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

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6 c

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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

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6 c

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Lip

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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