Embed Size (px)
Citation preview
stratégie expérimentalestratégie expérimentale
tempstemps
naissancenaissance
embryonembryon implantationimplantation fœtus/fœtus/placentaplacenta
Pilotage par Pilotage par « microfluidique »« microfluidique » Embolisation utérineEmbolisation utérine
perturbations spécifiques externesperturbations spécifiques externes
perturbations globales externesperturbations globales externes
ClonageClonage NutritionNutritionMaternelleMaternelle
Milieu in vitroMilieu in vitro
MétabolismesMétabolismesspécifiquesspécifiques
ActivationActivation transcriptionnelletranscriptionnelle
ontogénèseontogénèsetissulairetissulaire
EpithéliumEpithélium
Génomique et Génomique et organogénèseorganogénèse
OrganisationOrganisation noyaunoyau
différentiationdifférentiationgonadiquegonadique
méc
anis
mes
méc
anis
mes
pert
urba
tions
pert
urba
tions
perturbations spécifiques internesperturbations spécifiques internes
Tg additionnelleTg additionnelle RecombinaisonRecombinaisonhomologuehomologue
modèles animaux utilisés dans l’AIP modèles animaux utilisés dans l’AIP
tempstemps
naissancenaissance
embryonembryon implantationimplantation fœtus/fœtus/placentaplacenta
ActivationActivation transcriptionnelletranscriptionnelle
ontogénèseontogénèsetissulairetissulaire
EpithéliumEpithélium
Génomique et Génomique et organogénèseorganogénèse
OrganisationOrganisation noyaunoyau
différentiationdifférentiationgonadiquegonadique
lapinlapin
bovinbovin
ovinovin
Véronique Duranthon CR1 INRA
5000 ESTs banques SSH
Isabelle HUECR1 INRA
4 BACs banque dédiée
Corinne Cotinot DR2 INRA
7000 ESTs banques SSH
soutien AIP sequencage à BDR 2005-2007
modèle lapin Véronique Duranthon CR1 INRA
5000 ESTs 13 000 €
transition transition totipotence-pluripotencetotipotence-pluripotence
banques SSH
why using the rabbit as an alternative model to the mouse to study early development ?
• PhylogeneticsPhylogenetics
• Metabolism Metabolism
• 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: ex: - efficient use of glucose in rabbit and human, not in the mouse.- 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- 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 ofa more appropriate animal model than the mouse to study the embryonic origin ofepigenetic diseases evidenced afterhuman in vitro fertilisation 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
24h 48h 72hE1 E2 E3 E4
96h
S phase
fertilization
Xenopus,
Activation of the embryonic genomeActivation of the embryonic genome is progressive in the rabbit and the human species is progressive in the rabbit and the human species
EGAEGA
Transcriptional activation spans over several cell cycles offering a window of opportunity to Transcriptional activation spans over several cell cycles offering a window of opportunity to study interactions between maternal (oocyte) and embryonic gene productsstudy interactions between maternal (oocyte) and embryonic gene products
consequence:
POST-TRANSCRIPTIONAL REGULATIONS
PROTEINS
stability
NUCLEUS TRANSCRIPTS
TRANSCRIPTIONAL REGULATIONS
PROTEINStranslation
localisation
phosphorylation
EMBRYONIC INFORMATION
MATERNAL INFORMATION
TRANSCRIPTS
Véronique Duranthon Véronique Duranthon
2022 contigs2022 contigs-982 contain only EGA EST982 contain only EGA EST-937 contain only FD EST937 contain only FD EST-103 contain both EGA and FD EST 103 contain both EGA and FD EST
a first generation of dedicated rabbit cDNA arrays
construction of two subtracted libraries :
EGA libraryEGA library First differentiation libraryFirst differentiation library
_ _
In vivo + In vitro In vivo + In vitroIn vivo In vivo
EST sequencingContig assemblyPCR inserts spotting
Véronique Duranthon, Catherine Archilla
EGAEGA
A first description of embryonic transcriptomic variations at EGA and first differentiation in the rabbit embryo.
Roger Léandri, Nathalie Peynot Véronique Duranthon
Cell communicationIntra cell signaling cascadeRegulation of signal transductionAmino acid metabolismtRNA metabolism
RNA metabolismRNA processingEnergy storage and metabolism
Aromatic compound metabolismGeneration of precursor metabolites and energy
DNA integrity Checkpoint,Nucleotide and nucleic acid metabolism
ProteolysisUbiquitin cycle
Cellular biosynthesisProtein biosynthesis
Response to biotic stimulusCytoskeleton-dependent intrac. transport
Transcriptome analysis Transcriptome analysis through through
differential screeningdifferential screening
Effect of culture on early rabbit embryonic transcriptomeEffect of culture on early rabbit embryonic transcriptome
B2B2 B2+ FCSB2+ FCS ISM1/2ISM1/2
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(14.2
10(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=51ISM n=56
B2 n=57
B2S n=51ISM 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
EGAN=111
BlastocystN= 87
9102 78
A- In vitro culture does not affect the same genes at EGA and at the blastocyst stage
Genes commonly affected whatever the culture medium
EGAN=20
BlastocystN= 28
0 2820
results 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 don’t belong to any cluster
Low density lipoprotein 2 (LRP2)Peptidyl cis-trans isomerase AArginase 2 DEAD box polypeptide 3LIN 28 homologue
At blastocyst stage 12/87 genes affected by in vitro culture don’t belong to any cluster
Phophoserine aminotransferaseAsparagine synthetaseFatty acid binding protein E-FABPTransferrin receptorMethionyl-tRNA synthetaseHypoxia inducible factor 1 (HIF1A)
an assessment of embryo robustness? an assessment of embryo robustness?
D. Kitano, 2004Nat Rev Genet,5:826-37.
At EGA, in vitro culture differentially At EGA, in vitro culture differentially affect transiently expressed and long affect transiently expressed and long term induced genesterm induced genes
Are in vitro culture effect dependant on gene regulation ?
Distribution of genes with a significant cultureDistribution of genes with a significant cultureeffect among clusters at EGAeffect among clusters at EGA
05
101520253035
cluster1
cluster2
cluster3
cluster4
cluster5
cluster6
cluster7
variable vitro/vivo
under vitro/vivo
over vitro/vivo
Distribution of genes with a significant culltureDistribution of genes with a significant culltureeffect among clusters at the blastocyst stageeffect among clusters at the blastocyst stage
0
5
10
15
20
cluster
1
cluster
2
cluster
3
cluster
4
cluster
5
cluster
6
cluster
7
variable vitro/vivo
under vitro/vivo
over vitro/vivo
Roger Leandri, Véronique Duranthon,
Perspectives : Functional approaches :Perspectives : Functional approaches :
-Transient overexpressionTransient overexpression
- RNA interference in the preimplantation embryon- RNA interference in the preimplantation embryon
24h 48h 72h 96h
Injection Observation
- Following fetal growth of in vitro cultured embryosFollowing fetal growth of in vitro cultured embryos
- Mimicking transient gene deregulation, then following fetal growthMimicking transient gene deregulation, then following fetal growth
Soutien financier:Soutien financier:
AIP PhaseAIP PhaseAgence de BiomédecineAgence de BiomédecineFondation (RTRS) PremUpFondation (RTRS) PremUp
soutien AIP sequencage à BDR 2005-2007
modèle lapin Véronique Duranthon CR1 INRA
5000 ESTs 13 000 €
transition transition totipotence-pluripotencetotipotence-pluripotence
banques SSH
modèle bovin Isabelle HUECR1 INRA
4 BACs 15 000 €
croissance et differentiation croissance et differentiation de l’épithélium trophoblastiquede l’épithélium trophoblastique
banque dédiée
B : tubular50 – 60mm
C: filamentous140 – 160mm
A : ovoid10 – 18mm
(Barone, 1978)
Elongation Phases of Bovine Embryo
A B CA B C
embryonembryon implantationimplantation fœtus/fœtus/placentaplacenta
BAx10 X2.5 x1
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 : D18-19vascularized allantois present at D25
C
posterior pole anterior pole
Isabelle Hue, Michel Guillomot, Severine Degrelle
In Cattle nearly half of the embryonic mortalities occur before Implantation
D0 D5 D7-8D2 D14 D16 D18 D21 D23
embryo
D21 D23 D60 D90 term
50
30
60
40
70
80
90
100
6262
5454
4040
D0Em
bry
o s
urv
iva
l/ e
mb
ryo
fe
cu
nd
ed
(%
)
Dairy cow 7500 kg
Dairy cow 9000 kg
Dairy cow> 9000 kg
Evelyne CampionGeraldine TaghoutiIsabelle Hue
Orthologues murins (et humains) connusOrthologues murins (et humains) connus
profils d expressionprofils d expressiondifferents entre ces especesdifferents entre ces especespour Oct4, Nanog et Hand1 pour Oct4, Nanog et Hand1
In Cattle nearly half of the embryonic mortalities occur before Implantation
D0 D5 D7-8D2 D14 D16 D18 D21 D23
embryo
D21 D23 D60 D90 term
50
30
60
40
70
80
90
100
6262
5454
4040
D0Em
bry
o s
urv
iva
l/ e
mb
ryo
fe
cu
nd
ed
(%
)
Dairy cow 7500 kg
Dairy cow 9000 kg
Dairy cow> 9000 kgA first cDNA librairy at D14
AIP sequencage
Isabelle Hue with Severine Degrelle
ovoï
detu
bula
irefil
amen
teux
Chorio
n J2
4Villo
sités
term
ein
test
in
poum
onG
land
e m
amm
aire
mus
cleov
oïde
tubu
laire
filam
ente
uxCho
rion
J24
Villosit
és
term
ein
test
in
poum
onG
land
e m
amm
aire
mus
cle
ovoïde tubulaire
filamenteux
c12
c93
Nature ?Fonction ?
c12, c93:Pas d’orthologues(murins, humains) connus à ce jour
Isabelle Hue, Severine Degrelle, Evelyne Campion
c12
c93
Hand1
ovoide filamenteux
fibronectine
BAC pour FISH 3DBAC pour FISH 3D
Non (seq retrovirale)Non (seq retrovirale)
OUI, 2 BAC bovins OUI, 2 BAC bovins sequences sequences
OUI, BAC identifiéOUI, BAC identifié
OUI, BAC identifiéOUI, 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 3DEt Un BAC contenant une sondecentromérique
André Eggen et Hélène Hayes, LGBC
fecondation placentationimplantation termelongation
D14
1920 cDNA embryonnairesdont 1000 séquences uniques
Réseau MEM
soutien AGENAE
Collaboration MIG
Octobre 2002
D30 D60
Extension de la collection de séquences bovine
D18
banque bca2, 7972 ESTs
réseau 13 000 cDNA (13K)embryonaire, placentaire et foetal
Soutien Phaseet collaboration INRA- Illinois
Octobre 2004
14.000 Oligos+ endometre
Réseau 22K
Soutien Geneanimaljuillet 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
D15D16 D17 D18
D19D20
D24D25
trophoblast differentiation is related to the stage of the embryonic disc rather than to the day of recovery
2 3 4 5
stages of embryonic disc
st 2
st 3 st 4 st 5
C D
dedicated Bovine array 13360 long oligos Extra embryonic, fœtal, Extra embryonic, fœtal, placentalplacental
posterior pole
anterior pole
Gastrulation and tropholast differentiation : an highly regulated process
1 2 3 4 5 6 7
0
+
-
2 3 4 5
500 EST -> 138 GO
st 2
st 2
-3
st 2
-3-4
st
3
st 3
-4
st 4
st 5
22 33 44 55
regulation of biological process
13%
physiological process
36%
behavior 1% biological_process
unknown 5%
cellular process 29%
development16%
Severine Degrelle, Isabelle Hue,,
Transcrits sans orthologues murins/humains ?
Phylogenese
(P Pontarott, AgroBi)
1 2 3 4 5 6 7
0
+
-
2 3 4 5
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 2005-2007
modèle lapin Véronique Duranthon CR1 INRA
5000 ESTs 13 000 €
transition transition totipotence-pluripotencetotipotence-pluripotence
banques SSH
modèle bovin Isabelle HUECR1 INRA
4 BACs 15 000 €
croissance et differentiation croissance et differentiation de l’épithélium trophoblastiquede l’épithélium trophoblastique
banque dédiée
modèle ovin Corinne Cotinot DR2 INRA
7000 ESTs 15 000 €
entrée en méiose des ovogonies entrée en méiose des ovogonies -formation des follicules primordiaux-formation des follicules primordiaux
banques SSH
Chronologie de la différenciation précoce de l’ovaire de brebis
Objectif:Obtenir des données moléculaires sur les étapes précoces du Obtenir des données moléculaires sur les étapes précoces du développement des ovaires chez les ruminantsdéveloppement des ovaires chez les ruminants
-entrée en méiose des ovogonies -entrée en méiose des ovogonies -formation des follicules primordiaux-formation des follicules primordiaux
CONCEPTIONCONCEPTION
Folliculeprimordial
Folliculeprimaire
Folliculesecondaire
Folliculetertiaire
NAISSANCENAISSANCE
30-32 55 75 100 120 145 jpc0 135
FOLLICULOGENESE
35
multiplication des cellules germinales
Différenciation du sexe gonadique
PROPHASE I MEIOSEFEMELLE
82
Exposé Corinne Cotinot Exposé Corinne Cotinot mercredi 7 novembre 11h 11h30mercredi 7 novembre 11h 11h30
