Séquencage ESt et banques de BAC domaine animal INRA Jouy en Josas Jean-Paul Renard

stratégie expérimentale temps naissance embryonimplantation fœtus/placent a Pilotage par « microfluidique » Embolisation utérine perturbations spécifiques externes perturbations globales externes Clonage NutritionMaternelle Milieu in vitro Métabolismesspécifiques Activation transcriptionnelle transcriptionnelle ontogénèse ontogénèsetissulaireEpithélium Génomique et organogénèseOrganisation noyau noyaudifférentiationgonadique mécanismes perturbations perturbations spécifiques internes Tg additionnelle Recombinaisonhomologue

modèles animaux utilisés dans lAIP modèles animaux utilisés dans lAIP temps naissance embryonimplantation fœtus/placent a Activation transcriptionnelle transcriptionnelle ontogénèse ontogénèsetissulaireEpithélium Génomique et organogénèseOrganisation noyau noyaudifférentiationgonadique lapin bovin ovin Véronique Duranthon CR1 INRA 5000 ESTs banques SSH Isabelle HUE CR1 INRA 4 BACs banque dédiée Corinne Cotinot DR2 INRA 7000 ESTs banques SSH

soutien AIP sequencage à BDR modèle lapin Véronique Duranthon CR1 INRA 5000 ESTs transition transitiontotipotence-pluripotence banques SSH

why using the rabbit as an alternative model to the mouse to study early development ? PhylogeneticsPhylogenetics MetabolismMetabolism Placental propertiesPlacental properties Accessibility of the conceptus at specific developmental stagesAccessibility of the conceptus at specific developmental stages Ultrasonographic monitoring of fetal developmentUltrasonographic monitoring of fetal development Early development…Early development… ex: - efficient use of glucose in rabbit and human, not in the mouse. - requirement of high amino acid levels in human and rabbit, not in the mouse a more appropriate animal model than the mouse to study the embryonic origin of epigenetic diseases evidenced afterhuman in vitro fertilisation In vitro fertilisation in medicine: In vitro fertilisation in medicine: 3 time more premature birth (20% vs 6%) 3 time more premature birth (20% vs 6%) 2 time more BWS (2.2% vs 1.1%) 2 time more BWS (2.2% vs 1.1%)

Mouse: Rabbit Human 24h48h72h E1E2E3 E4 96h S phase fertilization Xenopus, Activation of the embryonic genome is progressive in the rabbit and the human species EGA EGA

Transcriptional activation spans over several cell cycles offering a window of opportunity to study interactions between maternal (oocyte) and embryonic gene products consequence: POST-TRANSCRIPTIONAL REGULATIONS PROTEINS stability NUCLEUS TRANSCRIPTS TRANSCRIPTIONAL REGULATIONS PROTEINS translation localisation phosphorylation EMBRYONIC INFORMATION MATERNAL INFORMATION TRANSCRIPTS Véronique Duranthon

2022 contigs -982 contain only EGA EST -937 contain only FD EST -103 contain both EGA and FD EST a first generation of dedicated rabbit cDNA arrays construction of two subtracted libraries : EGA library First differentiation library __ In vivo + In vitro In vivo EST sequencing Contig assembly PCR inserts spotting Véronique Duranthon, Catherine Archilla

EGA A first description of embryonic transcriptomic variations at EGA and first differentiation in the rabbit embryo. Roger Léandri, Nathalie Peynot Véronique Duranthon Cell communication Intra cell signaling cascade Regulation of signal transduction Amino acid metabolism tRNA metabolism RNA metabolism RNA processing Energy storage and metabolism Aromatic compound metabolism Generation of precursor metabolites and energy DNA integrity Checkpoint, Nucleotide and nucleic acid metabolism Proteolysis Ubiquitin cycle Cellular biosynthesis Protein biosynthesis Response to biotic stimulus Cytoskeleton-dependent intrac. transport

Transcriptome analysis through differential screening Effect of culture on early rabbit embryonic transcriptome B2 B2+ FCS ISM1/2

8 (8.1) 7 (8.13) 8 (6.1) 28 (21.9) 7 (11.5) 8 (11.5) 14 (15.4) 7 (4.3) 13 ( (15.8) 11 (11.8) 20 (13.7) 7 (12) 13 (13.4) 16 (16.1) 21 (13.9) Distribution of genes with a significant « in vitro culture effect » (paired T-test p<0.01) Genes with a significant « culture condition effect » (ANOVA FDR p<0.05) EGA: n= 111 Blastocyst: n= 87 Hypothesis of culture media independency B2 n=59 B2S n=51 ISM n=56 B2 n=57 B2S n=51 ISM n=57 Commonly deregulated genes : - Apoptosis - Post-translational modifications - Mitochondrial oxydative phosphorylation - Energetic metabolism - Cell differentiation - Cell migration - Apoptosis Rejected at EGA p<0.01 Not rejected at blastocyst

Genes affected by each of the culture media EGA N=111 Blastocyst N= A- In vitro culture does not affect the same genes at EGA and at the blastocyst stage Genes commonly affected whatever the culture medium EGA N=20 Blastocyst N= results Véronique Duranthon, Roger Léandri, with Alain Hénaut Agro ParisTech

B- In vitro culture affect genes which are not differentially expressed during development At EGA 23/111 genes affected by in vitro culture dont belong to any cluster Low density lipoprotein 2 (LRP2) Peptidyl cis-trans isomerase A Arginase 2 DEAD box polypeptide 3 LIN 28 homologue At blastocyst stage 12/87 genes affected by in vitro culture dont belong to any cluster Phophoserine aminotransferase Asparagine synthetase Fatty acid binding protein E-FABP Transferrin receptor Methionyl-tRNA synthetase Hypoxia inducible factor 1 (HIF1A) an assessment of embryo robustness? D. Kitano, 2004 Nat Rev Genet,5:

At EGA, in vitro culture differentially affect transiently expressed and long term induced genes Are in vitro culture effect dependant on gene regulation ? Distribution of genes with a significant culture effect among clusters at EGA cluster variable vitro/vivo under vitro/vivo over vitro/vivo Distribution of genes with a significant cullture effect among clusters at the blastocyst stage cluster variable vitro/vivo under vitro/vivo over vitro/vivo Roger Leandri, Véronique Duranthon,

Perspectives : Functional approaches : -Transient overexpression - RNA interference in the preimplantation embryon - Following fetal growth of in vitro cultured embryos - Mimicking transient gene deregulation, then following fetal growth Soutien financier: AIP Phase Agence de Biomédecine Fondation (RTRS) PremUp

soutien AIP sequencage à BDR modèle lapin Véronique Duranthon CR1 INRA 5000 ESTs transition transitiontotipotence-pluripotence banques SSH modèle bovin Isabelle HUE CR1 INRA 4 BACs croissance et differentiation de lépithélium trophoblastique banque dédiée

B : tubular 50 – 60mm C: filamentous 140 – 160mm A : ovoid 10 – 18mm (Barone, 1978) Elongation Phases of Bovine Embryo ABCABCembryonimplantation fœtus/placen ta

BA x10X2.5x1 ovoïde tubulaire filamenteux tubular.50–60mmovoïd:1 – 12mm filamentous140– 160mm D14 D21 cattle embryos gastrulate before implantation, concomitantly with a marked growth of their trophoblast apposition initiates at : D18-19 vascularized allantois present at D25 C posterior poleanterior pole Isabelle Hue, Michel Guillomot, Severine Degrelle

In Cattle nearly half of the embryonic mortalities occur before Implantation D0D5D7-8D2 D14D16D18D21D23 embryo D21 D23 D60D90term D0 Embryo survival/ embryo fecunded (%) Dairy cow 7500 kg Dairy cow 9000 kg Dairy cow> 9000 kg

Evelyne Campion Geraldine Taghouti Isabelle Hue Orthologues murins (et humains) connus profils d expression differents entre ces especes pour Oct4, Nanog et Hand1

In Cattle nearly half of the embryonic mortalities occur before Implantation D0D5D7-8D2 D14D16D18D21D23 embryo D21 D23 D60D90term D0 Embryo survival/ embryo fecunded (%) Dairy cow 7500 kg Dairy cow 9000 kg Dairy cow> 9000 kg A first cDNA librairy at D14 AIP sequencage Isabelle Hue with Severine Degrelle

ovoïde tubulaire filamenteux Chorion J24 Villosités terme intestin poumon Glande mammaire muscle ovoïde tubulaire filamenteux Chorion J24 Villosités terme intestin poumon Glande mammaire muscle ovoïdetubulaire filamenteux c12 c93 Nature ? Fonction ? c12, c93: Pas dorthologues (murins, humains) connus à ce jour Isabelle Hue, Severine Degrelle, Evelyne Campion

c12 c93 Hand1 ovoide filamenteux fibronectine BAC pour FISH 3D Non (seq retrovirale) OUI, 2 BAC bovins sequences OUI, BAC identifié Isabelle Hue avec André Eggen, LGBC

BTA29q22 Localisation de C93… BTA17q22 Un transcrit, 2 localisations = Un BAC chimérique mais de ce fait: Un BAC c93+ pour FISH 3D Et Un BAC contenant une sonde centromérique André Eggen et Hélène Hayes, LGBC

fecondation placentationimplantation term elongation D cDNA embryonnaires dont 1000 séquences uniques Réseau MEM soutien AGENAE Collaboration MIG Octobre 2002 D30D60 Extension de la collection de séquences bovine D18 banque bca2, 7972 ESTs réseau cDNA (13K) embryonaire, placentaire et foetal Soutien Phase et collaboration INRA- Illinois Octobre Oligos + endometre Réseau 22K Soutien Geneanimal juillet 2006 Avec Patrice Martin, jean François Hocquette, François Hattey

Severine Degrelle, Evelyne Campion, Isabelle Hue,, (see also Ushizawa 2004) D16 n=5 D17 n=8 D18 n=6 D20 n=6 D19 n=4 D24 n=8 D15 n=2 D25 n=5 days of recovery D15 D16D17 D18 D19 D20 D24 D25 trophoblast differentiation is related to the stage of the embryonic disc rather than to the day of recovery stages of embryonic disc st 2st 3st 4st 5 C D dedicated Bovine array long oligos Extra embryonic, fœtal, placental posterior pole anterior pole

Gastrulation and tropholast differentiation : an highly regulated process EST -> 138 GO st 2 st 2-3 st st 3 st 3-4 st 4 st regulation of biological process 13% physiological process 36% behavior 1% biological_process unknown 5% cellular process 29% development 16% Severine Degrelle, Isabelle Hue,,

Transcrits sans orthologues murins/humains ? Phylogenese (P Pontarott, AgroBi) Dynamique d expression extra-embryonnaire C0NCEPTUS Emb + Extra-Emb 3 tissus: Ect, End, Mesod UTERUS VACHE NUTRITION FISH 3D ?

- Lien tropho-uterus a l implantation (J18-J23). Profils d expression des 2 compartiments quels dialogues moléculaires ? - Integration moleculaire-tissulaire-morphogentique modlisation morphogentique en cours ( Programme AgroBi avec MIA ( Juhui Wang, Alain Trubill) - Réseaux de gènes inferes par reseaux bayesiens (avec Francoise Kjaffrezic SGQA et Sandra Rodriguez, University of Illinois) - Phylogense sur groupes d expression (avec Pierre Pontarotti CNRS Agrobi) Travaux en cours

soutien AIP sequencage à BDR modèle lapin Véronique Duranthon CR1 INRA 5000 ESTs transition transitiontotipotence-pluripotence banques SSH modèle bovin Isabelle HUE CR1 INRA 4 BACs croissance et differentiation de lépithélium trophoblastique banque dédiée modèle ovin Corinne Cotinot DR2 INRA 7000 ESTs entrée en méiose des ovogonies -formation des follicules primordiaux banques SSH

Chronologie de la différenciation précoce de lovaire de brebis Objectif: Obtenir des données moléculaires sur les étapes précoces du développement des ovaires chez les ruminants -entrée en méiose des ovogonies -formation des follicules primordiaux CONCEPTION Follicule primordial Follicule primaire Follicule secondaire Follicule tertiaire NAISSANCE jpc0135 FOLLICULOGENESE 35 multiplication des cellules germinales Différenciation du sexe gonadique PROPHASE I MEIOSE FEMELLE 82 Exposé Corinne Cotinot mercredi 7 novembre 11h 11h30