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First published online October 30, 2006
doi: 10.1242/10.1242/dev.02598
,
1 Vanderbilt University Program in Developmental Biology and Department of Cell
and Developmental Biology, Vanderbilt University Medical School, Nashville, TN
37232-8240, USA.
2 Department of Visual Science, Osaka University Graduate School of Medicine,
Suita, Osaka 565-0871, Japan.
3 Center for Advanced Biotechnology and Medicine and Dentistry of New Jersey,
Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854,
USA.
4 Department of Molecular Physiology and Biophysics, Vanderbilt University
Medical School, Nashville, TN 37232-0615, USA.
5 Department of Surgery and Surgical Basic Science, Kyoto University Graduate
School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan.
6 Umeå Center for Molecular Medicine, University of Umeå, SE-901 87
Umeå, Sweden.
7 Department of Molecular Biology, The University of Texas Southwestern Medical
Center, Dallas, TX 75390-9148, USA.
8 Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874,
Japan.
Authors for correspondence (e-mail:
yoshio-f{at}osb.att.ne.jp;
christopher.wright{at}vanderbilt.edu)
Accepted 30 August 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Retinal development, Basic helix-loop-helix, Amacrine cell, Horizontal cell, Ganglion cell, Lineage tracing, Ptf1a, Foxn4, Progenitor, Cell specification
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Young,
1985). It has been postulated that, in response to changes of
intrinsic and extrinsic cues, retinal progenitors undergo a series of changes
in competence to give rise to the various retinal cell types
(Cepko, 1999;
Harris, 1997;
Livesey and Cepko, 2001).
Recent advances in molecular genetic approaches have begun to unravel the
molecular bases that underlie the determination and differentiation of
different retinal cell types, including horizontal and amacrine cell.
Despite extensive studies, the precise molecular mechanism for horizontal
cell type specification is far from completely elucidated. In particular, the
bHLH genes that regulate horizontal cell differentiation remain to be
determined. Horizontal cell genesis is significantly impaired in
Mash1(Ascl1);Ngn2(Neurog2);Math3(Neurod4)
and Ngn2;Math3;Neurod1 triple mutant retinas, but not in single or
double mutant retinas (Akagi et al.,
2004). It is therefore likely that, in retinas containing single
or double mutations for bHLH genes, other known and unknown neurogenic bHLH
genes may compensate and allow horizontal cell development. The roles for two
other transcription factors during horizontal cell development are more
clearly defined. Prox1 is a crucial intrinsic factor that controls fate
commitment of horizontal cells (Dyer et
al., 2003). And we have shown that the forkhead/winged helix
transcription factor Foxn4 plays an essential role in horizontal cell
generation, as Foxn4-null retinas completely lack horizontal cells
(Li et al., 2004a).
The specification of amacrine cells is found to depend on several
transcription factors. Foxn4 appears to confer retinal progenitors with the
competence for an amacrine cell fate in part by activating the expression of
Math3 (Neurod4 - Mouse Genome Informatics) and
Neurod1 (Li et al.,
2004a). In mice deficient for both Neurod1 and
Math3, a complete loss of amacrine cells is accompanied by a
fate-switch of progenitors to ganglion and Müller cells
(Inoue et al., 2002), whereas
the formation of amacrine cells is essentially normal in single null mutants
for either Neurod1 or Math3
(Inoue et al., 2002;
Morrow et al., 1999).
Therefore, the current model is that Math3 and NeuroD1 are redundantly
required downstream of Foxn4 for specifying amacrine cells. Overexpression
studies have also confirmed the important role of these three factors during
amacrine cell development (Inoue et al.,
2002; Li et al.,
2004a; Morrow et al.,
1999). Aside from the bHLH factors, the Pax6 and Barhl2
homeodomain factors are expressed by differentiating and mature amacrine
cells, and have been implicated in the specification and/or differentiation of
glycinergic amacrine cells (Marquardt et
al., 2001; Mo et al.,
2004).
Ptf1a (pancreas transcription factor 1a, also known as
Ptf1a-p48), which encodes a bHLH factor, functions during pancreas
development to drive undifferentiated foregut endoderm cells towards the
pancreatic fate (Kawaguchi et al.,
2002; Krapp et al.,
1998). Recently, Ptf1a mutations were linked to the human
permanent neonatal diabetes mellitus associated with cerebellar ataxia
(Sellick et al., 2004). Mice
mutant for Ptf1a result in cerebellar hypoplasia caused by the
specific inhibition of GABAergic neuron production from the cerebellar
ventricular zone (VZ) (Hoshino et al.,
2005), and Ptf1a is also a crucial factor for the development of
GABAergic neurons in the spinal cord dorsal horn
(Glasgow et al., 2005).
Expression of Ptf1a in developing zebrafish retinas suggests a
possible role for Ptf1a in the specification and differentiation of retinal
neurons (Lin et al., 2004;
Zecchin et al., 2004).
Here, we report that Ptf1a is expressed by a subset of post-mitotic precursors in the developing mouse retina VZ and acts as a fate determinant to drive these precursors to differentiate into horizontal and amacrine neurons. Loss of Ptf1a function causes a conversion of horizontal/amacrine precursors into a ganglion cell fate. The essential role of Ptf1a in specifying horizontal and amacrine cells is thus non-redundant with other factors, and as such we have identified a key trigger factor that is a primary downstream target for Foxn4, which regulates progenitor competence for horizontal and amacrine neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
Ptf1aCitrine mutant mice also have a knock-in Ptf1a
null allele, which drives citrine expression, instead of Ptf1a, under the
control of the endogenous Ptf1a promoter. Details on construction of
this allele will be described elsewhere (J. Burlison and M.A.M., unpublished).
The reporter strains R26R and R26R-EYFP were used to
visualize cells and progeny that were exposed to Cre recombinase activity
(Soriano, 1999;
Srinivas et al., 2001).
X-gal staining
Embryo tissues were fixed for 1 hour in 4% paraformaldehyde/2%
glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at 4°C and washed
three times with 0.1 M sodium phosphate buffer (pH 7.4) for 30 minutes.
Tissues were sunk in 30% sucrose in 0.1 M sodium phosphate buffer (pH 7.4)
overnight at 4°C, embedded in OCT and cryosectioned at 16 µm. Adult
mice were perfused (10 minutes) with 0.1 M sodium phosphate buffer (pH 7.4)
containing 4% paraformaldehyde, 2% glutaraldehyde and 30% sucrose. Tissues
were embedded in OCT and cryosectioned at 16 µm. Cryosections were
incubated at 37°C for between 4 hours to overnight in X-gal staining
solution [0.1 M sodium phosphate buffer (pH 7.4), 5 mM potassium ferricyanide,
5 mM potassium ferrocyanide, 2 mM MgCl2, 1 mg/ml X-gal].
Immunohistochemical analysis
Appropriately staged embryos and 3-week-old retinas were fixed for 30
minutes to 1 hour at 4°C in 4% paraformaldehyde. Tissues were washed
several times with phosphate-buffered saline (PBS), sunk in 30% sucrose
overnight, embedded in OCT and cryosectioned. Immunofluorescence was carried
out on 14-16 µm sections using: mouse monoclonal antibodies specific to
BrdU (Megabase Research Products), calbindin-D-28K (Sigma), syntaxin (HPC-1;
Sigma), rhodopsin (RET-P1; Sigma), GAD65, Lim1 (Developmental Studies
Hybridoma Bank; Iowa) and CRALBP (ABR); rabbit polyclonal antibodies to Ptf1a
(Li and Edlund, 2001),
recoverin (Chemicon), calretinin (Chemicon), GABA (Sigma), tyrosine
hydroxylase (Chemicon), Prox1 (Chemicon) and GFP (Molecular Probes); goat
polyclonal antibodies to Brn3b (Santa Cruz) and Glyt1 (Chemicon); and a sheep
polyclonal antibody to Chx10 (Exalpha). Cy2- or Cy3-conjugated secondary
antibodies were from Jackson ImmunoResearch Laboratories. Antibody
concentrations and use available upon request (Y.F.). Some samples were
counterstained with the nuclear dye, YO-PRO-1 (Molecular Probes) or DAPI
(Vector Labs). Immunofluorescence was imaged on a Zeiss LSM 510 confocal
microscope. For BrdU experiments, BrdU (Sigma; 200 µg/g body weight) was
injected into pregnant mothers 1 hour before sacrifice. Double
immunofluorescence of Pft1a and BrdU was performed by completing
immunofluorescence of Ptf1a, followed by treating sections with 2.0 M HCl for
15 minutes, 0.1 M sodium borate (pH 8.5) for 20 minutes, before incubating
with the primary BrdU antibody. TUNEL assays were performed using the In
situ Apoptosis Detection Kit (Takara) according to the manufacturer's
instruction.
Quantification of Brn3b-positive cells
Embryonic day 18.5 (E18.5) retinal sections were immunostained with goat
anti-Brn3b antiserum. Five animals were analyzed for each genotype (wild type
versus Ptf1a-null). For each animal, cell counting was performed on
four nonadjacent optic-nerve-containing sections, to avoid double scoring.
Three non-overlapping fields (230 µm x 230 µm) of central retina,
with the inner-most edge of GCL included at one edge of each field were
photographed from each section using the confocal microscope and printed at
400x magnification to count Brn3b-positive cells. Comparison of
Brn3b+ cell numbers was made by unpaired t test.
Quantification of TUNEL-positive cells was also performed in the same
method.
In situ hybridization
RNA in situ hybridization (Sciavolino
et al., 1997) used digoxigenin-labeled riboprobes prepared
following the manufacturer's protocol (Roche Diagnostics). The full-length
cDNAs of Foxn4, Math3, Neurod1, Math5
(Li et al., 2004a), and a
partial 3' Ptf1a cDNA were used as probes. The Ptf1a
probe was subcloned after PCR amplification using the following primers:
5'-AGTCCATCAACGACGCCTTCGA-3' and
5'-ACAAAGACGCGGCCGACCCGATGTGAG-3'.
Real-time quantitative RT-PCR (qRT-PCR)
Total RNA was isolated from four each of E14.5 Foxn4+/-
and Foxn4-/- retinas as described
(Li et al., 2004b). qRT-PCR
was performed in duplicate for each RNA sample (100 ng) using the QuantiTect
SYBR green one-step RT-PCR kit (Qiagen). The following sequence-specific
primers were designed using the MacVector software (Accelrys): Foxn4,
5'-CGACAAGATGGAGGAGGAGAT-3' and
5'-CTTGTCCAACTCCTCAGGGTT-3'; Ptf1a,
5'-GCACTCTCTTTCCTGGACTGA-3' and
5'-TCCACACTTTAGCTGTACGGA-3'; ß-actin,
5'-AGAGGGAAATCGTGCGTGAC-3' and
5'-CAATAGTGATGACCTGGCCGT-3'; and Gapdh,
5'-TCACCACCATGGAGAAGGC-3' and
5'-GCTAAGCAGTTGGTGGTGCA-3'. PCR products were monitored in real
time (Mx4000 multiplex quantitative PCR system; Stratagene), and the threshold
cycles (Ct) were determined using the Mx4000 software. Relative quantities
were calculated for a target gene transcript in comparison to a reference gene
(ß-actin) transcript as described previously
(Pfaffl, 2001). All data were
tested for significance using two-sample Student's t-test with
unequal variances.
Retinal explant culture
The retinal explant culture was performed, as described
(Mo et al., 2004;
Tomita et al., 1996). The
neural retina, with pigment epithelium stripped away, was placed on a chamber
filter (Whatman: 25 mm diameter, 0.2 µm pore size) with the ganglion cell
layer upwards, which was then transferred to a six-well culture plate. Each
well contained 1 ml of culture medium (50% MEM with HEPES, 25% Hank's
solution, 25% heat-inactivated horse serum, 200 µM L-glutamine and 5.75
mg/ml glucose). Explants were cultured at 34°C in 5% CO2, with
medium changed every other day.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), for which the signal specificity was established by the
absence of signal on stage-matched retinal tissues from
Ptf1aCre/Cre (null) embryos
(Fig. 1E,H). Ptf1a expression
was not observed at E11.5 (Fig.
1A), and was detectable at E12.5 in the central region of the
retina (Fig. 1B). By E14.5,
Ptf1a expression had expanded from the center to the entire retina
(Fig. 1C and data not shown).
At this stage, the developing retina comprises two discrete zones: the outer
neuroblastic layer (onbl) and inner neuroblastic layer (inbl). Ptf1a
expression was rarely observed in the inbl. Between E16.5 and postnatal day 1
(P1), Ptf1a continued to be expressed strongly in a subset of cells within the
outer neuroblastic layer (Fig.
1D,E). Ptf1a expression began to be downregulated by P2
(Fig. 1F) and was undetectable
in P6 retinas (Fig. 1G). Ptf1a
expression was not detected in late postnatal and adult retinas. The observed
time-course of Ptf1a expression is consistent with the transient expression of
Ptf1a mRNA previously reported in the zebrafish eye
(Lin et al., 2004;
Zecchin et al., 2004),
suggesting an evolutionarily conserved temporal expression profile for this
gene.
The onbl of the developing retina consists of a mixture of dividing
progenitor cells and newly generated postmitotic neurons/glial cells. To
determine the cell-cycle status of Ptf1a-expressing cells, we pulse-labeled S
phase cells in the E14.5 retina with BrdU just before fixation and analysis (1
hour labeling period) and performed immunostaining using Ptf1a and BrdU
antibodies. Ptf1a-expressing cells appeared to have exited the cell cycle,
judged by the absence of co-labeling with BrdU
(Fig. 1I). The post-mitotic
status of Ptf1a-expressing cells is in contrast to the Prox1-expressing cell
population, in which a significant proportion of cells are still dividing at
E14.5 (Dyer et al., 2003).
Prox1 and Ptf1a most probably mark distinct progenitor/precursor populations,
as no co-expression of Ptf1a and Prox1 was observed in the E14.5 retina (data
not shown). At E18.5 and P1, newborn amacrine cells in the inner half of the
retina, which were identified based on expression of differentiation markers
such as calretinin, did not express Ptf1a (data not shown). Taken together,
these data indicate that the expression of Ptf1a is restricted to post-mitotic
cells in the VZ of the developing retina, and in a pattern suggestive of a
neuronal precursor population.
Ptf1a-expressing precursors are restricted to horizontal and amacrine cell lineages
To gain insight into the cell types in the adult retina that are derived
from Ptf1a-expressing precursors, we performed genetic lineage
tracing by Cre-mediated reporter-gene activation. We previously generated a
Ptf1aCre knock-in allele in which the Ptf1a
protein-coding region was precisely replaced by nuclear-targeted Cre
recombinase (Kawaguchi et al.,
2002). We crossed Ptf1aCre/+ with
Gt(ROSA)26Sortm1sor(R26R) mice, which carry a
modified lacZ gene driven by the cell type-independent
ROSA26 promoter (Soriano,
1999). In offspring obtained from this cross,
Ptf1a-driven expression of Cre excises a stop cassette upstream of
lacZ and activates cell-type-independent expression of
ß-galactosidase (ß-gal), which is maintained specifically in
Ptf1a-expressing cells and their progeny
(Kawaguchi et al., 2002).
R26R-EYFP reporter mice, which carry the enhanced YFP gene
driven by the Rosa26 promoter
(Srinivas et al., 2001), were
also used for lineage tracing studies. The Rosa26 promoter is active
in all retinal cell types in adult mice
(Rowan and Cepko, 2004).
|
).
Ptf1a-lineage-labeled cells in the GCL
(Fig. 2A) were not labeled with
the ganglion cell marker Brn3b (Fig.
2B), but were positive for amacrine markers, including calbindin
and GABA (Fig. 2E,F,H,I). Thus,
Ptf1a-lineage-labeled cells in the GCL were displaced amacrine cells,
which have been previously identified in the GCL
(Barnstable and Drager, 1984).
The glial markers Cralbp (cellular retinaldehyde-binding protein; Rlbp1 -
Mouse Genome Informatics) (Fig.
2L) (Bunt-Milam and Saari,
1983) and vimentin (data not shown) showed no colocalization with
YFP, indicating that glial cells were not derived from
Ptf1a-expressing precursors. By contrast, all amacrine markers
examined [calbindin, syntaxin, Gad65 (Gad2 - Mouse Genome Informatics), GABA,
glycine transporter 1 (Glyt1; Slc6a9 - Mouse Genome Informatics), calretinin,
tyrosine hydroxylase (Th), choline acetyltransferase (Chat) and the vesicular
glutamate transporter type 3 (Vglut3; Slc17a8 - Mouse Genome Informatics)]
showed variable but high proportions of colocalization with YFP
(Fig. 2E-K; data not shown).
For example, some of the amacrine markers labeled a few cells that were YFP
negative (e.g. arrowheads in Fig.
2E,H). The percentage of amacrine marker-positive cells that were
labeled with anti-YFP varied depending on the subclass of amacrine cells
(Table 1). Horizontal cells,
which are located on the outer border of the INL and are characterized by a
relatively large soma and calbindin immunoreactivity, were labeled with
anti-YFP without exception (Fig.
2E), suggesting that all horizontal cells were derived from
Ptf1a-expressing precursors. Thus, our lineage tracing data suggest
that Ptf1a-expressing cells represent a subset of precursors that are
restricted to production of the horizontal and amacrine cells.
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;
Krapp et al., 1998). We
therefore explanted E18.5 retinas and cultured them for up to 2 weeks to study
the role of Ptf1a in retinal differentiation and maturation. After 2 weeks of
culture, retinas from wild-type embryos exhibited well-developed laminar
structures containing the three cellular layers: GCL, INL and ONL
(Fig. 3). By contrast, explants
from Ptf1aCre/Cre embryos, although initially of the same
thickness as wild type, were significantly thinner after 2 weeks and often
exhibited substantial focal perturbations in laminar organization.
Rosette-like structures (e.g. Fig.
3L,P) were often observed in Ptf1aCre/Cre
retinas. Immunostaining by antibodies against the photoreceptor markers
rhodopsin (Fig. 3A) and
recoverin (data not shown) revealed normal generation of photoreceptor cells
in Ptf1aCre/Cre retinal explants. In addition, cells in
the rosette-like structures were labeled by photoreceptor markers (data not
shown), which strongly suggested a photoreceptor identity. These rosettes may
be analogous to similar structures previously identified in explant cultures
of Hes1-null mutant retinas
(Tomita et al., 1996). There
was no general deficiency in the emergence of Chx10-positive bipolar cells
(Fig. 3C,D) or Cralbp-positive
glial cells (Fig. 3O,P) in
Ptf1aCre/Cre retinas. Thus, Ptf1a is dispensable
for the genesis of bipolar cells and Müller glia, which is consistent
with the absence of Ptf1a-lineage labeling in these cell types.
Calbindin-positive cells in the outer border of the INL were entirely absent
in Ptf1aCre/Cre retinas
(Fig. 3E,F), indicating a
complete loss of horizontal cells. Lack of calbindin-positive and
syntaxin-positive cells in the mutant retina indicated also that amacrine
cells, including the displaced amacrine cells in the GCL, were profoundly
missing (Fig. 3E-H).
Immunofluorescence labeling for the specific amacrine subtypes (e.g. GABA and
Gad65 for GABAergic neurons; Th for dopaminergic neurons, Glyt1 for
glycinergic neurons, Chat for cholinergic neurons, and Vglut3 for
glutamatergic neurons) (Fig.
3E-N; data not shown) revealed a dramatic decrease of all amacrine
subtypes. Occasionally, very small numbers of residual amacrine cells were
observed in some sections of retinal explant, irrespective of which amacrine
marker was examined. Taken together, these findings provide evidence that
Ptf1a function is selectively required for proper specification of
the vast majority of horizontal and amacrine cells.
|
Loss of Ptf1a function results in mis-specification of Ptf1a-expressing retinal precursors as ganglion cells
Loss of horizontal and amacrine cells concomitant with the abnormal
location of Ptf1a-lineage labeled cells in the optic nerve fibers
suggested that precursors that would normally produce horizontal and amacrine
cells might have changed fate to become ganglion cells in the absence of
Ptf1a function. Consistent with this hypothesis, E18.5 mutant retinas
contained 54.7% more cells expressing Brn3b, which encodes a
POU-domain transcription factor specific for differentiated retinal ganglion
cells (RGCs) (Gan et al.,
1996; Xiang et al.,
1993), with no change in the total thickness of the retina
(Fig. 4K-M). The supernumerary
Brn3b-expressing cells were confirmed to be RGCs based on expression of the
additional ganglion markers Brn3a and islet 1 (data not shown).
Double-immunolabeling analysis on E18.5 Ptf1aCre;R26R-EYFP
heterozygous and mutant retinas provided some evidence that the additional
RGCs arose via a switch in fate of the postmitotic precursors. The ganglion
marker Brn3b was very seldom co-expressed with YFP in retinas heterozygous for
Ptf1a (Fig. 5E),
consistent with the results obtained from 3-week-old heterozygous mice
(Fig. 2B). In
Ptf1aCre/Cre;R26R-EYFP retinas, however, YFP and Brn3b
were often co-localized (Fig.
5F) providing direct evidence that the precursors that normally
produce horizontal and amacrine cells switch fate in the absence of Ptf1a and
contribute to the RGC lineage. This cell fate switch was observed as early as
E16.5 (data not shown).
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|
1 day compared
with endogenous Ptf1a expression (data not shown). Furthermore, considering
that RGCs are first specified in the onbl soon after exit from the cell cycle,
and then migrate inwards to the GCL, Brn3b+ cells within the onbl
should represent recently specified RGCs
(Xiang, 1998). As Ptf1a
expression was seen only post-mitotically
(Fig. 1I), recently specified
RGCs in the onbl may not have had enough time to activate the
R26R-EYFP reporter. To test this possibility, we used a novel
Ptf1a null allele, Ptf1acitrine, in which citrine
(another yellow variant of GFP) is directly expressed from the Ptf1a
locus. The onset of citrine expression in Ptf1aCitrine/+
mice coincided with the onset of endogenous Ptf1a protein expression, and
citrine protein was detectable for 2 days after disappearance of
endogenous Ptf1a (data not shown), presumably because of its slow turnover.
The sustained expression of citrine allows `partial lineage tracing', wherein
citrine protein presence marks Ptf1a-expressing cells and their progeny for
2 days after shutdown of Ptf1a expression. Although only a small
fraction of Brn3b+ cells in the onbl co-localized with YFP in
Ptf1aCre/Cre;R26R retinas, most of the Brn3b+
cells in the onbl of
Ptf1aCitrine/Citrine
retinas co-localized with Citrine (Fig.
5F,L). Based on these results, we conclude that the
Brn3b+ cells detected in the mutant onbl at E18.5 are derived from
precursors that can activate expression from the Ptf1a locus but are
unable to produce Ptf1a protein. Therefore, conversion of
Ptf1a-active amacrine/horizontal precursors into ganglion precursors
is likely to be the primary contributor to increased production of RGCs in
Ptf1a mutants.
Ptf1a as a downstream target for Foxn4
Targeted deletion of Foxn4 also causes the loss of all horizontal
cells and the great majority of amacrine cells
(Li et al., 2004a). Given the
similar retinal phenotypes in Ptf1a and Foxn4 mutant mice,
we investigated their inter-regulatory relationship. A microarray analysis
revealed that Ptf1a expression was dramatically downregulated in
E14.5 Foxn4-/- retina, by 37-fold compared with the wild
type (F.Q. and M.X., unpublished). To confirm this result, we performed
real-time qRT-PCR, using total RNA isolated from E14.5
Foxn4+/- and Foxn4-/- retinas
(Fig. 6A). We found that
Foxn4-/- tissue contained no Ptf1a mRNA and, as
expected, Foxn4 mRNA was absent from the Foxn4-/-
sample. RNA in situ hybridization with a Ptf1a riboprobe detected
prominent signals scattered within the onbl of wild-type retina at E14.5 and
E15.5 (Fig. 6B,D), and the
absence of these signals in Foxn4-/- tissue
(Fig. 6C,E), further confirming
the complete downregulation of Ptf1a expression by the loss of
Foxn4 function. In reciprocal experiments, in situ hybridization
revealed no alteration of Foxn4 expression in the Ptf1a null
retina (Fig. 7). We conclude
that Foxn4 functions upstream of Ptf1a to activate its expression,
thereby regulating the generation of amacrine and horizontal cells during
retinogenesis.
|
). Compared
with wild-type controls, Prox1 expression was severely downregulated in
Ptf1a-null retinas at E14.5 (Fig.
7K,L) and E16.5 (data not shown), whereas the strong lens
expression was not altered. Loss of horizontal cell specification was further
demonstrated by a loss of Lim1 expression in E18.5 Ptf1a-null retinas
(Fig. 7M,N). The bHLH factors
Math3 and Neurod1 are redundantly required for the determination of amacrine
cells (Inoue et al., 2002).
Unexpectedly, the expression of both genes did not exhibit any alteration in
the mutant retina (Fig. 7E-H).
Thus, Ptf1a does not appear to act upstream of Math3 or Neurod1 for the fate
determination of amacrine cells (Fig.
8C). Expression of Math5, a bHLH factor required for
ganglion cell genesis (Wang et al.,
2001), becomes downregulated at E18.5 in the wild-type retina.
Consistent with the increase of RGCs (Figs
4,
5) in the Ptf1a-null
retinas, Math5 expression in the mutant was moderately upregulated
compared with wild-type controls (Fig.
7I-J). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Livesey and Cepko, 2001),
there have been no studies that provide rigorous evidence for fate-restricted
precursors in vivo. By using recombination-based lineage tracing analysis, we
have demonstrated that Ptf1a-expressing cells represent a subset of
precursors that are restricted to the production of horizontal and amacrine
cells. Transient and/or low expression of Cre from the Ptf1a locus often
results in `escapers' in Cre-loxP lineage tracing systems
(Gu et al., 2002;
Hoshino et al., 2005;
Kawaguchi et al., 2002). The
variable penetrance of the lineage-labeling among the amacrine cell subtypes
(Table 1) could be most
probably explained by Cre not rising to high enough levels in all cells,
despite all precursors expressing the Ptf1a gene, or that for unknown
reasons the reporter gene locus is not equally sensitive in all cells. Our
lineage analysis has revealed that Ptf1a-expressing precursors contribute to
all amacrine subtypes analyzed. We cannot, however, formally rule out the
possibility that a minor population of Ptf1a-non-expressing precursor cells
can contribute to amacrine cells, or that some amacrine subclasses may be
generated in a Ptf1a-independent manner.
Ptf1a, a bHLH factor implicated in the cell fate determination in various organs
Originally, Ptf1a was identified as a transcriptional regulator of
pancreatic exocrine-specific genes such as elastase 1
(Krapp et al., 1996). Mice
null for Ptf1a exhibit a complete loss of exocrine tissue and severe
defects in the formation and spatial organization of endocrine cells
(Kawaguchi et al., 2002;
Krapp et al., 1998). Dramatic
phenotypes have also been observed in the development of the cerebellum and
dorsal spinal cord. The requirement for Ptf1a to specify multiple GABAergic
cell-types, including Purkinje, stellate and basket cells, all derived from
Ptf1a-expressing precursors, is responsible for the cerebellar hypoplasia in
Ptf1a mutants (Hoshino et al.,
2005). The Ptf1a-null mouse also develops a dorsal spinal
cord with nearly a complete loss of inhibitory GABAergic neurons. In dorsal
spinal cord, loss of Ptf1a function results in a reciprocal increase
of excitatory glutamatergic neurons. These findings lead to the conclusion
that Ptf1a functions as a selector molecule that determines GABAergic over
glutamatergic cell fate in these neural tissues
(Glasgow et al., 2005).
Defects in Ptf1a-null retinas, however, do not seem to be restricted
to GABAergic neurons. Our results that all amacrine subtypes examined were
overall affected in the absence of Ptf1a, suggesting the involvement
of Ptf1a in the genesis for all amacrine neurons. We are unable to rule out
completely the possibility that some amacrine subtypes were lost as an
indirect consequence of the massive loss of the cells in the INL during
explant culture. Horizontal and amacrine cells, which are deficient in
Ptf1a-null retinas, are two classes of interneurons that modulate and
integrate visual signals in the retinal circuitry. Thus, Ptf1a in the
developing retina may be involved in the determination of two interneuron cell
fates, rather than in regulating transmitter-subtype specification, as
proposed for a Ptf1a function in the cerebellum and the spinal cord
(Glasgow et al., 2005).
|
). Second, we have
demonstrated the requirement of Ptf1a in the generation of the overwhelming
majority of amacrine neurons and all horizontal cells. Lineage tracing
experiments based on Ptf1aCre-mediated activation of R26R
and R26R-EYFP revealed that Ptf1a expression marks lineage-restricted
precursors that exclusively produce horizontal and amacrine cells.
Furthermore, the Ptf1a-active precursors, normally fated to amacrine
and horizontal cells, switched to ganglion cell fates in the absence of Ptf1a
function. These observations clearly demonstrate that activation of
Ptf1a is tightly associated with the acquisition of the amacrine and
horizontal cell fate. The function of Ptf1a in switching cell fates in the
retina is similar to the role attributed to Ptf1a in the development of
pancreas and dorsal spinal cord (Glasgow
et al., 2005; Kawaguchi et
al., 2002). By using the same lineage tracing method, we showed
that Ptf1a is expressed by early progenitors for the pancreatic ducts, acinar
and endocrine cells, and that Ptf1a-deficient pancreatic progenitors adopt
duodenal cell fates (Kawaguchi et al.,
2002). Similarly, in the spinal cord dorsal horn, inactivation of
Ptf1a switches dI4 and dILA fates (GABAergic neuron
lineage) into the alternative fates dI5 and dILB (glutamatergic
neurons). It remains to be determined whether Ptf1a also codes for shared
characteristics in cell fate determination in other Ptf1a-expression domains,
such as cerebellum and the ventral hypothalamus
(Glasgow et al., 2005).
|
) and Lim1 (Liu et al.,
2000). As Prox1 is believed to be the major intrinsic factor that
is both necessary and sufficient for the promotion of horizontal cells from
competent progenitors (Dyer et al.,
2003), we tested whether Ptf1a controls specification of
horizontal cells by regulating Prox1 expression. As expected, our analyses
have indeed revealed that Ptf1a positively regulates the expression of Prox1
during horizontal cell ontogeny, beginning as early as E14.5
(Fig. 7). Given that Prox1
expression is regulated also by Foxn4 (Li
et al., 2004a), Foxn4 most probably controls Prox1 expression
through the activation of Ptf1a in the horizontal cell specification cascade
(Fig. 8). At E18.5, expression
of Lim1, which is exclusively confined within the horizontal cell type in the
adult retina, was entirely absent from Ptf1aCre/Cre
retinas, providing yet more evidence for the lack of specification of the
horizontal cells (Fig. 7).
A Foxn4-Ptf1a pathway determines horizontal and amacrine cell fate
The complete downregulation of Ptf1a expression in
Foxn4-null retinas, together with the unaltered expression of Foxn4
in Ptf1aCre/Cre retina, places Ptf1a downstream of Foxn4
in the transcriptional cascade that leads to amacrine/horizontal cell
specification in the developing retina. These findings are in agreement with
several of our observations. First, the timing of Ptf1a expression closely
follows the transient expression pattern of Foxn4 in progenitor cells.
Initiation of Ptf1a expression at E12.5 is 0.5-1 day later than that of
Foxn4 at E11.5, and expression of both genes is largely downregulated by about
the same time at P5-P6 (Fig. 1)
(Li et al., 2004a). Second,
Foxn4 is expressed by a subset of progenitor cells whose progeny are
significantly biased toward amacrine and horizontal cell fates
(Li et al., 2004a), whereas
Ptf1a is expressed by postmitotic precursors that are almost exclusively fated
for horizontal and amacrine cells. The onbl of E11.5-E13.5 embryonic retina
carries substantial numbers of Foxn4+ mitotic progenitors, many of
which have competence to preferentially differentiate to horizontal or
amacrine cells (Li et al.,
2004a). Our results suggest that these Foxn4+
progenitors require Ptf1a function after exit from the cell cycle to acquire
completely their fates as mature amacrine or horizontal cells; these ideas are
summarized in the model presented in Fig.
8. In Ptf1aCre/Cre retinas, these precursors
undergo reallocation to the ganglion fate relatively quickly after exit from
the cell cycle, otherwise presumably being pushed into an apoptotic program.
This possibility is supported by a modest increase of cell death in
Ptf1aCre/Cre retinas (48.6±5.8
cells/mm2) at E18.5 compared with wild-type (22.4±1.7
cells/mm2; P<0.01) littermates as assessed by TUNEL
analysis (Fig. 7).
The cell fate switch in Ptf1a-null retina is considered to be
analogous to the fate switch of amacrine to ganglion cells in
Math3-Neurod1 double mutant retinas, as reported previously
(Inoue et al., 2002). The
function of Ptf1a in postmitotic cells highlights another important example in
which cell fate allocation is regulated after cell cycle exit. This agrees
well with previous reports where postmitotic precursors fated to become
photoreceptor cells can be respecified to become amacrine cells in the absence
of Otx2 (Baas et al., 2000;
Nishida et al., 2003), or
become bipolar cells in the presence of Cntf
(Ezzeddine et al., 1997).
Given its necessity, we tested whether Ptf1a was also sufficient to promote
the fates of horizontal and amacrine cells from retinal progenitors by a
gain-of-function approach. Overexpression of Ptf1a in retinal progenitors by
an expression vector revealed that expression of Ptf1a alone was not
sufficient to promote the generation of horizontal and amacrine cells (data
not shown). The other intrinsic factors that are required together with Ptf1a
for horizontal and amacrine cell genesis remain to be elucidated. Recently
RBP-L (Rbpsuhl - Mouse Genome Informatics) was identified as one of the
subunits to constitute a heterotrimeric transcription factor Ptf1 together
with Ptf1a (Beres et al.,
2006). Although Ptf1a alone failed to activate its reporter gene,
addition of RBP-L significantly potentiated its transcriptional activity.
Thus, co-factors for Ptf1a, such as RBP-L, might be required for full
activation of Ptf1 to induce horizontal and amacrine cell genesis.
In spite of the selective deficiency in horizontal and amacrine cells that
is shared by Ptf1a and Foxn4 mutants, these mutants show
some phenotypical differences. Although Ptf1a-null cells
preferentially trans-differentiate to ganglion cells,
Foxn4-/- cells appear to primarily adopt a photoreceptor
fate (Li et al., 2004a). The
switch of the horizontal/amacrine precursors to a ganglion fate rather than a
photoreceptor fate in the Ptf1a-null retinas may reflect their
ongoing competence based upon the presence of Foxn4 within these cells
(Fig. 7). Foxn4 may have a
suppressive role over photoreceptor differentiation given that
Foxn4-/- cells preferentially differentiated to
photoreceptor lineages, and that the expression of the photoreceptor
differentiation factor Crx was upregulated in
Foxn4-/- retinas
(Furukawa et al., 1997;
Furukawa et al., 1999;
Li et al., 2004a). There is a
formal possibility that some Foxn4-/- cells may indeed
switch fates to become ganglion cells at early stages, but that they quickly
degenerate because of elevated apoptosis during postnatal stages in
Foxn4-/- retinas (Li
et al., 2004a).
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Center for Therapeutic Innovations in Diabetes and
Department of Medicine, Metabolism and Endocrinology, Juntendo University,
2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan
|
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