Elise C Lelièvre

Monkol Lek

Henrik Boije

L Houille-Vernes

Valérie Brajeul

A Slembrouck

Jérôme E Roger

José-Alain Sahel

L Houille-Verne

J M Matter

F Sennlaub

F Hallböök

Olivier Goureau
email: olivier.goureau@inserm.fr

Xavier Guillonneau
email: xavier.guillonneau@inserm.fr

Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina

Keywords: Cell specification, Horizontal cell, Atoh7/Ath5, retina, chick

INTRODUCTION

Ptf1a encodes a basic-helix-loop-helix (bHLH) transcription factor that drives undifferentiated cells in the foregut to differentiate into a pancreatic lineage during pancreas development [START_REF] Kawaguchi | The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors[END_REF][START_REF] Krapp | The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas[END_REF]. Ptf1a is also expressed during the development of many structures of the central nervous system [START_REF] Glasgow | Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn[END_REF][START_REF] Zecchin | Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates[END_REF]. In the cerebellum and the spinal cord, Ptf1a is expressed in the precursors of GABAergic neurons, and the loss of Ptf1a induced these GABAergic neurons to adopt a glutamatergic phenotype [START_REF] Glasgow | Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn[END_REF][START_REF] Hoshino | Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum[END_REF]Pascual et al., 2007). In the retina, Ptf1a acts as an HC and AC fate determination factor at the expense of ganglion cells in mice, zebrafish and Xenopus [START_REF] Dong | Graded levels of Ptf1a differentially regulate endocrine and exocrine fates in the developing pancreas[END_REF][START_REF] Dullin | Ptf1a triggers GABAergic neuronal cell fates in the retina[END_REF][START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF]Nakhai et al., 2007) and of photoreceptors in zebrafish and Xenopus [START_REF] Dong | Graded levels of Ptf1a differentially regulate endocrine and exocrine fates in the developing pancreas[END_REF][START_REF] Dullin | Ptf1a triggers GABAergic neuronal cell fates in the retina[END_REF]. These studies have thoroughly characterized the phenotypic modifications induced by Ptf1a loss-and gain-of-function in the retina, but the molecular mechanisms underlying Ptf1a activity are not as well understood. Loss-of-function studies reported a change in the expression of a set of retinogenic factors in the Ptf1a-null retinas [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF]Nakhai et al., 2007). Nevertheless, because some of these factors are persistently expressed in mature cells, it remains to be established if these changes were due to specific transcriptional regulations or qualitative changes in the cell populations expressing these factors.

Unlike other class II bHLH transcription factors, Ptf1a functions in the pancreas as part of a trimeric complex. The pancreas transcription factor 1 complex (PTF1) includes Ptf1a, a ubiquitous class I bHLH transcription factor (E-protein) and the mammalian Suppressor of Hairless protein, Rbpj (PTF1-J complex) or its paralog, Rbpjl (PTF1-L complex) [START_REF] Beres | PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L[END_REF][START_REF] Masui | Early pancreatic development requires the vertebrate Suppressor of Hairless (RBPJ) in the PTF1 bHLH complex[END_REF]Obata et al., 2001). The Ptf1a-Rbpj interaction is also required for Ptf1a to specify GABAergic cells in the dorsal spinal cord and the cerebellum [START_REF] Hori | A nonclassical bHLH Rbpj transcription factor complex is required for specification of GABAergic neurons independent of Notch signaling[END_REF]. However, the requirement of the Ptf1a-Rbpj interaction in the specification of retinal cell types remains to be elucidated.

The chick retina has proven to be a powerful model to study retinal differentiation and its genetic regulation. Moreover, in contrast to mice possessing only one axon-bearing HC subtype (Peichl and Gonzalez-Soriano, 1994), the chick retina has both axon-less and axonbearing HC subtypes, as found in the majority of vertebrate retinas [START_REF] Gallego | Chapter 7. Comparative studies on horizontal cells and a note on microglial cells[END_REF][START_REF] Genis-Galvez | Evidence of three horizontal cells in the chick retina[END_REF]. The chick retina contains H1 axon-bearing HCs, H2 axon-less "stellate" HCs and H3 axon-less "candelabrum" HCs [START_REF] Edqvist | Axon-bearing and axon-less horizontal cell subtypes are generated consecutively during chick retinal development from progenitors that are sensitive to follistatin[END_REF][START_REF] Tanabe | Cadherin is required for dendritic morphogenesis and synaptic terminal organization of retinal horizontal cells[END_REF]. Therefore, this model is more representative of Ptf1a activity on HC subtype determination in vertebrates.

This study aimed to gain a better understanding of the molecular regulation underlying the Ptf1a activity during retinal development using the chick retina model. In this study, we showed that the forced expression of Ptf1a leads to a massive disorganization of the differentiated retina and changes in retinal cell representation that complement the retinal phenotype of Ptf1a-null mice: an increase of ACs and all HC subtypes and a decrease of ganglion and photoreceptor cells. Using this model, we identified several retinogenic factors that were rapidly regulated by Ptf1a overexpression and reported that ectopic Ptf1a strongly downregulated Atoh7 expression in the chick retina. Finally, our study demonstrated that the interaction between Ptf1a and Rbpj cofactors was required for Ptf1a activity in the developing retina.

MATERIALS AND METHODS

Gallus gallus white leghorn embryos were obtained from Haas (France). The animal experimentation was conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in Ophthalmic and Vision Research and a protocol approved by our local animal care committee.

Animals

Chick genome sequences (V2.0 and V3.0) were obtained from The Genome Institute at Washington University (http://genome.wustl.edu/). The expressed sequence tags (accession numbers: BU487258 and BU347629) were ordered from Source Bioscience and sequenced (MWG-Operon). The alignments were performed using Basic Local Alignment Search Tool available at NCBI (http://blast.ncbi.nlm.nih.gov/) and VNTI software (Invitrogen).

DNA sequences

The mouse Ptf1a was subcloned using specific CDS primers into the pDONR221 vector (Invitrogen) using BP clonase (Invitrogen). The Ptf1a mutants were generated using Polymerase Chain Reaction (PCR)-based mutagenesis. The pDONR221-Ptf1a plasmid was recombined in the presence of LR recombinase (Invitrogen) into either RCAS-BP(A)-NHY, which allowed for the fusion of a HA-tag with the N-terminal part of the Ptf1a protein (gift from Dr. Loftus) or the pCIG gateway vectors [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF]. The RCAS viral stocks Retroviral stock and plasmid production with titers > 1x10 8 colony forming units per milliliter (cfu/ml) were prepared by transfecting DF1 cells with viral DNA constructs using FuGENE6 Reagent (Roche Diagnostics) [START_REF] Yang | Retrovirus-mediated gene expression during chick visual system development[END_REF]. Viruses were concentrated by centrifugation using 100K Centrifugal Filters Amicon Ultra (Millipore). All embryos were injected into the right optic vesicle at embryonic day 2 (E2). The openings in the eggs were sealed with scotch tape and further incubated at 37.5°C.

Parental RCAS-BP(A) viruses [START_REF] Hughes | Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors[END_REF] served as controls in the viral infection experiments.

The eyes were fixed in 4% paraformaldehyde (PFA) and incubated in 30% sucrose (Sigma) in phosphate-buffered saline (PBS) overnight followed by 1 hour incubation at 37°C in PGS (PBS, 7.5% gelatin (Sigma), and 10% sucrose). Eyes were embedded in PGS, frozen at -50°C in isopentane and stored at -80°C. Ten micrometer-thick cryosections were collected.

Cryosection

The retinas were collected in HBSS without Ca Cell dissociation 2+ /Mg 2+ , trypsinized (1 mg/ml) (Sigma) and incubated for 10 minutes at 37°C. The digestion was stopped with HBSS with Ca 2+ /Mg 2+ containing trypsin inhibitor (Sigma) (1 mg/ml), and the cells were mechanically dissociated in the presence of DNaseI (Sigma) (0.1 mg/ml). For flow cytometry, the suspended cells were washed with PBS, fixed for 15 minutes with 2% PFA at room temperature (RT) and washed in PBS before immunostaining. For the manual counting, 1x10 5 cells/ml were seeded on Poly-L-Lysine (Sigma) coated plates. After two hours, adherent cells were fixed 10 minutes with 2% PFA at RT before immunostaining.

Antibodies

Mouse anti-gag (3c2), anti-Ap2α (3B5), anti-Islet1 (39.4D5), anti-Lim1/2 (4F2), and anti-Visinin (7G4) antibodies were purchased from the Developmental Studies Hybridoma Bank.

Mouse anti-Brn3a (MAB1585), mouse anti-Glutamine Synthetase (MAB302), mouse anti-Prox1 (MAB5652), and rabbit anti-PhosphoHistone3 (07-145) antibodies were obtained from Millipore, rabbit anti-gag (p27) from Charles River, rabbit anti-Protein Kinase Cα (PKCα) (Sc-208) from Santa Cruz, and rabbit anti-Prox1 (DP3501P) from Acris. The rabbit anti-Ptf1a antibody was a gift from Dr. Edlund (Umeå University), and the rabbit anti-TrkA was a gift from Pr. Lefcort (Montana State University). Primary antibodies were detected using AlexaFluor488-, AlexaFluor594-, AlexaFluor633-(Invitrogen) or Phycoerythrine (PE)-(Beckman Coulter) conjugated goat antibodies.

Immunostaining was performed as previously described [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF]. Retinal section counterstaining was performed with 4',6'-diamidino-2-phenylindole (DAPI) (1:1200). Apoptotic cells were detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) labeling using the In Situ Cell Death Detection Kit (Roche Diagnostics). For S-phase cell labeling, 5 µg (E7 embryos) or 10 µg (E9 embryos) of 5ethynyl-2'-deoxyuridine (EdU) were injected into the intravitreal space two hours before being sacrificed. The Click iT Immunostaining and in situ hybridization TM Digoxigenin-labeled Rbpj and cAtoh7 probes were generated by cloning template DNA (the full length coding sequence of cAtoh7 and the cRbpj coding sequence from 123 to 873 bp) into pCRII-TOPO vector (Invitrogen). In situ hybridization was performed as previously described [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF].

EdU Imaging kit (Invitrogen) was used to visualize EdUpositive cells.

Images were captured with a DM5500 microscope (Leica) equipped with an ORCA ER Hamamatsu camera or a LSM710 confocal microscope (Zeiss) and analyzed with MetaMorph software (Molecular Devices). Dissociated cells were incubated in blocking buffer (PBS, 10% fetal calf serum (FCS), 2% goat serum, and 0.1% saponin) one hour at RT. Primary antibodies were diluted in blocking buffer, applied two hours at RT, and washed. The cell pellets were then incubated for one hour with 5 µl of PE-and/or AlexaFluor633-conjugated secondary antibodies (1:10) at RT. The data were collected using an LSRII cytometer (BD Biosciences) and analyzed using BD FACS DIVA software. For cell sorting, a total of 1x10 Flow cytometry and cell sorting 5 GFP-positive cells were collected in lysis buffer for RNA extraction using Vantage Sorter (BD Biosciences).

The eyes were collected at E5, and the pigmented epithelium was removed from the retina.

Whole retinas were positioned in an electroporation chamber (CUY520P5, Sonidel, Napagene, Japan) filled with PBS with Ca Electroporation 2+ /Mg 2+ containing plasmids (0.5 μg/μl).

Electroporation was performed using a CUY215C square wave generator (Sonidel) and consisted of 5 pulses at 30 V with 50 ms duration, 1 second interval and repeated twice.

Whole retinas were then cultured as floating explants at 37°C in DMEM, 10% FCS, and 1% Penicillin/Streptomycin. For confocal microscopy, the retinas were fixed 20 minutes in 4% PFA and rinsed for 24 hours in PBS. For inclusion, the fixed retinas were cryoprotected and embedded in PGS.

Total RNA was extracted using the Nucleospin RNAII Kit (Macherey Nagel).

Retrotranscription was performed as described previously [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF]. Real-time PCR was performed using 7300 Real-Time PCR System (Applied Biosystems) in a 20 µl final volume with Power SYBR Green PCR Master Mix (Applied Biosystems) and 0.25 µM primers. All samples were run in triplicate. Primers used for real-time PCR analysis are listed in Table S1 (See Supplementary Materials).

RNA isolation, reverse-transcription and quantitative PCR.

Protein extracts were collected in hypotonic buffer (20 mM HEPES pH 7.9, 1 mM Na Cell fractionation 3 VO 4 , 1 mM NaGlycerophoshate, 5 mM EDTA pH 7.5, 1 mM EGTA pH 7.5, 1 mM DTT, and protease inhibitors (Calbiochem, Merck4Biosciences)) plus 0.2% Nonidet P-40, incubated on ice for 15 minutes and centrifuged (20 seconds at 16000 g). For nuclear extracts, pellets were resuspended in saline buffer (120 mM NaCl and 20% glycerol in hypotonic buffer) supplemented with protease inhibitor cocktail, incubated for 30 minutes at 4°C and centrifuged for 20 minutes at 16000 g to collect the supernatant. For cytosolic extracts, NaCl (120 mM) was added to the supernatant of the first centrifugation, and the extract was centrifuged for 20 minutes at 16000 g to remove debris. Glycerol (10%) was then added to the supernatant.

Western Blotting was conducted as described previously [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF] using the following primary antibodies: mouse anti-HA (MMS 101R, Covance), goat anti-LaminB (sc-6216, Santa Cruz), mouse anti-Actin (Sigma), goat anti-Ptf1a (ab62818, Abcam), and rat anti-Rbpj (T6719, Institute of Immunology, Tokyo, Japan).

Western Blotting

Infected DF1 cells were lysed in immunoprecipitation buffer (50 mM Tris/HCl pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, and protease inhibitors) [START_REF] Rodolosse | p/CAF modulates the activity of the transcription factor p48/Ptf1a involved in pancreatic acinar differentiation[END_REF]. The lysates were incubated overnight and centrifuged for 20 minutes at 16000 g. Protein extracts (250 µg) were incubated with anti-HA antibody (Covance, 1/100) for 2 hours at RT to immunoprecipitate the HA-tagged proteins. The immune complex was then captured by incubating for 2 hours at RT with 40 µl of Protein-G Sepharose beads (Fast Flow, GE Healthcare). The complexes were pelleted by gentle centrifugation, washed four times with immunoprecipitation buffer and eluted in the loading buffer before western blotting.

Co-immunoprecipitation

The quantifications are expressed as the mean ± standard error of the mean (s.e.m). Analyses were conducted using the Student's t-test assuming equal variances (two groups) or one-way

RESULTS

The Ptf1a spatiotemporal expression pattern has been studied during retinogenesis [START_REF] Boije | Temporal and spatial expression of transcription factors FoxN4, Ptf1a, Prox1, Isl1 and Lim1 mRNA in the developing chick retina[END_REF][START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF]Nakhai et al., 2007). Ptf1a mRNA was detected from embryonic day 3 (E3) to E15 in the developing chick retina. Early, it is expressed in the inner neuroblastic layer (NBL) and then becomes restricted to the inner nuclear layer (INL) [START_REF] Boije | Temporal and spatial expression of transcription factors FoxN4, Ptf1a, Prox1, Isl1 and Lim1 mRNA in the developing chick retina[END_REF]. To gain insight into the function of Ptf1a during chick retinal development, a replication-competent retrovirus, RCAS, was used to drive ectopic and overexpression of Ptf1a in the retina. The chick Ptf1a gene sequence is not fully known. We have characterized its C-terminal using the available information at The Genome Institute and two ESTs. It contains the highly conserved bHLH DNA-binding and Rbpj/Rbpjl-interaction domains [START_REF] Beres | PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L[END_REF] (Fig. S1). However, the N-terminal of the chick Ptf1a gene has remained uncharacterized in recent chicken genome assemblies. In contrast, the mouse Ptf1a sequence is fully known, and the Rbpj/Rbpjl-interaction domains have been extensively studied [START_REF] Beres | PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L[END_REF]. Moreover, previous studies have reported that mouse Ptf1a blocks the differentiation of dIL B excitatory neurons and promotes dIL A During any developmental stage, the plexiform and nuclear layers had formed properly in the empty-RCAS (RCAS) control infected patches (Fig. 1F-G,J-K,N-O). At E7, no major structural defects were observed in the RCAS-Ptf1a-infected patches other than the presence of a thicker retina and a decreased cell density (Fig. 1H-I). In contrast, at E9 (Fig. 1L-M) and, more extensively, at E12 (Fig. 1P-Q), the lamination of the RCAS-Ptf1a-infected patches was severely disturbed with disorganized plexiform and nuclear layers. No changes in inhibitory neuron specification in the chick spinal cord, a phenotype that was consistent with Ptf1a loss of function studies in mouse [START_REF] Glasgow | Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn[END_REF][START_REF] Hori | A nonclassical bHLH Rbpj transcription factor complex is required for specification of GABAergic neurons independent of Notch signaling[END_REF]Mizuguchi et al., 2006;[START_REF] Wildner | dILA neurons in the dorsal spinal cord are the product of terminal and nonterminal asymmetric progenitor cell divisions, and require Mash1 for their development[END_REF].

These results suggest a conserved activity of mouse Ptf1a in avian and rodent models.

Therefore, the mouse Ptf1a cDNA was chosen to allow for misexpression experiments of Ptf1a in the chick retina and for genetic manipulations of the Ptf1a coding sequence. Effective wild type Ptf1a protein mis/overexpression from RCAS-HA-Ptf1a (RCAS-Ptf1a) was verified by western blotting for the HA-tagged proteins in DF1 cells (Fig. 1A) and by immunohistochemistry in E9 retinas (Fig. 1D). In the chick retina, viral injections resulted in a widespread infection. Retinal patches infected with the different RCAS viruses and infected retinal dissociated cells could be detected using either the p27 or 3c2 anti-gag antibodies (Fig. 1C,E). the retinal structure were observed outside of the RCAS-Ptf1a-infected areas (data not shown).

Forced expression of Ptf1a affects progenitor cell proliferation and migration.

These defects in retinal structure and lamination might arise from disturbed migration or modification of the cell cycle. Therefore, S-phase cells were pulse-labeled with 5-ethynyl-2'-deoxyuridine (EdU), and mitotically active progenitors were immunostained using a Phospho-Histone3 (P-H3) antibody. At E7, in the control retinas, EdU-positive cells were located in the NBL (Fig. 2A), whereas P-H3 positive cells were primarily found in the ventricular side (Fig. 2E), which was consistent with interkinetic nuclear movements (IKNM) of progenitor nuclei and free-cell migration [START_REF] Baye | Nuclear migration during retinal development[END_REF][START_REF] Boije | Horizontal cell progenitors arrest in G2-phase and undergo terminal mitosis on the vitreal side of the chick retina[END_REF][START_REF] Hinds | Differentiation of photoreceptors and horizontal cells in the embryonic mouse retina: an electron microscopic, serial section analysis[END_REF]. P-H3-positive (Fig. 2F) and numerous EdU-positive cells (Fig. 2B) were detected within the RCAS-Ptf1a-infected patches at E7. The EdU-positive cells were mislocalized in the ventricular half of the retina (Fig. 2B), and an increased number of ectopic P-H3 positive cells were detected in the NBL and in the vitreal side of the RCAS-Ptf1ainfected patches (Fig. 2F, arrows and Fig. S2). These results suggest that the movement of progenitors was altered in the RCAS-Ptf1a-infected retina at E7 and might partially account for the observed structural defects.

The EdU-positive and P-H3-positive cells were scored using flow cytometry. The percentage of EdU-positive cells among the infected cells was normalized by the percentage of EdU-positive cells among non-infected cells. At E7, the number of EdU-positive cells was significantly decreased following the forced expression of Ptf1a (p=0.031) (Fig. 2I). At E9, fewer EdU-(p=0.001) (Fig. 2D) and P-H3-positive cells (Fig. 2H) (0.14 ± 0.04%, s.e.m, in the RCAS-infected cells and 0.02 ± 0.01%, s.e.m, in the RCAS-Ptf1a-infected cells, p=0.0178) were detected after Ptf1a mis/overexpression (Fig. 2I,J). Together, these findings suggest that the forced expression of Ptf1a induced progenitors to withdraw from the cell cycle.

Ptf1a mis/overexpression is sufficient to induce a change in retinal cell type representation.

To investigate whether the structural and cell cycle changes were paired with alterations in the proportion of major retinal cell types, using immunocytochemistry, we analyzed the markers of the different cell classes in the infected areas of E12 retinas, the age when most cells have initiated their differentiation. Concomitantly, the number of ACs, HCs and photoreceptor cells among the gag-positive infected cells was scored by flow cytometry.

At E12, immunoreactivity for Ap2α was found in most AC nuclei in the INL (Fig. 3K) [START_REF] Edqvist | Early identification of retinal subtypes in the developing, pre-laminated chick retina using the transcription factors Prox1, Lim1, Ap2alpha, Pax6, Isl1, Isl2, Lim3 and Chx10[END_REF][START_REF] Fischer | Heterogeneity of horizontal cells in the chicken retina[END_REF]. Prox1 strongly labeled HCs and a small subset of ACs (Fig. 3P). The forced expression of Ptf1a induced a three-fold increase of Ap2α-positive cells compared to control cells (22.7 ± 2.2% in the RCAS-infected population and 63.0 ± 12.9% in the RCAS-Ptf1a-infected population, n=5) and a two-fold increase of Prox1-positive cells (18.0 ± 1.3% in the RCAS-infected population and 32.6 ± 4.7% in the RCAS-Ptf1ainfected population, n=5) (Fig. 3L,Q,W). Conversely, Brn3a labeling for ganglion cells was severely reduced in the RCAS-Ptf1a-infected patches (Fig. 3B), and the number of Visininpositive photoreceptors was decreased from 17.7 ± 2.6% in the RCAS-infected cells to 4.2 ± 0.5% in the RCAS-Ptf1a-infected cells (n=5) (Fig. 3G,U-W). For late born retinal cell subtypes, the number of Glutamine Synthetase (GS)-positive Müller glial cells was significantly decreased from 3.2 ± 0.2% in the RCAS-infected cells to 1.8 ± 0.3% in the RCAS-Ptf1a-infected cells (p=0.0083, n=4) (Fig. S3B,E), but Protein Kinase Cα (PKCα) immunoreactivity for rod bipolar cells revealed no significant difference (1.8 ± 0.1% among the RCAS-infected cells and 3.9 ± 0.8% among the RCAS-Ptf1a-infected cells, p=0.2651, n=4) (Fig. S3D,E).

Retinal lamination defects at E9 and later stages may result in a secondary alteration of cell type representation. Therefore, we conducted the same analysis prior to the structural defects, at E7. At this age, Ptf1a mis/overexpression also led to a significant increase of Prox1-(4.4 ± 0.3% among the RCAS-infected cells and 14.7 ± 1.5% among the RCAS-Ptf1ainfected cells, p=0.0004, n=4) (Fig. 4F,I) and Ap2α-positive cells (10.9 ± 0.1% among the RCAS-infected cells and 18.9 ± 1.2% among the RCAS-Ptf1a-infected cells, p=0.001, n=5) (Fig. 4H,I) at the expense of ganglion cells (Fig. 4B) and photoreceptor cells (10.2 ± 0.7% in the RCAS-infected retinas and 6.5 ± 0.4% in the RCAS-Ptf1a-infected retinas, p=0.0014, n=5) (Fig. 4D,I). These results indicated that the Ptf1a mis/overexpression effects on earlyborn retinal cell types occurred prior to the defects in retinal lamination and argued against the notion that the loss of photoreceptors and ganglion cells was secondary to the retinal lamination defects. Interestingly, a few Ap2α-positive cells (Fig. 4H) and the majority of Prox1-positive cells (Fig. 4F) were ectopically located in the vitreal side of the retina.

We performed TUNEL labeling to assess if the reduction of some cell subtypes could be due to early cell death. A significantly higher number of apoptotic cells, located throughout the entire thickness of the NBL, was detected in the RCAS-Ptf1a patches (43.3 ± 9.8 cells per area) compared to the patches infected with the control virus (8.5 ± 0.2 cells per area, n=3, p=0.0305) (Fig. 4J-L).

Ptf1a mis/overexpression is sufficient to induce all HC subtype specification.

Our results showed that Ptf1a mis/overexpression increased the size of the HC population. The chick HC population is composed of at least three HC subtypes that can be molecularly distinguished by the expression of Prox1, Lim1, Islet1, TrkA and GABA [START_REF] Edqvist | Axon-bearing and axon-less horizontal cell subtypes are generated consecutively during chick retinal development from progenitors that are sensitive to follistatin[END_REF][START_REF] Fischer | Heterogeneity of horizontal cells in the chicken retina[END_REF]. All three subtypes express Prox1. The H1 subtype (50% of all HCs) has GABA, is Lim1-positive, Islet1-negative, and TrkA-negative. The H2 (approximately 10% of all HCs) also has GABA, is Lim1-negative, Islet1-positive but TrkAnegative, and the H3 (approximately 40% of all HCs) does not have GABA, is Lim1-negative and Islet1/TrkA double-positive. We first investigated the normal Ptf1a expression in the HC subtypes in the developing chick retina. The retina was analyzed with respect to Ptf1a, Prox1, Lim1 and Islet1 immunoreactivity in E6.5 retinas when HCs migrated bi-directionally and, in E9, E12 and E16 retinas when the HC layer (HCL) was established [START_REF] Boije | Horizontal cell progenitors arrest in G2-phase and undergo terminal mitosis on the vitreal side of the chick retina[END_REF][START_REF] Edqvist | Newborn horizontal cells migrate bi-directionally across the neuroepithelium during retinal development[END_REF]. At E6.5, after the onset of Prox1 expression in HCs, 31 ± 5% (n=3) of the Ptf1a-immunoreactive cells were also Prox1-positive (Fig. 5A). The Ptf1a/Prox1 double-positive cells were localized on the vitreal side of the retina but also scattered in the prospective INL, which was consistent with the location of migrating HCs at this age. At E9, all Ptf1a/Prox1 double-positive cells were located in the developing HCL. Of the Prox1positive cells in the HCL, 64 ± 5% (n=3) were Ptf1a-positive HCs (Fig. 5B). This result implies that not all HCs expressed Ptf1a at this age. The Lim1-positive cells were Ptf1apositive, and this overlap was found at both E9 (96 ± 1%, n=3) in the HCL and at earlier ages on the vitreal side (Fig. 5E,F). In contrast, only a few Ptf1a/Islet1 double-positive cells could be seen at E6.5 and at E9 (5 ± 0.6%, n=3) (Fig. 5I,J).

To assess whether Ptf1a was able to direct the development of all HC subtypes, we next analyzed the presence of HC subtype markers in the RCAS-Ptf1a-infected patches. At E7, an accumulation of Prox1/Lim1 double-positive and Prox1/Islet1 double-positive cells (Fig. 6B,D) were found in the normal location of the GCL in the RCAS-Ptf1a-infected patches compared to the control patches, suggesting that both the H1 and H3 subtypes had been generated following Ptf1a mis/overexpression. These results were strengthened by the TrkA immunoreactivity of Islet1-positive cells in the RCAS-Ptf1a-infected areas (Fig. 6F).

Furthermore, the accumulation of some HCs in the vitreal side suggested that HC migration was either arrested or delayed by Ptf1a mis/overexpression. These results were similar at E9 when all HCs had normally migrated back to the HCL. Supernumerary Prox1/Lim1 doublepositive (Fig. 6H), Prox1/Islet1 double-positive (Fig. 6J), and Islet1/TrkA double-positive cells (Fig. 6L) were observed in the RCAS-Ptf1a-infected retinas. Islet1-positive and TrkAnegative cells were found within the clusters of Prox1-positive cells of the RCAS-Ptf1ainfected patches (Fig. 6l, arrows), suggesting the presence of H2 HCs. Moreover, TrkA and Lim1 were never expressed in the same cell, indicating that H1 and H3 HCs were correctly specified (Fig. 6N). We found that the proportion of non-GABAergic TrkA-positive cells among the Prox1-positive HCs decreased significantly from 43.3 ± 1.9% in the RCASinfected patches to 27.1 ± 2.8% in the RCAS-Ptf1a-infected areas (p=0.0007), whereas a slight, but not significant, increase of Lim1-positive HCs was observed (51.9 ± 2.2% in the RCAS-infected patches and 58.9 ± 2.1% in the RCAS-Ptf1a-infected patches, p=0.0898) (Fig. 6O). Moreover, the back-migration of ectopic HCs to the HCL was inhibited, and ectopic HCs remained on the vitreal side of the E9 RCAS-Ptf1a-infected retinas.

Ptf1a overexpression induces changes in retinogenic factor expression.

To gain knowledge about the molecular mechanisms underlying the changes in retinal cell specification, we studied early changes in the expression of a selected set of genes involved in retinal differentiation. The E5 whole chick retinas were electroporated ex vivo with pCIG-Ptf1a-GFP plasmids, which drive the green fluorescent protein (GFP) expression [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF] (Fig. 7A). The GFP-positive cells were isolated by fluorescence activated cell sorting as early as 36 hours after electroporation to study changes in gene expression that were as independent as possible from cell differentiation. The empty pCIG-GFP plasmids were used as controls. First, our quantification demonstrated that electroporation of pCIG-Ptf1a in the chick retinas resulted in a strong expression of mouse Ptf1a mRNA, compared to the reference level (the non-specific amplification in pCIG-electroporated retinal cells) (Fig. 7B). Surprisingly, endogenous chick Ptf1a mRNA levels were significantly decreased in the mouse Ptf1a-overexpressing cells (p=0.0002) (Fig. 7B). No effects were observed on either Ngn2, a direct target of Ptf1a in the spinal cord and the cerebellum [START_REF] Henke | Neurog2 is a direct downstream target of the Ptf1a-Rbpj transcription complex in dorsal spinal cord[END_REF], or Sox2. We found that Pax6 and Ap2α, coding for two transcription factors highly expressed in ACs [START_REF] Belecky-Adams | Pax-6, Prox 1, and Chx10 homeobox gene expression correlates with phenotypic fate of retinal precursor cells[END_REF][START_REF] De Melo | Dlx1, Dlx2, Pax6, Brn3b, and Chx10 homeobox gene expression defines the retinal ganglion and inner nuclear layers of the developing and adult mouse retina[END_REF][START_REF] Edqvist | Early identification of retinal subtypes in the developing, pre-laminated chick retina using the transcription factors Prox1, Lim1, Ap2alpha, Pax6, Isl1, Isl2, Lim3 and Chx10[END_REF], were upregulated in Ptf1a-overexpressing cells (p=0.009) (Fig. 7B). A significant 2.6-fold increase of NeuroAB mRNA, a transcription factor suggested to be involved in GABAergic AC development (Ohkawara et al., 2004), was also observed in Ptf1a-overexpressing cells (p=0.0167). Interestingly, the expression levels of Ascl1, a pro-amacrine transcription factor in chick retina [START_REF] Mao | Proneural gene ash1 promotes amacrine cell production in the chick retina[END_REF], was downregulated (p=0.0037), suggesting that Ptf1a might act downstream of Ascl1 and inhibit its expression in the retina as described in the spinal cord [START_REF] Hori | A nonclassical bHLH Rbpj transcription factor complex is required for specification of GABAergic neurons independent of Notch signaling[END_REF]. The expression level of Otx2, a key regulatory gene for photoreceptor development (Nishida et al., 2003), was decreased by 2.5-fold in Ptf1a-overexpressing cells (p=0.0076), and Atoh7, a transcription factor involved in ganglion cell specification [START_REF] Liu | The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development[END_REF][START_REF] Matter-Sadzinski | A bHLH transcriptional network regulating the specification of retinal ganglion cells[END_REF], was strongly repressed by 6.4-fold by Ptf1a overexpression (p=0.0071) (Fig. 7B). Furthermore, Atoh7 mRNA was nearly undetectable by in situ hybridization in RCAS-Ptf1a-infected patches (Fig. S5C). Consistently, ex vivo, ectopic Ptf1a downregulated the activity of the chick Atoh7 promoter. Indeed, a significant decrease in the number of GFP-positive cells was observed in retinas co-electroporated with RCAS-Ptf1a and pAtoh7-GFP, a plasmid that drives GFP expression under the control of the chick Atoh7 promoter, compared to RCAS and pAtoh7-GFP co-electroporated retinas (Fig. S5E,F,H, p anova =0.001, p tukey <0.01).

We further focused our analysis on transcription factors involved in HC genesis. The mRNA levels of Neurod4 (Fig. 7B) and Prox1 (Fig. 7C), two factors involved in HC development [START_REF] Dyer | Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina[END_REF][START_REF] Inoue | Math3 and NeuroD regulate amacrine cell fate specification in the retina[END_REF], were not altered, whereas Lim1, which is necessary for H1 specification in the chick retina [START_REF] Suga | LIM family transcription factors regulate the subtype-specific morphogenesis of retinal horizontal cells at post-migratory stages[END_REF], was induced following Ptf1a overexpression (Fig. 7C). Foxn4 is upstream of Ptf1a during AC/HC specification in mouse retina [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF][START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]. Interestingly, Ptf1a overexpression induced a two-fold decrease of Foxn4 expression levels (p=0.0030) (Fig. 7C). To investigate whether Foxn4 conversely regulates Ptf1a in the chick retina, we electroporated E5 retinas with pCIG-Foxn4-GFP vectors (Fig. 7A). Foxn4 overexpression resulted in a significant increase in Ptf1a expression (p=0.0498) and Lim1 and Prox1 mRNA expression levels (Fig. 7C).

Ectopic Ptf1a requires its interaction with endogenous RBP-J

Ptf1a forms the PTF1 complex with any one of the E-proteins and Rbpj (PTF1-J) or Rbpjl (PTF1-L) [START_REF] Beres | PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L[END_REF]. Ptf1a interacts with Rbpjl through its C1 domain and with Rbpj through its C2 and C1 domains (Fig. S4K). The C2 domain deletion was sufficient to abolish the formation of the PTF1-J complex [START_REF] Beres | PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L[END_REF]. In situ hybridization revealed that Rbpj mRNA was expressed throughout the NBL at early stages and in the INL at later stages up to E12 (see Fig. S6A-J). Conversely, Rbpjl mRNA was neither detected during chick retinal development (data not shown) nor in mature retinas by quantitative PCR (Fig. S6K). To test the requirement for Rbpj proteins for Ptf1a activity in the chick retina, we generated Ptf1a-∆C2 and Ptf1a-∆C1∆C2 (Ptf1a-∆C12) mutant forms of Ptf1a that interact with E-proteins but are unable to interact with Rbpj [START_REF] Beres | PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L[END_REF] (Fig. S4L). The Ptf1a-∆C2 and Ptf1a-∆C12 proteins were expressed at levels equivalent to Ptf1a full-length proteins (Fig. S4N). Moreover, subcellular fractionation revealed that deletion of the C1 and C2 domains did not prevent Ptf1a-∆C12 nuclear importation (Fig. S4O). The injection of RCAS-Ptf1a-∆C2 and RCAS-Ptf1a-∆C12 in the optic cup did not lead to gross structural defects of the retina (Fig. 3 and Fig. S4E-H). However, the outer plexiform and nuclear layers were disrupted with rosette-like structures (Fig. 3H,I). Early-born cell types were analyzed by immunochemistry and flow cytometry in the RCAS-Ptf1a-∆C2-and RCAS-Ptf1a -∆C12infected population of E12 retinas, but no alteration of the cell type distribution was observed following the forced expression of these two Ptf1a mutant forms (Fig. 3C-D,H-I,M-N,R-S,W). The requirement of Rbpj-Ptf1a interaction for gene regulation downstream of ectopic Ptf1a was assessed by evaluating the ability of ectopic Ptf1a-∆C12 to regulate the Atoh7 promoter activity. Co-electroporation of pAtoh7-GFP with the RCAS-Ptf1a-∆C12 plasmids did not decrease the number of GFP-positive cells compared to co-electroporation with the control RCAS vectors (Fig. S5G,H ).

Ectopic Ptf1a activity is independent from Rbpj pool depletion

Rbpj is a major downstream effector of the Notch pathway. Previous studies have suggested that the Ptf1a activity might be downregulated by sequestering Rbpj from Ptf1a [START_REF] Cras-Meneur | Presenilins, Notch dose control the fate of pancreatic endocrine progenitors during a narrow developmental window[END_REF] following Notch activation. Therefore, we examined if ectopic Ptf1a might function in the chick retina by conversely sequestering Rbpj. We generated a Ptf1a-∆basic mutant where the highly conserved RER-R amino acids (168-172) (Fig. S4L,M) in the basic domain were replaced by AVA-A. These mutations were shown to abolish MyoD and bHLHa15/Mist1 bHLH transcription factor binding to DNA [START_REF] Lemercier | The basic helix-loop-helix transcription factor Mist1 functions as a transcriptional repressor of myoD[END_REF][START_REF] Skowronska-Krawczyk | The basic domain of ATH5 mediates neuron-specific promoter activity during retina development[END_REF]. The Ptf1a-∆basic protein was still imported into the nucleus (See Fig. S4O) and retained its ability to interact with Rbpj (See Fig. S4P). The in vitro reporter assay confirmed that the transcriptional activity of Ptf1a-∆basic was strongly decreased (data not shown). No alteration of retinal structure and cell type representation (Fig. 3E,J,O,T and Fig. S4I-J) was detected within the E12 RCAS-Ptf1a-∆basic-infected patches. 20 This result argues against the hypothesis that the activity of wild-type ectopic Ptf1a might be completely mediated by a disruption of Rbpj function.

Our study demonstrated that Ptf1a was sufficient to affect the chick retinal cell composition in an Rbpj-dependent manner. Our "candidate-gene" approach showed that Ptf1a overexpression selectively upregulated NeuroAB, Ap2α and Lim1 while it downregulated Otx2 and Atoh7. This study provides a new molecular basis for Ptf1a activity in specifying the ACs and all HC subtypes and inhibiting photoreceptor and ganglion cell genesis.

DISCUSSION

Cellular and molecular mechanisms underlying ectopic Ptf1a activity during chick retinal development.

Several hypotheses may explain the changes in cell type representation induced by Ptf1a.

First, cell proliferation defects may change the final retinal cell composition. Indeed, early cell cycle exit has been shown to favor early-born retinal cell types. Conversely, late cell cycle exit biases progenitor cells toward later neuronal fates [START_REF] Austin | Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch[END_REF][START_REF] Dorsky | Regulation of neuronal diversity in the Xenopus retina by Delta signalling[END_REF][START_REF] Henrique | Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina[END_REF][START_REF] Jadhav | Notch 1 inhibits photoreceptor production in the developing mammalian retina[END_REF][START_REF] Livesey | Vertebrate neural cell-fate determination: lessons from the retina[END_REF][START_REF] Hayes | Notch signaling regulates regeneration in the avian retina[END_REF]Ohnuma et al., 2002;[START_REF] Silva | Regulation of ganglion cell production by Notch signaling during retinal development[END_REF][START_REF] Yaron | Notch1 functions to suppress cone-photoreceptor fate specification in the developing mouse retina[END_REF]. We found that the forced expression of Ptf1a in the chick retina induced a premature cell-cycle exit, but led to a dramatic decrease of early-born ganglion and cone photoreceptor cells. Thus, it is unlikely that cell proliferation defects alone account for the effect of ectopic Ptf1a activity in chick retinogenesis. How Ptf1a induced cell cycle withdrawal remains to be defined. The cell cycle exit could be linked to the defects in the IKNM of retinal progenitors observed following Ptf1a mis/overexpression. Indeed, in the chick retina, pharmacological perturbation of IKNM was shown to promote premature neurogenesis (Murciano et al., 2002) through a reduction of Notch-mediated lateral inhibition [START_REF] Del Bene | Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient[END_REF]Murciano et al., 2002). Consistent with this model, the expression of Hes5, a target of the Notch pathway in the retina (Nelson et al., 2006), was significantly decreased in electroporated Ptf1a-overexpressing cells (data not shown). Moreover, the expression level of cyclin D1 that is required for RPC proliferation [START_REF] Fantl | Mice lacking cyclin D1 are small and show defects in eye and mammary gland development[END_REF][START_REF] Godbout | Elevated levels of cyclin D1 mRNA in the undifferentiated chick retina[END_REF][START_REF] Sicinski | Cyclin D1 provides a link between development and oncogenesis in the retina and breast[END_REF] was also significantly downregulated in Ptf1a-overexpressing cells (data not shown), suggesting that ectopic Ptf1a might regulate the core of the cell cycle machinery.

Second, Ptf1a might bias progenitor cells toward the AC and HC fates. We found that the expression of Atoh7 [START_REF] Brown | Math5 is required for retinal ganglion cell and optic nerve formation[END_REF][START_REF] Wang | Requirement for math5 in the development of retinal ganglion cells[END_REF]Otx2 (Nishida et al., 2003), which specify ganglion cells and photoreceptors, respectively, were inhibited in cells that overexpressed Ptf1a. In contrast, Lim1, involved in HC specification [START_REF] Suga | LIM family transcription factors regulate the subtype-specific morphogenesis of retinal horizontal cells at post-migratory stages[END_REF], and NeuroAB, which is presumably involved in AC genesis (Ohkawara et al., 2004), were upregulated. These transcriptional regulations support a mechanism where ectopic Ptf1a might act cell-autonomously to drive retinal progenitors toward the AC and HC fates at the expense of ganglion cells and photoreceptors by regulating their transcriptional program. The effects of ectopic Ptf1a on cell fate might also be influenced by cell proliferation regulation. Indeed, early cell cycle exit was shown to enhance the activity of proneural factors (Atoh7, Ngn1, and Neurod4) that promote early cell fates (Ohnuma et al., 2002). Recently, the majority of Ptf1a-expressing cells were found to originate from Atoh7-positive progenitor cells, and Ptf1a expression drove their differentiation toward ACs/HCs at the expense of ganglion cells in the zebrafish retina [START_REF] Jusuf | Origin and determination of inhibitory cell lineages in the vertebrate retina[END_REF]. Our overexpression study showed that ectopic Ptf1a was a negative regulator of Atoh7. Therefore, it would be interesting to assess whether endogenous Ptf1a, which is turned on after Atoh7 in the Atoh7-positive lineage, is able to downregulate Atoh7 expression during retinogenesis, how it might regulate Atoh7, and if this regulation is involved in the fate switch toward ACs/HCs. Third, Ptf1a might also selectively induce the death of ganglion and photoreceptor cells, modifying the cell representation in favor of ACs/HCs. The number of apoptotic cells was increased in Ptf1a-infected retinas at E7, but the precise determination of their identity was difficult to assess because many apoptotic cells have lost their differentiation markers. To our knowledge, no report has demonstrated a direct induction of cell death either by Ptf1a or by the misexpression of transcription factors without apoptotic regulatory properties. [START_REF] Dullin | Ptf1a triggers GABAergic neuronal cell fates in the retina[END_REF] did not detect any apoptosis induction following Ptf1a overexpression in Xenopus retinas, suggesting that Ptf1a has no pro-apoptotic function per se. Secondary cell death is a common feature of transcription factor gain-and loss-of-functions and has often been associated with defects in cell trans-commitment in the retina [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF][START_REF] Mao | Proneural gene ash1 promotes amacrine cell production in the chick retina[END_REF]Qiu et al., 2008;[START_REF] Zheng | The transcription factor RBP-J is essential for retinal cell differentiation and lamination[END_REF]. Therefore, the increase in cell death is likely a secondary event that occurs following the late re-specification of already committed precursors. Alternatively, a lack of crucial trophic support and inappropriate cell-cell interactions may lead to the elimination of prematurely differentiated cells.

Transcriptional regulation upstream and downstream of Ptf1a

Our results showed that Foxn4 upregulated Prox1, Lim1 and Ptf1a in the chick retina, suggesting that Foxn4 might be sufficient to induce HC fate in the developing chick retina, which is in contrast to mice [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]. Together with the upregulation of endogenous Ptf1a by Foxn4, these data confirm that Ptf1a functions downstream of Foxn4 in the transcriptional cascade leading to HC specification as proposed in mice [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF].

Interestingly, we discovered that Ptf1a downregulated Foxn4, indicating that Ptf1a would be required to finely regulate Foxn4 expression in HC and AC precursors. In line with this hypothesis, Li et al. reported that in Foxn4 LacZ knock-in mice, β-galactosidase (LacZ) expression was upregulated and persisted longer than in wild type retinas [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]. This Foxn4 locus deregulation could result, at least partially, from the loss of Ptf1a expression in Foxn4 -/- An increase in photoreceptor cells was reported in addition to the decrease of ACs/HCs following Foxn4 and Ptf1a inactivation [START_REF] Dong | Graded levels of Ptf1a differentially regulate endocrine and exocrine fates in the developing pancreas[END_REF][START_REF] Dullin | Ptf1a triggers GABAergic neuronal cell fates in the retina[END_REF][START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF][START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]Nakhai et al., 2007). Consistently, our misexpression study demonstrated that Ptf1a inhibited photoreceptor cell production and decreased Otx2 expression. Inversely, the loss of Otx2 in the mouse retina decreased the number of photoreceptors in favor of ACs (Nishida et al., 2003). These gain-and loss-of-function studies highlight a possible connection between the production of HCs/ACs on the one hand and photoreceptors and ganglion cells on the other. This balance may rely, to some extent, on Notch-mediated lateral inhibition. Notch signaling was shown to specifically inhibit mouse photoreceptor specification [START_REF] Jadhav | Notch 1 inhibits photoreceptor production in the developing mammalian retina[END_REF][START_REF] Yaron | Notch1 functions to suppress cone-photoreceptor fate specification in the developing mouse retina[END_REF]. In various structures of the central nervous system, Ascl1 and Foxn4 have been implicated in the regulation of the Delta-like/Notch signaling pathway. Notably, in the ventral spinal cord, Foxn4 functions upstream of Ascl1, Delta-like and Notch expression to regulate the diversification of excitatory and inhibitory V2 cells by lateral inhibition [START_REF] Del Barrio | A regulatory network involving Foxn4, Mash1 and delta-like 4/Notch1 generates V2a and V2b spinal interneurons from a common progenitor pool[END_REF]. In the retina (Nelson et al., 2009;Nelson and Reh, 2008) and mouse dorsal spinal cord (Mizuguchi et al., 2006), Ascl1 activates Delta-like and Ptf1a expression. Thus, similar to the spinal cord, Foxn4 and/or Ascl1 might initiate lateral inhibition among a subset of retinal progenitors. These factors would then be required to sustain Notch signaling in AC/HC precursors that were activated by Delta-expressing neighboring cells that were prone to differentiate as photoreceptor cells. Ascl1 and Foxn4 expression would also contribute to turning off the photoreceptor and ganglion cell specification programs in AC/HC precursors through Ptf1a mice [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF]. activation. Further studies will be needed to determine to what extent Foxn4 and Ascl1 function together to regulate Notch signaling and Ptf1a expression in the chick retina. Ptf1a alone might not sustain Notch signaling because Hes5 was downregulated in Ptf1aoverexpressing cells (data not shown).

Surprisingly, endogenous chick Ptf1a mRNA levels were significantly decreased in mouse Ptf1a-overexpressing cells. These results were in contrast with the positive autoregulation of Ptf1a that occurs in the pancreas, spinal cord and cerebellum [START_REF] Masui | Transcriptional autoregulation controls pancreatic Ptf1a expression during development and adulthood[END_REF][START_REF] Meredith | Multiple transcriptional mechanisms control Ptf1a levels during neural development including autoregulation by the PTF1-J complex[END_REF]. Recently, Jusuf et al. [START_REF] Jusuf | Origin and determination of inhibitory cell lineages in the vertebrate retina[END_REF] reported that a feedback loop originating from HCs/ACs operated to limit the number of cells initiating Ptf1a expression in the zebrafish retina. Thus, supernumerary ACs/HCs might regulate the expression of pro-amacrine factors, such as Foxn4 and Ptf1a, in autocrine or paracrine feedback loops. Alternatively, our results demonstrated that Foxn4 was downregulated in mouse Ptf1a-overexpressing cells and that Foxn4 activated endogenous chick Ptf1a. Chick Ptf1a expression would not be triggered in mouse Ptf1a-overexpressing cells as a result of the absence of its upstream activator, Foxn4.

PTF1-J complex during retinogenesis

Previous studies have demonstrated that the Ptf1a/Rbpj/E-protein (PTF1-J) complex is the functional endogenous PTF1 complex required for GABAergic cell specification in mouse cerebellum and spinal cord [START_REF] Hori | A nonclassical bHLH Rbpj transcription factor complex is required for specification of GABAergic neurons independent of Notch signaling[END_REF]. Nonetheless, the formation of this functional trimeric complex during retinogenesis has not yet been assessed. In this study, we showed that the loss of the Rbpj-binding domains within the Ptf1a protein was sufficient to abolish the Ptf1a effects on chick retinal cell differentiation, indicating that ectopic Ptf1a was dependent on the Rbpj cofactors in the chick retina. Does the endogenous Ptf1a factor require Rbpj for the specification of ACs and HCs?

Several points suggest that this functional interaction occurs. First, we showed that Rbpj was expressed in the retina during retinogenesis, notably in the INL (Fig. S6A-J). Second, recent conditional loss-of-function studies (Riesenberg et al., 2009;[START_REF] Zheng | The transcription factor RBP-J is essential for retinal cell differentiation and lamination[END_REF] reported that retinas null for Rbpj exhibited a phenotype that mimics some features of the Ptf1a-null retinas [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF]Nakhai et al., 2007) and complement our gain-of-function study. Indeed, the loss of Rbpj in the retina induced an increase of ganglion cells together with an upregulation of Atoh7 (Riesenberg et al., 2009;[START_REF] Zheng | The transcription factor RBP-J is essential for retinal cell differentiation and lamination[END_REF]. Moreover, the loss of Rbpj in Chx10-positive retinal cells induced a significant decrease of ACs and HCs in P21 mouse retinas [START_REF] Zheng | The transcription factor RBP-J is essential for retinal cell differentiation and lamination[END_REF]. The ganglion cell increase was attributed to Notch3 signaling inhibition (Riesenberg et al., 2009). We found that the Ptf1a-Rbpj interaction was necessary to inhibit ganglion cell specification following Ptf1a overexpression. Based on our study, the increase in ganglion cell number in Rbpj-null mice might result from either Notch3 signaling inhibition or from endogenous PTF1-J complex inactivation. The increase of ACs/HCs was not observed following Rbpj inactivation in the alpha-positive peripheral retinal cells (Riesenberg et al., 2009).The discrepancies between the two conditional Rbpj knock-out mouse models might result from the inactivation of Rbpj in different progenitor pools or a genetic compensation by Rbpjl because Rbpj was shown to compensate for the loss of Rbpjl in the Rbpjl -/- It is noteworthy that, in humans, mutations of the Rbpj-interaction domain of Ptf1a protein caused optic nerve hypoplasy in addition to a cerebellar and pancreatic agenesis [START_REF] Sellick | Mutations in PTF1A cause pancreatic and cerebellar agenesis[END_REF]. The same phenotype was reported in Rbpj-null mice where ectopic ganglion cells underwent cell death during late developmental stages. Even if the late specific cell death of the supernumerary ganglion cells remained to be assessed in Ptf1a-deficient mouse pancreas [START_REF] Masui | Replacement of Rbpj with Rbpjl in the PTF1 complex controls the final maturation of pancreatic acinar cells[END_REF]. They could also be due to the use of different amacrine cell markers. retina, this supports a highly conserved Ptf1a-Rbpj functional interaction during vertebrate retinal development.

Horizontal cell subtype specification by Ptf1a

It has been shown that Ptf1a is required to specify GABAergic neurons over glutamatergic neurons in the mouse dorsal spinal cord [START_REF] Glasgow | Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn[END_REF] and cerebellum [START_REF] Hoshino | Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum[END_REF]. However, Ptf1a controls the specification of glutamatergic climbing fiber neurons of the inferior olivary nucleus [START_REF] Yamada | Origin of climbing fiber neurons and their developmental dependence on Ptf1a[END_REF], suggesting that Ptf1a is a neuronal fate determinant in this structure. In the retina, two hypotheses were formulated: either Ptf1a is involved in all AC/HC specifications [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF][START_REF] Jusuf | Ptf1a is expressed transiently in all types of amacrine cells in the embryonic zebrafish retina[END_REF], or Ptf1a favors only the GABAergic cell subtypes [START_REF] Dullin | Ptf1a triggers GABAergic neuronal cell fates in the retina[END_REF]. We found in this study that the number of both H1 GABAergic cells (and probably H2 cells) and H3 non-GABAergic HCs were increased in the RCAS-Ptf1a-infected patches. Thus, Ptf1a is likely to act primarily as a general HC determination factor.

We detected only a few Islet1-positive HCs (H2 or H3 cells) that expressed endogenous Ptf1a during chick retinogenesis, which argues against a direct exclusion between Islet1 and Ptf1a. One hypothesis is that H3 HCs, which are the majority of the Lim1-negative and Islet1-positive, may not express Ptf1a and that the few Ptf1a/Islet1 double-positive cells in the HCL were H2 HCs. Alternatively, Ptf1a expression could be transiently required for the specification of H2/H3 cells and turned off at the onset of expression of H2 and H3 markers in the late-born Islet1-positive HCs [START_REF] Edqvist | Axon-bearing and axon-less horizontal cell subtypes are generated consecutively during chick retinal development from progenitors that are sensitive to follistatin[END_REF]. Consistent with this hypothesis in the zebrafish retina containing both Lim1-positive and Islet1-positive HC subtypes (Hallböök, unpublished), the knock-down of Ptf1a expression caused a decrease of Prox1/Islet1-positive HCs [START_REF] Dong | Graded levels of Ptf1a differentially regulate endocrine and exocrine fates in the developing pancreas[END_REF].

We found that Lim1-positive HCs expressed Ptf1a and that Ptf1a remained during bidirectional migration and once the cells stopped their migration in the HCL, indicating that Ptf1a might be involved in several steps of H1 cell specification/differentiation beyond its implication in all HC fate. In contrast, we found that Ptf1a downregulated Islet1 expression, suggesting that Ptf1a should be turned off for Islet1 to be expressed (data not shown).

Therefore, secondary to the specification for all HCs, differential regulation of Ptf1a expression in the HC subtypes (sustained expression in H1 and downregulation in H2/H3) might be necessary to achieve their proper differentiation. In the context of forced and persistent Ptf1a expression, this regulation might fail and result in a decrease of the H3 proportion among HCs.

  analysis of variance followed by Tukey's multiple comparison tests (three or more groups) using Prism 5.0 (Graphpad software). (*) p<0.05, (**) p<0.01, (***) p<0.001, (ns) not significant. p Statistical analyses anova , p-value of the one-way analysis of variance; p Tukey , p-value of the Tukey post-hoc test. Forced expression of Ptf1a results in a loss of retinal lamination.

Fig. 1 :

 1 Fig. 1: Mis/overexpression of Ptf1a a ects retinal structure. (A) Lysates from DF1 cells transfected with the RCAS-Ptf1a plasmids or not (Control) were harvested, and the expression of exogenous Ptf1a proteins was monitored by western blotting using anti-Ptf1a and anti-HA antibodies. RCAS-Ptf1a-noTag: RCAS-Ptf1a without HA-Tag, RCAS-HA-Ptf1a: RCAS-Ptf1a with HA-tag. (B-E) The RCAS-infected (B-C) and RCASPtf1ainfected (D-E) retinal sections were stained with anti-Ptf1a (B,D) or anti-gag (p27) (C,E) antibodies at E9. (F-Q) The RCAS-infected (F-G,J -K,N-O) and RCAS-Ptf1a-infected (H-I,L-M,P-Q) retinal sections were co-stained with hemalun (F,H,J,L,N,P) and anti-gag (3c2) antibodies (G,I,K,M,O,Q). In B-E,G,I,K,M,O,Q, retinal sections were counterstained with DAPI. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NBL, neuroblastic layer. Bars: 50 μm (B-E in B and D), 25 μm (F-Q).

Fig. 2 :

 2 Fig. 2: E ects of Ptf1a mis/overexpression on retinal progenitor cell proliferation. (A-H) Sections from the RCAS-infected (A,C,E,G) and the RCAS-Ptf1a-infected (B,D,F,H) retinas were stained with EdU and anti-gag (3c2) antibodies (A-D) or with anti-P-H3 and antigag antibodies (E-H) at E7 and E9. In F, arrows point to P-H3 positive cells in an ectopic location in the RCAS-Ptf1a-infected retinas. (I-J) Quantitative analysis of EdU-positive (I) or P-H3-positive cells (J) among the RCAS-and RCAS-Ptf1a-infected cells at E7 and E9. Values represent the mean ± s.e.m. The percentage of EdU-positive cells among the infected cells was normalized by the percentage of EdU-positive cells among the non-infected cells for each embryo. NBL, neuroblastic layer; GCL, ganglion cell layer; INL, inner nuclear layer. Bars: 25 μm.

Fig. 3 :

 3 Fig. 3: E ects of wild-type and mutant forms of Ptf1a on retinal cell di erentiation. (A-T) The RCAS-infected (A,F,K,P), RCAS-Ptf1a-infected (B,G,L,Q), RCAS-Ptf1a-ΔC2infected (C,H,M,R), RCAS-Ptf1a-ΔC12-infected (D,I,N,S) and RCAS-Ptf1a-Δbasic-infected (E,J,O,T) patches from E12 retinas were immunostained using anti-Brn3a (A-E), anti-Visinin (F-J), anti-Ap2α (K-O), and anti-Prox1 (P-T) antibodies. For clarity, the gag labeling is not represented. Retinal sections were counterstained with DAPI. (U-V) Representative ow cytometry analysis after staining the RCAS-infected (U) and RCAS-Ptf1a-infected (V) dissociated cells with anti-gag (p27) and anti-Visinin antibodies at E12. (W) Quantitative analysis of Visinin-, Ap2α-and Prox1-positive cells among the infected cells at E12. Values represent the mean ± s.e.m of at least four separate eye counts. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; PE, Phycoerythrin; Alexa633, AlexaFluor633. Bars: 50 μm

Fig. 5 :

 5 Fig. 5: Ptf1a expression in subtypes of developing horizontal cells. Epi uorescence micrographs show Ptf1a, Prox1 (A-D), Ptf1a, Lim1 (E-H), and Ptf1a, Islet1 (I-L) double-labeling of E6.5, E9, E12 and E16 chick retinas, and the corresponding split uorescence images are to the right of each panel. Insets in b, f, and j are higher magni cations of the boxes in B, F and J. Arrows point at double-labeled cells. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Bars: 25 μm.

Fig. 6 :

 6 Fig. 6: Ptf1a mis/overexpression is su cient to increase the number of all horizontal cell subtypes. (A-F) The RCAS-infected (A,C,E) and RCAS-Ptf1a-infected (B,D,F) patches were double immunostained at E7 with either anti-Lim1 and anti-Prox1 antibodies for H1 cells (A-B), anti-Islet1 and anti-Prox1 antibodies for H2 and H3 cells (C-D) or anti-Islet1 and anti-TrkA antibodies for H3 cells (E-F). a-f are higher magni cations of the boxes in A-F, respectively. a'-f' and a''-f'' are split uorescence images of a-f. Arrows point to some double-positive cells. (G-N) The RCAS-infected (G,I,K,M) and RCAS-Ptf1a-infected (H,J,L,N) patches at E9 were double immunostained with either anti-Lim1 and anti-Prox1 (G-H), anti-Islet1 and anti-Prox1 (I-J), anti-Islet1 and anti-TrkA (K-L) or anti-TrkA and anti-Lim1 antibodies (M-N). g-n are higher magni cations of boxes in G-N, respectively. g'-n' and g''-n'' are split uorescent images of g-n, respectively. Arrows point to some Islet1-positive/TrkA-negative cells. (O) The quanti cation of Lim1-(H1), Islet1-(H2-H3) and TrkA-positive (H3) cells among the Prox1-positive HCs in the RCAS-and RCAS-Ptf1a-infected patches at E9. The values represent the mean ± s.e.m. NBL, neuroblastic layer; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; pHCL, prospective horizontal cell layer. Bars: 25 μm (A-F), 5 μm (a-f ), 50 μm (G-N), 10 μm (g-n).

Fig. 7 :

 7 Fig. 7: Changes in retinogenic factor mRNA expression following Ptf1a overexpression. (A) Schematic structure of pCIG expression vectors. IRES, internal ribosomal entry site. (B) The expression level of candidate genes was analyzed by quantitative PCR (qPCR) on mRNA from GFP-positive sorted retinal cells electroporated at E5 with either pCIG-GFP (Empty pCIG) or pCIG-Ptf1a-GFP (pCIG-Ptf1a). mmPtf1a, mouse Ptf1a; ggPtf1a, chick Ptf1a. (C) The expression level of a subset of genes involved in HC development was assessed by qPCR on mRNA isolated from GFP-positive sorted retinal cells electroporated at E5 with empty pCIG, pCIG-Ptf1a or pCIG-Foxn4-GFP (pCIG-Foxn4). Data represent the mean ± s.e.m of a least four retinas.

Fig. S1 :

 S1 Fig. S1: The partial chick Ptf1a protein sequence.

Table S1 : Summary list of primers used in this study.

 S1

	Name	Primer Sequence 5'->3'	Reference
	Actin Forward	CATCACCATTGGCAATGAGAG
	Actin Reverse	TGGACAGGGAGGCCAGGATAG
	Ap2 Forward	AGAGTTCACCGACCTGCT
	Ap2 Reverse	GCCGTGCGAGATGAGGTTGA
	ggPtf1a Forward	GAAGGCGAACGCCAAACCCT
	ggPtf1a Reverse	GTTGATTGCCCTTAGAGTCC
	mmPtf1a Forward	GAAGGTCATCATCTGCCATCG
	mmPtf1a Reverse	TCAGGACACAAACTCAAARGGTG
	Ascl1 Forward	AGGGAACCACGTTTATGCAG	(Hayes	et	al.,
			2007)
	Ascl1 Reverse	TTATACAGGGCCTGGTGAGC	(Hayes	et	al.,
			2007)
	Atoh7 Forward	CTGTGAACACTTTCATCCAGAG
	Atoh7 Reverse	CTAATTAGCTATTTGAAAGTGTTCAG
	Foxn4 Forward	GCTGCCATTCATAGGAGCAT	(Hayes	et	al.,
			2007)
	Foxn4 Reverse	CTGGGTCTGGATCTGGTGAT	(Hayes	et	al.,
			2007)
	GAPDH Forward	TGCCCAGAACATCATCCCAGCG
	GAPDH Reverse	TGGGGGTTGGCACACGGAAA
	Lim1 Forward	TTCTCCTCGATATCCGTCAACG
	Lim1 Reverse	CACCGTAACAACAAGCAGACTAG
	NeuroAB/bHLHb4 Forward ACCCTCAACCCCTTCGGACA
	NeuroAB/bHLHb4 Reverse	AGTGTTTGCAGAGCGCGGAG
	Ngn2 Forward	GCTGTCTCCGTGATTTACGAG
	Ngn2 Reverse	CCCTTCCCTTCCCTCTACAGTT
	Otx2 Reverse	TTAACTTCCAGGAGGCGGTTTGG
	Otx2 Forward	ATGGGTGCCAATGCGGTCACCA
	Pax6 Forward	TCCAGTTCAAGTTCCTGGCAG
	Pax6 Reverse	GTGTTACAGCGTAGGGCACAG
	Prox1 Forward	CAGGGCCCTGAACATGCACTA
	Prox1 Reverse	CTCTTGTAGGCAGTTGGGGGA
	Rbpjl Forward (TC_237853) CCCTCGCAGAAGAAGCAGTC
	Rbpjl Reverse	TGTGAGCGCAGGCGGTTGAA
	Sox2 Forward	AGGCTATGGGATGATGCAAG	(Hayes	et	al.,
			2007)
	Sox2 Reverse	GTAGGTAGGCGATCCGTTCA	(Hayes	et	al.,
			2007)
	Neurod4/Ath3 Forward	AACCCCCCCGACTGCACCACT	.
	Neurod4/Ath3 Reverse	CTGACCGAAGGGTAGTGAGCCA	.

Acknowledgments: We thank Pr. Lefcort for the anti-TrkA antibody, Pr. Edlund for the anti-Ptf1a antibody, Pr. Real for the Ptf1aK200R construct and Pr. Loftus for the RCAS-BP(A)-NHY plasmid. We are grateful to Prs. Martinerie, Laurent and Chiodini for their helpful support. We acknowledge Drs. Sadzinski-Matter, Thomasseau and Sandlung for technical assistance. This work was financed by INSERM, Retina France, EU (LSHG-CT-2005-512036, ERC-StG-210345), French ANR (ANR-Geno-031-03, ANR-08-MNPS-003), Swedish Research Council, Swedish SSMF and SSF.

(A-J) The E12 retinas infected with the indicated RCAS virus were immunostained using anti-gag (3c2) antibody to detect infected patches (B,D,F,H,J) and hemalun-stained (A,C,E,G,I) to label nuclei. The red line in J indicates the infected area. (K) Schematic diagrams of PTF1 complex formed by Ptf1a/p48, an E-protein (AbHLH) and either Rbpj or Rbpjl. Ptf1a interacts with Rbpj with its C2 and C1 domains, whereas only the C1 domain is required for its interaction with Rbpjl [START_REF] Beres | PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L[END_REF]. (L) The schematic structure of the Ptf1a mutant forms generated in this study. The Ptf1a protein contains a transactivation domain within its N-terminal [START_REF] Rodolosse | p/CAF modulates the activity of the transcription factor p48/Ptf1a involved in pancreatic acinar differentiation[END_REF] The level of HA-tagged Ptf1a proteins was monitored using anti-HA antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and LaminB were used as positive controls and standards for cytoplasmic and nuclear fractions, respectively. The Ptf1aK200R mutant, previously shown to be partially retained in the cytoplasm, was used as a control to assess the validity of cell fractionation protocol [START_REF] Rodolosse | p/CAF modulates the activity of the transcription factor p48/Ptf1a involved in pancreatic acinar differentiation[END_REF]. In this mutant, the lysine located 200 amino acids upstream of the start codon was replaced by an arginine. (P) The total extracts from DF1 cells infected with RCAS, RCAS-HA-Ptf1a and RCAS-HA-Ptf1a-∆basic were immunoprecipitated with an anti-HA antibody. Inputs and immunoprecipitates were tested for Ptf1a or Rbpj by western blotting using anti-Ptf1a and anti-Rbpj antibodies, respectively. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 25 µm (A-J). (A-J) Sections from the chick retina of the indicated embryonic stages were hybridized with antisense and sense Rbpj riboprobes. Rbpj mRNA was detected within the whole retinal epithelium at E6. At E9, Rbpj expression was restricted to the prospective inner nuclear layer and decreased at E12 in the central retina but was higher in E12 peripheral retina. At E16, Rbpj expression was not detectable. (K) Total RNA was extracted from various organs of E19 chick embryos. Rbpjl mRNA levels were quantified using qPCR. Rbpjl was highly expressed in the chick pancreas but was not detected in the mature retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; NBL, neuroblastic layer. Scale bars: 20 µm (A-J).


Storing events to retell them

Jean-Louis Dessalles ParisTech, Ecole Nationale Superieure des Telecommunications, F-75013 Paris, France. dessalles@enst.fr www.enst.fr/~jld Abstract: Episodic memory is certainly a unique endowment, but its primary purpose is something other than to provide raw material for creative synthesis of future scenarios. Remembered episodes are exactly those that are worth telling. The function of episodic memory, in my view, is to accumulate stories that are relevant to recount in conversation.

As the authors of the target article suggest, episodic memory (EM) can be seen as a "plug-in" device added to a standard vertebrate brain, and quite an expensive one, as much of our cortical mass seems devoted to it. Suddendorf & Corballis (S&C) are certainly right to say that it demands an evolutionary explanation. Their suggestion is that EM serves only the practical purpose of providing raw material for future planning. In a similar attempt to provide an evolutionary account for EM, [START_REF] Brown | Flashbulb memories[END_REF] highlighted the benefit of storing unexpected and highly emotional events, as "a marked departure from the ordinary in a consequential domain would leave [the individual] unprepared to respond adequately and endanger his survival" (p. 97). The problem with such accounts is that EM is badly designed for its alleged function. Does an optimal use of storage capacity leave room for the memory of instantiated episodes? In machine learning, rote learning is an inefficient strategy. The purpose of any learning task is to make generalization possible. A good way to perform induction is to aggregate experience into structures such as prototypes or clusters. Storing particular instances (e.g., cluster centers) generally makes sense if they are statistically representative. This function is implemented in living beings through semantic and procedural memory.

To delineate categories, it may be useful to remember borderline instances, as with support vectors [START_REF] Cornuejols | Apprentissage artificiel -Concepts et algorithmes[END_REF]. Also, in certain applications in which data are scarce and non-homogeneous, storing actual encountered examples, regardless of their representativeness, may be a viable strategy. The Case-Based Reasoning technique [START_REF] Kolodner | Case-based reasoning[END_REF] aims at solving new problems by matching them with memorized known examples. Superficially, episodic memory could be understood as a biological implementation of these principles, but its actual form does not match up to the assignment.

Episodic memory is highly selective. It retains a tiny fraction of all our daily experiences. One may come across dozens of people each day and remember only a few encounters per month. Selected episodes are, however, retained with a significant amount of detail, including what S&C call the www criterion. From an efficiency perspective, details such as the precise location in space and time, the weather conditions, the persons present, the words exchanged, and so forth, are most often irrelevant and yet are almost systematically remembered, even in the long term in cases when emotion is high (Brown Dessalles, J-L. (2007). Storing events to retell them (Commentary on Suddendorf & Corballis: 'The evolution of foresight'). Behavioral and Brain Sciences, 30 (3), 321-322. & Kulik 1977). From a computational perspective, not only do such details represent a waste of storage, they also hinder and mislead retrieval matching.

An alternative view is that EM is an outgrowth of the language faculty [START_REF] Dessalles | Human language in the light of evolution[END_REF]. It is not fortuitous that memorized episodes are exactly those which are narratable. People spend one fifth of their waking time in spontaneous conversation [START_REF] Dunbar | Theory of mind and the evolution of language[END_REF], and a significant share of this time is devoted to reporting past events (Tannen 1984, p. 99;Eggins & Slade 1997, p. 265). Interlocutors draw from their memory relevant episodes that they can relate to the current conversational topic and they systematically try to recount them. However, only a tiny fraction of past experiences may be recounted in this way. One crucial requirement is that reported stories must appear unexpected (Dessalles, in press).

The requirement of unexpectedness provides also a good prediction of the kind of episodes that are preferentially stored in memory. To appear unexpected, a situation must be less complex (i.e., more easily describable) than expected (Dessalles, in press). Witnessing a six-legged cow makes both a memorable event and a good story to tell, just because this cow, thanks to its unique peculiarity, requires a minimal description to be distinguished from all others. If, as we claim, the primary purpose of storing episodes is to offer material for future recounting, then systematically remembering details such as time and space location makes perfect sense. If the six-legged animal lives in the vicinity, interest is raised. Not specifying the location would leave the listener with the idea that that location requires a lengthy description, and interest drops down. By computing complexity differences, one can derive the way interest varies according to location and time, and according to various factors such as the persons involved (Dessalles, in press). For instance, the interest of coincidentally encountering someone increases with the complexity (e.g. remoteness) of the place and the simplicity (e.g. celebrity or closeness) of that person. It is thus crucial, when memorizing an episode, to remember every detail that may affect the cognitive complexity of the situation.

It may seem surprising that the expensive resources devoted to EM serve such a futile purpose as everyday chatter. This is only because one fails to see that casual conversation is an arena where much of our social existence is decided [START_REF] Dessalles | Why we talk -The evolutionary origins of language[END_REF]. Eliciting interest through conversational stories is a high-stakes game. Boring participants are rapidly ignored and may lose their friends. When it comes to establishing solidarity bonds, individuals prefer those who successfully demonstrate their informational capacity and their experience with unexpected events. In our species, those who know first or who can draw highly relevant events from their past experiences make potentially good allies. Natural selection favored not only this preference, but also the narrative skills that allow any of us to display these qualities [START_REF] Dessalles | Why we talk -The evolutionary origins of language[END_REF]. Episodic memory, in this context, is a crucial tool that enables us to produce the most relevant story at the right time. It has been tailored for this purpose, as demonstrated by the fact that the factors that favor memorization, such as unexpectedness and atypicality [START_REF] Shapiro | The role of typical and atypical events in story memory[END_REF][START_REF] Stangor | Memory for expectancy-congruent and expectancyincongruent information: a review of the social and social developmental literatures[END_REF][START_REF] Woll | Memory discrimination for information typical and atypical of person schemata[END_REF], are exactly the factors which increase tellability. This account explains why remembered episodes are communicated, instead of remaining private; why they remain coherent in memory (instead of being dismantled for creative synthesis of future scenarios); why they systematically involve various details and precision; why we keep on memorizing episodes throughout our entire life; and why even slight failures in episodic memory (as those that occur with aging or in certain pathologies) have dramatic influence on social relations. It also explains the uniqueness of EM, which was not selected for increasing planning efficiency, but as a tool in support of language performance.


Jean-Louis Dessalles

SPONTANEOUS NARRATIVE BEHAVIOUR IN HOMO SAPIENS: HOW DOES IT BENEFIT TO SPEAKERS?

The fact that human beings universally put much energy and conviction in reporting events in daily conversations demands an explanation. After having observed that the selection of reportable events is based on unexpectedness and emotion, we make a few suggestions to show how the existence of narrative behaviour can be consistent with the socio-political theory of the origin of language.

Spontaneous narratives: a fundamental component of language

Dozens of times everyday, human individuals feel the urge to signal current events or to report past events to their conspecifics. In doing so, they respond to specific stimuli such as departure from norm, coincidences or emotion. This event reporting behaviour seems to be unique in nature [START_REF] Boyd | The origin of stories: Horton hears a who[END_REF], as remote analogues such as bee dance and alarm calls do not compare with it. During conversational narratives, the speaker may hold the floor during several minutes, with no other interruption than minimal approval signals emitted by interlocutors. Within a Darwinian framework, the existence of such behaviour requires an explanation. How does this time-consuming activity, which deals most often with futile anecdotes that are unlikely to be of any direct interest for survival, benefit not only to listeners, but also to speakers?

We first show the importance of narratives by providing quantitative estimates. Then we outline a cognitive characterization showing that individuals respond to definite stimuli when selecting reportable events. Lastly, we look for plausible Darwinian explanations for the existence of narrative behaviour, in line with our socio-political account of the origins of language.

Narratives in daily speech

Human beings make extensive use of their language ability. Individuals have been observed to speak 15 000 words a day on average [START_REF] Mehl | Are women really more talkative than men?[END_REF]). This Dessalles, J-L. (2008). Spontaneous narrative behaviour in homo sapiens: how does it benefit to speakers?. In A. D. M. Smith, K. Smith & R. Ferrer i Cancho (Eds.), The evolution of language -Proceedings of the 7th International Conference (Evolang7 -Barcelona), 91-98. Singapore: World Scientific. http://www.enst.fr/~jld/papiers/pap.evol/Dessalles_07091501.pdf behaviour covers several activities which can be distinguished by the cognitive mechanisms involved. The two main components of spontaneous language use are discussion (argumentation) and narration (event reporting) (Dessalles 2007a). The proportion of event reporting may vary significantly from one corpus to the next. Suzanne Eggins and Diana Slade (1997) observed the repartition indicated in Table 1 in the three hours of casual conversation data they collected during coffee breaks in three different workplaces. Storytelling takes up the major share in this distribution. We made similar measures on our main corpus, composed of 17 hours of conversations, recorded during meals at family gatherings between 1978 and 1988 among educated individuals belonging to a French middle class family. The distribution of conversation types was explored through a sampling method (Figure 1). The corpus has been digitalized, and 150 excerpts of 120 s. have been automatically extracted at random positions. For each excerpt, the central utterance (occurring at time 60 s.) has been assigned a category ( The small relative size of the 'empty' category reveals that in this family meal context, language is used 89% of the time. Conversation proper, which excludes utilitarian (more or less ritualized) speech, occupies nearly 70% of the time (as most of inaudible utterances are likely to be conversational). Narratives and signalling together amount to 26% of conversation time. Though it is less than in Eggins and Slade's corpus, event reporting still represents a significant share of spontaneous language use. Quantitative data about conversational narratives are unfortunately lacking, especially for intercultural comparisons. We can only conjecture that no culture lacks spontaneous narrative behaviour, as such a people would have been well-known for its conversational specificity. Narrative speech contrasts with the other main discourse category, discussion. Superficially, the most manifest difference is that narratives deal with situated events, whereas discussion deals with problems and their solutions. When facts are mentioned during a discussion, they are most often not situated (e.g. 'Fabius [a potential Prime Minister] makes a better impression on TV'), though there are cases in which situated facts or genuine stories are recounted in support of an argument (two occurrences in the sample). But narration and discussion can be opposed more fundamentally on the cognitive ground. Discussion consists in settling an issue or solving an epistemic puzzle. It proceeds through a characteristic problem-abduction-revision procedure (Dessalles 2007a). By contrast, event reporting relies on unexpectedness and emotion elicitation (see below). Though narrative and argumentative moves may be intertwined in conversation, they often remain separate: arguments spark off arguments in a typical problem-solution alternation, whereas narratives spark off new narratives, generating 'story rounds' (Tannen 1984 p. 100).

Events reported in conversations are most often not fictional (only one example in the sample can be considered fictitious, as it consists in describing the content of a cartoon). Though fiction seems to obey definite patterns [START_REF] Hogan | The mind and its stories -Narrative universals and human emotion[END_REF], conversational narratives seem to be ruled by even more constraining imperatives. Any narrative must have an interesting point (Labov 1997), as otherwise speakers may be regarded as socially inept (Polanyi 1979 p. 211). In what follows, we propose a cognitive characterization of competent storytelling, before considering its possible biological role.

The selection of reportable events

The dozens of episodes that human beings tell each day through conversational narratives represent only a tiny fraction of their actual experience. The selection of reportable events (Labov 1997) obeys specific patterns. In what follows, we highlight two fundamental requirements that are deeply rooted in our cognition: reportable events must be unexpected and/or arouse emotion.

Unexpectedness

The property of unexpectedness covers various aspects of newsworthiness, including deviance, atypicality, rarity, proximity, remarkable structures, and coincidences. In former accounts, we equated unexpectedness with improbability [START_REF] Dessalles | La fonction shannonienne du langage : un indice de son évolution[END_REF]. Though, the model left important cases unexplained. Individuals consider situations that depart from the norm for some qualitative reason (e.g. jogging nuns) highly unexpected, what probability theory fails to explain. Our new model [START_REF] Dessalles | Complexité cognitive appliquée à la modélisation de l'intérêt narratif[END_REF], based on formal complexity [START_REF] Li | An introduction to Kolmogorov complexity and its applications[END_REF], makes correct predictions for all situations perceived as unexpected. We found that the relevant parameter is the contrast between the expected and the actual complexity of the situation. While a standard encounter with nuns is expected to be complex to individuate, encountering jogging nuns is maximally simple, thanks to their unique characteristics. Similarly, a narratable fortuitous encounter is all the more unexpected since the person one bumped into is simple (a close friend or a celebrity) and the place is remote enough to be complex. Complexity contrasts turn out to have a systematic influence on reportability [START_REF] Dessalles | Complexité cognitive appliquée à la modélisation de l'intérêt narratif[END_REF].

Assessing variations of individuation complexity is certainly not a trivial cognitive operation, but every human being seems to be equipped to perform it.

The universal sensitivity to unexpectedness is a fundamental component of narrative competence and must have been selected for definite reasons.

Emotion

Emotion is the other parameter that stimulates event reporting. Emotional situations are systematically shared [START_REF] Rimé | Le partage social des émotions[END_REF]). In the following conversation from Neal Norrick's corpus (2000 p. 64), a young man recalls an accident story his aunt told him (transcription details omitted).

Mark: you know what happened to my one of my aunt's friends out in Iowa?

Like when-when she was younger, she had a headgear from braces, and these two girls were wrestling around just playing around, wrestling. And one girl pulled her headgear off her mouth and let it snap back. And it slid up her face and stuck in her eyes and blinded her. Jacob: wow. Mark: isn't that horrid? That's horrid. Blinded her for life. Isn't that horrid.

That's just-I mean just from goofing around.

Unexpectedness and emotion are often combined to enhance reportability. Unexpectedness here lies in the contrast between standard play situations, which are complex to individuate because they are all alike, and the actual situation which is unique by its consequences. Mark's final remark points to this contrast.

Studies show that emotion is reactivated during recounts, and this can be seen as a paradox (Rimé 2005 p. 109). Quite surprisingly, bringing back memories about negative emotions and sharing them with listeners is experienced as enjoyable by both parties. People like to talk not only about positive events, but also about events that generated fear, sadness, anger, guilt, embarrassment, contempt and even shame. They also like to listen to others' corresponding experiences, despite the fact that the evocation of such events produces negative feelings similar to the original ones. It seems that the pleasure of sharing these feelings compensates for experiencing them again.

Why are conversational stories told?

The pervasive presence of stories in conversations is an embarrassment for most accounts of the evolutionary origin of language. If language has been selected because of its effect on the welfare of the group [START_REF] Victorri | Homo narrans : le rôle de la narration dans l'émergence du langage[END_REF]Castro et al. 2004 p. 734;Ritt 2004 p. 1-2) or as a fair exchange of information based on strict reciprocity (Pinker 2003 p. 28;[START_REF] Nowak | Five rules for the evolution of cooperation[END_REF]Nowak p. 1561)), then the efforts that speakers devote to tell stories for all to hear, most often with much emphasis to highlight interest, is incomprehensible. We would expect speakers to whisper minimal factual information to specific ears and then demand of listeners that they reciprocate. What may be true for crucial advice (such as which shares to buy on the stock exchange) does not apply to conversational stories.

Other accounts emphasize the educational value of language [START_REF] Fitch | Evolving honest communication systems: Kin selection and 'mother tongues[END_REF]Castro et al. 2004 p. 725). But stories are found to be spontaneously told, not only from adults to children, but also from adults to adults and even from children to adults. As soon as by nine months of age, children spontaneously point to unexpected stimuli [START_REF] Carpenter | Social cognition, joint attention, and communicative competence from 9 to 15 months of age[END_REF]. More generally, theories of language function that emphasize the practical value of information are at odds with the fact that most stories are about futile matters. Unexpected events are, by essence, unlikely to occur again. Any practical processing of information would concentrate on vital information (danger, food, mating opportunities) and would neglect the myriad of anecdotal facts that fill daily chatter. Animals do not care about situations just because they are unexpected: they show no interest four-leaf clovers, they would regard a unicorn as a mere horse and they do not care about coincidences. Human communication, on the other hand, is universally replete with details about inconsequential episodes, just because of their unexpectedness, and this requires an evolutionary explanation.

The key difference seems to lie in human sociality: we crave the attention of others and narratives are a major way to do so [START_REF] Boyd | The origin of stories: Horton hears a who[END_REF]. This makes sense within our political theory of the origin of language (Dessalles 2007a), which states that individuals use language to demonstrate qualities that are in demand in the establishment of solidarity bonds. To fit in with the model, however, the qualities shown when telling stories must have political significance. In what way does the ability to produce unexpectedness and elicit emotions correlate with being a valuable coalition partner? Though these issues have not been properly investigated yet, we may have some idea. First, from early age on, individuals are in competition to demonstrate that they knew first, as in the next exchange, observed between two children aged eight and ten: Being the first who noticed the presence of balloons is important for M, as his second utterance shows. The first-to-know phenomenon is the most obvious case in which unexpectedness is produced. Reclaiming authorship for the news is understandable if, as we suggest, language is display (Dessalles 2007a). The informational competence that individuals display by offering scoop stories to conspecifics reveals crucial in the specific political context of our species.

Hominins are 'apes with spears'. 1 The introduction of deadly weapons in an ape species dramatically changes the political game: physical strength becomes much less relevant. The best strategy to survive is to share one's fate with other individuals (here, we radically depart from Paul [START_REF] Bingham | Human evolution and human history: A complete theory[END_REF] theory, in which the main effect of weapons is to enforce cooperation through retaliation within coalitions). In a context in which every individual must choose allies, the problem is to determine the best ones. Those who are able to spot unusual goings-on are a good choice, as they are the first to warn for complex danger. Since that time, individuals crave for displaying their ability to notice unexpectedness, as they are descended from ancestors for whom it was a good way to be accepted as coalition partners. Still now, individuals who are able to bring unexpected news are perceived as 'interesting' and are preferentially chosen as friends. They get social success, whereas boring individuals are avoided. Of course, the political game is more complex in our species than in the first hominin species. However, those who are able to spot unexpected situations or activities remains a crucial asset in a species in which being taken by surprise may be fatal. The claim is thus that there is continuity in the signalling and narrative behaviour along the hominin lineage down to sapiens, as it remains a way for individuals to show off their value as coalition partner.

Why do individuals tell emotional narratives? Emotions displayed in stories are associated with political values, such as solidarity or courage. By showing that they are able to experience compassion, pity, concern, indignation at cowardice, cheating or unfairness, that they admire selfless love and feats, individuals try to appear as ideal friends. If we accept that successfully communicated emotions are hard to fake, then expressing them through stories is a reliable indication that one really values the corresponding qualities.

Human beings are information oriented animals, who exchange stories on a daily basis. We tried to indicate how this fact, which is hard to explain in traditional accounts of language origins, can be a natural outcome of the particular socio-political organization of our species, in which individuals must compete by demonstrating their information qualities in order to attract friends.

M:

  Did you see there are more [hot-air] balloons up there this morning? Q: Yes, I know. M: You, be quiet! I'm not talking to you, I'm talking to the others. [To his father] Did you see there are balloons up there this morning?

Table 1 :

 1 Distribution of conversational topic types inEggins and Slade's corpus (1997 p. 265)

	Conversation type	%
	Storytelling	43.4
	Observation/Comment	19.75
	Opinion	16.8
	Gossip	13.8
	Joke-telling	6.3

Table 2 )

 2 .

	Utilitarian		Discussion
	11%		74%
	Empty 11%	Conversation 60%
	Inaudible
	13%		Narratives
	5% Other		7% Signalling	19%
	overall time	conversation
	Figure 1. Distribution of utterance types in our own corpus, assessed through a sampling method.

Table 2 :

 2 Definition of utterance types in the family conversation corpus

	Utterance type	Definition	Example
	Narratives	Reports about past situated events	Dishwasher breakdown which turned out to be a mere leak from a plastic bottle
	Signalling	Drawing attention to current facts	Showing a new electronic personal organizer
	Discussion	Dealing with problems	Who's going to be the next Prime Minister
	Utilitarian speech	Offers, requests,… (mostly in relation with food)	Offering more foie gras
	Other	Songs, child screaming	Singing to bring a child to sing along
	Inaudible	Superimposed noise, simultaneous conversations
	Empty	No speech, no utterance in progress

Spears may be thought to have been the first efficient weapons used by hominins: they are easy to make and to carry (the use of spears may have contributed to make biped walk advantageous).