.

different time of anoxia for hyl-2(gnv1) (c, mean ± SD, 24h: n=7; 48h: n=44; 72h: n=10), hyl-2(tm2031) (d, mean ± SD, 24h: n=4; 48h: n=8; 72h: n=5), hyl-1(ok976) (e, mean ± SD, 24h: n=4; 48h:

n=7; 72h: n=6) and hyl-1(gk203) (f, mean ± SD, 24h: n=3; 48h: n=7; 72h: n=4). * is P<0.05; ** is P<0.01.

These results suggested either a toxic effect of hyl-1 or a protective effect of hyl-2.

To unravel this question, we constructed a GFP::hyl-1 fusion gene under the control of the promoter of hyl-2 and injected it in hyl-2(gnv1) animals (see material and method). This construct ensured that hyl-1 expression pattern would be similar to that of hyl-2. The GFP::hyl-2 fusion gene under the control of the promoter of hyl-2 that we already showed to rescue the hypersensitivity of hyl-2(gnv1) animal was used as positive control (figure 37 e, see material and method). Using three different

transgenic strains, we found that the rescue conferred by hyl-1 was significantly lower than that conferred by expression of hyl-2 (figure 47). Since expression of hyl-1 had a small effect on survival, it is likely that the absence of hyl-2 rather than a toxic effect of hyl-1 is responsible for the sensitivity of hyl-2 deficient worms to anoxia.

Moreover, recent studies showed that hyl-2 mRNA content was increased in hyl-1 deleted animals (267). Therefore, the better adaptation of hyl-1 deleted animals to anoxia could be explained by an increase of hyl-2 expression.

Figure 47: HYL-1 is not completely functionally identical to HYL-2 in C.elgans. Survival of hyl-2(gnv1) animal expressing either hyl-1::GFP under the control of hyl-2 promoter (gnvEx22, gnvEx23, gnvEx21) or hyl-2::GFP under the control of hyl-2 promoter (gnvEx16) to 48 h anoxia (mean ± SD, unless indicated, n=7). 1 and 2 are schematic representations of the transgenes used and their corresponding effect indicated on the graph. . **p<0.01 and ****p<0.001

IV.9. Ectopic hyl-2 expression fails to rescue the sensitivity to anoxia