excellent modèle d étude pour la biologie neuro-développementale La rétine: excellent modèle d étude pour la 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
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
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
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.
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. Injection au stade 16 cellules Dorsky et al., 1997
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. Injection au stade 16 cellules Lipofection au stade neurula 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
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 souches Neuroblastes Précurseurs en différenciation ZMC = zone marginale ciliaire ZMC Epithéliumpigmenté Rétine neurale Nerfoptique Cristallin
Expression of bHLH gene Xath5 Xath5 sexprime 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 Groucho
EXPRESSION DE HES vésicule otique oeil HES a une expression très restreinte au cours du développement
st. 30st. 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 Control 1244 cells 11 retinas Hes 939 cells 13 retinas % of retinal cells * * * * * * * * * * * * * * * GanglionnaireAmacrineBipolaireHorizontalePhotorécepteurMüller
XHes2 Groucho helix loop helix orange HC WRPW basique LES CONSTRUCTIONS DE HES XHes2-WRPW (dominant négatif) XHes2-WRPW-VP16 (antimorphe) domaine dactivation de VP16
WRPW VP16 Control XHes2 60 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_/_ Retinas Math3, 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 ? Ø 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
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 levolution? 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 lévolution. Salvini-Plawen and Mayr, 1961
têteaile 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 1995 Walter Gehring
Les gènes eyeless de drosophile et Pax6 de vertébrés sont interchangeables Lœil de drosophile et celui de vertébrés utilisent le même « gène maître ». Lœil de drosophile et celui de vertébrés auraient une origine commune... Conclusions
Quen est-il de la détermination des neurones rétiniens?
Rétinogenèse chez les vertébrés et la drosophile Cook 2003 Vertebrate Drosophila 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