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Atoh7 controls mitochondrial activity along the pathway converting pre-committed progenitors into retinal ganglion cells in retina adapted for high acuity vision

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Reference

Atoh7 controls mitochondrial activity along the pathway converting pre-committed progenitors into retinal ganglion cells in retina adapted

for high acuity vision

BRODIER, Laurent

Abstract

How metabolism is rewired during embryonic development of the nervous system is still largely unknown because tracing metabolic activities with spatiotemporal resolution during neurogenesis remains a major technical challenge. Here, we investigated changes in the number of active mitochondria in the developing avian retinas, focusing on the conversion of progenitors into retinal ganglion cells (RGCs). Our study uncovers how ATOH7 and HES5.3 influence the dynamic changes in the number of active mitochondria as cells transit from uncommitted to pre-committed progenitors and then to newborn RGCs. There is a transient decrease of mitochondrial activity few hours before cells become committed to the RGC fate.

While there is a general trend toward fewer mitochondria when retinogenesis begins, cells that enter the RGC lineage, as well as newborn RGCs, recover the high number of active mitochondria that characterizes uncommitted retinal progenitors. We assessed metabolic dynamics at the onset of cell differentiation by monitoring changes in metabolite concentration in vivo. Combined, our approaches identify changes in metabolic [...]

BRODIER, Laurent. Atoh7 controls mitochondrial activity along the pathway

converting pre-committed progenitors into retinal ganglion cells in retina adapted for high acuity vision. Thèse de doctorat : Univ. Genève et Lausanne, 2018, no. Neur. 222

DOI : 10.13097/archive-ouverte/unige:103549 URN : urn:nbn:ch:unige-1035492

Available at:

http://archive-ouverte.unige.ch/unige:103549

Disclaimer: layout of this document may differ from the published version.

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FACULTÉ DES SCIENCES

DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES

Docteur Jean-Marc MATTER, directeur de thèse

TITRE DE LA THÈSE

ATOH7 CONTROLS MITOCHONDRIAL ACTIVITY ALONG THE PATHWAY CONVERTING PRE-COMMITTED PROGENITORS INTO RETINAL GANGLION CELLS

IN RETINA ADAPTED FOR HIGH ACUITY VISION

THESE Présentée à la Faculté des Sciences de l’Université de Genève

pour obtenir le grade de Docteur en Neurosciences

par

Laurent BRODIER

de Bernex, Genève, Suisse Thèse N° 222

Genève

Editeur ou imprimeur: Université de Genève 2018

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Table of Contents

Aknowledgements ... 6

Résumé de thèse en français ... 7

Introduction ... 9

Metabolism in the developing retina... 9

Hypothesis and aim of the project ... 10

Summary of results ... 11

Literature review ... 13

Retina development ... 13

Eye field formation occurs in the specified anterior forebrain ... 13

Eye field transcription factors expression ... 14

Sonic hedgehog splits the eye field ... 15

Optic vesicle evagination ... 16

Optic vesicle patterning ... 17

Optic cup formation ... 20

Retinal progenitor cells of the neuroretina ... 22

Neurogenesis and focus on RGCs differentiation ... 27

Mitochondria and metabolism ... 33

Catabolism vs. anabolism ... 35

Regulation of metabolism ... 38

Mitochondria and neurogenesis ... 39

Mitochondria influence on cell fate ... 40

Bibliography ... 42

Research article ... 64

Abstract ... 65

Introduction ... 66

Results ... 68

1. Dynamic distribution of mitochondria in growing axons of RGCs ... 68

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Figure 1: dynamic distribution of mitochondria in growing axons ... 69

Figure S1: mitochondria detection ... 71

Figure S2: mitochondria dynamics in axons ... 73

2. Apical accumulation of mitochondria at the onset of neurogenesis ... 73

Figure 2: apical accumulation of mitochondria ... 75

3. Mitochondria relocate in newborn RGCs ... 76

Figure 3: dynamic redistribution of mitochondria in newborn RGCs ... 77

4. Mitochondria in the RGC lineage ... 78

Figure 4: mitochondria in pre-committed progenitors and in cells committed to the RGC fate ... 80

5. HES5.3 decreases the number of active mitochondria in pre-committed progenitors ... 81

Figure 5: MitoDsRed2-labelled mitochondria content vs mitochondria count in Hes5.3+ pre- committed progenitors ... 83

Figure S3: mitochondria in Hes5.3+ progenitors ... 85

6. Active mitochondria in cells that misexpress Atoh7 ... 86

Figure 6: ATOH7 and NGN2 promote the accumulation of MitoDsRed2-labelled mitochondria ... 87

7. Mitochondria content decreases as development proceeds ... 88

Figure 7: mitochondrial content in the chick and pigeon retinas ... 89

8. Neurogenesis is associated with changes in the concentration of metabolites ... 90

Figure 8: in vivo 1H-NMR for measuring metabolite concentrations in the chick and pigeon eyes ... 92

Figure S4: 1H-NMR for measuring metabolite concentrations in the chick and pigeon eyes ... 94

Figure 9: model of mitochondria activity during retina development ... 96

Discussion... 97

Different metabolic profiles underlay the production of RGCs in chick and mouse retinas ... 97

Metabolic shift between uncommitted and pre-committed retinal progenitors ... 99

Metabolic remodeling: mitochondrial mass vs. mitochondrial activity ... 100

Materials and methods ... 102

Animals ... 102

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Reporter and expression plasmids ... 102

MitoTracker ... 102

Retina electroporation ... 102

Tissue culture ... 103

Tissue dissociation ... 103

Fluorescence Activated Cells Sorting ... 103

RT-qPCR and mt-DNA quantification by qPCR ... 103

Confocal imaging ... 103

Time-Lapse imaging ... 104

Transmission Electron Microscopy (TEM) ... 104

Image processing and morphometry ... 104

In vivo 1H-NMR spectroscopy (1H-MRS) ... 105

High resolution 1H-NMR spectroscopy ... 105

Acknowledgements ... 107

Funding ... 107

Authors contribution ... 107

Supplementary material ... 108

Movie S1... 108

Movie S2... 108

Table S1 ... 109

References ... 110

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Aknowledgements

I am grateful to my thesis director, Dr. Jean-Marc Matter, for giving me the opportunity to complete my thesis project in his laboratory. I thank him for his availability to discuss my results and for his precious advices in heading my project in the right direction. Under his supervision, I had the opportunity to develop my scientific thinking and to develop my skills in molecular technics.

I want to thank the members of my thesis committee, Prof. Anthony Holtmaat, Prof. Jean-Claude Martinou and Prof. Stephan Neuhauss for reading and evaluating my work. I also want to thank them for their kindness and for their challenging questions that helped me to improve my work.

I thank Dr. Lidia Matter-Sadzinski for her precious advices and the attentive and rigorous reading of my thesis manuscript. I thank Dr. Tania Rodrigues for the help with experimental technics and for the discussion and advices. I thank Dr. Florence Chiodini for her help at the beginning of the project.

I am grateful to Antoine Cherix for the collaboration and for his expertise with the MRI spectroscopy experiments.

I am grateful to Dr. Christoph Bauer and Dr. Jérome Bosset from the bioimaging center of the University of Geneva for their teaching and help for live imaging, confocal and electron microscopy experiments. I am grateful to Mike Parkan for his help with computer and softwares for microscopy data analysis. I am grateful to Jean-Pierre Aubry, Cecile Gameiro and Grégory Schneiter for the help with flow cytometry experiments. I am grateful to Dr. Mylene Docquier and Dr. Didier Chollet for the help with qPCR and RTqPCR experiments.

Finally, I want to thank my family and friends for their support, in particular my parents, Micheline and Géry Brodier, and my partner Amanda Guerreiro, who encouraged me and who gave me confidence and the will to succeed.

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Résumé de thèse en français

La régulation du métabolisme durant le développement embryonnaire du système nerveux central est peu connue car elle est difficile à suivre avec une bonne résolution spatio-temporelle. La rétine est un tissu sensible à la lumière qui tapisse le fond de l’œil. Son activité requiert beaucoup d’énergie et les cellules ganglionnaires rétiniennes (RGCs) dont les axones forment le nerf optique ont une consommation en ATP qui se situe parmi les plus élevée des cellules de l’organisme. Diverses pathologies du nerf optique sont liées à des troubles métaboliques et à des altérations de l’activité des mitochondries. Les différentes étapes de la différentiation des RGCs sont connues et identifiables par des marqueurs génétiques spécifiques. Cela fait de la rétine un modèle de choix pour l’étude des changements métaboliques en lien avec la neurogène.

Dans ce travail nous montrons comment ATOH7, un facteur de transcription nécessaire à la production des RGCs, influence l’activité des mitochondries durant la conversion de cellules progénitrices pré- spécifiées en RGCs différentiées.

La neurogenèse commence plus tard dans la rétine de pigeon que dans celle du poulet, ceci nous permettant de faire la distinction entre la croissance de la rétine et la neurogenèse proprement dite.

Grâce à une comparaison inter-espèces, nous avons pu démontrer que l’accumulation des mitochondries au pôle apical des cellules de l’épithélium rétinien coïncide précisément avec l’initiation de la neurogenèse. L’imagerie en temps réel nous a permis d’associer l’accumulation des mitochondries aux étapes initiales de la différentiation des RGCs. En effet la localisation apicale des mitochondries commence dans les RGCs nouvellement spécifiées qui expriment Atoh7 à un niveau élevé, jusqu’à leur migration vers la surface basale pour former la couche des cellules ganglionnaires (GCL). A ce moment, les mitochondries sont redistribuées dans le soma, puis entrent dans l’axone lorsque la cellule achève sa migration.

En contradiction avec l’idée communément admise que les cellules en prolifération utilisent principalement la glycolyse et ont un nombre réduit de mitochondries, nous avons découvert que les cellules progénitrices non-spécifiées de la rétine aviaire contiennent une grande quantité de mitochondries actives. L’activité mitochondriale ainsi que la quantité d’ADN mitochondrial diminuent graduellement lors de la neurogenèse. Plusieurs indices indiquent que la rétine devient glycolytique.

L’analyse comparée, in vivo, de métabolites dans l’œil embryonnaire de pigeon et de poulet par spectrométrie du proton par résonnance magnétique a révélé que la neurogenèse s’accompagne d’une accumulation de lactate dans le corps vitré. En parallèle, nous observons la stimulation de l’expression du gène de la phosphofructokinase dans la rétine. Nous avons constaté que l’activité des mitochondries, mais pas leur nombre, est fortement réduite dans les cellules pré-spécifiées qui expriment Atoh7 à un bas niveau et un autre facteur de transcription, HES5.3. Cette situation dure

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seulement quelques heures car dès que les cellules expriment Atoh7 à un niveau élevé et sont spécifiées en RGCs, elles récupèrent un nombre élevé de mitochondries actives, comparable à celui mesuré dans les cellules progénitrices non-spécifiées. Nos résultats suggèrent que les RGCs se distinguent des autres types de cellules rétiniennes par un contenu en mitochondries élevé. Une phosphorylation oxydative élevée dans les axones des RGCs pourrait être la cause de la diminution de citrate dans le corps vitré.

Nous démontrons que l’expression de Hes5.3 est à elle seule suffisante pour diminuer l’activité des mitochondries dans les cellules progénitrices rétiniennes, et que la différentiation en RGC requiert le passage par l’état de pré-spécification définit par cette expression de Hes5.3. En résumé, nous avons identifié des changements d’activité métabolique étroitement lié aux différentes étapes qui jalonnent la conversion de cellules progénitrices non-déterminées en neurones rétiniens, en particulier, les RGCs.

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Introduction

Metabolism in the developing retina

The retina is part of the central nervous system (CNS) and represents a good model for the study of neurogenesis. The tissue is easily accessible and, contrary to other CNS regions, its development is highly independent. The retina develops as an outpocketing of the neural tube and its development does not involve extensive cell migration. Indeed, all cells that compose the adult retina derive from the same pool of progenitors present in the embryonic retina (Cepko, 2014). This unique feature is convenient for explant culture that develops highly similar to in vivo retina (Caffe et al., 1989; Sparrow et al., 1990). Therefore, retina explants are particularly well suited for live imaging studies. Another good illustration of the independence of retina development is that a bilayered optic cup with proper cell types and correct organization can form in vitro from cell aggregates (Eiraku et al., 2011).

Retina development has been studied in numerous vertebrate species, including fish, amphibians, mammals and birds, as well as non-vertebrates like Drosophila. The broad sample of species described makes interspecies comparisons possible. The retina is composed of 6 types of neurons and 1 type of glia with a laminar organization that is well conserved across vertebrates. Despite clear differences between the mammalian lens eye and the Drosophila compound eye, many genes are conserved and many interneurons present in the vertebrate eye are displaced in cerebral structures in Drosophila, but conserve a common general connection and organization plan (Sanes and Zipursky, 2010). Across vertebrates, disparities arise mainly in the ratio of the different cell types (e.g. rod and cones ratio, or photoreceptors to ganglion cells ratio), and they are responsible for interspecies differences in visual acuity (Hoon et al., 2014; Williams and Moody, 2003). Study of retina development is essential in order to understand how regulation of neurogenesis can produce these differences.

The retina is thought to recapitulate many features of cortical development. Its limited classes of neurons and relatively simple organization makes the retina a particularly suitable model for developmental studies. Neurogenesis in the retina is relatively well understood and the retina cell types are well described. The proliferating progenitors generate all cell types in a sequential and overlapping manner. Many transcriptions factors involved in retina neurogenesis in vertebrates, like homeobox or basic helix-loop-helix (bHLH) transcription factors, have Drosophila homologs and a lot of our knowledge comes from Drosophila studies. The studies that looked at bHLH dynamic expression and that uncovered genetic regulation of the production of different cell types ensure that we dispose of convenient markers for all steps of neurogenesis. These are valuable tools in order to study events with a high temporal resolution. Moreover, high heterogeneity of commitment progression at a given time requires the use of precise markers in order to fix our result in a comparable time window of differentiation.

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An aspect of retinal development that has not been much studied is metabolism. The retina and particularly the retinal ganglion cells (RGCs) that form the optic nerve are among the largest energy consumer of the whole body (Wong-Riley, 2010). Indeed, metabolic diseases often affect vision and mitochondria have been linked to many eye diseases (Jarrett et al., 2010; Wong-Riley, 2010). Dividing and differentiated cells have very different metabolic requirements that match specific needs for a given cell state. Very little is known about the metabolic transition that occurs during neurogenesis when the cell progresses from a proliferating progenitor with primarily anabolic needs, to an adult neuron that has to be energy efficient. The particular energy requirement of the eye, notably RGCs, as well as the numerous associated diseases makes the retina a good choice for study of metabolism.

Indeed, understanding the metabolic requirements at different stages of retina development is critical to determine the exact part the mitochondria dysfunction plays in many eye diseases.

Hypothesis and aim of the project

In this study, our goal is to describe mitochondria dynamics and regulation during neurogenesis in the developing retina.

Mitochondria are actively transported by kinesin and dynein molecular motors along microtubules, and their position reflects local energy demands (Cai and Sheng, 2009). Particular events occur at specific position relative to the apico-basal axis of the neuroepithelium: progenitors undergo interkinetic nuclear migration (INM) with mitosis always occurring on the apical surface, and differentiating RGCs pause apically after the last mitosis prior to basal migration. Moreover, RGC become polarized as they extend apical dendrites and basal axon (Chiodini et al., 2013). Our first aim is to describe mitochondria dynamic localization in the embryonic retina as well as in RGCs as they become committed and differentiate. We want to identify specific pattern of behavior that would reflect changes in energy requirement in different cellular compartments during neurogenesis.

Adult RGCs demand high amount of energy, and vision on the whole requires continuous action potential propagation. These cells have relatively long axons that grow on the vitreous surface of the retina and exit the eye to form the optic nerve. RGC axons are not myelinated until they exit the eye in order to avoid light scattering, thus requiring more ATP for each action potential (Andrews et al., 1999; Bristow et al., 2002; Wong-Riley, 2010). Moreover, axon extension involves actin and microtubule polarization as well as coordinated responses to different cues from the environment, processes that also require ATP. Given this high energy needs, we hypothesize that RGC axons require the presence of a high mitochondria density. We want to follow mitochondria dynamics in the growing axon by time-lapse imaging, in order to describe characteristics of their movement as well as the general density. We hope to understand how the growing axon ensures continuous supply of mitochondria and how it achieves correct repartition. In addition, trafficking defect can be the cause

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of optic nerve neuropathies, as mitochondria mislocalization could lead to lack of ATP in distal axonal compartment. Describing their normal movements is the first necessary step toward a model of such defects.

Progenitors and postmitotic cells have very different metabolic needs. We want to address the question of energy production and mitochondria requirement during neurogenesis, when the cells transit from proliferating progenitors to adult neurons. We want to understand how cells cope with the different metabolic requirements underlying neuronal differentiation, and what are the mechanisms that regulate mitochondria activity and biogenesis. We look at mitochondria content during eye development and RGC differentiation, in order to correlate mitochondria amounts with particular cellular states. We also want to check the current idea that committed cells rely highly on mitochondria respiration for efficient energy production, while progenitors are largely glycolytic. If this is true for the retina, we should see an increase of mitochondria in adult RGCs. A study suggested that mitochondria decreases prior to RGC commitment and that a peak of glycolysis is required for RGC fate (Esteban-Martinez et al., 2017). Our goal is to precisely define the amount of mitochondria at the different time points and at key steps of RGCs production. Atoh7 (atonal homolog 7, also known as Math5 in mice, Cath5 in birds, and Ath5 in amphibians and fish) is a bHLH transcription factor that is required for RGC production. Its expression at low level coincides with expression of Hes5.3 (homolog of hairy and enhancer of split 5.3) and defines a pre-committed state. Cells that up-regulate Atoh7 are committed to the RGC fate and express RGC markers like Chrnb3 (Chiodini et al., 2013;

Matter-Sadzinski et al., 2001; Matter-Sadzinski et al., 2005). We plan to analyze mitochondria content with high spatiotemporal resolution in different populations that express specific markers using a combination of fluorescence imaging and mitochondrial DNA quantification.

Finally, we want the address the question of how factors involved in neurogenesis regulate mitochondria content. We try to correlate action of factors that are known to promote cell cycle exit and RGC fate with mitochondria production. We want to test the effect of Atoh7, and Hes5.3 as well as other factors on mitochondria content and expect to uncover a relationship.

Summary of results

We started by investigating mitochondria in committed RGCs, and found that their axons growing on the basal surface of the retina are characterized by a high density of mitochondria. We describe how mitochondria movements and dynamics are defined by slow-moving, fast-moving or arrested organelles with predominant anterograde transport, and are coordinated to ensure a homogenous distribution.

We then focused on mitochondria distribution in the developing retina neuroepithelium. Comparing by fluorescence confocal imaging and transmission electron microscopy (TEM), mitochondria

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distribution in chick and pigeon retinas that initiate neurogenesis at different times, we found a strong apical accumulation of mitochondria that precisely coincides with the onset of neurogenesis.

Using time-lapse imaging, we found that mitochondrial apical localization is associated with initial steps of RGC commitment. We illustrate how mitochondria accumulate in the apical endfoot of newly committed RGCs that up-regulate Atoh7 prior to the last mitosis, a position where they remain in postmitotic RGCs that arrest apically for 15 hours. Finally, mitochondria are redistributed to the soma as RGCs migrate basally to establish the ganglion cell layer (GCL), and into the axon as cells reach their destination.

At odd with the idea that proliferating cells are mainly glycolytic with few mitochondria, we found that uncommitted proliferating progenitors display very high level of active mitochondria and high ratio of mitochondrial DNA (mt-DNA) to genomic DNA (gDNA). As neurogenesis proceeds, these levels gradually decrease, and evidences suggest that the retina becomes more glycolytic. We used 1H Magnetic Resonance Spectroscopy (MRS) to quantify metabolites in chick and pigeon retinas in vivo, and found that accumulation of lactate in the vitreous body coincides with initiation of neurogenesis.

At the same time, expression of the glycolytic enzyme phosphofructokinase increases in retina.

Interestingly, while mitochondria activity suddenly drops in pre-committed progenitors expressing Hes5.3 without decline of mitochondrial mass, activity is restored to the same proportion found in uncommitted progenitors as the cells up-regulate Atoh7 and commit to the RGC fate. Thus, mitochondrial content is high in both uncommitted progenitors and RGCs compared to other retina cell types. A high rate of oxidative phosphorylation in RGC axons could explain decreased citrate concentration in vitreous body.

Finally, we demonstrate that Hes5.3 expression alone is sufficient to decrease mitochondria activity in retinal progenitors. Moreover, we show that decreasing Atoh7 expression in pre-committed progenitors by overexpression of its negative regulator Hes1, or overexpression of Atoh7 are both sufficient to increase mitochondrial activity, suggesting that the drop of activity occurs specifically in pre-committed progenitors expressing low level of Atoh7 and high level of Hes5.3.

Our results demonstrate how Atoh7, a bHLH transcription factor essential for RGC production, influences mitochondria activity during its different phases of expression leading to commitment and RGC differentiation.

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Literature review Retina development

Eye field formation occurs in the specified anterior forebrain

Eye development starts in the forebrain, by specification of the eye field. Different levels of Wnt signaling and Wnt antagonists define the antero-posterior axis in the forebrain with the most anterior structures comprising telencephalon and eye field requiring low Wnt signaling. The most anterior forebrain structures diverge from the diencephalon that forms more caudally at higher Wnt levels.

Wnt antagonists are secreted from the anterior border of the neural plate (ANB) and are essential for establishing anterior identity (Wilson and Houart, 2004). In zebrafish, Tlc a member of secreted Frizzeled Related Protein (sFRP) known to antagonize Wnt is secreted by the ANB and is required for telencephalic and eye field fate (Houart et al., 2002). Similarly, zebrafish masterblind (mbl) mutant that harbors a mutation in Axin1, an intracellular inhibitor of Wnt pathway, shows reduced or absent telencephalon and eye field accompanied by expansion of the diencephalon (Heisenberg et al., 2001).

These data suggest that negative regulation of Wnt is required for anterior forebrain ontogenesis.

However the eye field requires fine tuning of Wnt, as suggested by the fact that increased expression of Wnt antagonists causes more telencephalic markers expression at the expense of the eye field, while increased Wnt causes extended diencephalon and reduced eye field (Houart et al., 2002;

Stigloher et al., 2006; Wilson and Houart, 2004). Similarly, in-vitro studies suggest that formation of Rx (retinal homeobox protein) positive optic vesicle-like structures from embryonic stem cells (ESCs) necessitates at least some level of Wnt (Sakakura et al., 2016). While canonical Wnt/β-catenin signaling inhibits eye fate and promotes diencephalon mainly through Wnt8b and Fz8a, non-canonical Wnt signaling mediated by Wnt11 and Fz5 promotes eye fate, partly by antagonizing canonical Wnt signaling in zebrafish (Cavodeassi et al., 2005). Another link between non-canonical Wnt and eye specification comes from Xenopus, where Wnt4/Fz3 non-canonical Wnt signaling acting via JNK can induce ectopic eyes and is required for eye formation as well as for maintenance of eye field genes Rx and Pax6 (Maurus et al., 2005).

It has been proposed that neural induction in the absence of caudalizing factors like Wnt, is sufficient to produce anterior neural identity. Neuronal induction is thought to be caused mainly by fibroblast growth factors (FGF). Furthermore, repression of Wnt in the anterior forebrain allows for Fgf3 and Fgf8 expression in ANB. Another factor that negatively influences anterior neural fate is bone morphogenetic protein (BMP), and presence of BMP antagonists like Chordin and Noggin in the anterior forebrain promote anterior neural development (Bachiller et al., 2000; Houart et al., 2002;

Wilson and Houart, 2004). A threshold of BMP is required anteriorly in the prospective telencephalon for inhibition of eye field fate (Bielen and Houart, 2012). BMP-4 has been shown to inhibit retina fate

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in Xenopus blastomere, while BMP antagonists promote retina (Zaghloul et al., 2005). Moreover, proper expression of the Wnt antagonist Tlc in ANB requires precise but low levels of BMP signaling and is abrogated when there is too few or too much BMP, thus linking low BMP levels to rostral production of Wnt antagonist (Houart et al., 2002). In addition, low level of Wnt induces Six3 expression, a transcription factor required for forebrain and eye field specification that in turn inhibits Wnt, reinforcing rostral identity. Six3 mediated inhibition of Wnt1 is required for telencephalic fate (Lagutin et al., 2003), and a reciprocal inhibition exists between Six3 and Wnt. Indeed, Wnt has been shown to inhibits Six3 expression and to promote Irx3 expression caudally to form posterior forebrain (Braun et al., 2003; Lagutin et al., 2003). Finally, Six3 and Irx3 undergo reciprocal inhibition and define regionally distinct competence that affects tissue response to FGF and sonic hedgehog (Shh), in order to confer anterior and posterior identities respectively (Kobayashi et al., 2002).

Eye field transcription factors expression

During subsequent anterior forebrain patterning, the eye field diverges from cells that will give birth to the presumptive telencephalon. The eye field is characterized by strong expression of eye field transcription factors (EFTF), including Rx genes, Pax6, Six3 and Lhx2 (Bailey et al., 2004; Graw, 2003;

Mathers and Jamrich, 2000; Stigloher et al., 2006). Rx genes play a central role in vertebrate eye formation, and Rx null mutants are characterized by anophthalmia or severe microphthalmia (Bailey et al., 2004; Mathers et al., 1997). Rx is activated by Otx2, in the absence of Wnt signaling and is able to induce expression of others EFTF (Bailey et al., 2004; Mathers and Jamrich, 2000). Interaction of Otx2 with Sox2 is required to induce Rx transcription in the Xenopus retina. Indeed, Otx2 can induce both Sox2 and Rx expression, while Sox2 alone has no effect on either Otx2 or Rx, implying that Otx2 is the main regulator of Rx expression in presumptive eye field (Danno et al., 2008). The ability of Rx to activate other EFTF suggests an upstream role of Rx. Indeed, Rx is necessary for Pax6 and Six3 activation, while Rx is independent of Pax6 for its expression, as Rx expression is not changed in Pax6 null embryos (Zhang et al., 2000). However, the precise role of each EFTF is difficult to assess because many of these factors undergo complex cross-regulatory interactions and are usually able to activate each other inside the Otx2 permissive domain (Loosli et al., 1999; Mathers and Jamrich, 2000). This is why many overexpression studies reporting the ability of one particular EFTF to induce ectopic eye may only reflect an effect of cross activation. A good illustration is the overexpression Pax6 that can induce formation of ectopic eyes. However, Pax6 overexpression also activates Rx, despite the fact that Rx does not require Pax6 for activation during normal development (Bailey et al., 2004). Some studies suggest that Rx may not be upstream of other EFTF during normal development, but is instead required for maintenance of these factors in later proliferating retinal progenitor cells (RPC). Even though Rx direct role for initial EFEF activation is still unclear, Rx remains necessary for their expression, possibly because of its role in blocking telencephalon and diencephalon expansion into presumptive eye field (Fish et al., 2014). Accordingly, in zebrafish, Rx3 is required to direct cells of the

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presumptive eye field into a retinal fate, as in the absence of Rx3 these cells adopt a telencephalic fate, and expansion of Tlc and the telencephalon markers foxg1 and emx3 into presumptive retina is observed (Stigloher et al., 2006). Similarly, in Xenopus, Rx is required to downregulate Hesx1 and fezf2, and to avoid the presumptive retina to adopt a telencephalic fate (Fish et al., 2014).

Figure 1: network of transcription factors that establish the eye field (Heavner and Pevny, 2012).

Sonic hedgehog splits the eye field

The eye field forms initially as a unique central domain in the anterior forebrain that later separates into two lateral optic vesicles. Splitting of the eye field is thought to be caused by a combination of cell movements and Shh signaling (Wilson and Houart, 2004). In zebrafish, cells expressing opl (odd- paired-like), a marker of retinal precursor cells, initially form a single domain that includes the midline, and tracing of cells positioned inside this domain reveals that they all contribute to eyes (Varga et al., 1999). Subsequent rostral movement of the ventral diencephalon, i.e. the prospective hypothalamus located posterior to the eye field, separates the unique medial eye field into distinct lateral domains.

Absence of such movement, as observed in zebrafish cyclops mutant, causes failure to split and produces a single optic vesicle (England et al., 2006; Varga et al., 1999). A similar movement has been reported in chick, and probably also occurs in other vertebrates (Dale et al., 1999; Varga et al., 1999).

In Xenopus, ET and Pax6 are expressed in the eye field and initially form a unique domain including the medial region that later splits in two primordia. However unlike in zebrafish, Xenopus midline cells do not seem to contribute to eyes, suggesting absence of lateral movement (Li et al., 1997).

Shh is required for eye field splitting, and embryos lacking Shh form a single central optic vesicle, resulting in cyclopia and holoprosencephaly phenotype (Chiang et al., 1996; Wilson and Houart, 2004).

Shh is expressed in prechordal plate and ventral midline of the neural tube (Dale et al., 1999; Varga et al., 1999), and splitting requires the prechordal plate that inhibits retinal fate and Pax6 expression in the central region in both Xenopus and chick (Li et al., 1997). Failure to split in Shh mutant can in part be explained by ventralization defects, since zebrafish cyclops mutants lacking Shh fail to form ventral diencephalon structures (Varga et al., 1999). However, strong evidences suggest that Shh also directly inhibits retina fate in central region, and a combination of direct inhibition and cell movements is likely to occur. In chick, absence of Shh from the prechordal plate causes improper ventralization of the

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neural tube, including ventral diencephalon (Pera and Kessel, 1997), and Shh is required for patterning of ventral midline cells of the neural tube (Dale et al., 1999). Shh secreted from the ventral midline induces Pax2 and Vax genes expression and decreases Rx and Pax6 in regions adjacent to the midline, thus promoting optic stalk versus retina identity of medially positioned eye field cells (Yang, 2004).

Similarly, mice lacking Shh lose Pax2 expression and do not form an optic stalk, a phenotype accompanied by loss of ventral forebrain structures (Chiang et al., 1996). Accordingly, high levels of Shh are present between the optic vesicles in chick embryo, and Shh expression continues later in the forebrain region close to the optic stalk at the base of the optic vesicles and optic cup (Zhang and Yang, 2001b).

Optic vesicle evagination

The two eye fields evaginate from the ventral forebrain to form the optic vesicles. Rx is required for the cell movements necessary for optic vesicle evagination (Rembold et al., 2006; Stigloher et al., 2006), and accordingly Rx mutants fail to form optic vesicles (Mathers et al., 1997).

In zebrafish, Rx regulates Eph/Ephrin dependent active segregation of eye field cells to ensure their clustering into a domain separated from other anterior neural plate (ANP) regions. Interfering with Eph/Ephrin signaling results in intermixing of cells with distinct identities and in optic vesicle evagination defects, suggesting that Rx dependent pre-sorting of eye field cells and their segregation from other ANP domains during ANP morphogenesis is essential for proper evagination (Cavodeassi et al., 2013). Similarly expression of Cxcr4a, an adhesion molecule involved in correct segregation of eye field cells from telencephalon is activated by Rx in the zebrafish eye field and is repressed by BMP in telencephalon (Bielen and Houart, 2012). Despite morphogenetic differences between fish and mammals, Rx is also required cell autonomously for optic vesicles formation in mice, and chimera showed that Rx deficient cells cannot participate in retina, RPE or optic stalk formation, and that a pre-sorting of Rx positive cells occurs prior to evagination (Medina-Martinez et al., 2009).

In fish and frogs, the force generated to drive optic vesicle evagination involves individual cell movements (Fuhrmann, 2010). In medaka, Rx positive RPCs in the lateral eye fields first converge to the midline from where they migrate ventrally then laterally, pushing ventromedial RPCs laterally and contributing to optic vesicles evagination. Rx influences migratory proprieties of RPCs, as lateral RPCs adopt the behavior of forebrain cells and remain in the midline in the absence of Rx, resulting in the absence of evaginated optic vesicles (Rembold et al., 2006). Similar movements are also observed in zebrafish, where Rx affects RPC migration and optic vesicles evagination by downregulating Nlcam, an IG-domain cell adhesion molecule, resulting in slower midline convergence of lateral RPCs compared to neighboring telencephalic precursors that express high levels of Nlcam (Brown et al., 2010).

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In zebrafish, cell proliferation only partially contributes to the increase in cell number and size of the optic vesicles, and migrating cells entering the vesicles largely participate in expansion. Furthermore, cell proliferation is not required for optic vesicles formation, as blocking proliferation only affects optic vesicles size but not morphogenesis in zebrafish (Kwan et al., 2012) and Xenopus (Harris and Hartenstein, 1991).

In zebrafish, apico-basal polarization and epithelialization of the eye field occurs prior to adjacent regions of the neural plate and coincide with optic vesicles evagination. Indeed, Laminin1 is first detected basally in evaginating optic vesicles, and causes basal cells to elongate and arrange into a pseudostratified epithelium before such organization can be detected in surrounding regions. Then, apical cells displaying a round mesenchymal morphology elongate and integrate within the basal epithelized domain, contributing to evagination (Ivanovitch et al., 2013). Thus, epithelialization could be a prerequisite and lead to optic vesicles evagination. Consistently, downregulation of Laminin1 and the polarity protein pard6γb interferes with epithelialization and optic vesicles morphogenesis (Ivanovitch et al., 2013). EFTF are likely to play a role in this precocious epithelialization, as suggested by the fact that the polarity protein pard6γb is no longer detected earlier in the eye field compared to the rest of the neural plate in the absence of Rx (Ivanovitch et al., 2013). The process is less studied in mammals and birds. Nonetheless, in vitro formation of optic vesicle from 3D culture of mice ESCs requires matrigel that is rich in extracellular matrix (ECM) proteins and Laminin1 (Eiraku et al., 2011;

Kwan, 2014).

Optic vesicle patterning

During evagination, the optic vesicle is partitioned into distinct domains. The distal optic vesicle forms the presumptive neuro-retina, while the dorsal part differentiates into presumptive retinal pigment epithelium (RPE), and the ventral part into optic stalk. The neuroretina is characterized by Vsx2 (formerly Chx10) expression, the RPE by Mitf expression and the optic stalk by Pax2 expression (Fuhrmann, 2010).

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Figure 2: signaling networks involved in optic vesicle patterning (Heavner and Pevny, 2012).

The EFTF Lhx2 is required for optic vesicle regionalization and in mutants, eye development arrests prior to optic cup formation (Porter et al., 1997). In mice, Lhx2 is required cell autonomously for the expression of Vsx2 and Mitf that are essential for neuroretina and RPE identities respectively (Yun et al., 2009). In addition, Lhx2 acts via activation of BMP signaling (Bmp4, Bmp7) to influence dorso- ventral patterning of the optic vesicle and to induce the surface ectoderm into lens. Lhx2 dependent ventral expression of Vax2 that represses Pax6 ventrally is abrogated in mutant, resulting in increased Pax6 domain and reduced Pax2 expression. Accordingly, BMP addition in Lhx2 mutant is sufficient to rescue Sox2 expression in the surface ectoderm and ventral optic vesicle expression of Pax2, but not Vsx2 and Mitf (Yun et al., 2009).

Signals from the induced surface ectoderm play a major role for optic vesicle regionalization.

Consistently, optic cup do not form in the absence of lens placode (Hyer et al., 1998). In mice, Mitf is initially expressed in the whole optic vesicle, and is later downregulated in the presumptive neuroretina while it remains expressed in the presumptive RPE (Fuhrmann, 2010; Hodgkinson et al., 1993; Nguyen and Arnheiter, 2000). FGF1 and FGF2 secreted from the surface ectoderm are responsible for Mitf inhibition in the distal optic vesicle, and Mitf downregulation is sufficient to induce RPE to neuroretina conversion (Nguyen and Arnheiter, 2000). Activation of the MAPK FGF pathway in chick is also able to induce transdifferentiation of RPE into retina, and blocking FGF inhibits neuroretina formation (Pittack et al., 1997). FGF repress Mitf via Vsx2 activation, as Mitf is ectopically expressed in the neuroretina in the absence of Vsx2 (Horsford et al., 2005; Rowan et al., 2004), and FGF cannot induce RPE to neuroretina conversion in Vsx2 mutant (Horsford et al., 2005). Similarly, oj-

/- mice that lack Vsx2 show transdifferentiation of the retina into RPE together with activation of RPE genes, a phenotype that can be rescued by Vsx2 expression (Rowan et al., 2004). Moreover, chromatin immunoprecipitation (ChIP) showed that Vsx2 binds Mitf promoter, suggesting a direct inhibition

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(Bharti et al., 2008). Antagonistic relationship between Vsx2 and Mitf defines the border between neuroretina and RPE, as loss of function of Mitf causes conversion of RPE to neuroretina, and loss of Vsx2 causes increased Mitf and expansion of RPE at the expense of neuro retina (Bharti et al., 2008;

Heavner and Pevny, 2012; Horsford et al., 2005). Moreover, phenotype of Mitf or Vsx2 mutant is partially rescued in double mutant (Horsford et al., 2005).

In chick, Mitf is not initially expressed in whole optic vesicle, but restricted to presumptive RPE (Mochii et al., 1998). Nevertheless, FGF1 is also required for neuro retina specification in the chick, and is able to induce neuroretina in the presumptive RPE domain (Hyer et al., 1998). In mice, removal of the surface ectoderm as source of FGF causes the distal optic vesicle to retain Mitf expression, to lose Vsx2 and to form RPE instead of neuroretina, a phenotype that can be rescued by FGF addition (Nguyen and Arnheiter, 2000). In chick, removal of the surface ectoderm together with FGF1 addition causes the optic vesicle to produce neuroretina always close to the source of FGF. Likewise, addition of FGF1 in the mesoderm close to RPE causes the neuroretina and RPE domain to be inverted (Hyer et al., 1998). In addition, FGF production in the neuroepithelium itself cannot be excluded. Indeed, FGF9 expressed in distal optic vesicle also promotes retina fate by Vsx2 activation, and FGF9 deletion causes RPE expansion into neuroretina domain (Fuhrmann, 2010; Heavner and Pevny, 2012). Moreover, overexpression of FGF9 in mice RPE leads to transdifferentiation into neuroretina (Zhao et al., 2001).

At the same time, Otx2 is down-regulated in the neuroretina domain of the optic vesicle but persists in the presumptive RPE. Otx1 and Otx2 follow the same pattern of expression as Mitf and are important for Mitf induction, as loss of both causes RPE to neuroretina conversion (Fuhrmann, 2010;

Heavner and Pevny, 2012; Martinez-Morales et al., 2003; Martinez-Morales et al., 2004; Martinez- Morales et al., 2001). Moreover, Otx2 participates with Mitf to activate RPE genes, and both proteins interact in vitro (Martinez-Morales et al., 2003). Rx is also required for RPE specification, as it plays a permissive role allowing the presumptive RPE to respond to signals from the extraocular mesenchyme (Rojas-Munoz et al., 2005) that are required for Mitf expression and RPE specification (Fuhrmann, 2010). In cultured chick explants, removal of extraocular mesenchyme causes a loss of Mitf and other RPE markers (Wnt13, MMP115) as well as expansion of Vsx2, Pax6 and Optx2 into the whole vesicle.

The TGF-β family member Activin produced in the extraocular mesenchyme is a possible effector, as its addition increases expression of RPE markers and downregulates neuroretina markers (Fuhrmann et al., 2000). Wnt/β-catenin signaling in the presumptive RPE is required for RPE fate, as inactivation of β-catenin in RPE causes loss of Mitf and Otx2, expansion of Vsx2, and loss of the ability to form optic cup in mice (Hagglund et al., 2013; Westenskow et al., 2009). Wnt/β-catenin is activated in RPE of fish, chicks and frogs (Hagglund et al., 2013) and is required in 3D ESC culture for RPE formation (Eiraku et al., 2011). Six3 expressed in neuro retina inhibits Wnt (Liu et al., 2010).

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Shh has a role in ventralization of the optic vesicle and in optic stalk formation. Pax6 and Pax2 show reciprocal inhibition and establish boundary between neuroretina and optic stalk. Pax2 deletion causes Pax6 and neuroretina expansion into presumptive optic stalk, and Pax6 deficient embryos fail to develop neuroretina and RPE, and show Pax2 and optic stalk expansion (Heavner and Pevny, 2012;

Schwarz et al., 2000; Torres et al., 1996). However, Pax2 and Pax6 domains are not always exclusive, since they are initially co-expressed in the whole optic vesicle like Mitf, and become restricted to optic stalk and neuro retina respectively at a later time, when Mitf decreases in the presumptive neuro retina. Pax2 and Pax6 are redundant for Mitf activation, as both can bind and activate Mitf promoter in vitro, and the double mutants lose Mitf expression and show co-expression of neuroretina marker Vsx2 with RPE marker Otx2 (Baumer et al., 2003). Finally, maintaining Pax6 expression in the optic stalk, i.e. the normal Pax2 expression domain, when Pax6 expression is normally downregulated, results in ectopic Mitf induction and transformation of presumptive optic stalk into RPE (Baumer et al., 2003; Schwarz et al., 2000).

Optic cup formation

Optic vesicles eventually contact the surface ectoderm that becomes induced by this contact and develops the lens placode that will invaginate to form the adult lens. Reciprocal induction in turn causes the optic vesicle to invaginate and to form a bilayered optic cup, from which the inner layer will develop into neural retina while the outer layer will form RPE. Ventrally, the external layer in the most proximal part of this optic cup will turn into the optic stalk (Fuhrmann, 2010).

Figure 3: optic cup formation in vertebrates (Fuhrmann, 2010).

In chick, optic cup invagination does not require presence of lens placode or lens, but requires pre- lens surface ectoderm at earlier stages. The exact role of this ectoderm involves more than solely neuroretina specification, since addition of FGF that promotes neuroretina specification is unable to rescue optic cup formation in the absence of pre-lens ectoderm (Hyer et al., 2003). As well, pressure generated by the developing lens was excluded as the cause for optic vesicle invagination in amphibians (Lewis, 1904, 1907). Moreover, ectopic optic cup can be induced by overexpression of Six3

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without formation of ectopic lens, suggesting two separated morphogenetic events (Loosli et al., 2001). In vitro 3D culture of ESCs can spontaneously form optic vesicle that invaginate into an optic cup without requiring physical presence of any other tissues like lens or mesenchyme, suggesting that internal forces are sufficient for invagination (Eiraku et al., 2012; Eiraku et al., 2011). The distal part of evaginated optic vesicle undergoes a decrease of phosphorylated Myosin Light Chain 2 (pMLC2) when it starts to express the neuroretina marker Vsx2, while pMLC2 remains high in the proximal presumptive RPE region (Eiraku et al., 2012; Eiraku et al., 2011; Hagglund et al., 2013). In epithelium, pMLC2 has been associated with increase tissue rigidity. The relaxation expansion model postulates that decreased pMLC2 in neuroretina causes invagination of the expanding neuroretina since it is more flexible than the proximal RPE with high pMLC2 (Eiraku et al., 2012; Eiraku et al., 2011). Other studies suggested an important role of the ECM protein Laminin. Mice Laminin mutants display disrupted polarity and impaired optic cup morphogenesis (Bryan et al., 2016). The importance of ECM is confirmed by requirement of Matrigel that is rich in Laminin, for the formation of optic vesicle and optic cup in 3D culture (Eiraku et al., 2011). Optic cup folding requires basal constriction (Martinez- Morales et al., 2009), in opposition to neural tube formation where constriction occurs apically (Wallingford, 2005). Medaka opo mutants display RPCs with enlarged basal processes and reduced actin content that are unable to produce basal constriction, resulting in optic cup invagination failure.

The opo protein localizes basally and is required for correct basal localization of focal adhesion proteins integrin-beta1 and paxillin. Interfering with integrin functions causes a similar phenotype, suggesting that integrin adhesive function and focal contacts are necessary to produce basal tension and for optic cup morphogenesis (Martinez-Morales et al., 2009).

The optic cup is able to invaginate without surrounding tissues, however in vivo formation of transient filopodia from lens placode cells that contact the distal optic vesicle basal lamina has been observed in mice. This physical contact is thought to coordinate invagination of the presumptive lens with optic cup invagination, and filopodia formation is dependent upon the Rho small GTPase Cdc42, IRSp53.

IRSp53 mutant shows increased inter-epithelial distance and lens mispositioning (Chauhan et al., 2009). Deletion of focal adhesion kinase (FAK) also impairs filopodia formation, suggesting that integrin adhesion to ECM, a process requiring FAK, is necessary (Chauhan et al., 2009; Partridge and Marcantonio, 2006). On the other hand, the restricted expansion model (Hendrix and Zwaan, 1975) proposes that attachment of the lens placode to the ECM constrains expansion of lens territory and causes the force generated by proliferation in this restricted space to be responsible for lens invagination. This hypothesis is supported by the fact that fibronectin (Fn1), an essential component for ECM assembly, is required to limit expansion of lens placode and is necessary for its thickening and for lens invagination. Pax6 that is required for lens formation also regulates many components of the ECM including Fn1, and accordingly, loss of Pax6 in surface ectoderm results in decreased ECM accumulation, thinner and expended lens placode and defect of lens invagination (Huang et al., 2011).

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At the optic cup stage, Shh present ventrally inhibits BMP4, restricting it dorsally, and Shh and BMP4 are required to establish proper dorso-ventral compartments. BMP4 induces the dorsal marker Tbx4 while Shh is necessary for the ventral determinant Vax. The ventral and dorsal compartments show differential response to altered level of Shh, with reciprocal expansion/reduction of expression domains of the optic stalk marker Pax2, and the neuroretina and RPE marker Pax6, specifically in the ventral region (Zhang and Yang, 2001b).

Retinal progenitor cells of the neuroretina

The vertebrate retina is composed of six types of neurons and one type of glia. All cells that compose the adult retina originate from a single pool of multipotent proliferating progenitors that form a pseudostratified neuroepithelium. Birth order of the different cell types is very well conserved among species, with RGCs always being the first cell type to differentiate, followed at approximately the same time by horizontal cells, cones and amacrine cells, and later by rods and bipolar cells, and finally by Muller glia (Cepko et al., 1996; Livesey and Cepko, 2001). Retinal progenitors must generate each cell type at the right time at the proper position and in appropriate number. Variations in cell type ratio can influence the functional characteristics of the adult eye, and can explain some interspecies differences.

Rx is expressed in undifferentiated retinal progenitor cells where it promotes proliferation by the activation of Six6 (Optx2) and Six3, and inhibits differentiation (Bailey et al., 2004; Li et al., 2002). In Xenopus, overexpression of Xrx results in a higher number of retinal cells (Mathers et al., 1997), and result showing a longer expression of cyclin D1, a marker of proliferating cells, strongly suggests that this effect is due to an increased proliferation and delayed differentiation. This delay in differentiation does not affect the potential of retinal progenitor to produce all cell type (Bailey et al., 2004; Casarosa et al., 2003). Accordingly, in mouse, Mrx downregulation coincides with the loss of proliferation and progressive cell differentiation (Mathers et al., 1997). Finally, Rx has been shown to inhibit X-ngnr-1 that promotes cell differentiation and p27Xic1 a cell cycle inhibitor (Andreazzoli et al., 2003). Vsx2 initially required for neuroretina specification in the optic vesicle, also plays a role in RPCs proliferation, as Vsx2 mutant mice have smaller eyes and reduced RPC proliferation (Burmeister et al., 1996). More recently, it was shown that loss of Vsx2 in mice RPCs induces an increase of the cyclin- dependent kinase inhibitor p27Kip1 that blocks cell cycle progression, and which normally is expressed at high levels only in post-mitotic retinal cells. Moreover, in double mutant of Vsx2 and p27Kip1, the loss of proliferation observed in Vsx2 mutant is rescued. Vsx2 is thought to regulate p27Kip1 through the cell cycle regulator Cyclin-D1 by post transcriptional mechanism (Green et al., 2003). Conditional deletion of Pax6 in mice retina also leads to decrease of RPC proliferation, and Pax6 is thought to be necessary to maintain multipotency since upon Pax6 loss RPCs differentiate only into amacrine cells (Marquardt et al., 2001). The notch-delta pathway is involved in maintaining RPCs undifferentiated via

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expression of the notch effector bHLH Hes1 that acts as a negative regulator of neurogenesis (Jarriault et al., 1995; Nelson et al., 2006; Tomita et al., 1996a). Hes1 normally decreases as RPCs differentiate, and mice retinas where Hes1 expression was prolonged exhibit inhibition of neuronal differentiation, while Hes1 null mutants display premature differentiation and smaller eyes (Tomita et al., 1996a).

Hes1 inhibitory function is conserved in cerebral cortex (Ishibashi et al., 1994). Delta-1 is expressed transiently in newborn neurons and lateral inhibition to adjacent progenitors expressing notch inhibits RPC differentiation and avoid precocious progenitor exhaustion in the chick retina (Austin et al., 1995;

Henrique et al., 1995). Notch signaling is active in RPCs but not in newborn neurons of the chick retina (Austin et al., 1995; Nelson et al., 2006). Altering level of Notch in chick showed that the number of RGCs produced was inversely proportional to the level of Notch signaling (Austin et al., 1995). In Xenopus retina, Notch is also restricted to undifferentiated cells, and expression of activated Notch blocks differentiation (Dorsky et al., 1995). Differentiation is thought to occur as the delta-notch mediated inhibition of neurogenesis is released. By delaying differentiation, Notch enables RPCs to adopt alternate fates and to produce late retinal cell types (Dorsky et al., 1997).

Data imply that RPCs first undergo rapid cell division for progenitor pool expansion, followed by differentiation. Quantitative analysis in rat developing retina suggests that the progenitors first have a higher proportion of symmetrical divisions giving birth to two other progenitors, thus increasing the pool of proliferating progenitors. As development proceeds, there is a change in this proportion in favor of asymmetric divisions generating a progenitor and a post-mitotic neuron (Alexiades and Cepko, 1996; Livesey and Cepko, 2001), like in the cerebral cortex (Chenn and McConnell, 1995; Mione et al., 1997). These variations overtime result in a decrease of proliferating cells, itself accompanied by a lengthening of cell cycle as development proceeds (Alexiades and Cepko, 1996; Livesey and Cepko, 2001). A recent study in zebrafish retinas indicates that until 24 hpf progenitor cells have a very slow cell cycle and their number remains constant (He et al., 2012; Li et al., 2000). This is followed by a wave of proliferation from approximately 24 hpf to 48 hpf, where progenitors rapidly divide and generate more progenitors by symmetric division, as suggested by clone size analysis revealing rare odd clone size, as well as by time-lapse imaging. During a second phase, progenitor cells are thought to stochastically undergo either asymmetric divisions or symmetric divisions (proliferative or differentiative), because the proportion of odd clone size is increased. A stochastic model of equivalent RPCs fits well with the variable clone sizes. Subsequently cells undergo symmetric terminal divisions (He et al., 2012). Likewise, study of RPC clones in culture suggests that the mode of cell division of RPCs in the rat is also largely stochastic (Gomes et al., 2011). Recently, the symmetric terminal division of RGC committed progenitors was demonstrated (Chiodini et al., 2013).

How RPCs generate all cell types of the adult retina is still a matter of debate. One hypothesis is that all RPCs are equivalent and overtime they go through a series of competence states at which they

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produce specific cell types. Transition from one competence state to another could be regulated by intrinsic program or extrinsic factors. Alternative hypothesis is that RPCs are not equivalent and separate very early, each producing distinct cell types. Another possibility is that RPCs would have the ability to produce every cell type at any time and that decision depends exclusively on extrinsic factors (reviewed in Cepko, 2014).

Figure 4: the competence model of retina development (Livesey and Cepko, 2001).

The competence model of retina development proposes that at each of the successive stages the retinal progenitors are intrinsically competent to produce only a subset of retinal cell types. These progenitors will generate all retinal cell types in an ordered manner with some overlap. Some extrinsic signals then influence cell fate within the competence state, or might even act to induce transition from one competence state to another (Belliveau and Cepko, 1999; Cepko et al., 1996; Livesey and Cepko, 2001). The competence model is different from the model of progressive restriction of fate potential, that propose that a totipotent cell progeny becomes progressively restricted to produce more and more restricted cell types. In the progressive fate restriction model, the postulate is that cells progressively lose their potency rather than change their competence overtime (Cepko et al., 1996). The fact that progenitor competence state is intrinsically defined has been demonstrated by in vitro heterochronic transplants in chick and rodent, in which transplanted cells give birth to cell types corresponding to their stage of development, without being influenced by the age of their environment. That is, early retinal progenitors generate only early cell types when transplanted into late environment, and late progenitors generate late fates when transplanted into early environment.

These studies also demonstrated that extrinsic signals can alter fate choice, but only within a restricted range of cell types defined by the competence state of progenitor (Austin et al., 1995; Belliveau and Cepko, 1999; Livesey and Cepko, 2001). Similarly, mixing of embryonic progenitor with an excess of postnatal retinal cells is able to increase proportion of rods, but do not influence timing of opsin expression that normally displays a lag of 4 to 8 days after cell birth. This suggests that a specific

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competence state enables progenitors to respond to extrinsic cues promoting rod production, but that the transition to that state cannot be altered by environment (Watanabe and Raff, 1990).

Moreover, rat RPCs isolated in culture, i.e. without influence from retinal environment, produce cell types that correspond to their development stage (Cayouette et al., 2003; Reh and Kljavin, 1989), with clone size and composition similar as observed in retinal explant. No particular cell type is required for differentiation of any other cell type, suggesting that intrinsic factors play a predominant role in defining the competence state (Cayouette et al., 2003).

The question of how progenitors transit from one competence state to another remains open. One hypothesis might be that post-mitotic neurons produce a signal that either induces the competence transition or inhibits the production of a given type of neuron, thus acting like a negative feedback to regulate the proportion of each cell type (Livesey and Cepko, 2001). In rat retina, post-mitotic amacrine cells have been shown to have the capacity to inhibit the production of amacrine cells by progenitors when co-cultured in vitro (Belliveau and Cepko, 1999). Inversely, co-culturing progenitors with a population depleted of amacrine cells causes an increase in amacrine cells production. This effect is observed only during a very limited period, that is, during a specific competence state of the progenitor, and might contribute to the generation of a correct number of each cell type (Belliveau and Cepko, 1999). Similarly, when a drug is used to kill a specific population of amacrine cells in frog, progenitor fate specification is biased toward this population (Reh and Tully, 1986), again suggesting a role of feedback inhibition from post-mitotic cells. Further evidences of feedback inhibition come from chick retinal progenitors that produce less RGCs when cultured in presence of older retinal cells containing a high proportion of RGCs, while there is no effect if the older population has been depleted of RGCs. This effect is thought to be mediated by a diffusible factor as conditioned medium is also able to induce a reduction of RGC production (Waid and McLoon, 1998). This factor has been later identified as Shh (Zhang and Yang, 2001a). Thus, it seems that a combination of intrinsic changes in competence state of progenitors and modification of environment by post-mitotic cells act together to produce all cell types at the right time and in proper ratio (Cepko et al., 1996; Livesey and Cepko, 2001).

It is not clear how the specific birth order of the different cell types is achieved. A population of poorly synchronized RPCs that produce each cell type in a specific order could explain the overlapping birth sequence. In the zebrafish retina, in vivo analysis of BrdU incorporation in clones suggests that the different cell types are produced following a strict sequence in each single RPC lineage. Each RPC goes through sequential and unidirectional changes in fate potential, with very different intrinsic schedules, as almost any cell type can be produced at a given time (Wong and Rapaport, 2009). However, following lineage of single rat RPCs in vitro suggests that the birth order is not strictly defined, and that late fates can sometimes be produced before early ones in the same lineage. The authors propose

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that RPC differentiation does not follow a fixed sequence and that the fate choice is largely stochastic.

The probability to produce each cell type could be intrinsically determined and vary overtime, thus explaining the sequence of birth order at population level (Gomes et al., 2011). This study was conducted close to the end of neurogenesis and results are not necessarily incompatible with the competence model, since the stochastic model could apply for a restricted number of fates inside one particular competence state. Finally, the apparently contradictory results between amphibians and mammals could reflect interspecies differences or variation between in vitro and in vivo systems.

Single cells transcriptome analysis in mice argues in the favor of a high level of heterogeneity in the progenitor population (Trimarchi et al., 2008). However, it is unclear whether this variability arises from equivalent RPCs at different competence state, or whether it reflects separate classes of progenitors. Vitorino et al. (2009) suggest the existence of different RPC lineages producing distinct fates in zebrafish, and showed that RPCs that maintain Vsx2 expression are restricted to differentiate into a specific subtype of bipolar cells or into Muller glia. Differentiation into other cell types and the associated expression of fate determinants like Vsx1, Ath5 or foxrn4, require Vsx2 downregulation, and those factors are thought to be directly repressed by Vsx2 (Vitorino et al., 2009). Some progenitors already express VC1.1 and syntaxin-1a which are respectively markers of amacrine and horizontal cells, and are strongly biased toward these fates (Alexiades and Cepko, 1997). Moreover, Mash1 (achaete-scute homolog 1, also known as Ascl1) and Math5, two bHLH transcription factors, are expressed in different subsets of progenitors (Brown et al., 1998; Jasoni and Reh, 1996; Livesey and Cepko, 2001; Matter-Sadzinski et al., 2001). In the same way, two classes of progenitors have been described that rely either on p57Kip2 or on p27Kip1 cyclin kinase inhibitors for cell cycle exit (Dyer and Cepko, 2000a, b; Levine et al., 2000; Livesey and Cepko, 2001).

The cells might be committed to a particular cell fate already before being post mitotic, with fate decision occurring already in the progenitor and transmitted either asymmetrically or symmetrically to its progeny. For example, amacrine fate specification seems to occur already in G2 (Belliveau and Cepko, 1999; Livesey and Cepko, 2001), and some RGC markers, like CHRNb3 a nicotinic acetylcholine receptor subunit, are already expressed prior to the last mitosis (Matter et al., 1995). Moreover, in chick two distinct populations of progenitors defined by mutually exclusive expression of either Ash1, or Ngn2 and Atoh7/Ath5 exist very early (Matter-Sadzinski et al., 2001). Not all cells seem to be committed before their last division. This is the case for rods photoreceptors, which display a lag between terminal division and rhodopsin expression, and where decision seems to occur in cells that are already post-mitotic. CNTF addition to rat retinal explants reduces the number of rods, and is able to switch fate of presumptive rods that are post-mitotic until they express opsin, suggesting that presumptive rods are not committed at the time they are born (Ezzeddine et al., 1997). Altogether, these data suggest that some classes of terminally dividing RPCs produce very specific progeny,

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nevertheless the question remains whether distinct RPC lineages are specified very early or only close to terminal division (Cepko, 2014).

Neurogenesis and focus on RGCs differentiation

In many species studied including chick, mouse and zebrafish, differentiation occurs as a wave that begins in the central retina and expands to the periphery as development proceeds (Hu and Easter, 1999; McCabe et al., 1999; Prada et al., 1991). However, a wave of differentiation is probably not a general rule among vertebrates, as it does not occur in the pigeon retina (Rodrigues et al., 2016). The optic stalk serves as a signaling center that secretes FGF and is required for the initiation of neuronal differentiation in the neuroretina (Martinez-Morales et al., 2005). In zebrafish oep mutant that lacks the optic stalk, initiation of neurogenesis and Atoh7/Ath5 expression do not occur. Atoh7/Ath5 expression always initiates close to optic stalk-neuroretina boundary even when its position is manipulated or Pax2 positive optic stalk cells are transplanted (Masai et al., 2000). Remarkable exception to this rule is the onset of cell differentiation all over the retina in pigeon (Hufnagel and Brown, 2013). In the chick and zebrafish retinas, FGF from the optic stalk is required for the initiation of neurogenesis and RGC production. Ectopic Fgf8 addition is sufficient to induce neuronal differentiation and RGC production in chick, and FGF addition rescues neurogenesis and Atoh7/Ath5 expression in the absence of the optic stalk in zebrafish oep mutant (Martinez-Morales et al., 2005).

Recent study in pigeon suggested that Fgf3 rather than fgf8 could activate neurogenesis (Rodrigues et al., 2016).

In drosophila, hedgehog is responsible for both initiation and propagation of neurogenesis (Dominguez and Hafen, 1997). In chick retina, evidences suggest that Shh secretion by post-mitotic RGCs have a dual role. Behind differentiation front, high level of Shh acts on progenitors to inhibit RGC fate, thereby serving as a negative feedback (Zhang and Yang, 2001a). On the other hand, lower Shh levels at the edge of the differentiation front could promote expansion of the wave of Shh and RGC differentiation (Neumann and Nuesslein-Volhard, 2000; Zhang and Yang, 2001a). Another study suggested that Shh signal would set an intrinsic timer before neurogenesis, and that Shh is not required anymore at the time of neurogenesis wave. This interpretation is supported by the fact that transplanted zebrafish cells express Atoh7/Ath5 at a time that correspond to their original position independent of their grafted position. Moreover, blocking Shh just prior to the onset of the wave does not abolishes it, while earlier Shh blockage does (Kay et al., 2005).

In the developing retina, proneural genes instruct the progenitor to acquire a general neuronal fate, at the same time specifying neuronal subtype identities. Basic Helix Loop Helix (bHLH) transcription factors play a central role in controlling cell differentiation in the developing retina. (Bertrand et al., 2002). bHLH transcription factors contain a basic domain for interaction with DNA at conserved hexanucleotide motif E-box, and a helix-loop-helix domain for dimerization. They form homo- or

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Jean Piere Helfer et Jack Orsoni, le marketing, edition vubert, paris ( sans année), p6..  ﺀﺍﺮﺸﻟﺍ ﺔﻔﻴﻇﻭ : ﺓﺪﺣﺍﻭ ﺔﺑﺎﻗﺭﻭ ﺓﺪﺣﺍﻭ ﺔﻴﻜﻠﻣ ﺖﲢ ﻊﻠﺴﻟﺍ ﻊﻴﻤﺠﺘﻟ ﻡﺯﻼﻟﺍ

Using a series of calibration and characterization (C&C) tests at a range of different chamber gas compositions, pressures and temperatures, this research aimed

Finally, polarization-entangled photon pairs were used in conjunction with a trapped-atom quantum memory to create a quantum optical communication system capable

A rapid numerical method for solving Serre-Green-Naghdi equations describing long free surface gravity waves.. Nonlinearity, IOP