1 European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117
Heidelberg, Germany
2 Institute of Toxicology and Genetics, Research Center Karlsruhe, PO Box 3640,
76021 Karlsruhe, Germany
* Author for correspondence (e-mail: carl.neumann{at}embl-heidelberg.de)
Accepted 4 May 2004
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SUMMARY |
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Key words: Sonic hedgehog, Atonal homolog 5, Retina, Differentiation, Neurogenesis, Zebrafish
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Introduction |
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The Hedgehog (Hh) family of secreted signaling proteins participates in
patterning the zebrafish retina. One of the earliest functions of Hh signaling
is to induce optic stalk tissue at the expense of neural retina
(Ekker et al., 1995;
Macdonald et al., 1995
;
Perron et al., 2003
). Slightly
later, neurogenesis is initiated in the retina by a signal originating from
the optic stalk (Masai et al.,
2000
). The first neurons to be born are retinal ganglion cells
(RGCs), and these express sonic hedgehog (shh). From this
nucleation point, a wave of RGC differentiation sweeps through the retina
(Hu and Easter, 1999
), and this
wave requires hh signaling for its propagation
(Neumann and Nuesslein-Volhard,
2000
). This role of hh signaling in zebrafish retinal
neurogenesis is reminiscent of the role of Hh in directing the retinal
neurogenic wave in Drosophila (reviewed by
Jarman, 2000
;
Kumar, 2001
). A distinct wave
of shh expression occurs in the retinal pigmented epithelium (RPE)
and is associated with neurogenesis of photoreceptors in the outer nuclear
layer (ONL) (Stenkamp et al.,
2000
; Stenkamp et al.,
2002
).
The bHLH transcription factor atonal homolog 5 (ath5;
atoh7 Zebrafish Information Network) is required for the
generation of RGCs in the mouse and the zebrafish retina
(Brown et al., 2001;
Kay et al., 2001
;
Wang et al., 2001
). In the
zebrafish, ath5 mutants lack RGCs completely, but have a thin
ganglion cell layer (GCL) that is composed of `misplaced' amacrine cells
(Kay et al., 2001
).
ath5 is expressed in a wave that precedes the wave of RGC
differentiation (Masai et al.,
2000
), reminiscent of the way in which Drosophila atonal
is expressed during eye development
(Jarman et al., 1994
;
Dominguez, 1999
).
Here, we show that shh expression spreads in an additional wave in the inner nuclear layer (INL), giving rise to a subpopulation of amacrine cells that express shh. By performing in vivo timelapse analysis, we show that this wave spreads almost simultaneously with the first wave in the GCL. However, the second wave is independent of the first wave, as it spreads normally in the absence of RGCs in ath5 mutants. We also show that the differentiation of cell types found in the INL and ONL, as well as the laminar organization of the retina, depends on shh activity. By using mosaic analysis, we test which cells are the source of Shh in this context, and find that Shh directs these events as a short-range signal secreted by amacrine cells.
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Materials and methods |
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Generation of shh-GFP transgenic line
Line 2.2shh:gfp:ABC#15 was produced by microinjection of the
linear fragment of the construct p2.2shh:gfp:ABC
(SalI/KpnI) containing the 2.2 kb upstream promoter
(SalI/XhoI fragment) from the shh locus
(pshh7) (Chang et al.,
1997), inserted upstream of gfp with a polyA signal
followed by a NotI/KpnI fragment of the shh locus
(between +553 to +5631, AF124382) containing intron 1, exon 2 and intron 2.
The promoter and intronic sequences contain all four enhancer regions required
for activity in the floor plate and notochord
(Müller et al., 1999
).
The integrated transgene is stably expressed in six subsequent generations,
and is composed of head to tail concatemers of the transgene construct in an
unknown number of copies (Y. Hadzhiev, unpublished).
Histochemical methods
In situ hybridization was performed on wholemounts, using shh as a
probe (Krauss et al., 1993), a
fluorescent signal was obtained by using Fast Red as a substrate for alkaline
phosphatase (Roche), followed by confocal analysis. Antibody labeling was
carried out on 12 µm thick cryosections and analyzed with a Leica confocal
microscope. The following antibodies were used: rabbit anti-GFP (Torrey Pines
Bioloabs, San Diego; 1:1000); mouse anti-Isl1 (Developmental Studies Hybridoma
Bank; 1:50); mouse anti-Zn5 (University of Oregon; 1:500); mouse anti-Zpr1
(University of Oregon; 1:200); mouse anti-zpr3 (University of Oregon; 1:200);
mouse anti-Glutamine Synthetase (BD Biosciences; 1:500); rabbit anti-cPKC
ßI (Santa Cruz Biotechnology; 1:200); and rabbit anti-GAD67 (Chemicon;
1:50). F-actin staining was carried out as described previously
(Pujic and Malicki, 2001
),
using Alexa Fluor 568-conjugated phalloidin (1:40; Molecular Probes).
Transplantation
Transplantations were performed as described in Ho and Kane
(Ho and Kane, 1990). Donor
embryos transgenic for shh-GFP were injected at the one- to
eight-cell stage with a 2.5% mixture of biotin- and rhodamine-conjugated
dextrans (Molecular Probes) in a 1:9 ratio. Approximately 5-30 donor cells
were transplanted into the animal pole of host embryos from a syu
incross at late blastula stage. Host embryos were cryosectioned and analyzed
by immunofluorescence, as described above. The biotin tracer was detected with
Alexa Fluor 647-conjugated streptavidin (1:500; Molecular Probes) during the
secondary antibody incubation.
Time-lapse analysis
Prior to imaging, shh-GFP transgenic embryos were manually
dechorionated and transferred to embryo medium with 0.02% (w/v) tricaine.
Embryos were then imbedded in a coverslip-bottomed petri dish (MatTek
Corporation), in 0.5% low melting point agarose dissolved in embryo medium
with 0.02% tricaine. After agarose polymerization, embryos were covered with
embryo medium with 0.02% tricaine. Time-lapse imaging was performed on a
LeicaNT confocal microscope using a HCPL APO 10x/0.040 IMM objective. Z
series were obtained at 5 minute intervals with 8.8 µm steps over a total
distance of 35.2 µm and then imported into ImageJ. A single z-plane was
chosen and movies were compiled using the ImageJ software.
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Results |
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We further compared shh-GFP expression with Isl1, a
LIM/homeodomain transcription factor that is expressed in a range of neuronal
cells, and has been detected in the GCL and in a subpopulation of amacrine
cells in the rodent retina (Thor et al.,
1991; Galli-Resta et al.,
1997
). We find that there are four types of RGCs detectable with
this approach: those that express either shh-GFP or Isl1, those that
express both, and those that express neither
(Fig. 1G-I). We also find that
the amacrine cells expressing shh-GFP in the INL are distinct from
the amacrine cells expressing Isl1 (Fig.
1D-I).
These results indicate that shh is expressed in the GCL and in a
subset of amacrine cells located in the proximal INL. This is in agreement
with the observation that murine Shh is expressed both in the GCL and
in the proximal INL (Jensen and Wallace,
1997). The amacrine cells expressing shh are distinct
from the amacrine cells expressing Isl1.
In vivo imaging reveals that shh expression spreads in a wave in the INL that accompanies the RGC wave
In order to compare the spread of shh expression in the GCL and
INL of the living retina, we imaged eyes of live transgenic shh-GFP
embryos using confocal microscopy. shh expression is initiated at 28
hours post-fertilization (hpf) in the first RGC cells that differentiate in
the ventronasal retina (Neumann and
Nuesslein-Volhard, 2000). Although we detect shh-GFP
protein with an anti-GFP antibody at 30 hpf (data not shown), GFP fluorescence
is first detectable at 35 hpf (Fig.
2A, see movie at
http://dev.biologists.org/supplemental/),
due to the long folding time of GFP.
|
shh is expressed in a wave in amacrine cells in the absence of ath5 activity
To test whether the shh wave in the INL depends on the
shh wave in the GCL, we examined the spread of shh
expression in the lakritz (lak) mutant, which disrupts the
ath5 gene and leads to the complete loss of RGCs
(Kay et al., 2001). Although
all shh-expressing RGCs are absent in lak mutants, we still
detect shh-GFP expression in the inner retina of lak mutant
embryos (Fig. 3A-D). These
cells do not express the RGC marker Zn5 and are similar in morphology to the
shh-GFP-expressing amacrine cells located in the wild-type INL,
consistent with the observation that the formation of amacrine cells is not
impaired by the loss of ath5 (Kay
et al., 2001
). We observe the first shh-GFP-expressing
amacrine cells in lak mutant embryos at 32 hpf, with an anti-GFP
antibody (Fig. 3C). At 32 hpf,
we also detect a small number of shh-GFP-expressing cells in the
wild-type retina that are not Zn5 positive
(Fig. 3A, inset). These cells
may be early-born amacrine cells that express shh, although it is
also possible that they are a subset of RGCs that express shh earlier
than Zn5. The shh-GFP wave sweeps through the lak retina by
48 hpf (data not shown), and shh-GFP-expressing amacrine cells are
present both in the lak INL and the lak GCL
(Fig. 3D), the latter
corresponding to `misplaced' amacrine cells that make up the lak GCL
(Kay et al., 2001
).
|
shh signaling is required for the differentiation of cell types found in the INL
As shh is expressed in the INL, does it also have a function in
this layer of the retina? To address this question, we first examined cell
types found in the INL in wild-type embryos and shh mutants.
In the rodent retina, Isl1 is expressed both in a subset of RGCs and in a
subset of amacrine cells in the proximal INL
(Thor et al., 1991;
Galli-Resta et al., 1997
). We
find similar domains of Isl1 expression in the zebrafish retina
(Fig. 1D,
Fig. 4A). In addition, we also
detect two distinct groups of Isl1-positive cells in the distal INL at 64 hpf
that have not been described before, and that appear to be bipolar cells and
horizontal cells (Fig. 4A). In
shh mutant embryos, we observe an overall reduction of Isl1-positive
cells at 64 hpf, and most of these cells are found in the proximal retina
(Fig. 4B). The number of
Isl1-positive cells in the GCL of shh mutant embryos is greatly
reduced compared with the wild-type GCL
(Fig. 4A,B), consistent with
the observation that the number of Zn5-positive RGCs is also strongly reduced
in shh mutant embryos (Neumann
and Nuesslein-Volhard, 2000
). In addition, the other
Isl1-expressing cell types are also strongly reduced or absent, and the
laminar organization of Isl1-expressing cells is severely disrupted in
shh mutants (Fig.
4B).
|
Bipolar cells are found in the distal INL, and we used protein kinase C (PKC) as a marker to detect the presence of differentiated bipolar cells (Fig. 4E). We find no PKC-positive cells in the retina of shh mutant embryos at 72 hpf (Fig. 4F). This correlates well with the strong reduction of the Isl1-positive bipolar cells in shh mutant embryos (Fig. 4B).
Next we examined the expression of GAD67, which is a marker for differentiated amacrine cells (Fig. 4G). We detect only few GAD67-postive cells in the shh mutant retina, found in scattered locations, at 72 hpf (Fig. 4H).
Taken together, these results indicate that the differentiation of all cell types in the INL, including amacrine cells, bipolar cells, horizontal cells and Mueller glia, is dependent on shh activity.
shh is required for the generation of photoreceptors in the ONL, and for the formation of plexiform layers in the retina
It has previously been shown that the number of rod and cone photoreceptors
is reduced in shh mutant embryos
(Stenkamp et al., 2000;
Stenkamp et al., 2002
). As
described, we see a dramatic reduction of Zpr1-expressing red/green double
cones in shh mutant embryos at 72 hpf
(Fig. 5C,D). Similarly, we also
see a dramatic reduction of Zpr3-expressing rod photoreceptors at 72 hpf
(Fig. 5E,F). These cells are
present as a small cluster in a ventronasal patch, which is the region where
photoreceptor differentiation commences in the zebrafish retina. The
photoreceptor cells in shh mutants are sometimes restricted to their
normal position in the ONL, but are also often found scattered in more
proximal locations of the retina (data not shown). At 96 hpf, we detect a
small increase in the number of photoreceptors in shh mutants (data
not shown), which could be due to the activity of twhh.
|
We observe a similar disruption of retinal differentiation and lamination
when we treat embryos with the small molecule SANT-1 (data not shown), which
has been shown to act as an antagonist of the Hh signal transduction pathway
(Chen et al., 2002), further
supporting the proposal that Hh signaling is crucial for these events.
These results indicate that shh activity is required for the differentiation of photoreceptors, as well as for the formation of plexiform layers in the retina.
shh acts as a short-range signal in the neural retina to direct differentiation and lamination
To determine which shh-expressing cells direct differentiation in
the INL and the ONL, as well as the formation of plexiform layers, we
transplanted wild-type cells into shh mutant embryos. We marked
wild-type cells by injecting biotin-conjugated dextran into donor embryos; the
donor embryos were simultaneously transgenic for shh-GFP, to permit
the identification of shh-expressing cells in the wild-type
clones.
Wild-type cells are able to restore the differentiation of Mueller Glia in the shh mutant retina (compare Fig. 6 with Fig. 4D). The rescued Mueller Glia express glutamine synthetase and show the elongated morphology of this cell type, spanning the whole retinal epithelium. We only observed rescue in shh mutant cells that were located in close vicinity to wild-type cells (Fig. 6B-D). Also, we only observed rescue in those regions of the retina in which wild-type cells expressing shh-GFP were found (Fig. 6A-D), indicating that shh functions as a short-range signal (n=16 eyes). Due to this observation, we also performed transplantations in which donor cells were transgenic for shh-GFP, but were not labeled with biotin-dextran. Wild-type cells were thus identified by virtue of their shh expression in the host retina. Again, we only observed rescue in the immediate vicinity of cells expressing shh-GFP in the neural retina (n=20 eyes) (Fig. 6E,F). In most cases, wild-type clones gave rise to both RGCs and amacrine cells expressing shh, but in a few cases, only amacrine cells expressing shh-GFP were present. We also observed rescue of Mueller Glia in those cases (Fig. 6D). We never observed rescue when shh-GFP-expressing cells were located in the pigmented retina (Fig. 6G).
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Discussion |
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shh spreads in a wave in the INL following the GCL wave very rapidly
By performing in vivo timelapse recording on shh-GFP transgenic
embryos, we were able to follow the spread of shh expression in the
retina of living embryos. The most surprising result of this experiment was
the observation that the first shh-expressing amacrine cells are
already present one to two hours after the first shh-expressing RGCs
can be detected, and that the wave of shh expression in the INL
occurs almost simultaneously with the wave of RGC differentiation. Previous
studies based on BrdU labeling suggested that the first amacrine cells are
born 10 hours after the first RGCs (Hu and
Easter, 1999), and that the wave of INL differentiation is
temporally distinct from the RGC wave. By contrast, our results indicate that
the subset of amacrine cells that express shh are born very soon
after the first RGCs, and that these cells spread in a wave that is temporally
linked to the RGC wave. This conclusion is further supported by the
observation that we observe the first shh-expressing amacrine cells
in ath5 mutants, which lack all RGCs, at 32 hpf, only a few hours
after the RGC wave starts in wild-type embryos. Consistent with this, we also
observe the first shh-GFP-expressing cells that are not Zn5 positive,
and that are therefore not RGCs, at 32 hpf in the wild-type retina.
The wave of shh in the INL is independent of RGCs and independent of ath5 activity
We observe the activation and spread of shh expression in amacrine
cells in the absence of ath5 activity, indicating that this wave of
shh expression is independent of ath5 and of RGCs. This is
consistent with the finding that all cell types of the INL are formed in
ath5 mutant embryos (Kay et al.,
2001). This wave of shh expression has already started in
a ventronasal patch at 32 hpf, and spreads through the proximal region of the
retina in the absence of ath5 activity, giving rise to both the
misplaced amacrine cells that are found in the GCL of ath5 mutants,
and the shh-expressing amacrine cells in the INL. These cells are
likely to be the source of the Shh ligand that is required for the
differentiation of INL cell types, as INL cell types differentiate normally in
the absence of ath5 activity, but not in the absence of shh
signaling.
Control of cell differentiation in the INL by shh signaling
The work presented here indicates that shh signaling is required
for the differentiation of all cell types found in the INL, including amacrine
cells, bipolar cells, Mueller glia and horizontal cells. In fact, these cell
types appear to be more sensitive to a reduction in shh signaling
than RGCs are, as we have shown that the markers for differentiated bipolar
cells (PKC) and Mueller glia (glutamine synthetase) are completely absent in
shh mutant embryos, and a marker for differentiated amacrine cells
(GAD67) is almost absent in shh mutant embryos. RGCs, however, are
only partially depleted in shh mutant embryos and are completely lost
only upon further reduction of Hh signaling with cyclopamine, probably due to
the activity of twhh, which is also expressed in amacrine cells
(Neumann and Nuesslein-Volhard,
2000) (and data not shown).
As shh is required for the differentiation of all major cell types in the retina, including glial cells, it does not appear to impart any information concerning which cell fate is adopted by the responding cells, and hence it appears to function simply as a differentiation promoting factor in the retina. We observe the loss of differentiated cells both in the central and peripheral regions of the retina in shh mutants (Fig. 4, and data not shown), indicating that Shh is required in both of these domains.
An important question in this context is how directly Shh signaling
influences any of these cell types? An indirect mechanism of induction is
suggested by the observation that several other signaling pathways have been
implicated in controlling the differentiation of distinct cell types in the
vertebrate retina. For example, activation of the Notch pathway has been shown
to promote the differentiation of glial cells in the retina of frogs, rodents
and zebrafish (Ohnuma et al.,
1999; Furukawa et al.,
2000
; Scheer et al.,
2001
). This effect is cell-autonomous in the zebrafish, as even
single cells in which the Notch pathway is activated assume a glial fate
(Scheer et al., 2001
). One
possibility is thus that Shh signaling induces the differentiation of Mueller
glia by regulating the expression of Notch pathway ligands.
A further question that remains to be answered is at which stage Shh influences the transition from precursor cells to fully differentiated cells. Based on morphology, the cells remaining in shh mutants resemble precursor cells (data not shown), suggesting that they are blocked at a very early stage in differentiation. Unfortunately, we do not have molecular markers for intermediate stages of differentiation of the cells in the INL and ONL, and so cannot address this issue more directly.
Control of cell differentiation in the ONL and plexiform layer formation by shh signaling
Photoreceptor development in the zebrafish is severely impaired in the
absence of shh activity (Stenkamp
et al., 2000; Stenkamp et al.,
2002
) (this study). At 72 hpf, only a small, disorganized patch of
cells expressing the photoreceptor markers Zpr1 and Zpr3 are found in a
ventronasal patch in shh mutants, whereas the whole ONL is positive
for these markers in wild-type embryos at the same stage. These findings
complement those of Levine et al. (Levine
et al., 1997
) and Jensen and Wallace
(Jensen and Wallace, 1997
),
who showed an increase in the expression of photoreceptor markers when retinal
cultures of rat and mouse embryos were treated with Shh protein.
Our results also indicate that the plexiform layers fail to form in shh mutant embryos. The failure to form plexiform layers in shh mutant embryos is probably due to the almost complete absence of INL cell types in these embryos, as the axonal extensions of INL cells make important contributions to the plexiform layers.
Shh acts as a short-range signal in the neural retina to control retinal differentiation and lamination
To investigate which cells expressing shh are important for
directing retinal differentiation, and to determine the range at which Shh
controls differentiation and lamination, we performed mosaic experiments in
which we transplanted wild-type cells into shh mutant embryos. We
find that wild-type cells can rescue differentiation of Mueller glia and of
photoreceptors in nearby mutant cells, but only when wild-type cells are
located in the neural retina, and when they include shh-expressing
cells in the GCL and/or INL. It has been suggested that shh
expression in the RPE is responsible for directing photoreceptor
differentiation (Stenkamp et al.,
2000; Stenkamp et al.,
2002
), but our results do not support this proposal, because we
observed rescue of photoreceptor development only when wild-type cells were
located in the neural retina, but never when wild-type cells were found only
in the RPE.
We have never observed a lateral spread of rescued cells more than a few cell diameters from cells expressing shh. Although this does not address the issue of whether the effect of Shh is direct (and it may well be indirect, caused by the activation of an intermediate signal or cell state), it indicates that this event is not propagated far from the source of Shh in the lateral plane of the retina. The further spread of rescued cells in the apical/basal plane of the retina may be either due to the fact that Shh, secreted by amacrine cells, affects precursor cells, which span the whole epithelium, or that the direct or indirect range of Shh is greater in this axis. In this context, it would be very useful to determine the range of Shh signaling in the retina. Unfortunately, because of the rapid spread of the differentiation wave, patched, which is a general target gene of Hh signaling, is only transiently expressed in the zebrafish retina, and as it is very difficult to obtain a robust signal by in situ, at the moment we cannot directly assay the range of Shh signaling in the apical/basal axis of the retina.
Most clones of transplanted wild-type cells form radial columns in the host retina, and thus include both RGCs and amacrine cells. However, in very rare cases, we observed the presence of wild-type shh-expressing amacrine cells only in the INL, and found that these cells are able to rescue nearby mutant cells. Taken together with the observation that the ath5 mutant, which completely lacks RGCs, is able to generate all other cell types of the retina, this finding indicates that Shh secreted by amacrine cells is sufficient to direct differentiation in the zebrafish retina.
Formation of the IPL is rescued by wild-type cells expressing shh
in the immediate vicinity, indicating that this effect of Shh on retinal
lamination acts at very short range. The effect of Shh on plexiform layer
formation is probably indirect, via its effect on cell differentiation.
Restoration of lamination is thus likely to be due to the rescue of
differentiated cell types in the INL and GCL that contribute to the IPL with
their neurites. In this context, it is interesting to note that Shh has been
shown to promote laminar organization in the murine retina by signaling to
Mueller glia (Wang et al.,
2002).
These results indicate that Sonic hedgehog plays a crucial role in the control of differentiation in the zebrafish retina. It will be interesting to determine the interplay between Sonic hedgehog signaling and other signals in orchestrating the birth and assembly of neurons and glia in the retina.
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ACKNOWLEDGMENTS |
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Footnotes |
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