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Détermination de la destinée cellulaire des neurones de la rétine de vertébrés Muriel Perron...
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Transcript of Détermination de la destinée cellulaire des neurones de la rétine de vertébrés Muriel Perron...
Détermination de la destinée cellulaire des neurones de la
rétine de vertébrés
Muriel PerronJanvier 2005
Master 2Module Neuro-Evo-Devo
Développement de l’œil de vertébrés
La rétine: excellent modèle d ’étude excellent modèle d ’étude pour pour
la biologie neuro-développementalela biologie neuro-développementale
accessibilité nombre limité de neurones organisés en couches
Les cellules rétiniennes• Photoreceptors, rods and cones, are found in
the outer layer of the retina. Their outer segments are membranous structures that capture light and carry out phototransduction.
• They form synapses with bipolar and horizontal cells, found in the inner nuclear layer.
• Also in the inner nuclear layer are amacrine cells, which synapse with bipolar cells and the output cell type, ganglion cells.
• Ganglion cells then send the result of all of this processing to the brain via the optic nerve.
Klassen et al., 2004
Neuronal genesis in the retina
Pluripotent retinal progenitors
Clonal analysis:
the daughters of a single progenitor
injected with horseradish peroxidase contribute
many different cell types.
Holt et al., 1988
Mu and Klein, 2004
What is the importance of the lineage?
Cell lineage analysis
• Fluorescent dextran was injected into single cells of the embryonic optic vesicle.
– Labeled descendants were observed in all three layers of the larval retina.
– Furthermore, different clones were composed of various combinations of all major cell types, including the glial Muller cells.
Hence, single optic vesicle cells have the potential to form any type of retinal cell, suggesting that the interactions that specify the differentiation pathway of retinal cells must occur late in development.
Wetts R, Fraser SE. 1987
Cell lineage analysis
• Retrovirus-mediated gene transfer was used to mark cell lineages in vivo in the postnatal rat retina.
– Labelled clones contained up to three different cell types: three types of neurons or two types of neurons and a Muller glial cell.
This indicates that a single retinal progenitor can generate remarkably diverse cell types near the end of development.
Turner and Cepko 1987
Lineage-independent determination of cell type in the embryonic mouse
retina
Model for the generation of retinal
cell types in which the cessation of mitosis and cell type determination
are independent events.
Turner and Cepko, 1987
Turner et al., 1990
Wetts and Fraser, 1988
Holt et al., 1988
Models for the cell fate choice
Are extrinsic or intrinsic cues important for cell fate determination?
Extrinsic factors
Testing retinal cells for cell fate choices in different environments, e.g. after adding factors to cultures
Growth factors
Fibroblast growth factor-2• Addition of FGF-2 to cultured optic vesicles causes presumptive
pigmented epithelium to undergo neuronal differentiation whereas neutralizing antibodies to FGF-2 block neural differentiation in the presumptive retina. FGF-2 also accelerates the appearance of differentiated ganglion cells in retinal explants.
Transforming growth factor-alpha• In vitro, low concentrations of TGF-alpha stimulate
proliferation, whereas high concentrations inhibit rod differentiation and promote Müller cell differentiation.
Transforming growth factor-ß• Transforming growth factor-ß stimulates production of retinal
amacrine cells while photoreceptor production remains unchanged.
Pittack et al., 1997; Zhao et al., 1996; Lillien, 1995; Anchan et al., 1995; Harris, 1997
Hormonal factors
Retinoic acid• Application of RA to zebrafish causes precocious
differentiation of rods in postmitotic cells. When the synthesis of endogenous RA is inhibited, rod differentiation is impeded. RA treatment of dissociated rat cell cultures specifically increases the number of progenitors that develop as photoreceptors and decreases the number of cells that develop as amacrine cells.
Thyroid hormone• TH induces an increase in the number of cone
photoreceptors in embryonic rat retinal cultures.
Hyatt et al., 1996 ;Kelley et al, 1994 ; Stenkamp et al., 1993 ; Kelley et al., 1995
Neurotrophic factors
Ciliary neurotrophic factor
• Addition of CNTF to postnatal rat retinal explants results in a dramatic decrease in rod differentiation and an increase in bipolar differentiation, suggesting that postmitotic cells which would normally differcntiate into rods switch their fate and differentiate as bipolar cells in response to CNTF; consistent with this, more cells differentiate as rods in mouse retinal explants lacking a functional CNTF receptor.
Ezzeddine et al. 1997
Ezzeddine et al. 1997
Neurotrophic factors
• CNTF can drive cells fated to be rods to express features of the bipolar neuron phenotype and fail to express rod markers. In this case, although the cells are specified to become rods, an extrinsic signal can change the fate of these cells.
Feedback inhibition
• There are soluble factors produced by postmitotic neurons that provide feedback inhibition to progenitors to regulate cell-fate choices
Belliveau et Cepko, 1999
Placing cells in new cellular environments:
heterochronic transplant experiments
progenitors from different stages of development were placed in an environment of a different age (either earlier or later).
• early retinal progenitors, when
cocultured with cells from the late stage of histogenesis, failed to give rise to late-born
retinal cells
Morrow et al., 1998 Belliveau and Cepko, 1999 Rapaport et al.,
2001
• conversely, late retinal progenitors
failed to generate early-born retinal neurons
when cultured with cells from the late stage of
histogenesis
Morrow et al., 1998
Changes in competence progenitors over time
The competence model
Livesey and Cepko 2001
All these findings led to the development of the COMPETENCE model of retinal development, which proposed that progenitors pass through a series of competence states, during each of which the progenitors are competent to produce a subset of retinal cell types.
Cell-cell interactions
Notch/Delta signaling pathway
Jun Hatakeyama, Ryoichiro Kageyama 2004
The development of photoreceptor polarity in the eye-antennal imaginal
disc of Drosophila
Blair 1999
Changes in photoreceptor specification induced by the loss or
gain of Notch activity
Blair 1999
C20
Rôle de la cascade Notch dans le choix de la destiné des
précurseurs rétiniens chez les vertébrés?
Technique de micro-injection chez le xénope
DNA à étudier
Analyse du phénotype
Stade 2-32 cellules
Delta misexpression in the retina
f. Control.g. When the blastomere is injected with Delta (green), almost all the retinal descendants are in the ganglion cell layer and the photoreceptor layer. Almost all the Delta+ photoreceptors are double labeled with a cone marker (inset).
Dorsky et al., 1997
Delta misexpression in the retina
• Delta-misexpressing cells adopt earlier fates, primarily becoming ganglion cells and cone photoreceptors.
Inje
ctio
n a
u s
tad
e 1
6 c
ellu
les
Dorsky et al., 1997
DNA+
GFP DNA+
Dotap
Lipofection in vivo
Lipofection in vivo avec la GFP
Delta misexpression in the retina
• Delta-misexpressing cells adopt earlier fates, primarily becoming ganglion cells and cone photoreceptors.
• Progenitors transfected with Delta later in development also produce rod photoreceptors.
importance of timing in Delta function.
Inje
ctio
n a
u s
tad
e 1
6 c
ellu
les
Lip
ofe
ctio
n
au
sta
de n
eu
rula
Dorsky et al., 1997
Model to generate cellular diversity through Notch/Delta
signaling
Delta signaling in the vertebrate retina is a basic regulatory mechanism that can be used to generate neuronal diversity.
Perron and Harris, 1999
Intrinsic factors
Identifying and testing transcription factors that might play a role in retinal cell fate decision
Les facteurs bHLH
Bertrand et al., 2002
Vertebrate proneural genes fall into two groups: Ash and Ath
genes
• The Ash group (Ash1, Ash2 and Ash3) is composed of bHLH proteins with homology to Drosophila AS-C complex genes.
• The Ath genes have well-conserved bHLH amino acid sequences homologous to another Drosophila proneural gene, atonal. These include the Ath, Ngn and NeuroD subfamilies.
Reviewed in Vetter and Brown, 2001
bHLH factors in the nervous system
In the nervous system, bHLH factors have been proposed to coordinate the acquisition of both general neuronal properties and subtype-specific features of differentiated neurons.
bHLH factors in the nervous system
Different neural bHLH proteins, expressed in distinct dorsoventral progenitor domains of the spinal cord, control the specification of different interneuron subtypes
Role of bHLH factors in retinal cell fate determination?
Cellules souchesCellules souches
NeuroblastesNeuroblastes
Précurseurs enPrécurseurs endifférenciationdifférenciation
ZMC = zone marginale ciliaire
ZMCZMC
EpithéliumEpithéliumpigmentépigmenté
Rétine neuraleRétine neurale
NerfNerfoptiqueoptique
CristallinCristallin
Expression of bHLH gene Xath5
Xath5 s’exprime dans les précurseurs rétiniens, mais ni dans les cellules souches, ni dans les cellules différenciées
Kanekar et al., 1997
0
5
10
15
20
25
30
35
40
45
50
Ganglionnaire
Amacrine
Bipolaire
Horizontale
Photorécepteur
Müller
GFP
Xath5
*
*
*
La surexpression de Xath5 conduit à une augmentation des cellules ganglionnaires et une diminution des cellules de Müller et bipolaires
Overexpression of bHLH gene Xath5
Absence of retinal ganglion cells in lak mutants
Loss of function of bHLH gene Xath5
Kay et al., 2001
Role of bHLH factors in retinal cell fate determination
RGCs require Ath5
amacrine cells and photoreceptors require NeuroD
bipolar cells require Ash1 and Ath3
photoreceptor cells and bipolar cells require Ngn2
Reviewed in Vetter and Brown, 2001
Hatakeyama et Kageyama, 2004
bHLH repressor factors
The Hes/Her class of antagonistic genes is named for their sequence and functional homology with Drosophila hairy and E(spl) genes. These proteins inhibit neurogenesis through direct transcriptional repression of proneural genes.
helix loop helix
orange HC
WRPW
basique
LA PROTEINE HES
GrouchoGroucho
EXPRESSION DE HES
vésiculeotique
oeil
HES a une expression très restreinte au cours du développement
st. 30 st. 40st. 35
EXPRESSION DE HES AU COURS DE LA RETINOGENESE
La surexpression de Hes conduit à une augmentation des cellules gliales de Müller ganglionnaires et une inhibition de la neurogenèse
0
20
40
60
80
Control1244 cells11 retinas
Hes939 cells
13 retinas
% of retinal cells
***
**
***
***
*** *
Ganglionnaire Amacrine Bipolaire Horizontale Photorécepteur Müller
XHes2 GrouchoGroucho helix loop helix
orange HC
WRPW
basique
LES CONSTRUCTIONS DE HES
XHes2-∆WRPW (dominant négatif)
XHes2-∆WRPW-VP16 (antimorphe) domaine d’activationde VP16
∆WRPW
VP16
Control
XHes260
70
80
2
4
Cellules de Müller
Generation of Müller glia by bHLH repressors
HES
GLIOGENESE
NEUROGENESE
Role of other transcription factors in retinal cell fate
determination?
Prox1, an homeodomain transcription factor, is both necessary and sufficient for the
formation of horizontal cells
Dyer et al., 2003; Cook 2003
Requirement of the bHLH genes Mash1/Math3 and the
homeodomain gene Chx10 for bipolar cell fate specification
(a) Misexpression of Mash1 or Math3 alone predominantly generates photoreceptors.
(b) Misexpression of Chx10 alone generates INL cells, but they are Müller glia or undifferentiated cells.
(c) In contrast, misexpression of Mash1 or Math3 with Chx10 generates many bipolar cells.
Hatakeyama et Kageyama 2004
Cooperation of bHLH and homeodomain genes for retinal cell type specification
Hatakeyama et Kageyama 2004
Role of the homeodomain transcription factor Foxn4
Foxn4 is expressed solely by a subset of mitotic progenitors.
Liu et al., 2004
Defect in the Genesis of Amacrine and Horizontal Cells in
Foxn4_/_ Retinas
Liu et al., 2004
Reduction of Math3, NeuroD1, and Prox1 Expression in
Foxn4_/_ RetinasMath3, NeuroD1, and Prox1, which are retinogenic genes required for the generation of amacrine and horizontal cells, are reduced in Foxn4 -/- retinas.
Other retinogenic genes are not affected in Foxn4 -/- retinas.
Overexpressed Foxn4 Promotes the Formation of Amacrine Cells
Liu et al., 2004
What factor define the competence state of retinal progenitors ?
This transcription factor defines the amacrine-generating competence state.
Proposed Mechanism by which Foxn4 Controls the Genesis of Amacrine and Horizontal Cells by
Retinal Progenitors
Nouvelles approches
Molecular differences among progenitor cells using the microarray technology
Harris, 1997
Résumé
There are several subtypes of each class of
neurons…
For example: twenty or more different types of amacrine cells
Control of late off-center cone bipolar cell differentiation by the homeobox gene Vsx1
The transcription factor Bhlhb4 is required for rod bipolar cell maturation
Bramblett et al., 2004; Chow et al., 2004
Differentiation of a particular subtype of cell
Conservation des molécules impliquées dans la détermination des neurones rétiniens au
cours de l’evolution?
Kumar 2001
‘It requires little persuasion to be convinced that the lens eye of a vertebrate and the compound eye of an insect
are independent evolutionary events.’
Ernst Mayr
1961
Comparaisons de structures de rétines, de leur origine embryonnaire, position des axones…..
Les yeux auraient évolués indépendamment entre 40 et 65 fois au cours de l’évolution.
Salvini-Plawen and Mayr, 1961
tête aile
antenne patte
Surexpression de eyeless dans divers tissus
eyeless Small eye
Perte de fonction eyeless/Pax6
Surexpression chez la drosophile du
gène Pax6 de souris
1995Walter Gehring
Les gènes eyeless de drosophile et Pax6 de vertébrés sont
interchangeables
L’œil de drosophile et celui de vertébrés utilisent le même
« gène maître ».
L’œil de drosophile et celui de vertébrés auraient une origine
commune...
Conclusions
Qu’en est-il de la détermination des
neurones rétiniens?
Rétinogenèse chez les vertébrés et la drosophile
Cook 2003
Vert
eb
rate
Dro
sop
hila
As in the vertebrate eye, the commitment of each retinal cell type within the fly eye occurs from a pool of pluripotent retinal precursors in a sequential, stereotyped order.
Conservation of pathways that regulate retinogenesis across
evolution
• role for Prox1 transcription factor in specifying horizontal cells in the mouse retina
• Prospero, the Drosophila homolog of Prox1, also participates in retinal cell specification