Guillaume Calvet
email: calvet@irit.fr

Pascal Salembier
email: salembier@irit.fr

Moustapha Julien Kahn
email: kahn@rd.francetelecom.com

Etude empirique de l'interaction multimodale en mobilité : approche méthodologique et premiers résultats

Keywords: Mobilité, Multimodalité, Plateforme d'évaluation, Jeu collaboratif H.5.2 [Information Interfaces and Presentation]:User Interfaces---Evaluation/methodology, Voice I/O, Ergonomics, Interaction Styles Human Factors ; Experimentation Mobility, Multimodality, Evaluation plateform, Collaborative game

Cet article présente les premiers résultats d'une étude de l'usage d'un système multimodal en mobilité dans le contexte d'une situation de jeu collaboratif. Après avoir rappelé les caractéristiques du dispositif de recueil et d'analyse des données, nous donnons quelques éléments de résultats centrés sur la gestion de la multimodalité.

INTRODUCTION

La multiplication des systèmes interactifs mobiles contribue à renouveler le champ des problématiques investiguées dans le domaine de l'interaction Hommemachine. La prise en compte de la mobilité peut être traitée de plusieurs points de vue : gestion coordonnée du mouvement [1], de la navigation dans un espace physique [START_REF] Pirhonen | Gestural and audio metaphors as a means of control for mobile devices[END_REF] et de l'interaction avec le système ; effet des changements de contexte induits par l'extension de l'espace d'activité potentiel [START_REF] Salembier | Just follow me". Examining the use of a multimodal mobile device in natural settings[END_REF] ; conséquences de la mobilité sur les processus sociaux de constitution de réseaux d'acteurs [START_REF] Licoppe | Les usages émergents d'un jeu multijoueurs sur terminaux mobiles géolocalisés[END_REF]. Les artefacts mobiles posent également de nouveaux problèmes relatifs à la conception des dispositifs et des méthodes d'évaluation à mettre en oeuvre pour l'étude empirique de leur usage en situation naturelle. On distingue ainsi par convention deux approches paradigmatiques : les études menées en laboratoire et les études de terrain. Les études en laboratoire se focalisent sur le contrôle de variables susceptibles d'influer sur l'interaction ; elles requièrent de recréer artificiellement une situation de mobilité [1] qui se traduit généralement par un appauvrissement du contexte. Les études naturalistes insistent quant à elles sur le critère de validité écologique et s'efforcent de préserver la richesse contextuelle des activités mobiles. Récemment certains auteurs ont proposé sous les termes de « field experiments » [START_REF] Pirhonen | Gestural and audio metaphors as a means of control for mobile devices[END_REF] et de « quasi-experiments » [START_REF] Roto | Examining mobile phone use in the wild with quasiexperimentation[END_REF] une voie médiane qui tentent de concilier contrôle expérimental des variables et réalisme de la situation. Cette approche demeure essentiellement quantitative et s'inscrit dans la tradition orthodoxe de la psychologie expérimentale appliquée à l'interaction Homme-machine. Le travail présenté ici se rattache à la catégorie des études naturalistes, mais il se donne des contraintes de rigueur quant aux phases de recueil et d'analyse des observables. Il s'efforce en outre d'articuler les données quantitatives (qui constituent des éléments de synthèse commodes et permettent de résumer les tendances générales des résultats) avec des données contribuant à donner du sens aux comportements observés (verbalisations des sujets).

OBJECTIFS

Cette étude s'inscrit dans la continuité d'un premier travail mené sur l'utilisation d'un système portable multimodal en laboratoire [START_REF] Calvet | Etude empirique de l'usage de la multimodalité avec un ordinateur de poche[END_REF]. Notre objectif est cette fois de tester empiriquement les effets potentiels de la mobilité sur l'usage d'un système de ce type en situation réelle, et de documenter empiriquement en quoi la versatilité des systèmes multimodaux, souvent présentée comme un de leurs avantages principaux [START_REF] Oviatt | Multimodal System Processing in Mobile Environments[END_REF], peut aider l'utilisateur à gérer les contraintes induites par les changements de contexte inhérents à la mobilité (modification de l'environnement physique et social notamment). Dans Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. To copy otherwise, or republish, to post on servers or to redistribute to lists, requires prior specific permission and/or a fee. IHM 2005, September 27-30, 2005, Toulouse, France. Copyright 2005 ACM X-XXXXX-XXX-X/XX/XXXX $5.00 cet article nous décrirons le dispositif de recueil et d'analyse des données mise en oeuvre au cours d'une étude menée dans l'espace visiteurs de la Cité des Sciences de La Villette dans le cadre de la plateforme LUTIN [START_REF] Lutin | [END_REF]. Quelques uns des premiers résultats obtenus seront également présentés.

DESCRIPTION DU SYSTEME

Pour cette expérimentation, une application de messagerie multimodale et multimédia a été développée sur des PDAs de type Ipaq© équipés d'une carte réseau sans fil 802.11. Les messages échangés peuvent contenir conjointement un certain nombre de médias tels que du son, des images, du texte dactylographié ou encore du texte manuscrit ou des dessins. Trois modalités d'interaction ont été implémentées : la modalité tactile « stylet » classique qui permet de pointer sur les boutons correspondants à des commandes et sur les zones de texte. La modalité vocale : l'application est capable de reconnaître une trentaine de mots clés. Il est possible de combiner certaines commandes vocales, par exemple « Ouverture du message 5 ». La modalité geste 2D : l'application est également capable de reconnaître une demi-douzaine de gestes en deux dimensions, effectués à l'aide du stylet sur l'écran du PDA. Ces gestes sont prédéfinis et limités essentiellement à quelques commandes d'édition (copier, coller, supprimer, nouveau message,…). En sortie, la messagerie dispose d'une synthèse vocale qui peut être activée et désactivée. Lorsque les commandes sont vocales, il y a systématiquement un feedback vocal de réussite ou d'erreur. La synthèse peut également, à la demande, lire les informations affichées dans l'écran courant, l'en-tête des messages ou leur contenu.

METHODE Tâche et consigne.

La tâche consistait pour des couples de sujets à répondre collaborativement aux questions d'un jeu de type quiz. Les réponses devaient être trouvées dans des espaces distincts couvrant chacun approximativement 500 m2 (les sujets ne se rencontraient donc pas physiquement). Chacun des sujets avait cinq questions sur son PDA, et disposait de cinq indices correspondant aux questions de l'autre sujet. D'un point de vue expérimental l'objectif était ici double : favoriser les interactions entre les sujets via le PDA et provoquer un engagement réel dans l'exploration de l'environnement.

Sujets et protocole expérimental

Après une session de familiarisation avec le système où l'ensemble des fonctions et des modalités leur étaient montrées, 6 sujets ont effectué trois sessions d'interaction de 40 à 50 minutes, toutes espacées de plusieurs jours. Les sujets ont été recrutés et rémunérés pour les besoins de l'expérimentation. Ils étaient tous familiarisés avec l'usage du téléphone portable et des PDAs. Chaque session était suivie d'une autoconfrontation individuelle.

Dispositif de recueil des données

Le dispositif est composé de trois modules de recueil 1 : un système de prise de vue large qui permet de situer l'utilisateur dans son contexte global d'usage (vue contextuelle observateur) ; un système de prise de vue du champ visuel de l'utilisateur (vue « subjective ») réalisé au moyen de lunettes-caméra) (figure 1) ; un système de capture automatique des actions de l'utilisateur sur le système et les sorties de celui-ci (capture log).

Nous avons utilisé pour coder les données audio-vidéo une version optimisée du système d'analyse de données

Actogram TM [START_REF] Kerguelen | Quels outils concevoir pour aider au relevé d'observation sur le terrain ? XXXIIIème Congrès de la SELF[END_REF] qui ouvre plusieurs fenêtres vidéo actives simultanément, et permet de piloter et de visualiser de façon synchrone les deux prises de vue (contextuelle et subjective). Cet outil permet en outre de réaliser directement le codage des observables définis au préalable par l'analyste ; il permet également de construire des chroniques d'activité. Les données qui ont fait l'objet de ce codage systématique comprennent : l'usage des modalités, les conditions de mobilité (statique, micromobilité, déplacement), les directions de regard lors de l'utilisation du système, le type d'activité en cours, les informations contextuelles (selon une catégorisation inspirée de [START_REF] Quéré | La situation toujours négligée?[END_REF] qui distingue entre environnement, contexte et situation).

PREMIERS RESULTATS

Les premiers résultats présentés ici s'appuient uniquement sur des données quantitatives. La grille de lecture utilisée dans ce premier temps de l'analyse s'efforce de donner des éléments de réponse aux questions suivantes : quel est le poids respectif de chaque modalité dans l'utilisation du système par les sujets ? Y'a-t-il des différences interindividuelles marquées ? Constate-t-on une évolution entre la première et la dernière session expérimentale ? Observe-t-on un début de spécialisation (association récurrente d'une modalité et d'une commande) ? La mobilité joue-t-elle un rôle sur l'usage des modalités ?

Usage global des modalités (stylet, vocal, g2d)

La figure 2 indique un usage prédominant de la modalité tactile stylet (75,6%, n=4077) ; la modalité vocale ne représentant que 22,7% des commandes envoyées par les sujets. On retrouve ici des résultats classiques [START_REF] Oviatt | Multimodal System Processing in Mobile Environments[END_REF] qui 1 Pour plus de détails sur le dispositif d'observation et d'analyse voir [START_REF] Zouinar | Observation et capture de données sur l'interaction multimodale en mobilité[END_REF].

viennent contredire des positions « naïves » relatives à la primauté supposée du vocal dans les interfaces multimodales. La part du geste 2D est quant à elle quasi négligeable (1,7%).

Evolution des usages par session

Les résultats obtenus sur les trois sessions permettent de distinguer 2 catégories de sujets ; les sujets pour lesquels les choix de modalité sont quasiment acquis dès la première séance, et les sujets pour lesquels on observe une évolution importante entre la première et la dernière session. Il s'agit essentiellement d'un accroissement significatif de l'usage de la modalité vocale au détriment de la modalité tactile « stylet » . Si l'on considère par exemple le sujet 4, on note une inversion totale de la modalité dominante : l'usage de la modalité tactile « stylet » passant de 83,7% à la première interaction à 36,9% à la der-nière session. Corrélativement, la modalité vocale passe de 13,6 à 63,1%.

Usage spécialisé global de modalités par commandes

Les résultats mettent en évidence qu'un nombre important de commandes sont effectuées uniquement avec la modalité stylet (figure 4). Pour l'ensemble des sujets on n'observe pas de spécialisation intégrale de la modalité vocale pour une commande donnée. Il existe, cependant, une certaine tendance à la préférence vocale pour quelques commandes telles que « envoyer », « insérer », « ouvrir », etc.

Figure 1 :

 1 Figure 1 : Vues « contextuelle » et « subjective » Analyse et codage des données

Figure 2 :Figure 3 :

 23 Figure 2 : Usage global des modalités toutes commandes, toutes sessions et tous sujets confondus Usage des modalités par sujets La figure 3 indique une forte variabilité inter individuelle dans l'usage des modalités. On peut néanmoins distinguer trois groupes de sujets. Certains sujets (1 et 2) utilisent très peu la modalité vocale (en dessous des 4%). A l'inverse les sujets 4 et 6 l'ont beaucoup exploitée (46,3% pour le sujet 6 et 42,5% pour le sujet 4).

  présentés dans cet article devront être affinés et complétés. La catégorisation des informations contextuelles permettra notamment de tester certaines hypothèses sur l'effet possible des facteurs physiques d'ambiance et de la présence de tiers sur la gestion de la multimodalité. Plus globalement l'analyse des verbalisations des sujets permettra la mise en perspective systématique des données quantitatives de synthèse et de leur signification du point des vue des sujets. L'objectif étant ici de réduire la fracture entre approche externaliste de l'évaluation des systèmes (sur des critères tels que temps de réalisation, nombre d'errreurs) et point de vue en première personne (jugement du sujet sur son vécu expérientiel) qui donnent parfois des résultats contradictoires[START_REF] Cahour | Methodologies for evaluating the affective experience of a mediated interaction[END_REF]. Beck, E., Christiansen, M., Kjeldskov J.,Kolbe, N. & Stage, J. Experimental Evaluation of Techniques for Usability Testing of Mobile Systems in a Laboratory Setting. In Proceedings of OzCHI 2003, Brisbane, Australia, 2003.

	m u m
	100%	Nombre de Commande
	90%
	80%
	30% 40% 50% 60% 70%			déplacement micromobilité statique Mobilité
	20%
	10%
	0%
		G2D	Stylet	Vocale
		Modalité
	Figure 5 : Usage des modalités en mobilité	Sujet (Tous) Interaction (Tous)
	Les données disponibles (figure 5) indiquent comme on	900	Nombre de Commande
	pouvait s'y attendre que les modalités qui requièrent l'utilisation du stylet (G2D et Tactile Stylet) et consé-quemment un niveau de contrôle visuel précis de l'action sont effectuées à 80% en statique alors que la modalité vocale est utilisée quasiment à 80% en mobilité (« déplacement » plus « micromobilité »).	100 300 400 500 600 800 700 200	Modalité Vocale Stylet G2D
				0	Activer Aller Aller Aller reçus Annuler Bouton Non Bouton OK Bouton Oui Coller Copier Corps Couper Déselection Envoyer Inserer Inserer son Non Nouveau Objet OK Oui Ouvrir Precedent Repondre selection de Selection Selection Selectionne Suivant Supprimer Transferer
				Commande
				Figure 4: Usage global des modalités par commandes tous su-
				jets et sessions confondus
				Si l'on considère les résultats sujet par sujet on constate
				que le phénomène de spécialisation est davantage mar-
				qué. Ainsi le sujet 4 montre par exemple une préférence
				marquée pour la modalité vocale (commandes <En-
				voyer>,…) bien que certaines commandes ne sont jamais
				effectuées avec cette modalité (<Précédent >, <Répon-
				dre>, <Annuler>, <Sélection>,…). Cette tendance se
				confirme au cours des sessions : le G2D est ainsi totale-
				ment abandonné et certaines commandes réalisées prin-
	REMERCIEMENTS Cette étude a bénéficié d'un financement France Tele-com R&D (convention MOTIVE) et d'un financement	cipalement en vocal dans la première session, le sont ex-clusivement à la dernière session.
	RNRT (plateforme LUTIN).	Effets de la mobilité sur l'utilisation des modalités
				Les résultats sont encore incomplets, le dépouillement
				des données étant en cours (plus du tiers des résultats dé-
				jà codés). Cependant, des tendances intéressantes se dé-
				gagent déjà. Lors du codage de la mobilité, nous avons
				distingué trois catégories : déplacement (déplacement
				rapide d'un point à un autre avec une destination éta-
				blie), micromobilité (piétinement, déambulation sans but
				clair), statique (aucun déplacement).

CONCLUSION

Les premiers résultats

BIBLIOGRAPHIE 1.


Lorane Le Franc

Bruno Petton

Pascal Favrel

Guillaume Rivière
email: guillaume.riviere@unicaen.fr

N 6 -Methyladenosine Profile Dynamics Indicates Regulation of Oyster Development by m 6 A-RNA Epitranscriptomes

Keywords: RNA methylation, Transcription, Embryo, Metamorphosis, Evo-devo

The N 6 -methylation of RNA adenosines (N 6 -methyladenosine, m 6 A) is an important regulator of gene expression with critical implications in vertebrate and insect development. However, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq), we generated transcriptome-wide m 6 A-RNA methylomes covering the whole development of the oyster from oocytes to juveniles. Oyster RNA classes display specific m 6 A signatures, with messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) exhibiting distinct profiles and being highly methylated compared to transposon transcripts. Epitranscriptomes are dynamic and correspond to the chronological steps of development (cleavage, gastrulation, organogenesis, and metamorphosis), with a minimal mRNA and lncRNA methylation at the morula stage followed by a global increase. mRNA m 6 A levels are correlated to transcript levels and shifts in methylation profiles correspond to expression kinetics.

Differentially methylated transcripts cluster according to embryo-larval stages and bear the corresponding developmental functions (cell division, signal transduction, morphogenesis, and cell differentiation). The m 6 A level of transposon transcripts is also regulated and peaks during the gastrulation. We demonstrate that m 6 A-RNA methylomes are dynamic and associated to gene expression regulation during oyster development. The putative epitranscriptome implication in the cleavage, maternal-tozygotic transition, and cell differentiation in a lophotrochozoan model brings new insights into the control and evolution of developmental processes.

Introduction

The success of development of metazoan organisms is conditioned by the precise temporo-spatial regulation of gene expression. The N 6 -methylation of RNA adenosines (N 6 -methyladenosine, m 6 A) has recently emerged as a critical layer of the gene expression regulatory network. Indeed, m 6 A-RNA methylation is required in vertebrates for the proper regulation of developmental processes such as cell differentiation [START_REF] Zhang | Roles of N 6 -methyladenosine (m 6 A) in stem cell fate decisions and early embryonic development in mammals[END_REF][START_REF] Chang | Region-specific RNA m 6 A methylation represents a new layer of control in the gene regulatory network in the mouse brain[END_REF], X chromosome inactivation [START_REF] Coker | The role of the Xist 5' m 6 A region and RBM15 in X chromosome inactivation[END_REF][START_REF] Patil | m 6 A RNA methylation promotes XIST-mediated transcriptional repression[END_REF], maternal-to-zygotic transition (MZT) [START_REF] Zhao | m 6 A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition[END_REF][START_REF] Sui | METTL3-mediated m 6 A is required for murine oocyte maturation and maternal-to-zygotic transition[END_REF] and neurogenesis [START_REF] Yoon | Temporal control of mammalian cortical neurogenesis by m 6 A methylation[END_REF][START_REF] Wu | A novel m 6 A reader Prrc2a controls oligodendroglial specification and myelination[END_REF][START_REF] Edens | FMRP modulates neural differentiation through m 6 A-dependent mRNA nuclear export[END_REF]. Besides, the invalidation of enzymes which deposit m 6 A on RNA is lethal during the early development [START_REF] Geula | m 6 A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation[END_REF][START_REF] Wang | N 6 -methyladenosine modification destabilizes developmental regulators in embryonic stem cells[END_REF].

The m 6 A-RNA is regulated by an enzymatic machinery comprising writers and erasers. The METTL3/METTL14/WTAP writer core complex [START_REF] Yang | Dynamic transcriptomic m 6 A decoration: writers, erasers, readers and functions in RNA metabolism[END_REF] deposits methyl marks at the consensus sequence DRACH (D = A/G/T, R = A/G, H = A/C/T) [START_REF] Ke | A majority of m 6 A residues are in the last exons, allowing the potential for 3' UTR regulation[END_REF][START_REF] Meyer | Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons[END_REF][START_REF] Batista | m 6 A RNA modification controls cell fate transition in mammalian embryonic stem cells[END_REF][START_REF] Xiao | The RNA N 6methyladenosine modification landscape of human fetal tissues[END_REF].

Erasers like ALKBH5 [START_REF] Zheng | ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility[END_REF] and the controversial FTO [START_REF] Jia | N 6 -methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO[END_REF] remove methylation. The combined action of m 6 A writers and erasers makes m 6 A profiles highly dynamic during the development of investigated species [START_REF] Sui | METTL3-mediated m 6 A is required for murine oocyte maturation and maternal-to-zygotic transition[END_REF][START_REF] Wang | RNA m 6 A modification functions in larval development and caste differentiation in honeybee (Apis mellifera)[END_REF][START_REF] Zhang | Dynamic epigenomic landscapes during early lineage specification in mouse embryos[END_REF].The biological effects of RNA methylation are mediated by reader proteins able to bind m 6 A. For example, the YTH protein family of readers is involved in RNA stability, translation level [START_REF] Wang | N 6 -methyladenosine modulates messenger RNA translation efficiency[END_REF][START_REF] Wang | N 6 -methyladenosinedependent regulation of messenger RNA stability[END_REF][START_REF] Hsu | Ythdc2 is an N 6methyladenosine binding protein that regulates mammalian spermatogenesis[END_REF][START_REF] Shi | YTHDF3 facilitates translation and decay of N 6 -methyladenosine-modified RNA[END_REF], splicing [START_REF] Xiao | Nuclear m 6 A reader YTHDC1 regulates mRNA splicing[END_REF], and nuclear export [START_REF] Roundtree | YTHDC1 mediates nuclear export of N 6 -methyladenosine methylated mRNAs[END_REF]. Other readers like hnRNPA2B1 [START_REF] Alarcón | HNRNPA2B1 is a mediator of m 6 A-dependent nuclear RNA processing events[END_REF], IGF2BP [START_REF] Huang | Recognition of RNA N 6methyladenosine by IGF2BP proteins enhances mRNA stability and translation[END_REF], and Prrc2a [START_REF] Wu | A novel m 6 A reader Prrc2a controls oligodendroglial specification and myelination[END_REF] mediate m 6 A influence on RNA stability, and eIF3a guide capindependent translation of m 6 A-RNAs [START_REF] Meyer | 5' UTR m 6 A promotes cap-independent translation[END_REF]. This whole machinery allows to guide the methylation on specific RNA targets and modulates their cellular fate. As a result, m 6 A is a chemical modification that influences gene expression without modifying the associated transcript nucleic acid sequence and is therefore referred to as "epitranscriptomic" [START_REF] Saletore | The birth of the epitranscriptome: deciphering the function of RNA modifications[END_REF].

Many RNA classes are subjected to m 6 A methylation, which is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs). m 6 A in mRNAs are mostly found in the 3' untranslated transcribed region (UTR) at the vicinity of the stop codon [START_REF] Ke | A majority of m 6 A residues are in the last exons, allowing the potential for 3' UTR regulation[END_REF][START_REF] Meyer | Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons[END_REF][START_REF] Dominissini | Topology of the human and mouse m 6 A RNA methylomes revealed by m 6 A-seq[END_REF]. However, m 6 A is also present at lower levels in 5' UTRs [START_REF] Meyer | 5' UTR m 6 A promotes cap-independent translation[END_REF][START_REF] Coots | m 6 A facilitates eIF4Findependent mRNA translation[END_REF], long internal exons [START_REF] Batista | m 6 A RNA modification controls cell fate transition in mammalian embryonic stem cells[END_REF][START_REF] Dominissini | Topology of the human and mouse m 6 A RNA methylomes revealed by m 6 A-seq[END_REF] and introns [START_REF] Xiao | The RNA N 6methyladenosine modification landscape of human fetal tissues[END_REF][START_REF] Louloupi | Transient N 6 -methyladensosine transcriptome sequencing reveals a regulatory role of m 6 A in splicing efficiency[END_REF]. While m 6 A modification is well described in mRNA targets, it also affects most non-coding RNA classes (ncRNAs) including microRNAs (miRNAs) [START_REF] Alarcón | HNRNPA2B1 is a mediator of m 6 A-dependent nuclear RNA processing events[END_REF], circular RNAs (circRNAs) [START_REF] Yang | Extensive translation of circular RNAs driven by N 6 -methyladenosine[END_REF]. small nuclear RNAs (snRNAs) [START_REF] Warda | Human METTL16 is a N 6 -methyladenosine (m 6 A) methyltransferase that targets pre-mRNAs and various non-coding RNAs[END_REF] and small nucleolar RNAs (snoRNAs) [START_REF] Huang | Control of box C/D snoRNP assembly by N 6 -methylation of adenine[END_REF]. In vertebrates, more than 300 long non-coding RNAs (lncRNAs) are m 6 A-methylated [START_REF] Meyer | Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons[END_REF]. Such modification alters the RNA structure towards an increased protein accessibility [START_REF] Liu | N 6 -methyladenosine alters RNA structure to regulate binding of a low-complexity protein[END_REF], as well as subcellular localization [START_REF] Wu | m 6 A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1[END_REF]. The m 6 A of lncRNAs has critical developmental outcomes, as indicated by the requirement of m 6 A of the Xist lncRNA for the transcriptional silencing of the inactivated X chromosome [START_REF] Patil | m 6 A RNA methylation promotes XIST-mediated transcriptional repression[END_REF]. Besides, the m 6 A in RNA encoded by transposable element genes (repeat RNAs) increases their stability and promotes chromatin compaction [START_REF] Liu | N 6 -methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription[END_REF].

Aside from vertebrates, the m 6 A was described in a wide diversity of organisms, such as insects [START_REF] Lence | m 6 A modulates neuronal functions and sex determination in Drosophila[END_REF][START_REF] Jiang | The role of N 6 -methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism[END_REF][START_REF] Kan | The m 6 A pathway facilitates sex determination in Drosophila[END_REF], yeasts [START_REF] Bodi | Yeast targets for mRNA methylation[END_REF], and plants [START_REF] Luo | Unique features of the m 6 A methylome in Arabidopsis thaliana[END_REF]. However, despite the developmental significance of m 6 A being conserved across the evolution, several differences exist between animal groups. Indeed, in fruit flies, m 6 A is mostly present in the 5' UTR of mRNAs throughout head and embryo development [START_REF] Kan | The m 6 A pathway facilitates sex determination in Drosophila[END_REF][START_REF] Worpenberg | Ythdf is a N 6 -methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila[END_REF], whereas methylation enrichment at 5' UTRs promotes cap-independent translation for transcript selection during the stress response in vertebrates [START_REF] Meyer | 5' UTR m 6 A promotes cap-independent translation[END_REF][START_REF] Coots | m 6 A facilitates eIF4Findependent mRNA translation[END_REF][START_REF] Zhou | Dynamic m 6 A mRNA methylation directs translational control of heat shock response[END_REF]. In the silkworm Bombyx mori, mRNA is mostly methylated in coding sequences (CDS) but not UTRs, and a high methylation is associated with a high gene expression [START_REF] Li | Transcriptome-wide analysis of N 6 -methyladenosine uncovers its regulatory role for gene expression in lepidopteran Bombyx mori[END_REF]. Besides, a higher m 6 A content and gene expression regulation is found upstream the diapause [START_REF] Jiang | The role of N 6 -methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism[END_REF], a development phase which is reflective of a highly plastic phenotype. Finally, in Caenorhabditis elegans, where only few actors of the m 6 A machinery are present, methylation is mostly found in ribosomal RNAs (rRNAs) [START_REF] Sendinc | Analysis of m 6 A RNA methylation in Caenorhabditis elegans[END_REF]. This situation opens crucial questions about the evolution of m 6 A-related molecular pathways, target genes, and developmental significance that require investigations in divergent models.

However, despite being of upmost importance for our understanding of the evolution of the molecular control of developmental processes, there is a critical lack of knowledge in lophotrochozoan organisms where epitranscriptomes were not investigated to date to our knowledge.

The Pacific oyster Crassostrea gigas (i.e., Magallana gigas) is a bivalve mollusk whose great ecological and economical significance allowed it to emerge as a model species within lophotrochozoan organisms. As such, an important amount of genetic, transcriptomic, and epigenetic data has been generated in this model [START_REF] Zhang | The oyster genome reveals stress adaptation and complexity of shell formation[END_REF][START_REF] Riviere | DNA methylation is crucial for the early development in the oyster C. gigas[END_REF][START_REF] Dheilly | Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns[END_REF]. Besides, the embryo-larval development of C. gigas is under the strong epigenetic influence of DNA methylation [START_REF] Riviere | Dynamics of DNA methylomes underlie oyster development[END_REF][START_REF] Sussarellu | Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos[END_REF][START_REF] Saint-Carlier | Regulation of Hox orthologues in the oyster Crassostrea gigas evidences a functional role for promoter DNA methylation in an invertebrate[END_REF] and histone marks [START_REF] Fellous | The Jumonji gene family in Crassostrea gigas suggests evolutionary conservation of Jmj-C histone demethylases orthologues in the oyster gametogenesis and development[END_REF][START_REF] Fellous | Histone methylation participates in gene expression control during the early development of the Pacific oyster Crassostrea gigas[END_REF]. Moreover, it has recently been demonstrated that m 6 A and its complete associated machinery are conserved in oysters, with features strongly indicating an epitranscriptomic regulation of development [START_REF] Franc | A functional m 6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development[END_REF]. Furthermore, oysters develop as pelagic larvae in sea water before they metamorphose into fixed adult specimen. Therefore, the early life stages of oysters are directly subjected to the variations of external environmental conditions such as temperature changes, ultraviolet (UV) exposure, or endocrine disruptor contamination, which are described to influence m 6 A-RNA in investigated species [START_REF] Meyer | 5' UTR m 6 A promotes cap-independent translation[END_REF][START_REF] Zhou | Dynamic m 6 A mRNA methylation directs translational control of heat shock response[END_REF][START_REF] Lu | The role of N 6 -methyladenosine RNA methylation in the heat stress response of sheep (Ovis aries)[END_REF][START_REF] Xiang | m 6 A RNA methylation regulates the UV-induced DNA damage response[END_REF][START_REF] Cayir | Exposure to environmental toxicants reduces global N 6 -methyladenosine RNA methylation and alters expression of RNA methylation modulator genes[END_REF]. These elements are highly suggestive of a functional significance of m 6 A-RNA in oyster development, which remains unknown to date.

To investigate these questions, we characterized the transcriptome-wide dynamics of m 6 A-RNA methylomes across the entire development of the pacific oyster from the oocytes to the completion of the organogenesis. We used methylated RNA immunoprecipitation sequencing (MeRIP-seq) to investigate and monitor the dynamics of m 6 A levels, localization, target RNA subsets, and functional implications in a lophotrochozoan model to provide a better understanding of the evolution of developmental mechanisms and their epigenetic regulation.

Results

Oyster RNA classes display specific m 6 A signatures

In developing oysters, m 6 A methylation affects a vast majority of RNA classes, and is detected in 91.0% of mRNAs, 65.7% of lncRNAs, 87.0% of RNA transposable elements (TEs) (retrotransposons), and 99.4% of DNA TEs. In contrast, oyster transfer RNA (tRNA) are mostly unmethylated (rRNA were depleted during library preparation and not investigated here). In addition, 35.8% of mRNAs and 11.1% of lncRNAs are significantly m 6 A-enriched over background levels (Figure 1A). The methylation pattern depends on the RNA class considered (Kolmogorov-Smirnov [KS] test P < 7.55 × 10 -11 ). While m 6 A in mRNA is found from the 5' UTR to the 3' UTR, and is more abundant around the stop codon, it is more randomly spread along lncRNAs (Figure 1B). Methylation affects adenosines located within the "AGUGA*C" sequence (where * marks the modified adenosine) (Figure 1C). The mean methylation level differs between RNA classes and DNA TE transcripts are less methylated than mRNAs, lncRNAs, and RNA TE (Figure 1D). The MeRIP-seq procedure was validated by investigation of the status of five transcripts exhibiting contrasted m 6 A vs. mRNA profiles in other species (c-myc, klf, mettl3, hnrnpa2b1, and oct4) by targeted methyl-RNA immuno-precipitation followed by quantitative polymerase chain reaction (MeRIP-qPCR) [START_REF] Batista | m 6 A RNA modification controls cell fate transition in mammalian embryonic stem cells[END_REF][START_REF] Huang | Recognition of RNA N 6methyladenosine by IGF2BP proteins enhances mRNA stability and translation[END_REF][START_REF] Coots | m 6 A facilitates eIF4Findependent mRNA translation[END_REF][START_REF] Kwon | Functional roles of hnRNPA2/B1 regulated by METTL3 in mammalian embryonic development[END_REF]. All the examined candidates displayed similar patterns between the two techniques and those patterns are consistent with the literature (Figure S1).

Oyster m 6 A epitranscriptomes are dynamic and correspond to defined steps of the development chronology

The principal component analysis (PCA) of MeRIP-seq data indicates that the samples group together according to the embryo-larval stages and are overall organized chronologically along the X-axis (PC1) (Figure 2A). The PCA plot can be divided into four areas defining four developmental phases: cleavage (from oocytes to 2-to 8-cell stage), gastrulation (blastula and gastrula stages), tissue differentiation (trochophore and D-larvae stages), and metamorphosis (pediveliger and spat stages). The morula stage is clearly individualized along the PC2, suggesting peculiar m 6 A methylation and development features. This segregation is confirmed by the pairwise correlation matrix (Figure 2B). The chronological segregation of developmental stages according to m 6 A methylation indicates that m 6 A-RNA epitranscriptomes are an important component of the developmental process. The m 6 A epitranscriptomes are dynamic during oyster development. There is an important decrease in the number of methylated transcripts at the morula stage followed by a general shift towards increased m 6 A mRNAs and lncRNAs onwards, which is particularly noticeable at the late development stages (i.e., pediveliger and spat stages) (Figure 2C). The same tendency is also observed, although to a lesser extent, for RNA TE, whose unmethylated population becomes reduced in the late developmental phases (Figure 2D).

mRNA m 6 A profiles are regulated and correlated to gene expression level and kinetics throughout oyster development

The mRNA m 6 A methylation is highly dynamic during oyster development. Indeed, among the significantly methylated mRNAs (analysis of variance (ANOVA) P < 0.05, n = 8404), 17.8% of m 6 A-mRNA (n = 1494) display a significant variation in their m 6 A level (Figure 3A), and this m 6 A level is significantly correlated to their expression level (Figure 3B). The differentially methylated (DM) m 6 A-mRNAs define 4 clusters according to their methylation (and expression) profiles, corresponding to the developmental steps defined previously. Cluster1 includes mRNAs with a strong methylation and transcript content in the early stages (up to the morula stage) which decreases afterwards. By contrast, the mRNAs within the three other clusters display poor methylation during the early stages, which strongly increases from the gastrulation (cluster 2), the trochophore stage (cluster 3), and the pediveliger stage (cluster4), and remain strongly methylated afterwards. Overall, there is a marked decrease of m 6 A methylation at the morula stage.

Besides the level of methylation, the localization of m 6 A within mRNAs can also vary between clusters (KS test, 0.31 < P < 5.11 × 10 -12 depending on cluster pairwise comparison). Indeed, cluster 1 mRNAs, which are essentially maternal mRNAs, are methylated mostly around their stop codon in oocytes and thereafter. By contrast, mRNAs within the three other clusters, that are essentially expressed from the zygotic genome, display a marked biphasic profile, with an increased 5' m 6 A content at the vicinity of the CDS start (Figure 3C). However, this pattern shifts towards a less biphasic profile with m 6 A becoming relatively more dominant around the stop codon in the later development stages when the methylation and expression levels increase (KS test, 5.87 × 10 -7 < P < 5.35 × 10 -10 depending on the cluster). Furthermore, these m 6 A-mRNAs have their methylation and expression dynamics strongly correlated (clusters 2, 3, and 4), whereas early DM transcripts without 5' CDS methylation do not (cluster 1) (Figure 3D). This indicates that mRNA expression dynamics are correlated with m 6 A profile dynamics after, but not before, the zygotic genome activation.

Together these results bring to light a regulation of gene expression by both the level and localization of m 6 A methylation across oyster developmental stages.

Differentially m 6 A methylated mRNAs bear the corresponding developmental functions

Gene Ontology (GO) analyses of DM m 6 A-mRNAs shows an overall term enrichment related to developmental processes, like morphogenesis and mesoderm development (Figure 4; Table S1). More specific functions correspond to each methylation cluster.

Cluster 1 (cleavage) is enriched in terms related to the cell division whereas later clusters (gastrulation, tissue differentiation, and metamorphosis) bear terms that may be more a reflection of signal transduction associated to cell differentiation such as the TGFβ/SMAD pathway. These results point to a functional implication of the m 6 A in the regulation of oyster developmental processes.

ncRNA transcript content is also associated to m 6 A profiles

Similar to mRNAs, lncRNA can be grouped into clusters according to the developmental chronology of their m 6 A dynamics, with the highest methylation levels during the early stages for a cleavage cluster (cluster 1), during the gastrulation and tissue type differentiation (cluster 2), and at the pediveliger and spat stages for a metamorphosis cluster (cluster 3), respectively. A marked decrease of m 6 A levels at the morula stage is also observed for lncRNAs (Figure 5A). The methylation and expression levels are positively correlated overall (Figure 5B), although the methylation and expression dynamics are correlated only for the "late" clusters 2 and 3, but not for the "early" cluster 1 (Figure 5C).

RNA TE m 6 A methylation is regulated across oyster development

The methylation of RNATE is mostly represented by the methylation of long terminal repeat (LTR) and long interspersed nuclear elements (LINE) groups (62.92% and 31.46%, respectively). With 85.5% and 100% of transcripts being methylated, respectively. Their methylation levels display a peak at the blastula and trochophore stages for LTR and LINE, respectively (Figure 6A). Both DNA and retrotransposon methylation levels were dynamic during oyster development (ANOVA P < 0.05).

Terminal inverted repeat (TIR) (37.13%), Helitron (32.50%), and Crypton (20.25%) are the three main DNA TE types whose transcripts are the most methylated and represented 92.6%, 88.5%, and 100% of transcripts in each type, respectively. Their m 6 A levels are the highest around the gastrulation, and Helitron TEs exhibit an additional peak in pediveliger larvae (Figure 6B). These results show that the m 6 A methylation of transposon transcripts displays class-specific dynamics during oyster development that are different from mRNAs and lncRNAs.

Discussion

This work presents the profiling of m 6 A epitranscriptomes during the development of the oyster C. gigas from the egg to the completion of organogenesis. We demonstrate that the methylation of RNA is dynamic throughout the oyster's early life and specific to the RNA population considered. The methylation kinetics of mRNAs defines clusters corresponding to the chronology of development, which is correlated to gene expression level and dynamics, and the genes whose transcripts display m 6 A regulation bear developmental functions. lncRNAs display similar profiles while TE transcripts have a specific signature with a peak in m 6 A levels during gastrulation. Our study provides evidence for a developmental significance of m 6 A epitranscriptomes at various levels in oysters.

The distinct oyster RNA classes present specific m 6 A signatures and vary in terms of methylation level and location. Indeed, while mRNAs are m 6 A-modified from the CDS start but mostly around the stop codon, lncRNAs do not present a biased methylation enrichment at their 3' end. This result, suspected from our previous measurements of global m 6 A content in polyA vs. total RNAs [START_REF] Franc | A functional m 6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development[END_REF], is more consistent with what is observed in human embryonic cell types [START_REF] Xiao | The RNA N 6methyladenosine modification landscape of human fetal tissues[END_REF] than in insects where methylated adenosines are mostly lying in the 5' UTR and CDS [START_REF] Wang | RNA m 6 A modification functions in larval development and caste differentiation in honeybee (Apis mellifera)[END_REF][START_REF] Kan | The m 6 A pathway facilitates sex determination in Drosophila[END_REF][START_REF] Li | Transcriptome-wide analysis of N 6 -methyladenosine uncovers its regulatory role for gene expression in lepidopteran Bombyx mori[END_REF]. This finding is rather surprising because insects are ecdysozoans which are considered the "sister clade" of lophotrochozoans within protostomes. Nevertheless, this assumption is increasingly moderated by the closer resemblance of molecular features in mollusks and annelids to vertebrates, i.e., deuterostomes, rather than to ecdysozoan features [START_REF] Saint-Carlier | Regulation of Hox orthologues in the oyster Crassostrea gigas evidences a functional role for promoter DNA methylation in an invertebrate[END_REF][START_REF] Cho | Evolutionary dynamics of the wnt gene family: a lophotrochozoan perspective[END_REF][START_REF] Nederbragt | Hedgehog crosses the snail's midline[END_REF]. Overall, m 6 A are found in oysters within the motif "DRA*C" which contains the GAC consensus sequence which is widely conserved across the evolution [START_REF] Meyer | Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons[END_REF][START_REF] Wang | RNA m 6 A modification functions in larval development and caste differentiation in honeybee (Apis mellifera)[END_REF][START_REF] Lence | m 6 A modulates neuronal functions and sex determination in Drosophila[END_REF][START_REF] Bodi | Yeast targets for mRNA methylation[END_REF][START_REF] Luo | Unique features of the m 6 A methylome in Arabidopsis thaliana[END_REF]. The repetitive nature of TE sequences only allows a less precise analysis of their m 6 A features compared to other RNA classes. However, we show that oysters' DNA TE transcripts are significantly less methylated than RNA TEs, which is in line with observations made in Arabidopsis [START_REF] Wan | Transcriptome-wide high-throughput deep m 6 A-seq reveals unique differential m 6 A methylation patterns between three organs in Arabidopsis thaliana[END_REF].

Oyster epitranscriptomes are dynamic and associated to defined successive developmental steps, namely cleavage, gastrulation, tissue differentiation, and metamorphosis. The similarity of m 6 A profiles in eggs before and after fertilization suggests a poor contribution of sperm to embryonic m 6 A methylomes, which is consistent with the sperm scarce RNA content regarding the oocyte stock.

Epitranscriptomes clearly shift during the developmental chronology, with a marked decrease of m 6 A levels in mRNA and lncRNA transcripts at the end of cleavage followed by a gradual increase, and a maximum methylation reached at the premetamorphosis stage that remains high afterwards in juveniles. The morula stage constitutes a pivot point where the methylation of mRNA and lncRNA, but not of RNA TEs, is strongly depleted. Interestingly, based on transcriptomic [START_REF] Riviere | GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the Pacific oyster Crassostrea gigas[END_REF] and 5mC-DNA methylation data [START_REF] Riviere | Dynamics of DNA methylomes underlie oyster development[END_REF], this stage could be considered an important window of MZT in the oyster, when most maternal RNA resources are consumed and early transcriptional events occur [START_REF] Mclean | RNA synthesis during the early development of the pacific oyster, Crassostrea gigas[END_REF]. Interestingly, the marked epitranscriptomic features at cleavage and metamorphosis may remind of the important shifts in 5mC-DNA profiles (decrease during cleavage and important levels prior to metamorphosis) [START_REF] Riviere | Dynamics of DNA methylomes underlie oyster development[END_REF] and could suggest an interplay between the epigenetic layers in the regulation of oyster development, possibly through competition for methyl donor [START_REF] Shima | Sadenosylmethionine synthesis is regulated by selective N 6 -adenosine methylation and mRNA degradation involving METTL16 and YTHDC1[END_REF] or histone-related pathways [START_REF] Huang | Histone H3 trimethylation at lysine 36 guides m 6 A RNA modification co-transcriptionally[END_REF][START_REF] Wang | N 6methyladenosine RNA modification regulates embryonic neural stem cell selfrenewal through histone modifications[END_REF]. This observation is reminiscent of the crosstalk between the 5mC-DNA status of the Pou5f1 pioneer stem cell factor promoter and the m 6 A methylation of its transcript in humans [START_REF] Xiao | The RNA N 6methyladenosine modification landscape of human fetal tissues[END_REF], hence raising the question of m 6 A-modified transcripts in the oyster development.

The m 6 A content and transcript abundance of DM mRNAs are positively correlated across oyster development, which surprisingly contrasts with the case of the honeybee where highly methylated transcripts exhibit downregulated expression [START_REF] Wang | RNA m 6 A modification functions in larval development and caste differentiation in honeybee (Apis mellifera)[END_REF], thus further indicating an evolutionary divergence between mollusks and insects regarding epitranscriptomic mechanisms. Overall, oyster DM mRNAs have functions related to developmental processes, which further confirms the developmental significance of the epitranscriptomic regulation in the oyster. More precisely, DM mRNA transcripts group into 4 clusters that match the development steps described above, whose GO annotations assess the functional relevance in the cognate developmental processes (cluster 1, cleavage; cluster 2, gastrulation; cluster 3, tissue differentiation; cluster 4, metamorphosis). However, these clusters do not always display similar methylation profiles neither correlations between m 6 A and expression level dynamics.

Cluster 1 mRNA transcripts are highly methylated and abundant during the cleavage. They are present in oocytes and therefore correspond to maternal transcripts.

While their expression and methylation levels are dramatically reduced at the onset of MZT (morula stage), their m 6 A and transcript content dynamics are not significantly correlated, and they display predominant methylation around the stop codon throughout oyster development. Such m 6 A profiles promote mRNA degradation via interaction with readers such as YTHDF proteins during cleavage in other species [START_REF] Zhao | m 6 A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition[END_REF][START_REF] Lee | Molecular mechanisms driving mRNA degradation by m 6 A modification[END_REF], and we showed that an oyster YTHDF orthologue is present at high levels in this window [START_REF] Franc | A functional m 6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development[END_REF].

Besides, cluster 1 DM mRNAs bear functions in the cytoskeleton processes of cytokinesis. Together, these results indicate that the oyster MZT may be triggered by YTHDF-mediated decay of maternal mRNAs, whose translation is important for cell proliferation during cleavage. We hypothesize that the depletion of the cytokinesis machinery mRNA templates of maternal origin below a certain threshold, and/or the putative associated slowdown of cell divisions, may participate in triggering the zygotic genome activation. It remains to be deciphered whether and how such signals could be interpreted in terms of cell fate decisions. Clusters 2, 3, and 4 respectively correspond to the gastrulation, the setting up of differentiated tissues, and the metamorphosis. In contrast to cluster 1, their m 6 A pattern is dynamic and shifts from bimodal profiles in early stages to a late mostly unimodal m 6 A distribution around the stop codon. Such 5'-enriched m 6 A profiles are observed in plants [START_REF] Luo | Unique features of the m 6 A methylome in Arabidopsis thaliana[END_REF], Xenopus [START_REF] Sai | Distinct m 6 A methylome profiles in poly(A) RNA from Xenopus laevis testis and that treated with atrazine[END_REF], mice [START_REF] Chang | Region-specific RNA m 6 A methylation represents a new layer of control in the gene regulatory network in the mouse brain[END_REF], and fruit flies [START_REF] Worpenberg | Ythdf is a N 6 -methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila[END_REF], and may be associated to increased RNA turnover and fast transcript processing depending on the positioning of methylation in the CDS or 5' UTR. Consistently, in developing oysters, the early bimodal m 6 A distribution is associated with a low transcript abundance. The 3' patterns are associated to the recruitment of readers such as YTHDC1 [START_REF] Xiao | Nuclear m 6 A reader YTHDC1 regulates mRNA splicing[END_REF][START_REF] Kasowitz | Nuclear m 6 A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development[END_REF][START_REF] Xu | Structural basis for selective binding of m 6 A RNA by the YTHDC1 YTH domain[END_REF], IGF2BP [START_REF] Huang | Recognition of RNA N 6methyladenosine by IGF2BP proteins enhances mRNA stability and translation[END_REF],

or Prrc2a [START_REF] Wu | A novel m 6 A reader Prrc2a controls oligodendroglial specification and myelination[END_REF], and mediate translation initiation during gastrulation, as well as splicing and RNA trafficking during cell type differentiation. In oysters, the later sequential shift of clusters towards more 3'-dominant methylation is correlated to an increased expression level of the DM mRNA transcripts. Furthermore, those transcripts that become successively methylated and expressed bear the cognate developmental functions such as mesoderm specification, morphogenesis, and signal transduction related to cell differentiation. In addition, readers including YTHDCs, IGF2BP, and Prrc2a are functional in oysters and present in stage-specific sets after the MZT [START_REF] Franc | A functional m 6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development[END_REF], and METTL3 expression is also dramatically increased after the MZT and sustained later on. Together, this strongly suggests that m 6 A regulates gene expression towards cell differentiation upon the zygotic genome activation, possibly by promoting translation via transcript selection and increased mRNA stability. As suggested by GO annotation, m 6 A likely participates in mesoderm formation during gastrulation via a METTL3-dependent regulation of epithelial to mesenchymal transition, as described in human cancer cells [START_REF] Lin | RNA m 6 A methylation regulates the epithelial mesenchymal transition of cancer cells and translation of Snail[END_REF][START_REF] Chen | YTH domain family 2 orchestrates epithelial-mesenchymal transition/proliferation dichotomy in pancreatic cancer cells[END_REF]. Our findings (Table S1) are also consistent with the METTL3/YTHDF2-dependent osteoblast differentiation by SMAD inhibitor downregulation in mammalian cells [START_REF] Li | METTL3 modulates osteoclast differentiation and function by controlling RNA stability and nuclear export[END_REF][START_REF] Zhang | METTL3 regulates osteoblast differentiation and inflammatory response via smad signaling and mapk signaling[END_REF]. Regarding the conservation of the TGFβ pathway in oyster development [START_REF] Herpin | Structural and functional evidence for a singular repertoire of BMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests a shared ancestral BMP/activin pathway[END_REF][START_REF] Quéré | Structural and functional characterizations of an Activin type II receptor orthologue from the pacific oyster Crassostrea gigas[END_REF], these results may also suggest feedback loops between TGFβ signaling and m 6 A regulation in the control of (pluri)potency during differentiation [START_REF] Bertero | The SMAD2/3 interactome reveals that TGFβ controls m 6 A mRNA methylation in pluripotency[END_REF]. lncRNAs methylation levels display similar developmental dynamics to mRNAs, although m 6 A are not preferentially located at their 3'-end. Like mRNAs, the methylation levels in lncRNAs are also correlated to their transcript content and dynamics after the MZT. How lncRNA epitranscriptomes might participate in oyster development is less clear because oyster lncRNAs are poorly conserved and annotated.

Besides, their limited number compared to mRNAs in addition to the limited resolution of MeRIP-seq did not allow the detection of m 6 A peak localization shifts. Nevertheless, the vertebrate writer complex cofactor RBM15/15B and the hnRNPA2B1 nuclear complex are conserved in oysters [START_REF] Franc | A functional m 6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development[END_REF]. Therefore, we speculate that oyster lncRNA epitranscriptomes could regulate the chromatin state and transcription in an m 6 Adependent manner, which is somewhat reminiscent of the Xist-mediated silencing [START_REF] Coker | The role of the Xist 5' m 6 A region and RBM15 in X chromosome inactivation[END_REF][START_REF] Patil | m 6 A RNA methylation promotes XIST-mediated transcriptional repression[END_REF], MALAT1-mediated stimulation [START_REF] Brown | Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA[END_REF], and/or chromosome-associated regulatory RNA (carRNA) switches in mammals [START_REF] Liu | N 6 -methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription[END_REF]. However, Xist is not conserved in oysters and Although additional studies would be required to determine the interplay of m 6 A-RNA with the epigenetic network and the underlying mechanisms, this first evidence of an epitranscriptomic regulation of development in a lophotrochozoan species allows for a better understanding of developmental processes and their evolution.

Materials and methods

Animals

Broodstock oysters, embryos, larvae, and spat were obtained at the Ifremer marine facility (Argenton, France) as previously described [START_REF] Riviere | Dynamics of DNA methylomes underlie oyster development[END_REF][START_REF] Petton | Factors influencing diseaseinduced mortality of Pacific oysters Crassostrea gigas[END_REF]. Briefly, gametes of mature broodstock oysters were obtained by stripping the gonads and filtering the recovered material on a 60 µm mesh to remove large debris. Oocytes were collected as the remaining fraction on a 20 µm mesh and spermatozoa as the passing fraction on a 20 µm mesh. Oocytes were pre-incubated in 5 l of UV-treated and 1 µm mesh filtered sterile sea water (SSW) at 21 °C until germinal vesicle breakdown. Fertilization was triggered by the addition of ca.10 spermatozoids per oocyte. After the expulsion of the second polar body was assessed by light microscopy, embryos were transferred in 150 l tanks of oxygenated SSW at 21 °C. The embryonic stages were determined by light microscopy observation. The embryonic stages collected were oocytes (E, immediately before sperm addition), fertilized oocytes (FE, immediately before transfer to 150 l tanks), two to eight cell embryos (2-8 C, ca. 1.5 h post fertilization [hpf]), morula (M, ca. 4 hpf), blastula (B, ca. 6 hpf), gastrula (G, ca. 10 hpf), trochophore (T, ca. 16 hpf), and D larvae (D, ca. 24 hpf). For pediveliger (P) and spat (S) stages, D-larvae were collected and reared in a flow-through rearing system at 21.5 °C in SSW. At the end of the pelagic stage, competent larvae were collected on a 100 µm mesh to allow the larval settlement. At 20 days post fertilization (dpf) the pediveliger stage was sampled as the remaining fraction on a 400 µm mesh. The post-larvae were maintained in a downwelling system. Then, at 25 dpf the spat stage was sampled after metamorphosis as the remaining fraction on a 400 µm mesh.

For each embryonic stage, 3 million embryos were collected as the remaining fraction on a 20 µm mesh and centrifuged at 123 g for 5 min at room temperature. The supernatant was discarded and samples of 1 million embryos were then snap-frozen in liquid nitrogen directly after resuspension in TRI Reagent (Catalog No. T9424, Sigma-Aldrich, St Louis, MO) (1 ml/10 6 embryos). For pediveliger and spat samples, 100 mg of each stage were resuspended in TRI Reagent (Catalog No. T9424, Sigma-Aldrich, St Louis, MO) (1 ml/100 mg) and snap-frozen in liquid nitrogen. All samples were stored at -80 °C. Two distinct experiments were realized (February and March 2019) using the gametes of 126 and 140 broodstock animals, respectively (Figure S2).

RNA extraction and fragmentation

RNA of each development stage was extracted using phenol-chloroform followed by affinity chromatography as previously described [START_REF] Riviere | A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE[END_REF]. Briefly, embryos were ground in TRI Reagent (Catalog No. T9424, Sigma-Aldrich, St Louis, MO) and RNA was purified using affinity chromatography with NucleoSpin RNA Clean Up Kit (Catalog No. 740948, Macherey-Nagel, Duren, Germany). Potential contaminating DNA was removed by digestion with rDNase (Catalog No. 740963, Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions for 15 min at 37 °C and then RNA was purified using Agencourt AMPure XP solid-phase reversible immobilization (SPRI) paramagnetic beads (Catalog No. A63880, Beckman Coulter, Brea, CA) according to the manufacturer's instructions. Briefly, paramagnetic beads and RNAs were mixed slowly and incubated 5 min at room temperature followed by 2 min on a magnetic rack. The cleared supernatant was removed, and beads were washed three times with 70% ethanol. After 4 min of drying at room temperature, RNAs were mixed slowly with RNase free water and incubated for 1 min at room temperature on the magnetic rack. The eluted total RNA was stored at -80 °C.

The RNA was heat-fragmented as previously described [START_REF] Zeng | Refined RIP-seq protocol for epitranscriptome analysis with low input materials[END_REF]. Briefly, for each RNA sample (development stages and pools of development stages), 5 µg of total RNA was suspended in 18 µl of RNase free water. The RNA was fragmented by the addition of 2 µl of fragmentation buffer (100 mM Tris-HCl pH 7.0, 100 mM ZnCl2, diethylpyrocarbonate-treated (DEPC) water) and incubated for 2 min at 70 °C. After the incubation, 2 µl of ethylenediamidetetraacetic acid (EDTA) 0.5 M was immediately added and RNAs were incubated on ice for 2 min to stop the reaction. Then, RNAs were purified using Agencourt AMPure XP SPRI paramagnetic beads (Catalog No. A63880, Beckman Coulter, Brea, CA) as previously described and eluted in 50 µl of RNase free water. The fragment size (ca. 100 nucleotides) was verified by fluorescent capillary electrophoresis using an Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit (Catalog No. 5067-1513, Agilent, Santa Clara, CA) according to the manufacturer's instruction.

MeRIP

The MeRIP-seq of total RNA was performed on fragmented RNA. Of the 50 µl fragmented RNA solution, 5 µl (i.e., corresponding to 500 ng of starting RNA before fragmentation) was non immunoprecipitated and used as the input fraction (Input), and 44 µl (i.e., corresponding to 4.5 µg of starting RNA before fragmentation) were subjected to m 6 A-immunoprecipitation using m 6 A antibody-coupled Pierce TM Protein A/G Magnetic Beads (Catalog No. 88803, Thermo Fisher Scientific, Waltham, MA) following the low/high salt procedure by Zeng and collaborators [START_REF] Zeng | Refined RIP-seq protocol for epitranscriptome analysis with low input materials[END_REF]. For each sample, 30 µl of protein A and 30 µl of protein G magnetic beads were incubated on a magnetic rack and washed twice with IP buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, IGEPAL 0.1%, DEPC water) and resuspended in 500 µl of IP buffer. The magnetic beads were incubated overnight at 4 °C under gentle shaking with 5 µg of anti-m 6 A antibody (Catalog No. ABE572, Millipore, Burlington, MA). Then magnetic beads were washed twice with IP buffer and resuspended in RNA mixture composed of 100 µl of 5× IP buffer, 44 µl of fragmented RNAs, and 200 units of RNasin Plus Ribonuclease Inhibitor (Catalog No. N2611, Promega, Madison, WI), and the mixture was completed to 500 µl with IP buffer. The obtained MeRIP solution containing magnetics beads coupled to antibody and fragmented RNAs was incubated for 2 h at 4 °C with gentle shaking. After incubation, the MeRIP solution was incubated on a magnetic rack and the supernatant was removed. Beads were washed twice in IP buffer, twice in low salt buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1% IGEPAL, DEPC water), and twice in high salt buffer (500 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1% IGEPAL, DEPC water) for 10 min at 4 °C with gentle shaking for each washing step.

After extensive washing with IP buffer, RNAs hybridized to the antibody-coated magnetic beads were resuspended in 200 µl of RA1 buffer supplied in the NucleoSpin RNA Clean Up Kit (Catalog No. 740948, Macherey-Nagel, Duren, France) and incubated 2 min at room temperature then 1 min on a magnetic rack. The supernatant containing immunoprecipitated RNA was collected and mixed with 400 µl of 100% ethanol. Then the immunoprecpitated RNA was purified using affinity chromatography with NucleoSpin RNA Clean Up Kit (Catalog No. 740948, Macherey Nagel, Duren, France) and eluted twice with 14 µl of RNase free water. These immunoprecipitated RNAs correspond to the immunoprecipitated fraction (IP).

Reverse transcription-quantitative polymerase chain reaction

To validate the immunoprecipitation of m 6 A methylated RNA, a Reverse transcriptionquantitative polymerase chain reaction (RT-Qpcr) was performed on immunoprecipitated RNA pools. These pools are composed of equal amounts of RNA from each developmental stage for the two distinct experiments. Input and IP fractions were obtained as described above. The equivalent amount of 1.75 ng of Input and IP RNAs were used as starting template for the RT-qPCR protocol previously described [START_REF] Riviere | A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE[END_REF]. Targets included one reference transcript (ef1α) and five conserved genes exhibiting distinct transcript m 6 A methylation in vertebrates (c-myc, klf, mettl3, hnrnpa2b1, and oct4). Briefly, RNAs were reverse-transcribed using 200 U of M-MLV Reverse Transcriptase (Catalog No. M1701, Promega, Madison, WI) and 100 ng random hexamers. Resulting cDNAs were assayed for target gene expression using the Input expression as a reference. SYBR Green quantitative PCR was performed on a CFX96 apparatus (Catalog No. 1845096, Bio-Rad, Marnes-la-Coquette, France). Gotaq qPCR Master Mix (Catalog No. A6002, Promega, Madison, WI) was used in 40 cycles (95 °C/15 s and 60 °C/15 s) reactions. Target genes included one reference gene and 5 target genes with distinct transcript m 6 A methylation in vertebrates, and were amplified with the following primers: Cg-ef1 α (CGI_10012474, LOC105338957; forward: 5'-ACCACCCTGGTGAGATCAAG-3', reverse: 5'-ACGACGATCGCATTTCTCTT-3') as reference transcript of gene expression in C.gigas [START_REF] Dheilly | Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns[END_REF], genes described as methylated in vertebrates: Cg-c-myc (CGI_10002799, LOC105341025; forward: 5'-CGGTCTCTCCCAAATTCTCCC-3', reverse: 5'-TGCTACTTCCACTTGCCCTG-3') [START_REF] Batista | m 6 A RNA modification controls cell fate transition in mammalian embryonic stem cells[END_REF][START_REF] Huang | Recognition of RNA N 6methyladenosine by IGF2BP proteins enhances mRNA stability and translation[END_REF] , Cg-klf (CGI_10019173, LOC105329817; forward: 5'-GAAATCTCCGATGTTGCTGG-3', reverse: 5'-CTTTCCACGGTATTTGCGAG-3') [START_REF] Batista | m 6 A RNA modification controls cell fate transition in mammalian embryonic stem cells[END_REF], Cg-mettl3 (CGI_10023713, LOC105329074; forward: 5'-TGGAACCAAAGAAGAGTGTCAGA-3', reverse: 5'-AGAAATGAACAGTCTCCAAGGGA-3') [START_REF] Coots | m 6 A facilitates eIF4Findependent mRNA translation[END_REF], and Cg-hnrnpa2b1 (CGI_10028469, LOC105333434; forward: 5'-CCAGGGAGGCTACAATGAAGG-3', reverse: 5'-ACACCACCACCAAAGCTGTT-3') [START_REF] Kwon | Functional roles of hnRNPA2/B1 regulated by METTL3 in mammalian embryonic development[END_REF], and a gene described as not methylated in vertebrates:

Cg-oct4 (XM_034479449, LOC117680409; forward: 5'-GTGAAAGGTGCGCTAGAAAA-3', reverse: 5'-GGACCACTTCTTTCTCCAGT-3') [START_REF] Batista | m 6 A RNA modification controls cell fate transition in mammalian embryonic stem cells[END_REF]. The methylation level was calculated as the normalized ratio of the IP and the Input signals by the formulae: 2^(-(Ct IP-Ct Input)). The gene expression level was assayed on the Input fractions, normalized on the reference transcript ef1α [START_REF] Dheilly | Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns[END_REF], and given by the formula 2^(-ΔCt).

Library preparation and sequencing

Amounts equivalent to 1.125 µg of starting RNA of the IP fraction (i.e., 3.5 µl of the

MeRIP-seq data analyses

Read quality was evaluated using FastQC (v.0.11.7) and MultiQC (v.1.7) and adaptor sequences and low-quality reads were removed using Trimmommatic (v.0.38). The remaining reads were aligned to the oyster genome v.9 (GCF_000297895.1) and uniquely mapped reads were counted using STAR (v.2.7.3a) with the parameter "-quantMode GeneCounts" [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]. mRNA and ncRNA were identified from the gene annotation and TEs were identified using the RepeatMasker annotation output provided with assembly data. The expression level in all Input samples was expressed in transcripts per million (TPM) [START_REF] Li | RNA-seq gene expression estimation with read mapping uncertainty[END_REF]. The identification of m 6 A-enriched peaks was performed on uniquely mapped reads of the IP samples using SAMtools (v.1.9) and MeTPeak R package [START_REF] Cui | A novel algorithm for calling mRNA m 6 A peaks by modeling biological variances in MeRIP-seq data[END_REF] [false discovery rate (FDR) < 5%] with the cognate Input

GO analysis

The RNA sequences identified as DM across oyster development were identified using BLASTN [START_REF] Camacho | BLAST+: architecture and applications[END_REF][START_REF] Cock | NCBI BLAST+ integrated into Galaxy[END_REF][START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] against the GigaTON reference transcriptome database [START_REF] Riviere | GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the Pacific oyster Crassostrea gigas[END_REF] with default settings. GO analyses were carried out with the GO annotations obtained from the GigaTON database gene universe [START_REF] Riviere | GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the Pacific oyster Crassostrea gigas[END_REF]. GO term enrichment tests were performed using ClueGO plugin (v.2.5.7) [START_REF] Bindea | ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks[END_REF] on Cytoscape (v.3.8.0). The hypergeometric test with a FDR < 5% was used to consider significant GO term enrichment.

Statistical analyses and graph production

The m 6 A level variation across oyster development was analyzed using one-way ANOVA (factor: development stage) followed by Bonferroni's post hoc test when required, unless otherwise stated. m 6 A levels variations were computed on significantly methylated mRNAs and lncRNAs (e.g., "enriched" RNAs according to MeTPeak results) and on RNA TE displaying detectable methylation because of limitations in their accurate location within the genome due to their repetitive sequences. The values were log-centered and reduced for heatmap production when stated. Methylation profiles were compared using KS tests (CDS regions were divided into 20 bins, while start and stop codon intervals as well as 5' UTR and 3' UTR regions were left unmodified from the default MetPeak output). The correlation between methylation level and transcript content across oyster development, as well as methylation variation and transcript content variation between stages, were estimated using linear regression.

A P value < 0.05 was considered significant. All bioinformatic analyses (unless otherwise stated) were performed using R (v.3.6.3) and RStudio (v.1.0.153) softwares.

The R packages eulerr [START_REF] Larsson | eulerr: area-proportional Euler and Venn diagrams with ellipses[END_REF], ComplexHeatmap [START_REF] Gu | Complex heatmaps reveal patterns and correlations in multidimensional genomic data[END_REF], ggplot2 [START_REF] Gómez-Rubio | ggplot2 -elegant graphics for data analysis (2nd edition)[END_REF], Guitar [START_REF] Cui | Guitar: An R/Bioconductor package for gene annotation guided transcriptomic analysis of RNA-related genomic features[END_REF],

PCAtools [START_REF] Blighe | PCAtools: everything Principal Components Analysis[END_REF], and Prism v.6 (GraphPad) software were used for figure production.

  further work is required to test this hypothesis. Transposon transcripts display class-specific dynamics during oyster development that are different from mRNAs and lncRNAs. Both RNA TEs and DNA TEs, which are poorly m 6 A modified during cleavage, exhibit higher methylation during gastrulation and cell differentiation. Although the role of epitrancriptomes in transposon control remains largely unknown, this kinetics may suggest the participation of m 6 A within the widely conserved RNA mechanisms of TE silencing required for cell differentiation [82-84].Altogether, our results demonstrate that oyster m 6 A epitranscriptomes are dynamic and RNA-class specific, and reveal their implication in oyster MZT and sequential expression of genes required for gastrulation, cell differentiation, and metamorphosis.

  eluted IP fraction) and to the 50 ng of starting RNA of the Input fraction for each sample, respectively, were used for library construction using the SMARTer Stranded Total RNA-Seq Kit v.2 (Catalog No. 634418, Pico Input Mammalian, Takara/Clontech, Saint-Germain-en-Laye, France) according to the manufacturer's protocol without RNA fragmentation. The ribosomal cDNA depletion step and a final cDNA amplification of 16 cycles were performed. Paired-end 150-bp sequencing of Input and IP cDNA libraries of each sample were conducted on an Illumina HiSeq 4000 platform (Catalog No. SY-401-4001, Illumina, San Diego, CA) at the Genome Quebec Innovation Center (McGill University, Montréal, Canada).

  samples as controls. The methylation level of these m6 A-enriched RNAs corresponds to the mean of the IP/Input fold change provided by MeTPeak from the two development experiments. The distribution of m6 A across mRNAs and ncRNAs was visualized using Guitar plots[START_REF] Cui | Guitar: An R/Bioconductor package for gene annotation guided transcriptomic analysis of RNA-related genomic features[END_REF]. The methylation level of TEs was assessed as the ratio of IP/Input with reads per gene expressed in TPM. Transcript variants were pooled for each gene and only the transcripts that were present in the two distinct development experiments were considered expressed or methylated in Input and IP data, respectively. The gene expression and m 6 A level are expressed as the mean of the two replicates of development stages from the independent experiments The m6 A motif was searched in the 1000 m6 A peaks presenting the lowest FDR and the highest IP/Input fold change using HOMER (v.4.10.4). The motif length was restricted to 5-6 nucleotides. All peaks mapped on mRNAs and ncRNAs were used as the target sequences and the background sequences were constituted of 5% of the Input reads (pool of February development experiment sample) selected randomly using SAMtools (v.1.9).

Figure 1 m 6 A

 6 Figure 1 m 6 A signatures of oyster RNA classes A. m 6 A distribution among RNA classes. Diagram diameter was normalized between RNA classes. The internal circles in diagrams represent the proportion of RNA displaying significant m 6 A methylation over background (light color) and significant m 6 A enrichment (dark color), respectively. B. m 6 A localization along mRNAs and ncRNAs (5' to 3'). The mean m 6 A density and confidence interval are represented. C.

Figure 2

 2 Figure 2 Epitranscriptome dynamics during oyster development A. PCA of MeRIP-seq results. PCA plot of the first two principal components shown the MeRIP-seq results for each developmental sample of the two distinct experiments. B. Similarity (pairwise Pearson's correlation matrix) of m 6 A methylation between samples based on IP/Input signal (see methods). C. Dynamics of the m 6 A methylation level of the mRNA (purple) and lncRNA (blue) transcripts. All the transcripts found m 6 A-enriched over background using MetPeak in at least one development stage are represented. Areas under the curve are normalized for each RNA class, and undetectable methylation at certain stages was arbitrarily affected a -5 value for representation purpose. D. Dynamics of the m 6 A methylation level of the DNA TE (green) and RNA TE (yellow) transcripts. All the transcripts found m 6 A-enriched over background using MetPeak in at least one development stage are represented. Areas under the curve are normalized for each RNA class, and undetectable methylation at certain stages was arbitrarily affected a -5 value for representation purpose. E, egg (oocyte); FE, fertilized egg; 2-8C, two to eight cell-stage; M, morula; B, blastula; G, gastrula; T, trochophore; D, D-larva; P, pediveliger; S, spat/juvenile; PCA, principal

Figure 3

 3 Figure 3 Dynamics of mRNA expression and m 6 A methylation during oyster development A. Normalized expression (TPM, purple) and m 6 A content (IP/Input, pink) of significantly differentially methylated mRNA genes during oyster development (n =1494). The development stages are indicated below. B. Correlation between expression (Y-axis, TPM) and methylation (X-axis, IP/Input, divided into twelve quantiles). C. Methylation profiles of mRNAs displaying high (pink) or low (grey) expression within each cluster. The development stages in high and low expression groups are indicated (mean m 6 A density; confidence intervals were omitted for clarity). D. Correlation of expression vs. methylation dynamics throughout development. The linear correlation of the methylation variation vs. expression variation was assessed for each gene across oyster development per cluster and the results are given as surface plots (R-square, X-axis; P value, Y-axis; density of genes, Z-axis). Yellow color in the right bottom corner (i.e., P value < 0.05 and R 2 > 0.5) indicates strong correlation. TPM, transcripts per million.

Figure 4

 4 Figure 4 Functional annotation of differentially methylated mRNAs during oyster development Enriched Gene Ontology terms (hypergeometric test, FDR < 0.05) associated to differential m 6 A mRNA methylation across oyster development. Enriched terms corresponding to specific clusters are colored, and common terms between clusters are black. Color intensity is relative to cluster specificity, with color limit set to 51% of genes inside the respective clusters. Circle diameter is inversely proportional to the P value of the enrichment. FDR, false discovery rate.

Figure 5

 5 Figure 5 Dynamics of lncRNA expression and m 6 A methylation during oyster development A. Normalized expression (blue) and m 6 A content (turquoise) of significantly methylated lncRNA genes during oyster development. Transcript variants were pooled for each gene. B. Correlation between expression (Y-axis) and methylation (X-axis) levels. The methylation level was divided into ten quantiles. C. Correlation plot of

  Consensus sequence of the m6 A motif in the oyster identified by HOMER. D. Mean methylation level of the methylated transcripts of mRNA, lncRNA, DNA TE, and RNA TE classes during oyster development. Letters discriminate significantly different methylation levels (ANOVA followed by Bonferroni's post hoc test, P < 0.05). mRNA,

messenger RNA; lncRNA, long non-coding RNA; DNA TE, DNA transposable elements; RNA TE, retrotransposons; misc_RNA, miscellaneous RNA; tRNA, transfer RNA; ncRNA, non-coding RNA; ANOVA, analysis of variance; UTR, untranslated transcribed region; CDS, coding sequence; kb, kilobase.

Acknowledgments

This work was supported by the French national program CNRS EC2CO ECODYN (Ecosphère Continentale et Côtière 'HERITAGe', Grant No. 2019-200919362L) awarded to Guillaume Rivière and the Council of the Normandy Region (RIN ECUME Grant No. 18E01643 -18P02383) awarded to Pascal Favrel. ORCID 0000-0002-4051-4415 (Lorane Le Franc) 0000-0002-4169-2811 (Bruno Petton) 0000-0002-2504-9957 (Pascal Favrel) 0000-0001-8372-8715 (Guillaume Rivière)

Data availability

All data relative to this study are publicly available at the National Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GEO: GSE180388, and in the Genome Sequence Archive [100] at the National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation (GSA: CRA007976), and are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

CRediT author statement

Lorane Le Franc: Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing -original draft, Writing -review and editing, Visualization.

Competing interests

The authors have declared no competing interests. expression vs. methylation dynamics throughout development. The linear correlation of the methylation vs. expression variation was assessed for each gene across oyster development per cluster and the results are given as surface plots (R-square, X-axis; P value, Y-axis; density of genes, Z-axis).

Supplementary material