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First published online 14 February 2007
doi: 10.1242/dev.02781
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1 II. Medizinische Klinik, Klinikum rechts der Isar, Technische
Universtität München, Ismaninger Str. 22, D-81675 Munich,
Germany.
2 Department of Ophthalmology, Martin-Luther-University Halle-Wittenberg,
Ernst-Grube-Strasse 40, D-06120 Halle/Saale, Germany.
3 Institute of Human Genetics, GSF-National Research Center for Environment and
Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany.
4 Department of Anatomy and Cell Biology, Martin-Luther-University
Halle-Wittenberg, Grosse Steinstrasse 52, D-06097 Halle/Saale, Germany.
Author for correspondence (e-mail:
hassan.nakhai{at}lrz.tum.de)
Accepted 14 December 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Amacrine cells, Cre-loxP method, Development, Ptf1a, Retina, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Both the
chronological sequence of cell generation and the spatial expansion of these
diverse cell types are strictly regulated. In mice, retinal cell
differentiation starts with the generation of ganglion cells at embryonic day
11.5 (E11.5), followed in a temporally overlapping sequence by horizontal
cells, cone photoreceptors, amacrine cells, rod photoreceptors, bipolar cells,
and finally Mueller glia cells (Marquardt,
2003). Retinogenesis in mice is completed at approximately
postnatal day 10 (P10). These functionally different cells form three cellular
(nuclear) layers and two synaptic (plexiform) layers in the mature retina. The
outer nuclear layer (ONL) contains rod and cone photoreceptor cell bodies. The
inner nuclear layer (INL) is comprised of horizontal, bipolar, amacrine and
Mueller cell bodies. The ganglion cell layer (GCL) includes both nuclei of
displaced amacrine cells and ganglion cells. The synaptic connections of these
neuronal cells are localized in the outer plexiform layer (OPL) and the inner
plexiform layer (IPL).
The molecular basis of pattern formation and cell-type specification in the
vertebrate retina has been intensively investigated. Accumulating evidence
indicates that cell-intrinsic regulators, such as transcription factors, and
cell-extrinsic signals, such as neurotrophic factors, play important roles in
progenitor cell fate determination and their subsequent differentiation
(Malicki, 2004;
Yang et al., 2003). Recent
studies have demonstrated that basic helix-loop-helix (bHLH) transcription
factors regulate the determination and differentiation of multiple neuronal
cell types during retinogenesis (Akagi et
al., 2004; Hatakeyama and
Kageyama, 2004; Vetter and
Brown, 2001). The gene Ptf1a (also known as
PTF1-p48) encodes a bHLH protein of 48 kD. Ptf1a, together
with two proteins [RBP-L and a common (type I) bHLH protein] comprises the
three subunits of the pancreas transcription factor (PTF1)
(Roux et al., 1989;
Beres et al., 2006).
Ptf1a plays a fundamental role in exocrine and endocrine pancreas
development in mice (Kawaguchi et al.,
2002). In a recent study the expression of Ptf1a has also
been detected in the retina of developing zebrafish embryos by in situ
hybridization (Zecchin et al.,
2004). Using a positional candidate gene approach, Sellick et al.
(Sellick et al., 2004) have
identified mutations in the Ptf1a gene of patients with pancreatic
and cerebellar agenesis, and Hoshino and colleagues
(Hoshino et al., 2005) could
demonstrate that Ptf1a is involved in GABAergic neuronal cell
specification in the cerebellum. Very recently, it has been shown that
Ascl1 (previously Mash1) controls the expression of
Ptf1a. Furthermore, Mash1 is expressed in sensory
interneuron progenitors and is involved in the switch between excitatory and
inhibitory cell fates in the developing mouse spinal cord
(Mizuguchi et al., 2006).
In the present study, we show that the bHLH transcription factor Ptf1a is expressed in the neuroretina of developing mice. Furthermore, inactivation of the Ptf1a gene produces severe cellular defects of the inner retina as a result of inhibition of differentiation of GABAergic and glycinergic amacrine precursor cells and horizontal precursor cells.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Ptf1a-Cre(ex1) R26R and Ptf1a-Cre(ex1) Z/EG transgene
The Gt(ROSA)26Sortm1Sor (R26R) reporter
mouse line was purchased from The Jackson Laboratory (Bar Harbor, Maine, USA)
and the Tg(ACTB-Bgeo/GFP)21Lbe (Z/EG)
reporter mouse line was kindly provided to us by Dr Mori Tetsuji, Institute of
Stem Cell Research, GSF-National Research Center for Environment and Health
(Soriano, 1999;
Novak et al., 2000).
Heterozygous R26R and Z/EG mice were crossed with
Ptf1a-Cre(ex1)
(Ptf1a+/Cre(ex1))
to generate
Ptf1a+/Cre(ex1)
R26R and
Ptf1a+/Cre(ex1)
Z/EG mice.
Genotyping of transgenic mice
All offspring were genotyped by PCR of genomic DNA from mouse tail clips
with primers specific for the Ptf1a, Cre, R26R and Z/EG
transgenes as described by Krapp et al.
(Krapp et al., 1996), Gu et
al. (Gu et al., 1993), Soriano
(Soriano, 1999) and Novak et
al. (Novak et al., 2000)
respectively.
Southern blot analysis
Genomic DNA from ES clones were digested with SacI, separated by
agarose gel electrophoresis, blotted to nylon membranes, and detected with the
radioactive labeled 5' flanking external probe.
Quantitative TaqMan PCR and data analysis
Real-time PCR was performed in a Perkin-Elmer 7700 Sequence Detection
System. Total RNA was isolated by RNeasy Mini Kit (Qiagen) according to the
manufacturer's manual. Two micrograms of total RNA was first reverse
transcribed with SSIII (Invitrogen) and random primer in a total volume of 20
µl for 2 hours at 50°C. SSIII was inactivated by heating at 75°C
for 15 minutes. The cDNA was diluted fivefold, and 5 µl was used for each
30 µl PCR using the SYBR GREEN PCR Master Mix (Applied Biosystems). The
Taqman primers were designed using Primer Express Software (Applied
Biosystems). The primer sequences are listed in
Table 1. The PCR conditions for
all genes were as follows: 50°C for 2 minutes hold, 95°C for 2 minutes
hold and 40 cycles of 95°C, for 15 seconds and 60°C for 30 seconds.
For each gene, the real-time PCR assay was performed four times with four
different batches of total RNA. The cyclophilin gene served as an RNA
input control. Relative gene expression ratio (ER) was calculated on the basis
of differences between expression level of cyclophilin and genes
analyzed using the following formula: ER=2-
Ct, where
Ct is the difference of threshold cycles between the gene of interest
and the control gene (cyclophilin).
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).
X-Gal staining
Sections were air-dried for 20 minutes, fixed in paraformaldehyde, washed
in PBS and stained with X-Gal reaction buffer (2 mmol/l MgCl2, 5
mmol/l potassium ferrocyanide, 5 mmol/l potassium ferricyanide, 0.5 mg/ml
X-Gal in PBS) at 37°C overnight.
BrdU labeling
Pregnant mice were injected with 0.14 mg/g body weight BrdU 2 hours before
they were sacrificed. BrdU incorporation was detected on 10 µm cryostat
sections by antibody directed against BrdU (Sevotech), and at least four
different eyes from wild-type embryos were analyzed.
Samples, histology, immunohistochemistry, tunnel assay, microscopy and imaging
After enucleating the mouse eyes, we quickly removed the anterior segment
(cornea and lens) leaving half eyecups. We analyzed sections, which were
through the central retina. Histological analysis of retinal explant cultures
was as previously described (Hatakeyama and
Kageyama, 2002). Tissues and whole mounts were fixed in 4%
paraformaldehyde for 30 minutes on ice, equilibrated overnight in 30% sucrose
at 4°C, frozen in OCT compound (Leica) and stored at -80°C. Tissues
were sectioned (8-10 µm) with a cryostat (MICROM, Laborgeraete GmbH,
Walldorf, Germany).
Immunohistochemical and immunofluorescence analyses were carried out as
previously described (Hsu,
1990). Briefly, sections were incubated with cell-type-specific
antibodies in blocking serum overnight at 4°C. We used the following
primary antibodies: anti-Ptf1a (a kind gift from R. J. MacDonald), anti-Thy1.2
(CD90; Promega), anti-syntaxin HPC 1 (Sigma), anti-
-amino butyric acid
(GABA; Sigma), anti-glycine transporter 1 (GlyT1; Chemicon), anti-Lhx1
(anti-Lim1; Chemicon), anti-Hu/D (Elavl4; Molecular Probes), anti-Brn3 (clone
C-13; Santa Cruz) anti-Pax6 (Developmental Studies Hybridoma Bank),
anti-N-cadherin (Transduction Laboratories), anti-recoverin (Chemicon),
anti-ß-galactosidase (from rabbit; ICN), anti-ß-galactosidase (from
mouse; Developmental Studies Hybridoma Bank), anti-GFP (Molecular Probes),
anti-calbindin-D28K (Sigma), anti-PKC
(Zymed) and biotin-conjugated
secondary antibodies (Dianova), as well as the avidin-biotin-peroxidase and
avidin-biotin-alkaline phosphatase complex (Vectorstain ABC and ABC-AP),
according to the manufacturer's instructions (Vector Labs). Antibodies were
detected by BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitro blue
tetrazolium), TSA (Tyramide Signal Amplification) (Molecular Probes). The cell
nuclei were stained with DAPI
(4',6-diamidine-2-phenylindol-dihydrochloride; Boehringer, Mannheim).
Tunnel assay was performed using the In Situ Cell Death Detection Kit TMR red
(Roche Diagnostics) following the manufacturer's protocol, except that all
sections were counterstained with DAPI. At least three to five independent
retinae from knockout and wild-type mice were collected and analyzed. For each
retina, positive cells were counted under the microscope from four different
sections in an area extending from the outer segments of the photoreceptor
cells (osp) to the inner limiting membrane (ilm). The length of osp and ilm
was 200 µm. Images were acquired using a Zeiss ApoTome microscope (Axiovert
200M) and captured by a CCD cool digital camera (Zeiss). Software module
AnxioVision (Zeiss) was used for subsequent 3D reconstruction of the
images.
Statistical analysis
Data from quantitative PCR and immunohistochemistry were analyzed by
Student's t-test or Mann-Whitney U-test, as appropriate. A
P-value of <0.05 was considered statistically significant. All
analyses were performed using SPSS 12.0 software (SPSS, Chicago, IL).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and obtained a Ptf1a null allele
[Ptf1atm1(Cre)Nak
or Ptf1a-Cre(ex1)] (see Fig. S1 in the supplementary
material). After verifying germline transmission, we crossed
Ptf1a+/Cre(ex1)
heterozygous mice with Gt(ROSA)26Sortm1Sor
(R26R) or Tg(ACTB-Bgeo/GFP)21Lbe
(Z/EG) reporter mouse lines
(Soriano, 1999;
Novak et al., 2000) to
determine Cre activity. The R26R mice carry a modified lacZ
gene driven by the cell-type-independent ROSA26 promoter and the
Z/EG reporter mouse line contains a CMV enhancer/chicken ß-actin
promoter. In
Ptf1a+/Cre(ex1)
R26R animals, Ptf1a-driven expression of Cre excises a stop
cassette upstream from the lacZ gene and thereby activates the
expression of ß-galactosidase. Expression of ß-galactosidase
therefore marks all the cells in which Ptf1a is activated and the
active ß-galactosidase expressing locus is inherited by their descendant
cells. These cells can be efficiently detected by staining with
5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal). In offspring from the
cross between
Ptf1a+/Cre(ex1)
and Z/EG mice line, lacZ expression is replaced with the
enhanced green fluorescent protein (GFP) expression in cells expressing
Cre.
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),
neural tube (Obata et al.,
2001) and cerebellum (Hoshino
et al., 2005; Sellick et al.,
2004) (see Fig. S2A,B in the supplementary material). Moreover, we
detected ß-galactosidase-expressing cells in the neuroretina of
developing mice (see Fig. S2C-F in the supplementary material). These X-Gal
positive cells were first detectable at E14.5 (see Fig. S2C,E in the
supplementary material). At this early stage of embryonic development, the
mouse retina consists of an inner and an outer neuroblastic layer (NBL), which
contains RPCs (Dyer et al.,
2003). X-Gal labeled cells of the Ptf1a lineage were
located within the inner NBL. At E18.5, ß-galactosidase expressing cells
were primarily found in the inner NBL of the developing retina. Small numbers
of X-Gal positive cells were also detected in the GCL and the outer region of
the neuroretina (data not shown). In adult mice, most of the cells expressing
ß-galactosidase were localized in the innermost zone of the INL. Some
ß-galactosidase-expressing cells were found in the GCL and in the
outermost zone of the INL (see Fig. S2D,F in the supplementary material).
These data led us to a detailed analysis of the expression and function of the
Ptf1a gene during retinal development in mice. To identify the cells expressing Ptf1a in the developing retina, we immunolabeled retinal cryosections of wild-type mice at embryonic and postnatal stages of development. Ptf1a-expressing cells were first detected at E12.5 (Fig. 1A,D). From E12.5 to P1, Ptf1a was expressed in the nuclei of retinal precursor cells scattered in the NBL (Fig. 1A-F). The number of Ptf1a-expressing cells increased from E12.5 to P1. After P1, the number of Ptf1a-expressing cells decreased gradually, and from P3 onward, Ptf1a expression was not discernible in the retina.
Ptf1a is expressed in precursors of amacrine, displaced amacrine and horizontal cells
To identify the specific neuroretinal cells that are derived from
Ptf1a-expressing cells in the developing mouse, we analyzed the adult
retina of
Ptf1a+/Cre(ex1)
R26R mice by double-labeling experiments using antibodies against
ß-galactosidase and cell-type-specific markers: Thy1.2 (ganglion cell
marker), syntaxin (amacrine cell marker), GABA (GABAergic amacrine and
displaced amacrine cell marker), glycine transporter 1 (GlyT1, glycinergic
amacrine cell marker), calbindin (horizontal cell marker) and PKC-
(bipolar cell marker). These results showed that Ptf1a expression, as
assayed by Cre-mediated ß-galactosidase expression, was observed
in the amacrine, displaced amacrine and horizontal cells
(Fig. 2A-R).
ß-Galactosidase was not coexpressed with vimentin, a marker for Mueller
glia (data not show). These lineage-tracing data, taken together with the
immunolocalization results of Ptf1a, indicate that Ptf1a is expressed
in precursors of amacrine, displaced amacrine and horizontal cells.
The Ptf1a-expressing cells are postmitotic
To determine whether cells expressing Ptf1a were actively dividing, we
pulse-labeled retinae of wild-type mice with the thymidine analog BrdU for 2
hours and carried out double-immunofluorescence staining using anti-Ptf1a and
anti-BrdU antibodies at E13.5 and P1. We found that neither E13.5 nor P1
S-phase cells were Ptf1a immunopositive (see Fig. S3 in the supplementary
material). Thus, in summary, Ptf1a expression is initiated in the
postmitotic amacrine, displaced amacrine and horizontal cell precursors.
Ptf1a-deficient retinae lack the IPL
To study the biological role of the Ptf1a gene in retinal
development, we then investigated the retina of
Ptf1aCre(ex1)/Cre(ex1)
R26R mice. In these mice, all cells are homozygous for the
Ptf1a null mutation, but the retinal cells in which Ptf1a is
transcriptionally activated can be identified by their expression of
lacZ derived from Ptf1a-Cre-mediated R26R
activation. As the
Ptf1aCre(ex1)/Cre(ex1)
R26R pups die approximately three hours after birth, we examined the
morphology of the retina in
Ptf1aCre(ex1)/Cre(ex1)
R26R embryos at E18.5. After verifying the absence of Ptf1a protein
in
Ptf1aCre(ex1)/Cre(ex1)
R26R retinae (Fig.
3A,E) by immunohistochemistry, we compared cryosections of
Ptf1a+/Cre(ex1)
R26R with
Ptf1aCre(ex1)/Cre(ex1)
R26R retinae. In retinae of heterozygous
Ptf1a+/Cre(ex1)
R26R mice, the IPL formed a continuous layer between the GCL and NBL
(Fig. 3B,C,D). X-Gal staining
of
Ptf1a+/Cre(ex1)
R26R retina showed that the majority of
ß-galactosidase-expressing cells were localized in the innermost zone of
the NBL (Fig. 3B,C). By
contrast, in
Ptf1aCre(ex1)/Cre(ex1)
R26R retina, the GCL and NBL were fused, resulting in loss of the IPL
(Fig. 3F-H). We confirmed these
results using immunostaining with anti-N-cadherin antibody and 3D
reconstruction by ApoTome microscopy (Fig.
3D,H). In addition, the ß-galactosidase-expressing cells were
scattered in the inner retina of
Ptf1aCre(ex1)/Cre(ex1)
R26R mice (Fig. 3G).
These data suggested a misplacement of ß-galactosidase-expressing cells
in
Ptf1aCre(ex1)/Cre(ex1)
R26R toward the inner retina.
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At E18.5, Pax6 is mainly expressed in differentiated amacrine cells and to
a lower level in differentiated ganglion cells, as well as in RPCs
(Marquardt et al., 2001). We
found that Pax6 and ß-galactosidase were coexpressed in differentiated
amacrine cells of
Ptf1a+/Cre(ex1)
R26R mice (Fig. 4A-C).
By contrast, in Ptf1a-deficient retinae, the expression of Pax6 was
restricted to cells in the NBL and showed no coexpression with
ß-galactosidase (Fig.
4D-F). These data indicate that retinae of
Ptf1a-deficient mice fail to generate differentiated amacrine
cells.
During retinal development, the RNA-binding protein Hu/D is expressed in
differentiating amacrine and ganglion cells, but not in differentiated
amacrine cells (Link et al.,
2000). At E18.5, immunostaining with anti-Hu/D antibody of
Ptf1a+/Cre(ex1)
R26R retinae showed Hu/D expression mainly in the GCL, which was not
co-localized with ß-galactosidase-expressing cells
(Fig. 4G-I). However, in
Ptf1a-deficient retinae we found that the large majority of the cells
expressing ß-galactosidase also were positive for Hu/D
(Fig. 4J-L). These results
suggest that ß-galactosidase-expressing cells are amacrine cell
precursors and/or ganglion cells in Ptf1a knockout retina.
Based on these data, we hypothesized that in Ptf1a knockout retina, the ß-galactosidase-expressing cells, which would differentiate to amacrine or horizontal cells in the course of normal retinal development, were either inhibited in their differentiation or transdifferentiated to ganglion cells.
A proportion of Ptf1a-deficient precursor cells transdifferentiated to ganglion cells
To test the hypothesis that Ptf1a-deficient precursor cells
transdifferentiated to ganglion cells, we analyzed the cell fate of these
cells by using the Z/EG reporter mouse line, which expresses
ß-galactosidase before Cre excision and enhanced GFP after Cre excision.
The double immunostaining of the
Ptf1a+/Cre(ex1)
Z/EG retinae with anti-GFP and anti-Brn3 showed no co-labeling
(Fig. 5A-C). However, in
Ptf1aCre(ex1)/Cre(ex1)
Z/EG retinae, GFP and Brn3 were coexpressed in approximately 30% of
the cells (Fig. 5D-F,H). Taken
together, these data provide evidence that a proportion of
Ptf1a-deficient precursor cells transdifferentiate to ganglion cells.
In line with these results, we observed a significant increase of
Brn3-positive cells in
Ptf1aCre(ex1)/Cre(ex1)
(Cre/Cre) compared with wild-type retinae
(Fig. 5G).
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retinal explants were prepared from E18.5 embryos and cultured for 12 days
[E18.5+12 days in culture (DIC)]. We analyzed cryosections of retinal explants
from
Ptf1a+/Cre(ex1)
R26R and
Ptf1aCre(ex1)/Cre(ex1)
R26R mice using specific antibodies against each retinal cell type
(Fig. 6,
Table 2).
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To further characterize the defects of Ptf1a-deficient retinae, we investigated the genesis and differentiation of the remaining retinal cells. The presence of syntaxin- and calbindin-positive cells in Ptf1a-deficient retinal explants (Fig. 6J,L) indicates that not all of the amacrine precursor cells undergo apoptotic cell death or transdifferentiate to ganglion cells. Thus, we propose that a subtype of amacrine precursor cells do not need Ptf1a for differentiation.
Furthermore, we observed a disorganization of syntaxin-positive cells in
Ptf1aCre(ex1)/Cre(ex1)
R26R compared with
Ptf1a+/Cre(ex1)
R26R retinal explants (Fig.
6I,J). The same was true for calbindin (amacrine and horizontal
cells) and PKC (bipolar cells) (Fig.
6K-N) immunoreactive cells. Recoverin staining showed less evident
changes in Ptf1a-deficient retinal explants
(Fig. 6O,P).
In summary, retinal explant results show: (1) a small population of differentiated amacrine cells are present in the Ptf1a null mutant; (2) GABAergic and glycinergic amacrine cells are absent in the Ptf1a-deficient mice; (3) a disorganization of the inner neuroretina; and (4) an increase of apoptotic cells in Ptf1a knockout mice.
Genes involved in amacrine and horizontal cell genesis are differentially regulated in Ptf1a-deficient retinae
To consolidate our findings, we performed real-time PCR analysis on total
RNA extracted from wild-type and Ptf1a-deficient retinae at E18.5
using a set of marker genes that regulate amacrine [Foxn4, Neurod1
(formerly NeuroD), Neurod4 (formerly Math3),
Barhl2] and horizontal [Foxn4, Lhx1 (also known as
Lim1)] cell development plus glutamic acid decarboxylase 1
(Gad1), an enzyme that catalyzes the synthesis of the inhibitory
neurotransmitter GABA (Fig.
7I). Foxn4 controls the genesis of amacrine and
horizontal cells by activating the expression of Neurod1 and
Neurod4 (Li et al.,
2004). The expression level of Foxn4 remained unchanged
in wild-type and Ptf1a-deficient retinae, indicating that
Ptf1a acts chronologically later on amacrine cell genesis than
Foxn4. Both bHLH transcription factors, Neurod1 and
Neurod4, are expressed in differentiating amacrine cells and regulate
amacrine cell fate specification (Inoue et
al., 2002). The absence of Ptf1a does not alter
Neurod1 and Neurod4 expression, suggesting that the
involvement of Ptf1a to amacrine cell fate specification takes place
at later stages of amacrine cell development, as is the case for
Neurod1 and Neurod4. By contrast, in
Ptf1a-deficient retinae the expression of Barhl2 and
Gad1 transcripts were downregulated, indicating that Ptf1a
is involved in the molecular mechanism governing the specification of subtype
identity of amacrine cells. The result that the expression level of
Barhl2 in Ptf1a-deficient retinae is only slightly decreased
is consistent with the fact that Barhl2 is also expressed by ganglion
cells. Also in line with our immunohistochemical data was the finding that the
transcription factor Lim1 (horizontal cell marker) is not detectable
in Ptf1a-deficient retinae (Fig.
7II).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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35% and glycinergic amacrine cells 40% of
all amacrine cells (Mo et al.,
2004). We presented several lines of evidence that Ptf1a
is involved in the terminal differentiation of GABAergic and glycinergic
amacrine cells. First, birth-dating studies of retinal cells have revealed
that during mouse retinogenesis amacrine cells are generated over a time
period from E11 to P4 (Young,
1985). We demonstrated that Ptf1a expression starts at
around E12.5 and ceases gradually at P3 in the developing retina of wild-type
mice, which is concomitant to amacrine cell genesis. Second, we could show
that inactivation of the Ptf1a gene leads to downregulation of genes
(Barhl2, Gad1) contributing to mature amacrine cell phenotype and to
complete absence of GABAergic and glycinergic amacrine cells in retinal
explants.
Recent studies have shown that the bHLH transcription factor Ptf1a
is involved in driving neural precursors to differentiate into GABAergic
neurons in the cerebellum, and that Ptf1a is required for the
formation of GABAergic neurons in the dorsal horn of the spinal cord. The
absence of Ptf1a expression in this tissue causes a
transdifferentiation of GABAergic neurons to glutamatergic neural cells
(Glasgow et al., 2005;
Hoshino et al., 2005). By
contrast to spinal cord, lack of Ptf1a activity lead to
differentiation arrest of amacrine precursor cells and partial
transdifferentiation of amacrine precursor cells to ganglion cells, which
results in loss of GABAergic and glycinergic amacrine cells in mature retina.
The fact that in the Ptf1a-deficient retinae the number of
differentiated amacrine cells is significantly decreased and the number of
ganglion cells is increased could lead to loss of IPL. These data are in line
with the results that in Math3/NeuroD double-mutant retina the IPL is
not formed (Inoue et al.,
2002).
Horizontal cell development is impaired in Ptf1a- deficient retinae
The amacrine and horizontal cells are important interneurons that process
and transmit visual input in the retinal circuitry. It has been demonstrated
that amacrine and horizontal cells begin to exit the cell cycle at E11
(Young, 1985). In our
analysis, Ptf1a expression first appears at E12.5 in postmitotic
cells, indicating that Ptf1a is unlikely to be involved in initial
generation of amacrine and horizontal cells. We have also followed the cell
fate of horizontal cells in Ptf1a-deficient retina. Ptf1a is
important for differentiation of horizontal precursor cells. First, our
lineage-tracing analysis revealed that amacrine and horizontal cells express
Ptf1a. Second, in Ptf1a-deficient retina the expression of
Lim1 is totally absent, which indicates that horizontal cells are not
generated. Our observation of calbindin-positive cells in Ptf1a
mutant retinal explants, although the horizontal cells are deficient, may be
attributed to the presence of a subtype of amacrine cells as previously
reported (Uesugi et al.,
1992).
Foxn4 is involved in the genesis of horizontal cells
(Li et al., 2004). As the
absence of Ptf1a does not affect the expression level of
Foxn4 (Fig. 7I), we
conclude that Ptf1a acts downstream in the development of horizontal
cells, then Foxn4.
In Caenorhabditis elegans, genetic analyses of Lim and homeodomain
genes have demonstrated a prominent role for them in terminal differentiation
of specific neurons. For instance, mec-3 is required for
differentiation of mechanosensory neurons, and lim-6 regulates
neurite outgrowth and function of GABAergic motoneurons
(Hobert et al., 1999;
Way and Chalfie, 1988).
Inactivation of Ptf1a causes a switch in cell fate
In Ptf1a knockout retinae, we observed a co-localization of GFP
and Brn3 expression in approximately 30% of GFP-positive cells at E18.5
(Fig. 5). This strongly
indicates that a proportion of Ptf1a- deficient precursor cells
transdifferentiate to ganglion cells. As the number of apoptotic cells in
Ptf1a knockout retina is increased, we assume that
Ptf1a-deficient cells that do not transdifferentiate undergo
apoptotic cell death. Regarding the transdifferentiation of retinal cells,
Ptf1a knockout resembles Math3/NeuroD double-mutant retina
(Inoue et al., 2002). However,
there are several significant differences concerning amacrine cell
differentiation and the number of retinal cells. First, in INL of
Ptf1a-deficient retinal explants, some amacrine cells
(syntaxin+, calbindin+) were generated. By contrast, in
Math3/NeuroD null mutant retinal explants, amacrine cells
are completely missing (Inoue et al.,
2002). Second, whereas the number of ganglion cells was
significantly increased in Math3/NeuroD double-mutant retinal
explants, that of Ptf1a-deficient retinal explants was strongly
reduced. Third, in Ptf1a null mutant retinal explants, the number of
apoptotic cells was increased, bipolar cells were significantly reduced and
the architecture of the INL was disrupted. In Math3/NeuroD
double-mutant retinal explants, bipolar cells were normally generated and
positioned and the number of apoptotic cells was not increased. Therefore, we
conclude that there are more defects in Ptf1a-deficient retina
compared with Math3/NeuroD double-mutant retina.
To summarize previous results and our present study, we propose a
hypothetical model for cell differentiation in the retinae of embryonic mice
(Fig. 8). The transcription
factors Neurod1 and Neurod4 specify RPCs to amacrine
precursor cells. It has been demonstrated that the Barhl2 homeobox
gene is involved in the specification of glycinergic amacrine cells
(Mo et al., 2004). As the
inactivation of Ptf1a leads to loss of GABAergic and glycinergic
amacrine cells, as well as to the downregulation of Barhl2 and
Gad1 transcripts, Ptf1a promotes the differentiation of
amacrine cell precursors to GABAergic and glycinergic amacrine cells.
Furthermore, we found that in the Ptf1a null retina a small number of
amacrine precursor cells differentiated to amacrine cells. Thus, we conclude
that Ptf1a contributes to the specification of amacrine cell subtypes
rather than to the generation of amacrine cells. Ptf1a is expressed
in postmitotic horizontal precursor cells. As the expression level of
Foxn4 in Ptf1a-deficient retinae did not change
(Fig. 7I), we propose that
Ptf1a acts downstream to Foxn4 in the cell signaling cascade
regarding the generation of horizontal cells. Even though there are gaps in
our knowledge about retinogenesis, we provide evidence that Ptf1a
plays an important role in retinal cell differentiation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/6/1151/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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