Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina.
ABSTRACT During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix-loop-helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.
- SourceAvailable from: Joseph A Brzezinski[show abstract] [hide abstract]
ABSTRACT: The vertebrate retina contains seven major neuronal and glial cell types in an interconnected network that collects, processes and sends visual signals through the optic nerve to the brain. Retinal neuron differentiation is thought to require both intrinsic and extrinsic factors, yet few intrinsic gene products have been identified that direct this process. Math5 (Atoh7) encodes a basic helix-loop-helix (bHLH) transcription factor that is specifically expressed by mouse retinal progenitors. Math5 is highly homologous to atonal, which is critically required for R8 neuron formation during Drosophila eye development. Like R8 cells in the fly eye, retinal ganglion cells (RGCs) are the first neurons in the vertebrate eye. Here we show that Math5 mutant mice are fully viable, yet lack RGCs and optic nerves. Thus, two evolutionarily diverse eye types require atonal gene family function for the earliest stages of retinal neuron formation. At the same time, the abundance of cone photoreceptors is significantly increased in Math5(-/-) retinae, suggesting a binary change in cell fate from RGCs to cones. A small number of nascent RGCs are detected during embryogenesis, but these fail to develop further, suggesting that committed RGCs may also require Math5 function.Development 08/2001; 128(13):2497-508. · 6.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The diverse types of neurons and Müller glia in the vertebrate retina are believed to arise from common progenitor cells. To better understand how neural diversity is achieved during retinal neurogenesis, we examined the function of ash1, a proneural bHLH gene expressed in progenitor cells throughout retinal neurogenesis. Published studies using retinal explant culture derived from knockout mice concluded that ash1 is required for the production of late-born neurons, including bipolar cells. In this study, gain-of-function experiments were carried out in ovo in embryonic chick retina. In the developing chick retina, expression of ash1 temporally overlapped with, but spatially differed from, the expression of ngn2, also a proneural gene expressed in progenitor cells throughout retinal neurogenesis. Retrovirus-driven overexpression of ash1 in the developing chick retina decreased the progenitor population (BrdU+ or expressing ngn2), expanded the amacrine population (AP2alpha+ or Pax6+), and reduced bipolar (chx10 mRNA+) and Müller glial (vimentin+) populations. Photoreceptor deficiency occurred after the completion of neurogenesis. The number of ganglion cells, which are born first during retinal neurogenesis, remained unchanged. Similar overexpression of ngn2 did not produce discernible changes in retinal neurogenesis, nor in ash1 expression. These results suggest that ash1 promotes the production of amacrine cells and thus may participate in a regulatory network governing neural diversity in the chick retina.Developmental Neurobiology 01/2009; 69(2-3):88-104. · 4.42 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We have generated a mouse bearing a null allele of the gene encoding basic helix-loop-helix (bHLH) protein p48, the cell-specific DNA-binding subunit of hetero-oligomeric transcription factor PTF1 that directs the expression of genes in the exocrine pancreas. The null mutation, which establishes a lethal condition shortly after birth, leads to a complete absence of exocrine pancreatic tissue and its specific products, indicating that p48 is required for differentiation and/or proliferation of the exocrine cell lineage. p48 is so far the only developmental regulator known to be required exclusively for committing cells to an exocrine fate. The hormone secreting cells of all four endocrine lineages are present in the mesentery that normally harbors the pancreatic organ until day 16 of gestation. Toward the end of embryonic life, cells expressing endocrine functions are no longer detected at their original location but are now found to colonize the spleen, where they persist in a functional state until postnatal death of the organism occurs. These findings suggest that the presence of the exocrine pancreas is required for the correct spatial assembly of the endocrine pancreas and that, in its absence, endocrine cells are directed by default to the spleen, a site that, in some reptiles, harbors part of this particular cellular compartment.Genes & Development 01/1999; 12(23):3752-63. · 12.44 Impact Factor
Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all
horizontal cell subtypes in the chick retina
E.C. Lelièvrea,b,c,d,e, M. Lekf, H. Boijef, L. Houille-Vernesb,d,e, V. Brajeulb,d,e, A. Slembrouckb,d,e, J.E. Rogerd,
J.A. Sahelb,d,e, J.M. Matterg, F. Sennlauba,b,c, F. Hallböökf, O. Goureaub,d,e,⁎, X. Guillonneaua,b,c,⁎⁎
aCentre de Recherche des Cordeliers, INSERM UMR S872, 75006 Paris, France
bUniversité Pierre et Marie Curie, 75006 Paris, France
cUniversité Paris Descartes, 75006, Paris, France
dInstitut de la Vision, INSERM UMR S968, 75012 Paris, France
eCNRS UMR 7210, 75012, Paris, France
fDepartment of Neuroscience, Biomedical Centre, Uppsala University, Husargatan 3, Uppsala, Sweden
gDepartment of Biochemistry, Sciences II, University of Geneva, 1211 Genève 4, Switzerland
a b s t r a c ta r t i c l ei n f o
Received for publication 2 November 2010
Revised 19 July 2011
Accepted 25 July 2011
Available online 31 July 2011
cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-
helix–loop–helix transcription factor necessary for the specification of horizontal cells and the majority of
amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by
transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene
transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells
from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina.
into the molecular basis of Ptf1a activity on early cell specification in the chick retina.
© 2011 Elsevier Inc. All rights reserved.
The vertebrate neural retina is a laminar structure composed of six
types of neurons and one major type of glial cells, the Müller cells. The
seven cell types are derived from a common pool of multipotent retinal
progenitor cells (RPC) that differentiate in a conserved chronological
order. Retinal ganglion cells, cones, horizontal (HC) and amacrine (AC)
cells are produced first, whereas rods, Müller glial cells and bipolar cells
are generated last (Prada et al., 1991; Young, 1985). The RPC
differentiation pathway choice is determined by cell-intrinsic, i.e.,
transcription factors, and cell-extrinsic factors (Livesey and Cepko,
2001; Marquardt and Gruss, 2002; Ohsawa and Kageyama, 2008).
Ptf1a encodes a basic-helix–loop–helix (bHLH) transcription factor
that drives undifferentiated cells in the foregut to differentiate into a
Krapp et al., 1998). Ptf1a is also expressed during the development of
many structures of the central nervous system (Glasgow et al., 2005;
Zecchin et al., 2004). 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
(Glasgow et al., 2005; Hoshino et al., 2005; Pascual et al., 2007). In the
of ganglion cells in mice, zebrafish and Xenopus (Dong et al., 2008; Dullin
in zebrafish and Xenopus (Dong et al., 2008; Dullin et al., 2007). These
studies have thoroughly characterized the phenotypic modifications
factors in the Ptf1a-null retinas (Fujitani et al., 2006; 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
complex (PTF1) includes Ptf1a, a ubiquitous class I bHLH transcription
Developmental Biology 358 (2011) 296–308
⁎ Correspondence to: O. Goureau, Institut de la Vision, UMR S869, Equipe 3, 12, Rue
Moreau, 75012 Paris, France. Fax: +33 1 53 46 26 01.
⁎⁎ Correspondence to: X. Guillonneau, Centre de Recherche des Cordeliers, UMR S872,
Equipe 21, 15, Rue de L'Ecole de Médecine, 75006 Paris, France. Fax: +33 1 44 27 40 92.
E-mail addresses: email@example.com (O. Goureau),
firstname.lastname@example.org (X. Guillonneau).
0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/developmentalbiology
factor (E-protein) and the mammalian Suppressor of Hairless protein,
alsorequiredforPtf1a tospecifyGABAergic cellsinthedorsalspinalcord
and the cerebellum (Hori et al., 2008). However, the requirement of the
Ptf1a–Rbpj interactionin the specification of retinal cell types remains to
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-
subtypes, as found in the majority of vertebrate retinas (Gallego, 1986;
Genis-Galvez et al., 1979). The chick retina contains H1 axon-bearing
HCs, H2 axon-less “stellate” HCs and H3 axon-less “candelabrum” HCs
(Edqvist et al., 2008; Tanabe et al., 2006). Therefore, this model is more
representative of Ptf1a activity on HC subtype determination in
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
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
withthe Associationfor Research in Visionand Ophthalmology(ARVO)
statementonthe useof animals inOphthalmic and Vision Researchand
a protocol approved by our local animal care committee.
Chick genome sequences (V2.0 and V3.0) were obtained from
The Genome Institute at Washington University (http://genome.wustl.
BU347629) were ordered from Source Bioscience and sequenced
(MWG-Operon). The alignments were performed using Basic Local
and VNTI software (Invitrogen).
Retroviral stock and plasmid production
The mouse Ptf1a was subcloned using specific CDS primers into the
pDONR221 vector (Invitrogen) using BP clonase (Invitrogen). The Ptf1a
mutants were generated usingPolymerase Chain Reaction (PCR)-based
mutagenesis. The pDONR221-Ptf1a plasmid was recombined in the
presence of LR recombinase (Invitrogen) into either RCAS-BP(A)-NHY,
Ptf1a protein (gift from Dr. Loftus) or the pCIG gateway vectors (Roger
et al., 2006). The RCAS viral stocks with titersN1×108colony forming
viral DNA constructs using FuGENE6 Reagent (Roche Diagnostics)
(Yang, 2002). Viruses were concentrated by centrifugation using 100 K
Centrifugal Filters Amicon Ultra (Millipore). All embryos were injected
into theright 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 (Hughes et al., 1987) 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 h 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
The retinas were collected in HBSS without Ca2+/Mg2+, trypsinized
(1 mg/ml)(Sigma)andincubatedfor10 minat37 °C.Thedigestionwas
stopped with HBSS with Ca2+/Mg2+containing a 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 min with 2% PFA at
room temperature (RT) and washed in PBS before immunostaining.
For the manual counting, 1×105cells/ml were seeded on poly-L-lysine
(Sigma) coated plates. After 2 h, adherent cells were fixed 10 min with
2% PFA at RT before immunostaining.
Mouse anti-gag (3c2), anti-Ap2α (3B5), anti-Islet1 (39.4D5), anti-
Lim1/2 (4 F2), and anti-Visinin (7 G4) 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 and in situ hybridization
Immunostaining was performed as previously described (Roger
et al., 2006). 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 5-ethynyl-2′-deoxyuridine (EdU)
were injected into the intravitreal space 2 h before being sacrificed.
The Click iTTMEdU Imaging kit (Invitrogen) was used to visualize
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 (Roger et al., 2006).
Images were captured with a DM5500 microscope (Leica) equipped
withanORCA ER Hamamatsucamera ora LSM710 confocalmicroscope
(Zeiss) and analyzed with MetaMorph software (Molecular Devices).
Flow cytometry and cell sorting
Dissociated cells were incubated in blocking buffer (PBS, 10% fetal
calf serum (FCS), 2% goat serum, and 0.1% saponin) 1 h at RT. Primary
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
antibodies were diluted in blocking buffer, applied 2 h at RT, and
washed. The cell pellets were then incubated for 1 h 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
1×105GFP-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 Ca2+/Mg2+containing plasmids (0.5 μg/μl). Electropo-
ration was performed using a CUY215C square wave generator
(Sonidel) and consisted of 5 pulses at 30 V with 50 ms duration, 1 s
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 min
cryoprotected and embedded in PGS.
RNA isolation, reverse-transcription and quantitative PCR
Total RNA was extracted using the Nucleospin RNAII Kit (Macherey
Nagel). Retrotranscription was performed as described previously
(Roger et al., 2006). 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).
Protein extracts were collected in hypotonic buffer (20 mM HEPES
pH 7.9, 1 mM Na3VO4, 1 mM NaGlycerophoshate, 5 mM EDTA pH 7.5,
1 mM EGTA pH 7.5, 1 mM DTT, and protease inhibitors (Calbiochem,
Merck4 Biosciences)) plus 0.2% Nonidet P-40, incubated on ice for
15 min and centrifuged (20 s at 16,000 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 min at 4 °C and centrifuged for 20 min at 16,000 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 min at 16,000 g to remove debris. Glycerol
(10%) was then added to the supernatant.
Western blotting was conducted as described previously (Roger
et al., 2006) using the following primary antibodies: mouse anti-HA
(MMS 101R, Covance), goat anti-LaminB (sc-6216, Santa Cruz), mouse
(T6719, Institute of Immunology, Tokyo, Japan).
Tris/HCl pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, and protease
inhibitors) (Rodolosse et al., 2009). The lysates were incubated
overnight and centrifuged for 20 min at 16,000 g. Protein extracts
(250 μg) were incubated with anti-HA antibody (Covance, 1/100) for
2 h at RT to immunoprecipitate the HA-tagged proteins. The immune
complex was then captured by incubating for 2 h 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
The quantifications are expressed as the mean±standard error of
the mean (s.e.m.). Analyses were conducted using the Student's t-test
assumingequal variances (two groups) or one-wayanalysis of variance
followed by Tukey's multiple comparison tests (three or more groups)
using Prism 5.0 (Graphpad software). (*) pb0.05, (**) pb0.01, (***)
pb0.001, (ns) not significant. panova, p-value of the one-way analysis of
variance; pTukey, p-value of the Tukey post-hoc test.
Forced expression of Ptf1a results in a loss of retinal lamination
The Ptf1a spatiotemporal expression pattern has been studied
during retinogenesis (Boije et al., 2008; Fujitani et al., 2006; Nakhai
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) (Boije et al., 2008). 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
intheretina.ThechickPtf1a genesequenceis notfullyknown.Wehave
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 (Beres et al., 2006)
(Fig. S1). However, the N-terminal of the chickPtf1agenehas 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 (Beres et al., 2006). Moreover,
previous studies have reported that mouse Ptf1a blocks the differenti-
ation of dILBexcitatory neurons and promotes dILAinhibitory neuron
specification in the chick spinal cord, a phenotype that was consistent
with Ptf1a loss of function studies in mouse (Glasgow et al., 2005; Hori
et al., 2008; Mizuguchi et al., 2006; Wildner et al., 2006). These results
suggesta conservedactivity of mouse Ptf1ain avianand rodent models.
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
(Figs. 1C and E).
During any developmental stage, the plexiform and nuclear layers
had formed properly in the empty-RCAS (RCAS) control infected
patches (Figs. 1F–G, J–K and N–O). At E7, no major structural defects
were observed in the RCAS-Ptf1a-infected patches other than the
presenceofa thickerretinaandadecreasedcelldensity(Figs. 1HandI).
and Q), the lamination of the RCAS-Ptf1a-infected patches was severely
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 migrationormodification of thecell cycle.Therefore,S-phase
cells were pulse-labeled with 5-ethynyl-2′-deoxyuridine (EdU), and
mitotically active progenitors were immunostained using a Phospho-
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
Histone3 (P-H3) antibody. At E7, in the control retinas, EdU-positive
primarily found in the ventricular side (Fig. 2E), which was consistent
with interkinetic nuclear movements (IKNM) of progenitor nuclei and
free-cell migration (Baye and Link, 2008; Boije et al., 2009; Hinds and
Hinds, 1979). 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-Ptf1a-
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
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).
(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
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
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
foundinmost AC nuclei intheINL (Fig. 3K)(Edqvistetal.,2006; Fischer
et al., 2007). Prox1 strongly labeled HCs and a small subset of ACs
(Fig. 3P).The forcedexpressionofPtf1a 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-Ptf1a-infected population, n=5) (Figs. 3L, Q and W). Conversely,
Brn3a labeling for ganglion cells was severely reduced in the RCAS-
Ptf1a-infected patches (Fig. 3B), and the number of Visinin-positive
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) (Figs. 3G and
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) (Figs. S3B and 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)
(Figs. S3D and E).
Retinal lamination defects at E9 and later stages may result in a
secondary alteration of cell type representation. Therefore, we con-
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-Ptf1a-infected cells, p=0.0004, n=4) (Figs. 4F and I)
18.9±1.2% among the RCAS-Ptf1a-infected cells, p=0.001, n=5)
(Figs. 4H and I) at the expense of ganglion cells (Fig. 4B) and
Fig. 1. Mis/overexpression of Ptf1a affects 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 and C) and RCAS-Ptf1a-infected (D and E) retinal sections were stained with anti-Ptf1a (B and D) or anti-gag (p27) (C and E)
antibodies at E9. (F–Q) The RCAS-infected (F–G, J–K and N–O) and RCAS-Ptf1a-infected (H–I, L–M and P–Q) retinal sections were co-stained with hemalun (F, H, J, L, N and P) and
anti-gag (3c2) antibodies (G, I, K, M, O and Q). In B–E, G, I, K, M, O and 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).
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
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)
(Figs. 4D and I). These results indicated that the Ptf1a mis/over-
expression effects on early-born 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
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) (Figs. 4J–L).
Ptf1a mis/overexpression is sufficient to induce all HC subtype specification
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 (Edqvist et al., 2008;
Fischer et al., 2007). All three subtypes express Prox1. The H1 subtype
(50% of all HCs) has GABA, is Lim1-positive, Islet1-negative, and TrkA-
negative, Islet1-positive but TrkA-negative, 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
theHCsubtypes inthedevelopingchick retina.The retinawasanalyzed
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 (Boije et al., 2009;
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 Prox1-positive cells in the HCL, 64±5%
(n=3) werePtf1a-positive HCs (Fig.5B).This result implies that not all
HCs expressed Ptf1a at this age. The Lim1-positive cells were Ptf1a-
positive, and this overlap was found at both E9 (96±1%, n=3) in the
HCL and at earlier ages on the vitreal side (Figs. 5E and F). In contrast,
only a fewPtf1a/Islet1 double-positive cells couldbeseenatE6.5and at
E9 (5±0.6%, n=3) (Figs. 5I and J).
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 (Figs. 6B and D)
patches compared to the control patches, suggesting that both the H1
and H3 subtypes had been generated following Ptf1a mis/overexpres-
sion. These results were strengthened by the TrkA immunoreactivity of
Islet1-positive cells in the RCAS-Ptf1a-infected areas (Fig. 6F). Further-
more, the accumulation of some HCs in the vitreal side suggested that
HC migration was either arrested or delayed by Ptf1a mis/over-
expression. These results were similar at E9 when all HCs had normally
migrated back to the HCL. Supernumerary Prox1/Lim1 double-positive
(Fig. 6H), Prox1/Islet1 double-positive (Fig. 6J), and Islet1/TrkA double-
Fig. 2. Effects of Ptf1a mis/overexpression on retinal progenitor cell proliferation. (A–H) Sections from the RCAS-infected (A, C, E and G) and the RCAS-Ptf1a-infected (B, D, F and H)
retinas were stained with EdU and anti-gag (3c2) antibodies (A–D) or with anti-P-H3 and anti-gag 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 and 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.
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
specified (Fig. 6N). We found that the proportion of non-GABAergic
TrkA-positive cells among the Prox1-positive HCs decreased signifi-
cantly from 43.3±1.9% in the RCAS-infected 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
Togain knowledge aboutthemolecularmechanisms underlyingthe
changes in retinal cell specification, we studied early changes in the
expression of a selected set of genes involved in retinal differentiation.
GFP plasmids, which drive the green fluorescent protein (GFP)
expression (Roger et al., 2006) (Fig. 7A). The GFP-positive cells were
isolated by fluorescence activated cell sorting as early as 36 h 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
2009), or Sox2. We found that Pax6 and Ap2α, coding for two
Fig. 3. Effects of wild-type and mutant forms of Ptf1a on retinal cell differentiation. (A–T) The RCAS-infected (A, F, K and P), RCAS-Ptf1a-infected (B, G, L and Q), RCAS-
Ptf1a-ΔC2-infected (C, H, M and R), RCAS-Ptf1a-ΔC12-infected (D, I, N and S) and RCAS-Ptf1a-Δbasic-infected (E, J, O and 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 and V) Representative flow 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.
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
transcription factors highly expressed in ACs (Belecky-Adams et al.,
1997; de Melo et al., 2003; Edqvist et al., 2006), 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 (Mao et al., 2009), was downregulated (p=0.0037),
suggesting that Ptf1a might act downstream of Ascl1 and inhibit its
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
et al.,2005), wasstronglyrepressed by 6.4-fold byPtf1aoverexpression
(p=0.0071) (Fig. 7B). Furthermore, Atoh7 mRNA was nearly undetect-
able 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
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 (Dyer et al., 2003; Inoue et al.,
2002), were not altered, whereas Lim1, which is necessary for H1
specification in the chick retina (Suga et al., 2009), was induced
following Ptf1a overexpression (Fig. 7C). Foxn4 is upstream of Ptf1a
2004). Interestingly, Ptf1a overexpression induced a two-fold decrease
Foxn4 conversely regulates Ptf1a in the chick retina, we electroporated
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) (Beres et al., 2006). Ptf1a interacts with
Rbpjl through its C1 domain and with Rbpj through its C2 and C1
revealed that Rbpj mRNA was expressed throughout the NBL at early
stages and in the INL at later stages up to E12 (see Figs. 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-ΔC2 and Ptf1a-ΔC1ΔC2
and Ptf1a-ΔC12 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-ΔC12 nuclear importation (Fig. S4O). The injection of RCAS-
Ptf1a-ΔC2 and RCAS-Ptf1a-ΔC12 in the optic cup did not lead to gross
structural defects of the retina (Figs. 3 and S4E–H). However, the outer
Fig. 4. Retinal cell type representation is modified in E7 retinas prior to lamination defects. (A–H) The E7 RCAS-infected (A, C, E and G) and RCAS-Ptf1a-infected (B, D, F and H) retinal
patches were immunostained using an anti-gag antibody and either anti-Brn3a (A and B), anti-Visinin (C and D), anti-Prox1 (E and F) or anti-Ap2α (G–H) antibodies. Cell
type-specific labeling in panels A–H was represented without gag labeling in panels a–h, respectively. (I) Quantitative analysis of Visinin-, Ap2α- or Prox1-positive cells among the
infected cells. The values represent the mean±s.e.m of at least four separate retinal counts and are representative of two independent injections. (J–L) Cells undergoing apoptosis
were TUNEL-labeled at E7 in the RCAS-infected (J) and RCAS-Ptf1a-infected (K) patches detected using anti-gag (p27) antibody. Sections in J and K were counterstained with DAPI.
(L) Quantitative analysis of the number of apoptotic cells per area in the RCAS- and RCAS-Ptf1a-infected patches at E7. The values represent the mean±s.e.m. of at least three
separate retinas. NBL, neuroblastic layer; GCL, ganglion cell layer. Bars: 50 μm (A, C, E, and G in A and B, D, F, and H in B), 25 μm (J and K).
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
(Figs. 3H and I). Early-born cell types were analyzed by immunochem-
infected population of E12 retinas, but no alteration of the cell type
distribution was observed following the forced expression of these two
of Rbpj–Ptf1a interaction for gene regulation downstream of ectopic
Ptf1a was assessed by evaluating the ability of ectopic Ptf1a-ΔC12 to
regulatethe Atoh7promoter activity.Co-electroporationof pAtoh7-GFP
with the RCAS-Ptf1a-ΔC12 plasmids did not decrease the number of
GFP-positive cells compared to co-electroporation with the control
RCAS vectors (Figs. S5G and H).
Ectopic Ptf1a activity is independent from Rbpj pool depletion
Rbpj isa major downstreameffector of theNotchpathway. Previous
studies have suggested that the Ptf1a activity might be downregulated
by sequestering Rbpj from Ptf1a (Cras-Meneur et al., 2009) following
in the chick retina by conversely sequestering Rbpj. We generated a
Ptf1a-Δbasic mutant where the highly conserved RER-R amino acids
(168–172) (Figs. S4L and 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 (Lemercier et al., 1998;
Skowronska-Krawczyk et al., 2005). The Ptf1a-Δbasic 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-Δbasic was strongly decreased
(data not shown). No alteration of retinal structure and cell type
representation (Figs. 3E, J, O, T and S4I and J) was detected within the
E12 RCAS-Ptf1a-Δbasic-infected patches. 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 upregu-
lated NeuroAB, Ap2α and Lim1 while it downregulated Otx2 and Atoh7.
the ACs and all HC subtypes and inhibiting photoreceptor and ganglion
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 (Austin et al., 1995;
Dorsky et al., 1997; Henrique et al., 1997; Jadhav et al., 2006; Livesey
and Cepko, 2001; Nelson et al., 2007; Ohnuma et al., 2002; Silva et al.,
2003; Yaron et al., 2006). 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
Fig. 5. Ptf1a expression in subtypes of developing horizontal cells. Epifluorescence 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 fluorescence images are to the right of each panel. Insets in b, f, and j are higher magnifications 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.
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
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 (Del Bene et al., 2008; 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
Fig. 6. Ptf1amis/overexpressionissufficienttoincreasethenumberofallhorizontalcellsubtypes.(A–F)TheRCAS-infected(A,CandE)andRCAS-Ptf1a-infected(B,DandF)patcheswere
double immunostained at E7 with either anti-Lim1 and anti-Prox1 antibodies for H1 cells (A and B), anti-Islet1 and anti-Prox1 antibodies for H2 and H3 cells (C and D) or anti-Islet1 and
anti-TrkAantibodiesforH3 cells (EandF).a–fare highermagnificationsofthe boxesinA–F,respectively.a’–f’anda”–f”are splitfluorescenceimagesofa–f.Arrows pointtosomedouble-
positive cells.(G–N)TheRCAS-infected(G,I,Kand M)and RCAS-Ptf1a-infected (H,J,L and N) patches at E9weredoubleimmunostainedwitheitheranti-Lim1 and anti-Prox1(GandH),
anti-Islet1 andanti-Prox1 (Iand J), anti-Islet1 and anti-TrkA (Kand L)oranti-TrkA and anti-Lim1 antibodies(Mand N). g–n are highermagnificationsofboxes inG–N,respectively. g’–n’
and g”–n” are split fluorescent images of g–n, respectively. Arrows point to some Islet1-positive/TrkA-negative cells. (O) The quantification 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).
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
required for RPC proliferation (Fantl et al., 1995; Godbout and
Andison, 1996; Sicinski et al., 1995) was also significantly down-
regulated 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 (Brown et al., 2001;
Wang et al., 2001) and Otx2 (Nishida et al., 2003), which specify
ganglioncells and photoreceptors,respectively, wereinhibited in cells
that overexpressed Ptf1a. In contrast, Lim1, involved in HC specifica-
tion (Suga et al., 2009), 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 (Jusuf et al., 2011). 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
Third,Ptf1a might also selectively induce the deathof 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 demon-
strated a direct induction of cell death either by Ptf1a or by the
misexpression of transcription factors without apoptotic regulatory
properties. Dullin et al.(2007) 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
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 (Hayes et al., 2007; Matter-Sadzinski et al., 2001; Skowronska-Krawczyk et al., 2009).
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
in the retina (Fujitani et al., 2006; Mao et al., 2009; Qiu et al., 2008;
Zheng et al., 2009). 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
(Li et al., 2004). 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 (Fujitani et al., 2006). 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 Foxn4LacZ
knock-in mice, β-galactosidase (LacZ) expression was upregulated
and persisted longer than in wild type retinas (Li et al., 2004). This
Foxn4 locus deregulation could result, at least partially, from the loss
of Ptf1a expression in Foxn4−/−mice (Fujitani et al., 2006).
An increase in photoreceptor cells was reported in addition to the
decrease of ACs/HCs following Foxn4 and Ptf1a inactivation (Dong
et al., 2008; Dullin et al., 2007; Fujitani et al., 2006; Li et al., 2004;
Nakhai et al., 2007). Consistently, our misexpression study demon-
strated 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 (Jadhav et al., 2006; Yaron et al., 2006). In
various structures of the central nervous system, Ascl1 and Foxn4
have been implicated in the regulation of the Delta-like/Notch
upstream of Ascl1, Delta-like and Notch expression to regulate the
diversification of excitatory and inhibitory V2 cells by lateral
inhibition (Del Barrio et al., 2007). 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
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 Ptf1a-over-
expressing cells (data not shown).
Surprisingly, endogenous chick Ptf1a mRNA levels were signifi-
cantly 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 (Masui et al., 2008;
Meredith et al., 2009). Recently, Jusuf et al.(2011) 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
Foxn4activatedendogenous chick Ptf1a. Chick Ptf1aexpressionwould
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 (Hori et al., 2008). 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 (Figs. S6A–J). Second, recent
conditional loss-of-function studies (Riesenberg et al., 2009; Zheng
et al., 2009) reported that retinas null for Rbpj exhibited a phenotype
that mimics some features of the Ptf1a-null retinas (Fujitani et al.,
2006; 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; Zheng et al., 2009). Moreover, the loss of Rbpj in Chx10-
positive retinal cells induced a significant decrease of ACs and HCs in
P21 mouse retinas (Zheng et al., 2009). 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 compen-
sate for the loss of Rbpjl in the Rbpjl−/−mouse pancreas (Masui et al.,
2010). They could also be due to the use of different amacrine cell
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 (Sellick et al., 2004). 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 retina, this supports a
highly conserved Ptf1a–Rbpj functional interaction during vertebrate
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
(Glasgow et al., 2005) and cerebellum (Hoshino et al., 2005). However,
Ptf1a controls the specification of glutamatergic climbing fiber neurons
of the inferior olivary nucleus (Yamada et al., 2007), 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 (Fujitani et al., 2006; Jusuf and Harris, 2009), or Ptf1a
cells) and H3 non-GABAergic HCs were increased in the RCAS-Ptf1a-
E.C. Lelièvre et al. / Developmental Biology 358 (2011) 296–308
infected patches. Thus, Ptf1a is likely to act primarily as a general HC
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 (Edqvist et al., 2008). 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
(Dong et al., 2008).
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 down-
regulated 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
Supplementary materials related to this article can be found online
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.
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