Diapositive 1

1
Séquencage ESt et banques de BAC domaine animal
Jean-Paul Renard
INRA Jouy en Josas jean-paul.renard_at_jouy.inra.fr
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stratégie expérimentale
mécanismes
Activation transcriptionnelle
ontogénèse tissulaire Epithélium
Génomique et organogénèse
Organisation noyau
différentiation gonadique
naissance
embryon
implantation
fœtus/placenta
temps
Nutrition Maternelle
Clonage
Milieu in vitro
Pilotage par  microfluidique 
Métabolismes spécifiques
Embolisation utérine
perturbations
Recombinaison homologue
Tg additionnelle
perturbations globales externes
perturbations spécifiques externes
perturbations spécifiques internes
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modèles animaux utilisés dans lAIP
Activation transcriptionnelle
ontogénèse tissulaire Epithélium
Génomique et organogénèse
Organisation noyau
différentiation gonadique
naissance
embryon
implantation
fœtus/placenta
temps
lapin
Véronique Duranthon CR1 INRA
bovin
Isabelle HUE CR1 INRA
5000 ESTs
ovin
banques SSH
4 BACs
Corinne Cotinot DR2 INRA
banque dédiée
7000 ESTs
banques SSH
4
soutien AIP sequencage à BDR 2005-2007
modèle lapin
Véronique Duranthon CR1 INRA
5000 ESTs
13 000
banques SSH
transition totipotence-pluripotence
5
why using the rabbit as an alternative model to
the mouse to study early development ?

• Phylogenetics
• Metabolism
• Placental properties
• Accessibility of the conceptus at specific
  developmental stages
• Ultrasonographic monitoring of fetal development
• Early development

a more appropriate animal model than the mouse to
study the embryonic origin of epigenetic
diseases evidenced afterhuman in vitro
fertilisation
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
In vitro fertilisation in medicine
3 time more premature birth (20 vs 6) 2
time more BWS (2.2 vs 1.1)
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Activation of the embryonic genome is progressive
in the rabbit and the human species
EGA
S phase
fertilization
24h
48h
72h
96h
E1
E2
E3
E4
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consequence
Transcriptional activation spans over several
cell cycles offering a window of opportunity to
study interactions between maternal (oocyte) and
embryonic gene products
EMBRYONIC INFORMATION
stability
TRANSCRIPTS
translation
TRANSCRIPTS
PROTEINS
NUCLEUS
localisation
PROTEINS
phosphorylation
POST-TRANSCRIPTIONAL REGULATIONS
TRANSCRIPTIONAL REGULATIONS
Véronique Duranthon
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a first generation of dedicated rabbit cDNA
arrays
construction of two subtracted libraries
EGA library
First differentiation library
_
_
In vivo In vitro
In vivo In vitro
In vivo
In vivo
EST sequencing Contig assembly PCR inserts
spotting

• 2022 contigs
• 982 contain only EGA EST
• 937 contain only FD EST
• 103 contain both EGA and FD EST

Véronique Duranthon, Catherine Archilla
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A first description of embryonic transcriptomic
variations at EGA and first differentiation in
the rabbit embryo.
Cell communication Intra cell signaling
cascade Regulation of signal transduction Amino
acid metabolism tRNA metabolism
Aromatic compound metabolism Generation of
precursor metabolites and energy
RNA metabolism RNA processing Energy storage and
metabolism
DNA integrity Checkpoint, Nucleotide and nucleic
acid metabolism
Cellular biosynthesis Protein biosynthesis
Proteolysis Ubiquitin cycle
EGA
Response to biotic stimulus Cytoskeleton-dependent
intrac. transport
Roger Léandri, Nathalie Peynot Véronique
Duranthon
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Effect of culture on early rabbit embryonic
transcriptome
B2
B2 FCS
ISM1/2
Transcriptome analysis through differential
screening
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Distribution of genes with a significant in
vitro culture effect (paired T-test
plt0.01) Genes with a significant culture
condition effect (ANOVA FDR plt0.05)
Blastocyst n 87
EGA n 111
B2 n59
10 (15.8)
7 (8.13)
B2 n57
16 (16.1)
13 (13.4)
8 (11.5)
14 (15.4)
20 (13.7)
28 (21.9)
ISM n56
B2S n51
11 (11.8)
8 (8.1)
8 (6.1)
7 (12)
7 (11.5)
13 (14.2
ISM n57
B2S n51
21 (13.9)
7 (4.3)
Commonly deregulated genes - Apoptosis -
Post-translational modifications - Mitochondrial
oxydative phosphorylation

• - Energetic metabolism
• - Cell differentiation
• Cell migration
• Apoptosis

Hypothesis of culture media independency
Not rejected at blastocyst
Rejected at EGA plt0.01
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results
A- In vitro culture does not affect the same
genes at EGA and at the blastocyst stage
Blastocyst N 87
EGA N111
102
9
78
Genes affected by each of the culture media
Blastocyst N 28
EGA N20
28
20
0
Véronique Duranthon, Roger Léandri, with Alain
Hénaut Agro ParisTech
Genes commonly affected whatever the culture
medium
13
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 As
paragine 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,5826-37.
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Are in vitro culture effect dependant on gene
regulation ?
At EGA, in vitro culture differentially affect
transiently expressed and long term induced genes
Roger Leandri, Véronique Duranthon,
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Perspectives Functional approaches

• Following fetal growth of in vitro cultured
  embryos
• Mimicking transient gene deregulation, then
  following fetal growth

• Transient overexpression
• - RNA interference in the preimplantation embryon

Soutien financier AIP Phase Agence de
Biomédecine Fondation (RTRS) PremUp
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soutien AIP sequencage à BDR 2005-2007
modèle lapin
Véronique Duranthon CR1 INRA
5000 ESTs
13 000
banques SSH
transition totipotence-pluripotence
modèle bovin
Isabelle HUE CR1 INRA
4 BACs
15 000
banque dédiée
croissance et differentiation de lépithélium
trophoblastique
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Elongation Phases of Bovine Embryo
fœtus/placenta
embryon
implantation
A ovoid 10 18mm
B tubular 50 60mm
(Barone, 1978)
C filamentous 140 160mm
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cattle embryos gastrulate before implantation,
concomitantly with a marked growth of their
trophoblast
ovoïde
tubulaire
filamenteux
apposition initiates at D18-19 vascularized
allantois present at D25
posterior pole
anterior pole
B
A
C
x10
X2.5
x1
D14
D21
tubular.5060mm
ovoïd1 12mm
filamentous140160mm
Isabelle Hue, Michel Guillomot, Severine Degrelle
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In Cattle nearly half of the embryonic
mortalities occur before Implantation
100
90
Dairy cow 7500 kg
80
Dairy cow 9000 kg
Embryo survival/ embryo fecunded ()
70
62
Dairy cowgt 9000 kg
60
54
50
40
40
30
D21
D23
D60
D90
term
D0
20
Orthologues murins (et humains) connus
profils d expression differents entre ces
especes pour Oct4, Nanog et Hand1
Evelyne Campion Geraldine Taghouti Isabelle Hue
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In Cattle nearly half of the embryonic
mortalities occur before Implantation
100
90
Dairy cow 7500 kg
80
Dairy cow 9000 kg
Embryo survival/ embryo fecunded ()
70
62
A first cDNA librairy at D14 AIP sequencage
Dairy cowgt 9000 kg
60
54
50
40
40
30
D21
D23
D60
D90
term
D0
Isabelle Hue with Severine Degrelle
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c12
filamenteux
Glande mammaire
Villosités terme
tubulaire
Chorion J24
ovoïde
c12, c93 Pas dorthologues (murins, humains)
connus à ce jour
poumon
intestin
muscle
ovoïde
tubulaire
c93
filamenteux
Glande mammaire
Villosités terme
tubulaire
Chorion J24
ovoïde
poumon
intestin
muscle
Nature ? Fonction ?
filamenteux
Isabelle Hue, Severine Degrelle, Evelyne Campion
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ovoide
filamenteux
BAC pour FISH 3D Non (seq retrovirale) OUI,
2 BAC bovins sequences OUI, BAC
identifié OUI, BAC identifié
c12
c93
Hand1
fibronectine
Isabelle Hue avec André Eggen, LGBC
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Localisation de C93
Un BAC c93 pour FISH 3D Et Un BAC contenant une
sonde centromérique
Un transcrit, 2 localisations Un BAC chimérique
mais de ce fait
André Eggen et Hélène Hayes, LGBC
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Extension de la collection de séquences bovine
fecondation
term
placentation
implantation
elongation
D14
D30
D60
1920 cDNA embryonnaires dont 1000 séquences
uniques Réseau MEM soutien
AGENAE Collaboration MIG Octobre 2002
Avec Patrice Martin, jean François Hocquette,
François Hattey
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trophoblast differentiation is related to the
stage of the embryonic disc rather than to the
day of recovery
stages of embryonic disc
days of recovery
D19
D24
D15
D16
D17
D18
D20
D25
st 2
st 3
st 4
st 5
anterior pole
D15 n2
D
C
D16 n5
D17 n8
D18 n6
D19 n4
D20 n6
D24 n8
posterior pole
D25 n5
(see also Ushizawa 2004)
dedicated Bovine array 13360 long oligos Extra
embryonic, fœtal, placental
Severine Degrelle, Evelyne Campion, Isabelle
Hue,,
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Gastrulation and tropholast differentiation an
highly regulated process
500 EST -gt 138 GO
behavior
1

biological_process
regulation of
unknown
5
biological process
13
cellular process
29
physiological
process

development
36
16
Severine Degrelle, Isabelle Hue,,
28
C0NCEPTUS Emb Extra-Emb
FISH 3D ?
3 tissus Ect, End, Mesod
Dynamique d expression extra-embryonnaire
Phylogenese (P Pontarott,
AgroBi)
UTERUS
VACHE
Transcrits sans orthologues murins/humains ?
NUTRITION
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Travaux en cours
- 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)
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soutien AIP sequencage à BDR 2005-2007
modèle lapin
Véronique Duranthon CR1 INRA
5000 ESTs
13 000
banques SSH
transition totipotence-pluripotence
modèle bovin
Isabelle HUE CR1 INRA
4 BACs
15 000
banque dédiée
croissance et differentiation de lépithélium
trophoblastique
modèle ovin
Corinne Cotinot DR2 INRA
7000 ESTs
15 000
banques SSH
entrée en méiose des ovogonies -formation des
follicules primordiaux
31
Chronologie de la différenciation précoce de
lovaire de brebis
NAISSANCE
CONCEPTION
PROPHASE I MEIOSE FEMELLE
Différenciation du sexe gonadique
multiplication des cellules germinales
FOLLICULOGENESE
Follicule primordial
Follicule primaire
Follicule secondaire
Follicule tertiaire
30-32
55
75
100
120
145 jpc
0
135
35
82

• 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

Exposé Corinne Cotinot mercredi 7 novembre 11h
11h30