Roxane Marsac

Benoît Pinson

Christelle Saint-Marc

María Olmedo

Marta Artal- Sanz

Bertrand Daignan-Fornier

José - Eduardo Gomes

Dr Shohei Mitani

Purine homeostasis is necessary for developmental timing, germline maintenance and muscle integrity in C. elegans

Purine homeostasis is ensured through a metabolic network widely conserved from prokaryotes to humans. Purines can either be synthesized de novo, reused, or produced by interconversion of extant metabolites using the so-called recycling pathway. Although thoroughly characterized in microorganisms, such as yeast or bacteria, little is known about the regulation of the purine biosynthesis network in metazoans. In humans, several diseases are linked to purine metabolism through yet poorly understood etiologies. Particularly, the deficiency in Adenylosuccinate Lyase (ADSL), one enzyme involved both in the purine de novo and recycling pathways, causes severe muscular and neuronal symptoms. In order to address the mechanisms underlying this deficiency, we established Caenorhabditis elegans as a metazoan model organism to study purine metabolism, while focusing on ADSL. We show that the purine biosynthesis network is functionally conserved in C. elegans. Moreover, adsl-1 (the gene encoding ADSL in C. elegans) is required for developmental timing, germline stem cell maintenance and muscle integrity. Importantly, these traits are not affected when sole the de novo pathway is abolished, and we present evidence that germline maintenance is linked specifically to the ADSL activity in the recycling pathway. Hence, our results allow to ascribe developmental and tissue specific phenotypes to separable steps of the purine metabolic network in an animal model.

Introduction

Purine biosynthesis pathways ensure the production and homeostasis of AMP and GMP in the cell, and are widely conserved throughout evolution. The de novo pathway leads to the synthesis of IMP (Inosine monophosphate) using PRPP (5-Phosphoribosyl-1pyrophosphate) as precursor. The recycling pathway (a.k.a. salvage pathway) can then transform extant purines, including IMP synthesized through the de novo pathway, to produce AMP and GMP (Figure 1A; reviewed in [START_REF] Ljungdahl | Regulation of amino acid, nucleotide, and phosphate metabolism in Saccharomyces cerevisiae[END_REF]. Importantly, if available in the extracellular environment, purines can be recovered by specific transporters and subsequently be metabolized through the recycling pathway, which requires much less energy expenditure. Indeed, purine synthesis through the de novo pathway requires the hydrolysis of six more molecules of ATP than purine synthesis through the recycling pathway.

Imbalance in purine metabolism is involved in several diseases that have been extensively characterized, most of them associated with mutations in genes encoding for purine biosynthesis enzymes (reviewed in [START_REF] Nyhan | Disorders of purine and pyrimidine metabolism[END_REF][START_REF] Kelley | Disorders of purines and pyrimidines[END_REF]. The genetics and biochemistry of purine biosynthesis are well established in unicellular organisms like bacteria or yeast, and are highly conserved. Nonetheless, how the diseasecausing enzymatic deficiencies lead to the symptoms observed in patients remains elusive. It is particularly challenging to untangle the contribution of shortage in the final products -AMP and GMP -from mutation-caused accumulation of intermediate metabolites in the pathway. For instance, ZMP (5-amino-4-imidazole carboxamide ribonucleotide monophosphate; [START_REF] Bochner | ZTP (5-amino 4-imidazole carboxamide riboside 5'triphosphate): a proposed alarmone for 10-formyl-tetrahydrofolate deficiency[END_REF] and SZMP (succinyl-ZMP), two intermediates of the purine de novo pathway (Figure 1), are known to act as signal metabolites regulating gene expression transcriptionally in yeast [START_REF] Pinson | Metabolic intermediates selectively stimulate transcription factor interaction and modulate phosphate and purine pathways[END_REF]. Moreover, ZMP is known to activate AMPK (AMP activated kinase) by mimicking AMP [START_REF] Jin | Structural insight into AMPK regulation: ADP comes into play[END_REF] and SZMP has been shown to directly bind and interfere with pyruvate kinase activity in cancer cells [START_REF] Keller | SAICAR stimulates pyruvate kinase isoform M2 and promotes cancer cell survival in glucose-limited conditions[END_REF]. In addition, SAMP (succinyl-AMP), an intermediate metabolite in the recycling pathway, has been shown to stimulate Insulin secretion in pancreatic cells [START_REF] Gooding | Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism[END_REF]. Hence SZMP, ZMP or SAMP buildup could potentially disrupt homeostasis upon deficiencies in the enzymes using them as substrates -ADSL (SZMP and SAMP) and ATIC (ZMP, Figure 1A). Accordingly, mutations in the human genes encoding for these two enzymes are associated with ADSL deficiency and ATIC deficiency (a.k.a. AICAribosiduria), syndromes whose etiology remains unknown [START_REF] Race | Clinical, biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency[END_REF][START_REF] Marie | AICAribosiduria: a novel, neurologically devastating inborn error of purine biosynthesis caused by mutation of ATIC[END_REF][START_REF] Spiegel | Adenylosuccinate lyase deficiency[END_REF][START_REF] Jurecka | Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency[END_REF][START_REF] Zikanova | Biochemical and structural analysis of 14 mutant ADSL enzyme complexes and correlation to phenotypic heterogeneity of adenylosuccinate lyase deficiency[END_REF][START_REF] Jurecka | Adenylosuccinate lyase deficiency[END_REF]. Adding to the complexity of the issue, ADSL catalyzes two different reactions, conversion of SZMP in ZMP in the de novo pathway, and conversion of SAMP in AMP, a step required for AMP synthesis whether it's from the de novo or from the recycling pathway (Figure 1A). This raises the question of which of the two steps is associated with ADSL deficiency phenotypes.

The study of purine metabolic deficiencies in patients, faces yet other challenges, in comparison with microorganisms. One such challenge is tissue specificity, as different tissues may require different levels of purines, depending on their specialized function. For instance, in ADSL-deficiency patients, the most severe symptoms affect the muscles and the nervous system, suggesting these tissues have a larger requirement for purine metabolism, either for their function or during development. Another challenge is purine transport across tissues; the tissue affected by the deficiency not being necessarily the tissue where the enzyme is active. Moreover, purines can be available exogenously from the food source, therefore the consequences of purine metabolism deficiencies are not necessarily caused by limiting amounts of purines. Finally, purine metabolism might be subject to different regulations throughout development, in which case the impairments observed could be the consequence of a requirement for purine regulation at a specific developmental stage.

Project, Japan (NBRP), all strains carrying these alleles used in this study were outcrossed a minimum of four times. Nematode synchronization: Adult worms were collected with M9 buffer in 15 ml conical tubes, centrifuged 1 min at 1000 rpm and supernatant was discarded. Worm bleach solution (NaOH 1 M, NaOCl 0.25 M) was added until carcasses dissolve and eggs release (~8 min). Eggs were washed three times with M9 buffer transferred onto NGM seeded plates. R N A i : a d s l -1 P C R f r a g m e n t w a s a m p l i fi e d w i t h o l i g o m e r s 5 ' -CTTCTTCCGGCAGAATCATCCAGTG-3' and 5'-TGACTGTCGGACGTACTCATTACC-3', a n d a d s s -1 P C R f r a g m e n t w a s a m p l i fi e d w i t h o l i g o m e r s 5 ' -GACGTCTTCCAACAAGACTGTGTCC-3' and 5'-ACGAGACGGCGATACTTCTCCGAG-3'. Both fragments were inserted onto L4440 plasmid (EcoRV site) [START_REF] Timmons | Specific interference by ingested dsRNA[END_REF].

For RNAi experiments, HT115(DE3) bacteria transformed with either the adsl-1(RNAi) or adss-1(RNAi) feeding vectors, or L4440 plasmid (Control), were cultured overnight at 37°C in LB 150 mg/L Ampicillin and induced with 2 mM IPTG 1-2 h prior seeding onto NGM 2 mM IPTG 150 mg/L Ampicillin, in which synchronous populations of worms were cultured from hatching to adulthood (unless specified). Embryonic lethality in adsl-1(tm338)/+ progeny: adsl-1(tm338)/hT2 [bli-4(e937) let-?(q782) qIs48] hermaphrodites -expressing pharyngeal GFP -were crossed to N2 males, non-GFP hermaphrodites in the progeny were selected as adsl-1(tm338)/+ and allowed to lay eggs, one worm per plate, for ~24h. Upon removal of the adults plates were kept in culture for another 24h, upon which unhatched eggs were scored as dead embryos.

-Yeast heterologous rescue assay Yeast expression vectors were constructed by inserting full length cDNAs of ppat-1 ( a m p l i fi e d u s i n g o l i g o m e r s 5 ' -CACTAAATTACCGGATCAATTCGGGGGATCCATGTGTGGTATATTTGGGATTG-3' and 5'-CATAACTAATTACATGATGCGGCCCTCCTGCAGTTATAAATCGATAGCAACTGG-3'), [START_REF] Gari | A set of vectors with a tetracyclineregulatable promoter system for modulated gene expression in Saccharomyces cerevisiae[END_REF]. ade4∆ yeast strain, genotype ade4::KanMX4; his3∆1; leu2∆0; lys2∆0; ura3∆0, was used to test heterologous rescue by ppat-1, ade13∆ yeast strain, genotype ade13::kanMX4; his3∆; leu2∆; ura3∆, was used to test heterologous rescue by adsl-1 and ade16∆ ade17∆ double mutant, genotype ade16::KanMX4; ade17::KanMX4; his3∆1; leu2∆0; ura3∆0; lys2∆0 was used to test heterologous rescue with atic-1b.

a d s l -1 ( 5 ' -C G G G AT C C G TAT C AT G G C C T C C G A A G A C A A G T-3 ' a n d 5 ' - C C A AT G C AT G A AT C A A A C AT C C A G C T G A A C AT T T C C -3 ' ) a n d a t i c -1 b (5'- G A A G A T C T C G A C G A A T T G T G T G A A A T G A C C G A C -3 ' a n d 5 ' - ATAAGAATGCGGCCGCATCTAATGGTGGAAGAGACGAAGTC-3') onto pCM189 plasmid
-adsl-1(∆) rescue A genomic fragment containing the adsl-1 reference allele, ~1Kb upstream the start codon and ~1Kb downstream the stop codon, was amplified by PCR using oligonucleotides 5'-GCTCCCATGATATTCTACCG-3' and 5'-GAGGATTCAACGGTGCTTGCA-3', and as template the cosmid R06C7 (obtained from the Sanger Center). The PCR amplified fragment was cloned onto pBluescript on the EcoRV site, we named this plasmid pRM01. The insert sequence was verified upon cloning. Transgenic lines were obtained by standard microinjection [START_REF] Mello | Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences[END_REF], adsl-1/+ animals were co-transformed with pRM01 at 1ng/µl or 5ng/µl, pDD04 at 10ng/µl as transformation marker -pDD04 expresses GFP in tha pharynx, under the control of the myo-2 promotor [START_REF] Giordano-Santini | An antibiotic selection marker for nematode transgenesis[END_REF]) and pJM001 as carrier DNA at 30-90ng/µl (kind gift by Denis Dupuy, unpublished).

-Imaging For adult morphology, DIC (Differential Interference Contrast, a.k.a. Nomarski) images were taken with a Leitz Diaplan microscope. Adult worms were mounted in M9 buffer, on 2% agarose cushion. Images were acquired with VisiCam 5.0 camera, analyzed and processed with Fiji software. For imaging of pie-1p::GFP::H2B germline reporter, synchronized adult worms were mounted in M9 Buffer 5 mM Levamisole onto 2% agarose cushion. Images were acquired with an Olympus IX81 microscope under an 40X oilimmersion objective, with Hamamatsu Orca R2 camera. Images were analyzed with Fiji software. For size measurements synchronized populations of young adult worms were used, each worm was measured 3 times on three different pictures. Images were acquired on a Nikon SMZ18 stereo microscope with VisiCam 5.0 camera, analyzed and processed with Fiji software.

-Immunostainings Immunostaining was performed on synchronized populations of worms, N2 and de novo pathway mutants were fixed 72 h after synchronization, adsl-1(∆) and ppat-1(∆); adsl-1(∆) were fixed 96h after synchronization. Fixation protocol was adapted from Benian et al. [START_REF] Benian | The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains[END_REF]. Worms were collected in ice cold PBS buffer on 1.5 ml tubes, centrifuged and supernatant was discarded, and fixation solution was added (made fresh, 1ml of MRBW buffer KCl 320 mM, NaCl 80 mM, EGTA 40 mM, Spermidine 20 mM, Pipes 60 mM was added to 2.8 ml of Methanol and 200 µl of Formaldehyde 20%), followed by five cycles of freezing in liquid N2 and thawing and 1 h incubation at 4°C with gentle agitation, fixation solution was replaced by a permeabilization solution (PBS, Triton X-100 2%, ß-mercapto-ethanol 1%) and incubated 1h at room temperature, permeabilization solution was renewed and incubated 1h at room temperature, followed by three washes in PBSt (PBS, Triton X-100 0.5%), after which worms were incubated with primary antibody (diluted 1:1000 in PBSt 5% Fetal Calf Serum) overnight at 4°C with gentle agitation, washed three times in PBSt and incubated with secondary antibody (diluted 1:500 in PBSt 5%FCS). Monoclonal antibody 5-6 (Developmental Studies Hybridoma Bank) directed against muscle myosin was used as primary antibody, anti-mouse Alexa 555 (Molecular Probes) was used as secondary antibody, worms were mounted on Fluoroshield (Sigma). Images were acquired with a Leica DM6000 TCS SP5 confocal microscope using LAS AF (Leica) software.

-Metabolic profiling by HPLC For an extraction, ~7500 young adult worms, 72 h after synchronization, were collected with ice cold water, onto 15 ml conical tubes, and centrifuged at 1000 rpm, 1 min and 4°C, supernatant was discarded eliminating most bacteria. The nematode pellet was washed with 10 ml of cold Hepes 10 mM and centrifuged. The worm pellet was resuspended in Hepes 10 mM, 1 ml of worm suspension is transferred onto 4 ml of absolute ethanol at 80°C, incubated for 5 min and cooled on ice for 10 min. Samples were desiccated on a rotovapor apparatus, the dry residue was resuspended in 350 µl of HyClone Molecular Biology-Grade Water (GE Healthcare) and centrifuged at 21000 g, 5 min at 4°C, supernatant was transferred and centrifuged again at 21000 g, 1 h at 4°C. The last supernatant was used for HPLC. Metabolites were separated by ionic chromatography on an ICS3000 chromatography station (Dionex, Sunnyvale, CA), and measured by an UV diode array (Ultimate 3000 RS, Dionex). Metabolites separation was done on a carbopacPA1 column (250 × 2 mm; Dionex) using a sodium acetate gradient [START_REF] Ceballos-Picot | New biomarkers for early diagnosis of Lesch-Nyhan disease revealed by metabolic analysis on a large cohort of patients[END_REF]. Metabolite levels were normalized relative to dry weight; the same amount of worms used in the extractions were collected in ice cold water, 72h after synchronization, centrifuged, washed 5 times with water, allowed to evaporate for 4 days at 70°C and weighed.

-Lifespan Upon synchronization (see above), worms were grown on standard conditions until reach adulthood, upon which they transferred onto a new plate every 24 h, and scored. Immobile worms unresponsive to touch were considered dead. Three replicate experiments were performed on different days, typically with three different plates per genotype, per replicate and 10-40 worms per plate.

-Locomotion and Behavioral assays Locomotion speed measurement was adapted from [START_REF] Zheng | Neuronal control of locomotion in C. elegans is modified by a dominant mutation in the GLR-1 ionotropic glutamate receptor[END_REF]Hart, 2006); images were acquired on a Leica Z16 APO Macroscope and processed with Fiji software. Worm displacement was measured by tracking the anterior pharynx position, once per second. For each recording one single worm was placed on one intermediate plate without food, than transferred onto the recorded plate ~0,5cm away from the bacterial food source with the pharynx towards the bacteria lawn. Locomotion was recorded for one minute, or until the worm reached the bacteria if it did so in less than one minute. Aldicarb (Sigma) and levamisole (Sigma) assays were performed as described in [START_REF] Gendrel | A secreted complementcontrol-related protein ensures acetylcholine receptor clustering[END_REF], synchronized adult worms were placed on 1 mM Aldicarb or 0.4 mM Levamisole plates for 2 h, after which plates were tapped 10 times and worm movement was scored. Mechanosensory response assay was performed as described in [START_REF] Wormbook Hobert | A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans[END_REF]Hart, 2006), adult worms (~72h after synchronization) were isolated on plates without food for 3 min, and stimulated with an eyelash on the posterior pharynx, and scored for avoidance behavior, each worm was stimulated and score 10 times 3 minutes apart.

-Post embryonic development Monitoring of post-embryonic development was performed as described by [START_REF] Olmedo | A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans[END_REF]. In brief, upon synchronization, 20 embryos per µl were incubated in M9 buffer overnight with agitation, allowing L1 larvae to hatch. A single L1 larvae was added per well in 96-well plates with S-Basal media, 10 g/L E. coli OP50 (wet weight) and 100 µM D-Luciferin. The luminescence was measured with a Berthold Centro LB960 XS 3 during 1 second at 5-min intervals, within a temperature controlled Panasonic MIR-154 incubator.

-Statistics Statistical analysis was done using Graphpad, Excel, Numbers and R softwares. We consider statistical test to be significant when pValue <0.01.

Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Results

-Purine biosynthesis pathway is functionally conserved in C. elegans In order to establish C. elegans as a model to study purine metabolism, we first sought to investigate the conservation of the purine biosynthesis network. We identified, through sequence homology, genes encoding the putative nematode enzymes involved in purine biosynthesis (Figure S1A). We found that both de novo and recycling pathways are conserved in C. elegans, judged by sequence similarity. Specifically, for each enzyme we identified one single orthologue, in all but one case (three genes putatively encoding Xanthine Oxidase). Noteworthy, in S. cerevisiae the histidine pathway contributes to the purine de novo pathway, ZMP being a byproduct of histidine synthesis (reviewed in [START_REF] Ljungdahl | Regulation of amino acid, nucleotide, and phosphate metabolism in Saccharomyces cerevisiae[END_REF]. We have found no C. elegans orthologues for yeast HIS1, HIS4, HIS6 and HIS7 -required for ZMP synthesis through the histidine pathway -consistent with the histidine pathway loss in worms previously reported [START_REF] Payne | Retention and loss of amino acid biosynthetic pathways based on analysis of whole-genome sequences[END_REF], thus ZMP is most likely synthesized exclusively through the purine de novo pathway.

Given the sequence conservation, we undertook a functional analysis focusing on the ADSL enzyme, and including ATIC and GPPAT for comparison (see below). Importantly, sequence alignment showed that ADSL, ATIC and GPPAT are highly conserved (Figure S2). The genes encoding ADSL, ATIC and GPPAT in C. elegans were named respectively adsl-1, atic-1 and ppat-1 (wormbase.org). We first tested whether those three C. elegans genes were functionally conserved using an heterologous rescue assay. We transformed yeast Saccharomyces cerevisiae ade13∆, ade16∆ ade17∆ and ade4∆ mutants with plasmids containing the wild-type sequences of the predicted C. elegans homologous genes: adsl-1, atic-1 and ppat-1, respectively. Expression of adsl-1 restored adenine prototrophy of ade13∆ yeast (Figure 1B), likewise expression of splicing isoform atic-1b restored adenine prototrophy of ade16∆ade17∆ double mutant (Figure 1B). These results confirm adsl-1 encodes the ADSL enzyme and atic-1 encodes for the ATIC enzyme. By contrast, expression of ppat-1 alone was not sufficient to restore adenine prototrophy of yeast ade4∆ (Figure 1B), thus in our heterologous rescue assay we could neither confirm nor rule out ppat-1 encodes the GPPAT enzyme.

We next tested functional conservation of ADSL, ATIC and GPPAT activities in vivo. For our functional analysis, we used deletion mutant alleles, putative genetic nulls, henceforth referred to as adsl-1(∆), atic-1(∆) and ppat-1(∆) (see Materials and Methods). One would predict accumulation of ADSL substrates SZMP and SAMP upon adsl-1 lossof-function as well as ZMP buildup in atic-1(∆), and no detectable de novo pathway intermediate metabolites in ppat-1(∆) (Figure S1B). To test these hypotheses, we performed worm metabolic profiling by HPLC (Figure S1C). We observed a consistent accumulation of SZMP and its riboside form SAICAR upon adsl-1(RNAi), as well as a robust accumulation of SAMP, and its riboside form SAdo (Figure 1C), while neither metabolite was detected in the wild-type (N2) reference strain. Hence both de novo and recycling pathways are functionally conserved in C. elegans, and adsl-1 is required for ADSL activity in vivo. Furthermore, we observe a modest but consistent accumulation of ZMP both in atic-1(∆) mutants (Figure 1D), as well as atic-1(RNAi) animals (not shown), demonstrating atic-1 encodes ATIC enzyme. The accumulation of SZMP and ZMP in adsl-1(RNAi) and atic-1(∆) was rather modest, by contrast we observed a robust accumulation of SAMP in adsl-1(RNAi). These results suggest that C. elegans purine biosynthesis relies mostly on the recycling pathway, at least in standard culture conditions. Noteworthy, ATP levels are slightly lower in adsl-1(RNAi) compared to wild-type (Figure 1C), otherwise the abundance of ATP and GTP were not severely affected in the deficiencies analyzed.

As to the conservation of GPPAT, the HPLC analysis provided crucial functional evidence: neither SZMP was detected in ppat-1(∆); adsl-1(RNAi) (Figure 1C) nor was ZMP detected in ppat-1(∆); atic-1(∆) (Figure 1D), or ppat-1(∆); atic-1(RNAi) (not shown). This demonstrates that ppat-1 gene is indeed required for purine synthesis through the de novo pathway. Noteworthy, SAMP accumulation in ppat-1(∆); adsl-1(RNAi) is similar to adsl-1(RNAi) alone, providing an internal control for RNAi efficiency and further confirming SZMP accumulation in adsl-1(RNAi) is dependent on ppat-1. In summary, our metabolic profiling validates functional conservation of ADSL and ATIC, and demonstrates ppat-1 is required for the de novo pathway, consistent with the prediction that it encodes the GPPAT enzyme in C. elegans.

-Functional analysis of adsl-1 Because SZMP and SAMP are known to function as regulatory metabolites in other organisms [START_REF] Rebora | Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate[END_REF][START_REF] Pinson | Metabolic intermediates selectively stimulate transcription factor interaction and modulate phosphate and purine pathways[END_REF][START_REF] Keller | SAICAR induces protein kinase activity of PKM2 that is necessary for sustained proliferative signaling of cancer cells[END_REF][START_REF] Gooding | Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism[END_REF] and because they may play an important role in the etiology of the ADSL deficiency, we focused our functional analysis on adsl-1. In order to distinguish the effects of SZMP buildup from those due to the loss of de novo purine synthesis, we further include ppat-1 in our analysis. Moreover, in order to distinguish the effect of ADSL activity strictly in the de novo pathway, converting SZMP to ZMP, from the activity in converting SAMP to AMP, we used for comparison ATIC deficient worms. ATIC participates only in the de novo pathway accumulating ZMP, which shares some targets with SZMP, at least in yeast [START_REF] Rebora | Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate[END_REF][START_REF] Pinson | Metabolic intermediates selectively stimulate transcription factor interaction and modulate phosphate and purine pathways[END_REF]. Finally, in order to test whether phenotypes observed in adsl-1(∆) were due to SZMP buildup, we performed systematically epistasis analysis in the ppat-1(∆); adsl-1(∆) double mutant. Similarly, to test whether phenotypes in the atic-1(∆) mutant were caused by ZMP accumulation we performed epistasis in the ppat-1(∆); atic-1(∆) double mutant. Throughout the manuscript, we refer collectively as "purine mutants" to all three single mutant animals, plus the ppat-1(∆); adsl-1(∆) and ppat-1(∆); atic-1(∆) double mutants; and as "de novo" pathway mutants to ppat-1(∆), atic-1(∆) single mutants and ppat-1(∆); atic-1(∆) double mutant animals.

-adsl-1 is required for the maintenance of germline stem cells' (GSCs). The adsl-1(∆) homozygous animals display striking morphological defects, they are noticeably smaller than wild-type, sterile and often present a protruding vulva (Figure 2A). We observe a complete absence of oocytes and sperm cells in adsl-1(∆) and ppat-1(∆); adsl-1(∆) worms, only a reduced number of nuclei morphologically resembling the GSCs are detectable near the vulva, where the germline distal tip cell is found in wild-type animals (Figure S3A). By contrast, the de novo pathway mutants are viable and fertile, and the overall morphology is similar to wild-type worms. To better characterize the GSCs phenotype, we used a germline specific reporter transgene expressing H2B::GFP fusion in the GSCs, under the control of the pie-1 promoter [START_REF] Praitis | Creation of low-copy integrated transgenic lines in Caenorhabditis elegans[END_REF]. While in ppat-1(∆) single mutants the germline is indistinguishable from wild-type, in adsl-1(∆) and ppat-1(∆); adsl-1(∆) animals sterility is fully penetrant and the germline is barely detectable if at all (Figure 2B). Indeed we observed 52.2% of adsl-1(∆) (n = 23) and 52% of ppat-1(∆); adsl-1(∆) animals (n = 25) had no detectable H2B::GFP, 43.5% of adsl-1(∆) and 44% of ppat-1(∆); adsl-1(∆) had 10 or less H2B::GFP positive nuclei, and 4.3% of adsl-1(∆) and 4% of ppat-1(∆); adsl-1(∆) had barely above 10 H2B::GFP positive nuclei. Hence, adsl-1 is required for proliferation and/or maintenance of GSCs independently of ppat-1.

It has been shown that the sterility associated with a deficiency in the pyrimidine recycling pathway could be rescued by supplementation of the culture medium with pyrimidine nucleosides [START_REF] Chi | Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling in C. elegans[END_REF]. We, thus tested whether supplementation with purine nucleosides could rescue adsl-1(∆) sterility. When cultured with 20 mM Adenosine (Ado in Figure 1A) in the medium from hatching until adulthood, both adsl-1(∆) (n = 55) and ppat-1(∆); adsl-1(∆) (n = 54) were sterile, much like the non-supplemented controls (n = 80 and n = 88, respectively). As expected, 100 mM Inosine (Ino in Figure 1A) supplementation had no effect in adsl-1(∆) (n = 28) nor in ppat-1; adsl-1(∆) (n = 64) sterility. Under the same conditions wild-type (20 mM Adenosine n = 86; 100 mM Inosine n = 99) and ppat-1(∆) (20 mM Adenosine n = 76; 100 mM Inosine n = 100) worms were fertile, to the same extent as non-treated controls (wild-type n = 116; ppat-1(∆) n = 114). We further tested whether Adenosine supplementation could rescue adsl-1 partial loss of function. When we performed adsl-1(RNAi) with 20mM Adenosine in the culture medium we observed 30.8% sterility (n=182) compared to 17.0% in absence of Adenosine supplementation (n=141), while mock RNAi produced 0% sterility, in both 20mM Adenosine or no Adenosine supplementation (n=101 and n=103 respectively). In summary, we found no evidence that purine supplementation in the culture medium allows to recover the GSCs defects of adsl-1(∆).

As mentioned above, the de novo pathway mutants present a germline undistinguishable from wild-type, in contrast with the complete sterility of adsl-1(∆). In C. elegans, adult hermaphrodites reproduce through self-fertilization, with the number of progeny being modulated by food source and metabolic status [START_REF] Houthoofd | Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans[END_REF][START_REF] Watson | Interspecies systems biology uncovers metabolites affecting C. elegans gene expression and 24 life history traits[END_REF]. In order to investigate whether the de novo pathway could impact the level of fertility, we measured total number of eggs laid by ppat-1(∆), atic-1(∆) and ppat-1(∆); atic-1(∆) hermaphrodites throughout their reproductive life. We observed that ppat-1(∆) fertility is in the wild-type range, while atic-1(∆) fertility is significantly lower (Figure S3B). Importantly, ppat-1(∆) is epistatic to atic-1(∆) regarding fertility, suggesting that ZMP accumulation, rather than de novo pathway impairment, might reduce fertility. To test this hypothesis, we cultured ppat-1(∆); atic-1(∆) in the presence of ZMP precursor AICAR (0.1 mM), under this condition internal ZMP builds up (see below). Consistent with our hypothesis, we observed reduced fertility in ppat-1(∆); atic-1(∆) treated with AICAR compared to the control (Figure S3B). Furthermore, wild-type worms cultured with 0.5 mM AICAR also displayed reduced fertility. We conclude that ZMP buildup in atic-1(∆) causes reduced fertility, while the de novo pathway per se has no impact on this phenotype, as ppat-1(∆) is similar to wild-type. Taken together these data show that adsl-1 is essential for germline integrity, while de novo pathway genes ppat-1 and atic-1 are dispensable.

The strong germline defects in adsl-1(∆) prompted us to investigate whether adsl-1 is required during early embryonic development. We therefore examined the viability of ADSL deficient embryos. adsl-1(∆) sterility precludes the analysis of homozygous embryos devoid of maternal contribution. We first analyzed zygotic requirement by examining the progeny of adsl-1(∆)/+ heterozygotes (see Materials and Methods); if adsl-1 zygotic expression were strictly essential in the embryo one would expect no adsl-1(∆) homozygous progeny, and a mendelian ~25% proportion of embryonic lethality, instead we observed only 7.7% of dead embryos. Therefore, either adsl-1 is not essential for embryo development or maternal contribution is, at least in part, sufficient. To further investigate adsl-1 function in the early embryo we used RNAi, which depletes maternally expressed mRNAs. Because adsl-1 full penetrant loss-of-function leads to sterility, we performed adsl-1(RNAi) knock-down, starting at the L4 stage -resulting in a partial loss-of-function. Under these conditions, we observed 32.2% embryonic lethality in adsl-1(RNAi) (Figure S3C), suggesting adsl-1 is required for embryonic development, and maternal expression contributes to adsl-1 function at this stage. To test whether the de novo pathway is necessary for embryo development we performed adsl-1(RNAi) in the ppat-1(∆), resulting in 31.5% embryonic lethality. The observation that ppat-1(∆) does not enhance adsl-1(RNAi) hypomorphic phenotype suggests the de novo pathway does not contribute to embryonic development (see bellow). Consistent with this hypothesis, the de novo pathway mutants, display an embryonic development indistinguishable from wild-type: nearly 100% of the laid eggs hatched in all the de novo pathway mutants as in WT controls. Thus adsl-1 function in the recycling pathway is required for proper embryonic development.

-adsl-1(∆) leads to small body size phenotype. The overall morphology observed in adsl-1(∆) and ppat-1(∆); adsl-1(∆) (morphologically indistinguishable from adsl-1(∆) single mutants) suggests that cell lineage specification and morphogenesis are not affected. No defects were observed in somatic tissues of adsl-1(∆) other than the vulva, including pharynx, intestine or hypodermis, and animals are viable and mobile. Hence the smaller size does not seem to result from defects in fate specification. To better characterize this phenotype, we measured body length in adult worms. adsl-1(∆) are significantly smaller than wild-type and similar to ppat-1(∆); adsl-1(∆), while the de novo mutants are indistinguishable from the wild-type (Figure 2C). Given the volume occupied by the germline in adult C. elegans we wondered whether sterility could account for the small size in adsl-1(∆), we therefore compared adsl-1(∆) to glp-1(e2141) mutants, known to lack a germline when cultured at 25°C [START_REF] Priess | The glp-1 locus and cellular interactions in early C. elegans embryos[END_REF]. Given adult glp-1(e2141) length is in the wild-type range (Figure 2C), sterility cannot account for the small size of adsl-1(∆). Noteworthy, newly hatched adsl-1(∆) animals are the same size as wild-type and morphologically indistinguishable (not shown), hence size defects are likely to manifest during post-embryonic development. We conclude adsl-1 is requiredfor adult body size independently of the sterility phenotype. By contrast deficiencies in GPPAT or ATIC in the de novo pathway do not affect this trait.

-adsl-1 is required for normal developmental timing and lifespan. The small size of adsl-1(∆) prompted us to investigate whether post-embryonic development was impaired. Given newly hatched adsl-1(∆) are indistinguishable from wildtype, we hypothesized the growth defects were linked to abnormal larval development. Upon hatching, and before adulthood, C. elegans goes through four larval stages each followed by an inactive state known as lethargus during which a molt takes place [START_REF] Cassada | The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans[END_REF]. Importantly, this sequence follows a highly reproducible timing under standard culture conditions [START_REF] Byerly | The life cycle of the nematode Caenorhabditis elegans. I. Wild-type growth and reproduction[END_REF]). We therefore measured timing of post-embryonic development in purine mutants, using the method described by Olmedo et al [START_REF] Olmedo | A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans[END_REF]. Briefly, newly hatched L1 larvae carrying a transgenic luciferase are cultured in presence the of luciferin, emitting luminescence when active [START_REF] Kimble | Bridging the phenotypic gap: real-time assessment of mitochondrial function and metabolism of the nematode Caenorhabditis elegans[END_REF]. Since larvae cease feeding during lethargus, they stop taking the substrate and a drastic drop in luminescence is observed. Therefore, monitoring the luminescence of individual larvae allows to precisely time the occurrence of each larval (high luminescence) and lethargus (low luminescence) phases (Figure S4A; [START_REF] Olmedo | A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans[END_REF]. We observed that both adsl-1(∆) and ppat-1(∆); adsl-1(∆) present a strongly delayed post-embryonic development (Figure 3A, Supplemental Figure 4B). Although all larval and lethargus phases are affected in adsl-1(∆) mutants, the delay is more pronounced as post-embryonic development progresses. By contrast, the overall timing of post-embryonic development is not affected in de novo pathway mutants. We therefore conclude that adsl-1 is required for normal post-embryonic developmental timing, whereas de novo pathway genes ppat-1 and atic-1 are not.

Post-embryonic development, metabolism and the germline are all known to impact lifespan in C. elegans (reviewed in [START_REF] Antebi | Steroid regulation of C. elegans diapause, developmental timing, and longevity[END_REF]. Because of adsl-1(∆) sterility and developmental delay we measured the lifespan of the purine mutants. adsl-1(∆) mutant has a shorter lifespan that wild-type (Figure 3B). By contrast, the de novo pathway mutants' lifespan is indistinguishable from wild-type (Figure 3B). Importantly, ppat-1(∆) suppresses the adsl-1(∆) short-lifespan phenotype as ppat-1(∆); adsl-1(∆) double mutant survival profile is in the wild-type range (Figure 3B). These results strongly suggest that SZMP buildup in adsl-1(∆), rather than ADSL activity per se is responsible for the reduced lifespan. Moreover, we conclude that the developmental delay and sterility of adsl-1(∆) are not linked to lifespan.

-Ascribing adsl-1(∆) phenotypes to the de novo vs recycling pathway Most of defects we observed in adsl-1(∆) -germline, vulva, adult size, embryonic and post-embryonic development -are not observed in ppat-1(∆) or atic-1(∆) mutants, suggesting ADSL activity of converting SAMP in AMP is the cause of these phenotypes. However, the question of the relative contributions of the de novo and recycling pathways remains open as SAMP to AMP conversion by ADSL has the peculiarity of being required for the synthesis of AMP both through the de novo and recycling pathways (as is IMP to SAMP conversion by ADSS, Figure 1). Thus, two hypotheses can explain the adsl-1(∆) defects regarding SAMP to AMP conversion: either these defects are linked to the recycling but not the de novo pathway, or both the recycling and the de novo pathways contribute redundantly to the phenotypes, such that in ppat-1(∆) mutants the recycling pathway can compensate for the absence of the de novo pathway. Given ADSL functions both in the de novo and recycling pathways, the putative null adsl-1(∆) does not allow to discriminate between de novo and recycling pathway contributions. However, the analysis of adsl-1 or adss-1 partial loss-of-function does. In such condition, if the first hypothesis were true abolishing the de novo pathway would have no impact on adsl-1 or adss-1 partial loss-of-function phenotypes, while as if the second hypothesis were true the penetrance of the phenotypes should increase when adsl-1 or adss-1 partial loss-offunction is combined with ppat-1(∆).

We performed adsl-1(RNAi) and adss-1(RNAi) treatment -producing a partial lossof-function -in wild-type and ppat-1(∆) worms, and analyzed the germline and vulval defects. We observe no increase in penetrance of either phenotype when comparing wildtype and ppat-1(∆), both upon adsl-1(RNAi) and adss-1(RNAi) (Table 1). Similarly, as described above, adsl-1(RNAi) displays the same penetrance of embryonic lethality in wild-type and ppat-1(∆) backgrounds (Figure S3C). We conclude that adsl-1(-) defects, at least in the germline, vulva and embryonic development, are linked to the purine recycling pathway, while the de novo pathway has no detectable contribution.

-adsl-1(WT) transgene rescues adsl-1(∆) phenotypes Given adsl-1(∆) allele was generated by random mutagenesis, it is possible the phenotypes observed are caused by other mutations in the genetic background. In order to verify whether adsl-1 loss-of-function was indeed the cause for the observed phenotypes, we performed a transgene rescue assay. We generated a plasmid containing adsl-1(WT) allele genomic fragment including ~1Kb upstream the start codon and ~1Kb downstream the stop codon (see material and methods) and transformed adsl-1(∆)/+ animals through microinjection the. We obtained transgenic animals, carrying the adsl-1(∆) allele (either heterozygous or homozygous) and the adsl-1(WT) transgene. In seven independent transgenic clones we were able to observe rescue of adsl-1 function in adsl-1(∆) homozygous animals. Rescued adsl-1(∆) homozygous laid broods bearing viable embryos, hence transgenic adsl-1(WT) rescued the germline phenotype. Furthermore rescued adsl-1(∆) homozygous presented an overall wild-type like adult morphology, including WT-like vulva. Therefore transgenic adsl-1(WT) rescued body size and vulval defects as well. Given the extra-chromosomal arrays carrying the adsl-1(WT) transgene were poorly transmitted to the progeny, we could not characterize these lines further. We conclude that adsl-1(∆) phenotypes -at least sterility, vulval defects and body size -are caused by adsl-1 loss-of-function. Further supporting this conclusion adsl-1(RNAi) leads to sterility and vulval defects (see above, Table 1), matching what would be expected for a partial loss-of-function compared to adsl-1(∆), and adss-1(RNAi), depleting ADSS -the enzyme immediately upstream of ADSL in the recycling pathway -phenocopies adsl-1(RNAi) as would be expected.

-Imbalance in purine metabolism impacts locomotion Given the severe muscular and neuronal symptoms of ADSL deficiency patients [START_REF] Jaeken | Adenylosuccinase deficiency: an inborn error of purine nucleotide synthesis[END_REF][START_REF] Spiegel | Adenylosuccinate lyase deficiency[END_REF][START_REF] Jurecka | Adenylosuccinate lyase deficiency[END_REF], we sought to investigate muscle and neural defects in adsl-1(∆) worms. In order to address muscle function, we performed a locomotion assay (Figure 4A; [START_REF] Zheng | Neuronal control of locomotion in C. elegans is modified by a dominant mutation in the GLR-1 ionotropic glutamate receptor[END_REF]Hart, 2006), and observed that adsl-1(∆) mutants were slower than WT (Figure 4B). atic-1(∆) mutants, were also slower than wild-type, albeit to a lesser extent (Figure 4B), suggesting the de novo pathway could have a role in locomotion. However, ppat-1(∆) animals were indistinguishable from wild-type, thus de novo pathway per se does not affect locomotion. Moreover, the locomotion defect in adsl-1(∆) was not suppressed by ppat-1(∆), ppat-1(∆); adsl-1(∆) was actually slower than adsl-1(∆) single mutant. Thus, ADSL activity, independently of SZMP buildup, accounts for the adsl-1(∆) locomotion defect. By contrast, atic-1(∆) locomotion defect was suppressed by ppat-1(∆) as ppat-1(∆); atic-1(∆) speed was in the wild-type range. Hence, slower locomotion in atic-1(∆) is linked to ZMP build up, rather than the de novo pathway per se.

-AICAR treatment

The ADSL product ZMP is known to activate AMPK by mimicking AMP [START_REF] Jin | Structural insight into AMPK regulation: ADP comes into play[END_REF], and AICAR treatment is known to impact muscle function in mice, presumably through ZMP induced AMPK activation [START_REF] Narkar | AMPK and PPARd Agonists Are Exercise Mimetics[END_REF]. To test whether ZMP is responsible for the locomotion defects in atic-1(∆), we tested whether providing the worms with external AICAR would impact locomotion. We treated late L4 stage wild-type and de novo pathway mutants with AICAR, for 24 hours. Somewhat surprisingly AICAR treatment did not significantly affect locomotion in any genotype tested (Figure S5A). Given this surprising result, we performed metabolic profile analysis of AICAR treated worms. As expected, we observed higher levels of AICAR upon treatment, and noticeably a significant buildup of both ZMP and ZTP in all treated genotypes (Figure S5B), showing AICAR was efficiently internalized and phosphorylated. As expected ZMP levels are higher in atic-1(∆) than in other genotypes due to cumulative buildup of ZMP from the de novo pathway and from AICAR treatment (Supplementay Figure 5C). Therefore, buildup of ZMP in adult worms per se does not explain the reduced locomotion observed in atic-1(∆). Importantly, while AICAR treatment did not significantly affect ATP levels across genotypes, it led to an increase in uric acid, particularly in atic-1(∆) and ppat-1(∆); atic-1(∆). Moreover, we also observed, in all treated genotypes, an accumulation of ADSL substrate SZMP and its riboside form SAICAR. Therefore, AICAR treatment leads to increased levels of ZMP as expected, but also of uric acid, ZTP and SZMP (Figure S5B). The SAICAR and SZMP accumulation indicates that not only SZMP to ZMP conversion by ADSL is inhibited by AICAR treatment, this reaction can even be reversed, given SZMP was detected in ppat-1(∆) and ppat-1(∆); atic-1(∆) mutants (Figure S5B, see Figure S1B). Accordingly, in yeast, ADSL enzyme has been shown to convert ZMP in SZMP when ZMP is in excess [START_REF] Rebora | Revisiting Purine-Histidine Cross-Pathway Regulation in Saccharomyces cerevisiae: A Central Role for a Small Molecule[END_REF], which seems to also be the case for C. elegans ADSL.

-Muscle structure, but not the neuromuscular junction, is disrupted in adsl-1(∆) One possible explanation for the locomotion defects observed in atic-1(∆) and adsl-1(∆) is that the neuromuscular junction is compromised. We therefore tested whether purine mutants were sensitive to Levamisole and Aldicarb. Both drugs activate acetylcholine receptors at the neuromuscular junction in C. elegans, at either the presynapse (Aldicarb) or post-synapse (Levamisole) leading to paralysis of wild-type worms [START_REF] Lewis | The genetics of levamisole resistance in the nematode Caenorhabditis elegans[END_REF], while mutants with defective cholinergic neuromuscular junction are not affected [START_REF] Fleming | Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits[END_REF][START_REF] Gendrel | A secreted complementcontrol-related protein ensures acetylcholine receptor clustering[END_REF]). We found no significant differences among purine mutants and wild-type animals (Figure S6A andB), demonstrating that the neuromuscular junction remains functional upon purine metabolism deficiencies. We therefore tested whether other neuronal functions might be impaired in purine mutants. We performed a standard touch assay [START_REF] Wormbook Hobert | A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans[END_REF]Hart, 2006). We observed a similar response to touch in all purine mutants as in the wild-type, thus in our experiments the mechanosensory function as well as the behavioral response are intact in purine mutants (Figure S6C). In summary, we found no evidence for neuronal dysfunction in the purine metabolism mutants tested.

Given these results, we hypothesized that the locomotion defects we observed could be explained by muscle malfunction. We performed an analysis of muscle cell structure through immunohistochemistry and confocal microscopy. As expected, we observed regular parallel arrangement of myosin fibers in the wild-type (Figure 5A), while in adsl-1(∆) mutants (n = 13), muscle myosin fibers formed an irregular meshwork and muscle mass seemed reduced (Figure 5B). In atic-1(∆) (n = 14), by contrast, the structure of muscle myosin fibers was indistinguishable from wild-type controls (Figure 5C, n = 10), as are ppat-1(∆) (Figure 5D, n = 13). Furthermore ppat-1(∆); adsl-1(∆) (Figure 5E, n = 10) displayed the same muscle fiber defects as adsl-1(∆), while ppat-1(∆); atic-1(∆) were indistinguishable from ppat-1(∆) and atic-1(∆) single mutants (Figure 5F, n = 15), showing that de novo pathway deficiency does not affect muscle structure (Figure 5). These results show ADSL is required for muscle integrity, while deficiencies in the de novo pathway have no discernible effect on this trait. Muscle structure defects could account for the locomotion defects of adsl-1(∆).

Discussion

-A nematode model to study ADSL deficiency and, more broadly, purine metabolism We describe here the phenotypes and metabolic profiles associated with three deficiencies in the purine biosynthesis pathway in the nematode C. elegans, focusing on ADSL deficiency. This study provides a reference to future investigation of the underlying molecular mechanisms. The phenotypes observed are to some extent genetically separable. Particularly the most severe defects in the adsl-1(∆) mutant -muscle, germline and development -are not shared with ppat-1(∆) and atic-1(∆) mutants. The model we put forward links these defects to the ADSL activity in converting SAMP in AMP as the critical enzymatic step in ADSL deficiency (Figure 6). For instance, there is a long-standing debate concerning the etiology of the pathology associated with ADSL deficiency. It has been proposed that the SAICAR/S-Ado ratio correlated with specific symptoms [START_REF] Jaeken | Adenylosuccinase deficiency: an inborn error of purine nucleotide synthesis[END_REF][START_REF] Race | Clinical, biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency[END_REF], while more recent studies found no evidence for such correlation [START_REF] Jurecka | Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency[END_REF][START_REF] Zikanova | Biochemical and structural analysis of 14 mutant ADSL enzyme complexes and correlation to phenotypic heterogeneity of adenylosuccinate lyase deficiency[END_REF]. Studies in human subjects are of limited reach, while a genetically amenable model organism shall allow to investigate mechanisms underlying specific phenotypes. In our model, SAMP to AMP conversion is crucial, hence SAMP buildup could play a significant role in the ADSL deficiency. Notwithstanding the evolutionary distance between nematodes and humans, testing this hypothesis may be informative to the human pathology. However, at this point, caution is advised when comparing our results to the human pathology, more research is required to address this issue. Importantly, the reported mutations causing ADSL deficiency in humans are associated with a residual enzymatic activity (reviewed in [START_REF] Nyhan | Disorders of purine and pyrimidine metabolism[END_REF][START_REF] Kelley | Disorders of purines and pyrimidines[END_REF], contrasting with our analysis of putative null mutants. The use of null mutations allows to carefully establish the phenotypes associated with ADSL deficiency in the nematode model, and shall allow to further establish tissue specific contributions and perform epistasis analysis in order to ascribe phenotypes to different steps in the pathway. Only once this characterization is achieved, mutations comparable to human ADSL deficiency, can be modeled with C. elegans and provide further insight. In summary, although further investigation is required, C. elegans as metazoan model organism provides the means to investigate the metabolic regulation of developmental progression, muscle defects or cell proliferation in the context of ADSL deficiency, as well as in other deficiencies in the purine biosynthesis pathway.

-Purine recycling and GSCs maintenance One striking phenotype of adsl-1(∆) is the severe reduction in GSCs, leading to sterility. In adult C. elegans, the germline is the sole proliferating tissue. More precisely, GSCs in the distal gonad proliferate under the control of Notch signaling from neighboring somatic cells, while in the proximal region the germline nuclei enter meiotic differentiation (reviewed in [START_REF] Kimble | Bridging the phenotypic gap: real-time assessment of mitochondrial function and metabolism of the nematode Caenorhabditis elegans[END_REF][START_REF] Hansen | Stem cell proliferation versus meiotic fate decision in Caenorhabditis elegans[END_REF]. Interestingly, deficiencies in both the pyrimidine [START_REF] Chi | Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling in C. elegans[END_REF] and pyridine nucleotides (a.k.a. NAD+) recycling pathways [START_REF] Vrablik | Nicotinamidase modulation of NAD+ biosynthesis and nicotinamide levels separately affect reproductive development and cell survival in C. elegans[END_REF]) have also been linked to germline defects in C. elegans, suggesting nucleotide metabolism to play a critical role in maintaining the GSCs pool. Importantly however, adsl-1(∆) phenotype is more severe than in either case. In pnc-1(-) mutants, affecting the pyridines salvage pathway, animals are fertile despite germline defects. In cdd-1/2(-) mutants, deficient for the Cytidine Deaminase in the pyrimidine recycling pathway, GSCs proliferation could be restored with pyrimidine supplementation. By contrast, purine supplementation had no effect on either adsl-1(∆) or adsl-1(RNAi) -partial loss-of-function -suggesting the shortage in the final products of the purine pathway is not the main factor causing GSCs defect. While one cannot exclude the defects in pnc-1(-), cdd-1/2(-) and adsl-1(∆) are unrelated, the three metabolic pathways are closely connected. It is therefore plausible that germline defects in these three mutants share a common mechanism, through crosstalk among these metabolic pathways. In that case, the severity of the adsl-1(∆) phenotype remains to be explained. One tempting explanation is that the primary metabolic defect lies on the purine pathway, and that imbalance in the other nucleotide biosynthesis pathways impact purine metabolism as an indirect consequence. It is known that GSCs proliferation is influenced by environmental factors, particularly nutrient availability, and is likely regulated by metabolic signals (reviewed in [START_REF] Hubbard | Physiological control of germline development[END_REF]. Our results are consistent with the purine recycling pathway serving as sensor integrating nucleotide pathways' status, and providing a metabolic signal regulating GSCs proliferation.

Interestingly, the purine biosynthesis pathway has been shown to play a role in the balance between proliferation and differentiation in both stem cells and cancer cells, with inhibition of purine synthesis leading to increased differentiation at the expense of proliferation [START_REF] Oburoglu | Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification[END_REF]Tardito et al. 2015). A similar mechanism could explain adsl-1(∆) GSCs defect, in that scenario the ADSL deficiency would lead to germline nuclei entering meiosis at the expense of proliferation.

-Purine metabolism and development Metazoans often control their development in response to environmental conditions, namely nutrient availability. This control involves metabolic regulation, for instance through Insulin/Insulin-like or mTOR pathways (reviewed in [START_REF] Danielsen | Nutrient signaling and developmental timing of maturation[END_REF]. In C. elegans the adult worm size varies depending on the bacterial food source [START_REF] Samuel | Caenorhabditis elegans responses to bacteria from its natural habitats[END_REF], suggesting size is one of the developmental phenotypes subjected to metabolic regulation. This regulation could, in theory be due to metabolic regulation of cell fate decisions, or alternatively be a consequence of metabolic regulation of cell size. In adsl-1(∆), based on our analysis, only GSCs and vulval cells present cell fate abnormalities in the adult, which have no impact in overall adult size. Hence the small size we observe is most likely due to an effect of purine metabolism on cell size. In addition to size, adsl-1(∆) also displays a strong delay during post-embryonic development. In C. elegans progression through larval stage and molting cycles is highly regulated, involving oscillations of LIN-42, the orthologue the circadian protein PERIOD [START_REF] Monsalve | LIN-42/PERIOD controls cyclical and developmental progression of C. elegans molts[END_REF][START_REF] Edelman | Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing[END_REF]reviewed in Monsalve and Frand, 2012). The developmental changes associated with this progression involve a tight coordination of different cell lineages across the worm's body, and are very well characterized, as are the genetic pathways involved (reviewed in [START_REF] Rougvie | Developmental transitions in C. elegans larval stages[END_REF]. By contrast, the upstream mechanisms and signals triggering or modulating these developmental transitions are poorly understood. The quality and amount of food available certainly play a role, as they have been shown to impact the timing of post-embryonic development [START_REF] Houthoofd | Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans[END_REF][START_REF] Macneil | Diet-induced developmental acceleration independent of TOR and insulin in C. elegans[END_REF]. The dramatic developmental delay in adsl-1(∆) points at a metabolic regulation of these developmental transitions involving the purine recycling pathway.

Our results also suggest a role for ADSL in embryonic development. Presumably, a more penetrant adsl-1 loss-of-function would lead to higher levels of embryonic lethality, however the ensuing sterility precludes further study of early development. With the tools currently available we cannot determine conclusively the extent of adsl-1 requirement in embryonic development. Given most homozygous adsl-1(∆) embryos -where only zygotic adsl-1 expression is affected -are viable, and RNAi -depleting maternal mRNA -leads to embryonic lethality it is plausible that proper embryo development requires adsl-1 function, ensured, at least in part, by maternal contribution.

One possibility to explain the developmental phenotypes in adsl-1(∆) could be limiting ATP, causing namely slow development and smaller size. We have observed that in adsl-1(∆) food uptake is reduced compared to wild-type [START_REF] Rodríguez-Palero | An automated method for the analysis of food intake behaviour in Caenorhabditis elegans[END_REF], thus not only ATP but limiting overall bulk nutrients could account for the developmental defects observed. Although limiting nutrients and ATP levels are important factors, and certainly have a sizable impact, other elements suggest this hypothesis does not suffice to explain the adsl-1(∆) defects. In eat mutants, known to have feeding impairment, food uptake defects don't always correlate with developmental delay (M.O. and M.A.S. unpublished results). As to ATP levels, our metabolic profiles show them to be only slightly reduced in adsl-1(RNAi), compared to wild-type. This is not so surprising given adenine or adenosine are available from the bacterial food source could be used to produce ATP despite ADSL deficiency (see Figure 1A). An alternative hypothesis is that post-embryonic developmental and adult size are regulated by the wiring of the purine recycling pathway, rather than the synthesis of its final products. In this view, purine intermediate metabolites could function as metabolic sensors modulating developmental progression.

-Purine metabolism and neuro-muscular function Both neuronal and muscle tissues are severely affected in purine metabolism syndromes, suggesting these two tissues are particularly susceptible upon purine metabolism impairment in patients. In our nematode model, we observed locomotion defects associated with both ATIC and ADSL deficiencies. Somewhat surprisingly, no discernible neuronal dysfunction was observed in any purine mutant. All of the genotypes tested displayed sensitivity to Levamisole and Aldicard, similarly to wild-type controls, showing neuromuscular junction was still functional. Moreover, mechanosensory response in purine mutants was not affected either. Hence, we could not identify neuronal dysfunctions to account for the locomotion defects. Notwithstanding, given we did not investigate directly neuron integrity, it remains possible that purine mutants present neuronal impairments that the functional assays reported here do not allow to detect.

We did however find evidence for muscle abnormalities that could explain the locomotion defects. Noteworthy, the locomotion defects we observed in atic-1(∆) and adsl-1(∆) were linked to different mechanisms. Contrary to adsl-1(∆), the locomotion delay in atic-1(∆) was suppressed in the ppat-1(∆); atic-1(∆) double mutant, hence linked to ZMP buildup. One possible explanation is that ZMP accumulation leads to metabolic changes that in turn impact muscle function, possibly through AMPK activation [START_REF] Jin | Structural insight into AMPK regulation: ADP comes into play[END_REF]. Importantly, the structure of muscle myosin fibers in atic-1(∆) are undistinguishable from the wild-type, thus muscle integrity seems not to be affected in atic-1(∆). The ZMP effect on locomotion, however, is not straightforward; upon AICAR treatment of L4 larvae / young adults ZMP builds up as expected, nonetheless locomotion is not affected. Although the mechanism remains elusive, our results suggest that either ZMP effect on locomotion is developmental and occurs prior to the L4 stage, or ZMP accumulation from external AICAR does not affect the same tissues as endogenous ZMP build up in atic-1(∆).

On a side note, because ZMP is an AMPK agonist [START_REF] Jin | Structural insight into AMPK regulation: ADP comes into play[END_REF], its riboside form AICAR is often added to culture media in different model organisms, including C. elegans [START_REF] Moreno-Arriola | AMP-activated protein kinase regulates oxidative metabolism in Caenorhabditis elegans through the NHR-49 and MDT-15 transcriptional regulators[END_REF][START_REF] Ahmad | Metformin Attenuates Aβ Pathology Mediated Through Levamisole Sensitive Nicotinic Acetylcholine Receptors in a C. elegans Model of Alzheimer's Disease[END_REF], to trigger AMPK activation upon intracellular conversion to ZMP. Our results show that increase of ZMP is not the only metabolic consequence of AICAR treatment, therefore caution is recommended when attributing phenotypes to AMPK activation.

As to the locomotion defects in adsl-1(∆), our results point to ADSL activity in the purine recycling pathway, given ppat-1(∆) does not suppress the locomotion phenotype, quite to the contrary. Furthermore, adsl-1(∆) is associated with significant damage in the structure of muscle myosin fibers, suggesting that locomotion defects are caused by muscle defects. While the underlying cause for these defects is yet to be determined, we hypothesize that the developmental delay in adsl-1(∆) may contribute to muscle defects.

-Purine metabolic network in a metazoan model The results we present show the functional conservation of both the de novo and recycling purine biosynthesis pathways in C. elegans. Furthermore, we characterized phenotypes associated with the deficiencies in the critical enzymes ADSL and ATIC. While several phenotypes are genetically separable (Figure 6), it stands out that the most severe phenotypes -lack of GSCs, developmental delay and muscle integrity -are associated with ADSL activity, whilst deficiencies in the de novo pathway are viable and lead to rather subtle phenotypes -as reduced fertility and slower locomotion. Noticeably, the deletion of ppat-1 -which we show is required for the de novo pathway -produces no detectable phenotype in our culture conditions. The phenotypes observed in atic-1(∆), locomotion defects and reduced fertility, are associated with ZMP accumulation, rather than to shortage in purine synthesis through the de novo pathway, as though the de novo pathway per se is dispensable. It might seem paradoxical that the purine de novo pathway is conserved while having no essential function. Indeed, a number of biosynthesis pathways, namely amino-acids, have been lost during metazoan evolution [START_REF] Payne | Retention and loss of amino acid biosynthetic pathways based on analysis of whole-genome sequences[END_REF] likely because amino-acids are available from the food source, as are nucleotides. However, our culture conditions may not reflect the natural environment in which C. elegans evolved; it is plausible that the de novo pathway is essential in different environments where purines are scarce.

Contrasting with our results in C. elegans, Drosophila mutants in a GPPAT encoding genes are lethal prior to adulthood [START_REF] Malmanche | Drosophila melanogaster Prat, a purine de novo synthesis gene, has a pleiotropic maternal-effect phenotype[END_REF][START_REF] Ji | The purine synthesis gene Prat2 is required for Drosophila metamorphosis, as revealed by inverted-repeat-mediated RNA interference[END_REF]. These Drosophila mutants are purine auxotroph -lethality is higher in purine deprived food source -and lethality is observed during the pupal phase, a closed developmental stage were external nutrients are not available. These different consequences of GPPAT deficiency in C. elegans and Drosophila could owe to substantially different metabolic requirements in the two species for GPPAT, and the whole de novo pathway; they could also reflect different life cycles and different experimental conditions. Further highlighting the contrast between Drosophila and C. elegans, fly mutants deficient for ADSL and ADSS are also lethal, in this case at larval stages prior to pupation [START_REF] Stenesen | Adenosine nucleotide biosynthesis and AMPK regulate adult life span and mediate the longevity benefit of caloric restriction in flies[END_REF]. Interestingly, the authors show that ADSS and ADSL lower levels in heterozygous lead to increased lifespan through reduced AMP synthesis. Noteworthy, purine pathway mutants in yeast S. cerevisiae have been reported to increase chronological age [START_REF] Matecic | A microarray-based genetic screen for yeast chronological aging factors[END_REF]. In both cases purine supplementation of the culture media restores wild-type phenotype, presumably through raising AMP levels to a wild-type range. If the same scenario occurs in C. elegans, our results on the lifespan of purine mutants could be explained by the abundance of purines in the bacterial food source. This possibility emphasizes the contribution purine availability in the external medium to the phenotypic outcomes. Further investigation of the purine pathway in worms will certainly involve controlled manipulation of purine supplementation, which requires a suitable defined culture medium [START_REF] Flavel | Growth of Caenorhabditis elegans in Defined Media Is Dependent on Presence of Particulate Matter[END_REF].

Actually, if one draws a parallel with yeast, it's the severity of the adsl-1(∆) phenotypes that seems surprising, considering Ade13 null mutants in S. cerevisiae are viable when cultured with adenine [START_REF] Dorfman | The isolation of adenylosuccinate synthetase mutants in yeast by selection for constitutive behavior in pigmented strains[END_REF]. Given purines are available from the food source, one would have expected the worm to synthesize AMP and GMP despite ADSL deficiency. Indeed, external adenine and adenosine can be converted in AMP by Adenine phosphoribosyltransferase and Adenosine kinase respectively, both of which have orthologues in C. elegans (Figure S1A), and AMP can be converted in GMP (Figure 1A). The severity of adsl-1(∆) phenotypes, suggests that ADSL became essential even when external purines are available. Importantly, in humans, purines are also available from the food source, and all diseases affecting purine metabolism, but one, are linked to enzymes in the recycling pathway; with the notable exception being AICA-ribosiduria -deficiency in ATIC, in which ZMP buildup may be a crucial disease factor [START_REF] Marie | AICAribosiduria: a novel, neurologically devastating inborn error of purine biosynthesis caused by mutation of ATIC[END_REF][START_REF] Nyhan | Disorders of purine and pyrimidine metabolism[END_REF][START_REF] Kelley | Disorders of purines and pyrimidines[END_REF]. The emerging picture is that in metazoans the less energetically costly recycling pathway evolved to become the predominant form of purine metabolism, at least in favorable environmental conditions. Hence, specific functions evolved to strongly rely on the recycling pathway, independently of the abundance of external purine sources. The defects associated with ADSL deficiency, as other recycling pathway deficiencies, could be the consequence of insufficient synthesis of the final products AMP and GMP as much as unbalanced wiring of the pathway, namely deregulation caused by intermediate metabolites.

Figures

Figure 1 25 elegans genes adsl-1, atic-1b and ppat-1. Expression of S. cerevisiae genes ADE13, ADE17 and ADE4 was used as positive control, and expression vector without insert as negative control. A culture with adenine is shown as auxotrophy control. In all drop tests presented, four drops are shown per condition, corresponding to serial dilutions (1:10) of cellular suspensions, with deceasing cell concentrations from left to right. (C) Zoom in on HPLC chromatogram peaks of specific metabolites SAICAR (SZMP riboside form), SZMP, sAdo (succinyl-Adenosine, SAMP riboside form), SAMP, ATP and GTP, upon adsl-1(RNAi) and ppat-1(∆); adsl-1(RNAi). (D) Zoom in on HPLC chromatogram peaks of specific metabolites SAICAR (SZMP riboside form), SZMP, AICAR (ZMP riboside form), ZMP, ATP and GTP, in atic-1(∆) and ppat-1(∆); atic-1(∆). Abreviations: Ade -adenine, Adoadenonsine, SAdo -succinyl-adenosine, Ino -Inosine, Hypox -hypoxantine, Xant -Xantine, Gua -guanine, Guo -guanosine. In panels (C) and (D), scales are adjusted differently among metabolites, in order to highlight differences between genotypes. Table 1 -Germline and vulval phenotypes in adsl-1(RNAi) and adss-1(RNAi). All strains carry the germline specific pie-1p::H2B:GFP. Three classes of sterile phenotypes were defined based on the number of GFP positive germline nuclei. Vulval phenotypes were scored on DIC (Nomarski) microscopy.

ppat-1(∆) ppat-1(∆);atic-1(∆) ppat-1(∆);adsl-1(∆) atic-1(∆) adsl-1(∆)

Muscle Myosin

Figure 1 -

 1 Figure 1 -Purine metabolism in C. elegans. (A) Schematics of the Purine Biosynthesis pathways in C. elegans based on sequence homology. Enzymes subjected to functional analysis are represented in red, other enzymes in green and metabolites in black. (B) Drop test to assess adenine auxotrophy in yeast S. cerevisiae mutants deficient for enzymes ADSL (ade13∆), ATIC (ade16∆ ade17∆) or GPPAT (ade4∆) expressing respectively C. elegansgenes adsl-1, atic-1b and ppat-1. Expression of S. cerevisiae genes ADE13, ADE17 and ADE4 was used as positive control, and expression vector without insert as negative control. A culture with adenine is shown as auxotrophy control. In all drop tests presented, four drops are shown per condition, corresponding to serial dilutions (1:10) of cellular suspensions, with deceasing cell concentrations from left to right. (C) Zoom in on HPLC chromatogram peaks of specific metabolites SAICAR (SZMP riboside form), SZMP, sAdo (succinyl-Adenosine, SAMP riboside form), SAMP, ATP and GTP, upon adsl-1(RNAi) and ppat-1(∆); adsl-1(RNAi). (D) Zoom in on HPLC chromatogram peaks of specific metabolites SAICAR (SZMP riboside form), SZMP, AICAR (ZMP riboside form), ZMP, ATP and GTP, in atic-1(∆) and ppat-1(∆); atic-1(∆). Abreviations: Ade -adenine, Adoadenonsine, SAdo -succinyl-adenosine, Ino -Inosine, Hypox -hypoxantine, Xant -Xantine, Gua -guanine, Guo -guanosine. In panels (C) and (D), scales are adjusted differently among metabolites, in order to highlight differences between genotypes.

Figure 2 -

 2 Figure 2

Figure 3 Figure 3 -

 33 Figure 3

Figure 4 Figure 4 -

 44 Figure 4

Figure 5 Figure 5 -

 55 Figure 5

Figure 6 Figure 6 -

 66 Figure 6

Figure S3 Figure S3 -

 S3S3 Figure S3

  Figure S4

FigureFigure S6 -

 S6 Figure S5

Table 1 Genotype (+pie-1p::H2B::GFP)

 1

		Sterile	Sterile	Sterile	WT-like	n=	Protruding	WT-like	n=
		no germline	<10 germ cells	>10 germ cells	germline	(germline)	vulva	vulva	(vulva)
	WT	0%	0%	2,3%	97,7%	43	0%	100%	35
	ppat-1(∆)	0%	0%	0%	100%	57	0%	100%	57
	adsl-1(RNAi)	8,1%	0%	51,4%	40,5%	37	26,1%	73,9%	23
	ppat-1(∆); adsl-1(RNAi)	3,4%	0%	27,6%	69,0%	29	0%	100%	28
	adss-1(RNAi)	1,7%	1,7%	57,9%	38,8%	121	8,0%	92,0%	50
	ppat-1(∆); adss-1(RNAi)	0%	0%	7,7%	92,3%	52	0%	100%	39

C B

PRPP SZMP ZMP IMP SAMP AMP XMP Ade Hypox Gua PRA GAR FGAR FGAM AIR CAIR GMP Ado Ino Xant Guo Uric Acid GPPAT ADSL ATIC ADSL PRPP SZMP IMP SAMP AMP XMP Ade Hypox Gua PRA GAR FGAR FGAM AIR CAIR GMP Ado Ino Xant Guo Uric Acid GPPAT ADSL ATIC ADSL PRPP IMP SAMP AMP XMP Ade Hypox Gua GMP Ado Ino Xant Guo Uric Acid GPPAT ADSL ATIC ADSL adsl-1(∆) atic-1(∆) ppat-1(∆)

  Figure S1 -Purine biosynthesis pathway conservation in C. elegans. (A) Schematics of the Purine Biosynthesis pathways in C. elegans based on sequence homology, and table of comparison with both human and yeast (S. cerevisiae) orthologues. Enzymes subject to functional analysis are in red. (B) Schematics of predicted purine biosynthesis pathways in adsl-1(∆), atic-1(∆) and ppat-1(∆) mutants; large font represents intermediate metabolite accumulation. (C) Representative example of a chromatogram, absorbance at 260 worms, in mock (control) condition. Sequence alignment of C. elegans PPAT-1, ADSL-1 and ATIC-1 with closest orthologue in humans, rat (Rattus norvegicus), fly (Drosophila melanogaster) and yeast cerevisiae). Grey boxes indicate similar residues, black boxes indicate identical residues.

	Figure S2 -	Figure S2

Acknowledgements

Some C. elegans strains were provided by the CGC (https://cgc.umn.edu), which is funded

by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Wormbase, which is supported by National Institutes of Health (NIH) grant U41 HG002223. This work was supported by an AFM-Téléthon Trampoline grant (ref. 18313). RM is supported by AFM-Téléthon PhD scholarship (ref. 20450). RM stay in M. A-S. lab supported by a Short Term Scientific Mission financed by GENiE (COST Action BM1408). M.A-S and M.O are supported by the Ramón y Cajal program of the Spanish Ministerio de Economía y Competitividad, RYC-2010-06167 and RYC-2014-15551, respectively.

Confocal microscopy, and locomotion assay imaging were done in the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging. Deletion alleles adsl-1(tm3328), ppat-1(tm6344) and atic-1(tm6374) were generated by the National Bioresource Project, Japan, directed by Shohei Mitani. We thank Yuji Kohara (N.I.G., Mishima, Japan) for providing cDNAs. We thank the members of the B. D-F. lab for helpful discussions, and particularly M. Moenner for critically reading the manuscript. We thank D. Dupuy and Jonathan Millet (I.E.C.B.,

Supplementary Figures

Figure S1