Détermination de la destinée cellulaire des neurones de la rétine de vertébrés

1. Détermination de la destinée cellulaire des neurones de la rétine de vertébrés Muriel Perron Janvier 2005 Master 2 Module Neuro-Evo-Devo

2. Développement de l’œil de vertébrés

3. La rétine: excellent modèle d ’étude pour la biologie neuro-développementale • accessibilité • nombre limité de neurones • organisés en couches

4. 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.

5. Neuronal genesis in the retina Klassen et al., 2004

6. Pluripotent retinal progenitors • Clonal analysis:the daughters of a single progenitor injected with horseradish peroxidase contribute many different cell types. Holt et al., 1988

7. What is the importance of the lineage? Mu and Klein, 2004

8. 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

9. 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

10. 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

11. Models for the cell fate choice

12. Are extrinsic or intrinsic cues important for cell fate determination?

13. Extrinsic factors • Testing retinal cells for cell fate choices in different environments, e.g. after adding factors to cultures

14. 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

15. 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

16. 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

17. 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. Ezzeddine et al. 1997

18. 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

19. 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

20. Changes in competence progenitors over time

21. The competence model 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. Livesey and Cepko2001

22. Cell-cell interactions

23. Notch/Delta signaling pathway Jun Hatakeyama, Ryoichiro Kageyama 2004

24. The development of photoreceptor polarity in the eye-antennal imaginal disc of Drosophila Blair 1999

25. Changes in photoreceptor specification induced by the loss or gain of Notch activity Blair 1999

26. C20 Rôle de la cascade Notch dans le choix de la destiné des précurseurs rétiniens chez les vertébrés?

27. Technique de micro-injection chez le xénope DNA à étudier Analyse du phénotype Stade 2-32 cellules

28. 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

29. 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

30. Lipofection in vivo DNA + GFP DNA + Dotap

31. Lipofection in vivo avec la GFP

33. 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

34. 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

35. Intrinsic factors • Identifying and testing transcription factors that might play a role in retinal cell fate decision

36. Les facteurs bHLH Bertrand et al., 2002

37. 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

38. 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.

39. 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

40. Role of bHLH factors in retinal cell fate determination?

41. ZMC = zone marginale ciliaire Epithélium pigmenté Cellules souches Rétine neurale Neuroblastes Cristallin Précurseurs en différenciation ZMC Nerf optique

42. 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

43. Overexpression of bHLH gene Xath5 * 50 GFP 45 Xath5 40 35 30 25 20 * 15 10 * 5 0 Ganglionnaire Amacrine Bipolaire Horizontale Photorécepteur Müller La surexpression de Xath5 conduit à une augmentation des cellules ganglionnaires et une diminution des cellules de Müller et bipolaires

44. Loss of function of bHLH gene Xath5 • Absence of retinal ganglion cells in lak mutants Kay et al., 2001

45. 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 Hatakeyama et Kageyama, 2004 Reviewed in Vetter and Brown, 2001

46. 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.

47. WRPW helix loop helix basique orange HC Groucho LA PROTEINE HES

48. EXPRESSIONDE HES vésicule otique oeil HES a une expression très restreinte au cours du développement

49. st. 30 st. 35 st. 40 EXPRESSIONDE HES AU COURS DE LA RETINOGENESE

50. * * * * * * * * * * * * * * La surexpression de Hes conduit à une augmentation des cellules gliales de Müller ganglionnaires et une inhibition de la neurogenèse Control 1244 cells 11 retinas Hes 939 cells 13 retinas % of retinal cells 80 60 40 20 * 0 Ganglionnaire Amacrine Bipolaire Horizontale Photorécepteur Müller