1 Department of Physiology and Programs in Neuroscience, Genetics, and
Developmental Biology, University of California, 1550 Fourth Street, San
Francisco, CA 94158, USA
2 Department of Cell Biology, Neurobiology, and Anatomy, Medical College of
Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226-0509, USA
* Author for correspondence (e-mail: jnk{at}phy.ucsf.edu)
Accepted 22 March 2005
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SUMMARY |
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Key words: Zebrafish, ath5 (atoh7), Proneural genes, Atonal, Sonic Hedgehog
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Introduction |
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In the retina, neurogenesis spreads through the field of progenitors
following a precise spatiotemporal pattern, the essential features of which
are conserved across vertebrates (reviewed by
Vetter and Brown, 2001).
Retinal ganglion cells (RGCs), the first cell type to differentiate in the
vertebrate retina, initially form a small patch adjacent to the site where the
optic stalk attaches to the optic cup. Differentiation then progresses in a
manner that, while varying somewhat between species, generally fills first the
central retina and then the more peripheral retina in an orderly fashion that
resembles an advancing wave front. This feature of retinal differentiation
allows the timing of neurogenesis to be predicted from retinal location, an
important experimental advantage. In this study we use the zebrafish retina as
a model to understand how neuroblasts time their decision to become
neurogenically active.
Expression of the proneural gene atonal-homologue 5
(ath5; atoh7 Zebrafish Information Network) is
closely associated with the activation of retinal neurogenesis in all
vertebrates. The gene encodes a basic helix-loop-helix transcription factor
expressed in a wave-like pattern that prefigures the wave of RGC genesis
(Masai et al., 2000).
Retinoblasts that express ath5 during this wave do so just after
their final mitosis; immediately thereafter they begin to differentiate as
RGCs (Yang et al., 2003
). In
the absence of functional Ath5, these cells either ectopically re-enter the
cell cycle or fail to exit the cell cycle
(Kay et al., 2001
), causing
both a failure of RGC genesis and an overall delay in the formation of the
first retinal neurons (Brown et al.,
2001
; Kay et al.,
2001
; Wang et al.,
2001
). These findings indicate a requirement for ath5 in
activating neurogenesis.
As yet, little is known about the control of ath5 expression. A
signal from the optic stalk, presumably FGF
(Martinez-Morales et al.,
2005), appears to induce the first patch of
ath5-expressing cells (Masai et
al., 2000
) (but see Stenkamp
and Frey, 2003
). How ath5 spreads from that initial patch
to cover the rest of the retina is not known, but there is a dominant
hypothesis predicting the cellular and molecular mechanisms controlling this
process (Amato et al., 2004
;
Hsiung and Moses, 2002
;
Jarman, 2000
;
Kumar, 2001
;
Malicki, 2004
;
Neumann, 2001
). The hypothesis
originated with the observation that vertebrate retinal neurogenesis is
reminiscent of the wave-like progression of neurogenesis across the
Drosophila eye field. In the fly, the ath5 ortholog
atonal (ato) is expressed in a stripe just ahead of the
morphogenetic furrow, which contains the differentiating photoreceptors. Both
the ato stripe and the furrow advance across the eye imaginal disc
due to Hedgehog (Hh) secretion by newborn photoreceptors (reviewed by
Kumar, 2001
). Hh triggers
expression of ato in progenitor cells ahead of the furrow; Ato in
turn causes formation of the next group of photoreceptors, which secrete their
own Hh, thus forming a self-propagating wave that spreads by sequential
induction of new neurons. Loss of ato function blocks photoreceptor
formation, thereby removing the cellular source of Hh and bringing the wave to
a halt (Jarman et al.,
1995
).
By analogy with the mechanism in Drosophila, it has been
hypothesized that signals derived from newborn RGCs, particularly Sonic
hedgehog (Shh), might drive the ath5 wave. In support of this idea,
shh is expressed by newborn RGCs, and shh expression spreads
across the zebrafish retina in what appears to be a self-propagating wave
(Zhang and Yang, 2001;
Neumann and Nüsslein-Volhard,
2000
). Together, these findings have led to a model predicting
that Hh molecules, released by RGCs, should drive progression of the
ath5 wave and hence the wave of RGC differentiation. We call this, in
short, the `sequential-induction' model.
The central prediction of this model, that continuing production of RGCs
should require the presence of earlier-born RGCs, has been tested using
explant cultures, but results have been conflicting
(Masai et al., 2000;
McCabe et al., 1999
). Here, we
devised in-vivo tests of the sequential-induction model, using embryological
manipulations and mutant analysis in zebrafish. Our findings reveal that
cell-intrinsic factors are sufficient to activate neurogenesis in the
zebrafish retina, but also that cell-cell signals may act earlier in
development to establish these cell-intrinsic factors or to modulate their
activity in order to bring about retinotopic differences in the timing of
neurogenesis.
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Materials and methods |
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In order to accurately assay wave progression, we developed a protocol that
ensured developmental synchrony across individuals. Embryos were raised at low
density (no more than 50 embryos/100 mm Petri dish or 20 embryos/35 mm dish)
at 27°C. All times post-fertilization reported here (aside from those
cited in other works) refer to time at 27°C. For some experiments,
ath5:GFP carriers were raised at 24°C from 12 hours
post-fertilization (hpf). These embryos were staged either by counting somites
or by adjusting to hpf at 27°C based on the extent of ath5 wave
progression. As a result of raising the embryos at 27°C (rather than
28.5°C), the onset and progression of the ath5 wave in our
experiments was slightly later than previously reported
(Masai et al., 2000;
Masai et al., 2005
). In
control experiments (not shown), we reared embryos at 28.5°C and found
that the timing of the wave was identical to that previously reported.
Histology
Embryos were treated with phenylthiourea (PTU; 0.2 mM) to inhibit
pigmentation and fixed in 4% paraformaldehyde/1 x PBS overnight at
4°C. Whole-mount immunostains were performed as described
(Kay et al., 2001) using the
following primary antibodies: Mouse zn5, zn8 and zpr1 (Oregon monoclonal
bank); mouse anti-Hu (Molecular Probes); rabbit anti-GFP (Molecular Probes).
Secondary antibodies made in goat (Molecular Probes) were conjugated to
Alexa488, Alexa546 and Alexa405. Images were collected using a BioRad confocal
microscope and were processed with Image J and Adobe Photoshop.
An ath5 antisense DIG-labeled RNA probe of 700 bp was made
for in-situ hybridization by cloning the ath5 cDNA
(Kay et al., 2001
) into pCS2,
cutting with BamHI, and transcribing with T7 polymerase. This probe
also detected the ath5:GFP transgene mRNA due to the inclusion of the
ath5 3' UTR in the transgene construct. To identify
lak mutants, ath5 staining was followed by either RFLP
mapping (Kay et al., 2001
) or
by immunofluorescent labeling with the anti-Hu antibody (not shown). Templates
for synthesizing patched1 and patched2 riboprobes were a
gift of J. Eisen (Oregon). Brightfield images were collected using a CCD
camera (Spot).
Generation of lak/wild-type chimeras
Embryos from a lak/+ incross were used to generate chimeras by
transplantation of blastula cells at the 1000-cell stage, as described
(Ho and Kane, 1990;
Kay et al., 2004
). At 55 hpf,
hosts were fixed and stained with zn5 antibody and streptavidin:Alexa 546
(Molecular Probes) to reveal donor cells. Donors were genotyped at the
lak locus by RFLP (Kay et al.,
2001
). The absence of zn5 expression identified lak
mutant hosts.
Retinoblast transplants
Retina-to-retina transplants
Donor embryos hemizygous for ath5:GFP were labeled by injection at
the 1-4 cell stage with rhodamine- and biotin-dextran amine (RDA/BDA) in 120
mM KCl (5% w/v). Host embryos (also ath5:GFP/+) were uninjected
siblings. Cells were removed from the donor retina using a glass micropipette
attached to a microsyringe drive (Stoelting Co.). Transplants were begun when
the hosts were 24 hpf and continued until
26 hpf. In order to ensure
that GFP+ cells were not transplanted accidentally, cells were
never taken from the ventronasal patch itself, but rather from more dorsal
regions of nasal retina. We confirmed by in-situ hybridization that the
ath5:GFP transgene was not expressed there at this age (not shown).
Grafted cells did not begin expressing GFP for several hours, further
suggesting (as GFP folds and becomes fluorescent quickly) that GFP mRNA was
not present at the time of transplant. After transplantation, embryos were
examined periodically for GFP expression until 58 hpf, using a Zeiss Axioskop
II microscope and a 20 x air or 40 x water-immersion lens. Further
images were collected from live fish using the BioRad confocal microscope.
Depending on the exact location into which the donor cells settled, the
difference between the predicted differentiation time of the original location
of the donor cells and their new location could be quite small. In order to
increase this time window, and thereby make more of the transplants
informative, we delayed development of the hosts by maintaining them at
24°C starting at 12 hpf. This delayed start of the host wave relative to
donors by
5 hours.
Retina-to-brain transplants
RDA/BDA-labeled ath5:GFP hemizygous embryos were used as donors;
age-matched TL (wild-type) embryos were used as hosts. Retinoblasts were
removed (as described above) from the central or temporal retina and placed
into the head mesenchyme or brain ventricles of a host. Immediately after
transplant, and at various times thereafter until 58 hpf, the live hosts were
examined for the presence of GFP expression. Importantly, no grafts expressed
GFP at the time of transplantation. Donors ranged in age from 20 somites to 29
hpf, depending on the experiment.
RT-PCR methods
Embryos at the 22-24-somite stage, or 35 hpf as a positive control for
ath5 expression, were homogenized in Trizol reagent (Gibco). Total
RNA was isolated according to the manufacturer's instructions. Two separate
RNA samples were prepared for each time point, yielding identical results.
First-strand cDNA for ath5 or cdc16 (a ubiquitously
expressed positive control) (A. M. Wehman and H.B., unpublished) was
synthesized (Promega reverse transcriptase) using the gene-specific primers
5'-TTTCGTAGTGGTAGGAGAAAG for ath5 and
5'-TCCAACACAGAGGACACGAT for cdc16. A 300 bp ath5 PCR
product was generated using the `RFLP' primers described
(Kay et al., 2001). A
200
bp cdc16 PCR product resulted using the primers
5'-CATGGTTTGCTGTTGGATGT (forward) and 5'-GGCCTGGTCATGTTCACTCT
(reverse).
Laser ablation
The ablation method and equipment are described by Roeser and Baier
(Roeser and Baier, 2003). In
one set of experiments, animals were at the 22-28 somite stage; in another set
they were at 24-26 hpf. A single Pax6DF4:mGFP embryo was transferred
to the agarose-coated lid of a 35 mm Petri dish. The weight of the embryo
caused it to lie on its side, eye up. The liquid level (embryo medium + 0.02%
Tricaine as anesthetic) was adjusted so that the embryo was barely submerged.
The ventronasal region of one eye was irradiated with the laser until GFP
fluorescence in the targeted area was thoroughly quenched (
1-2
minutes/embryo). Following staining for ath5, we verified that
ablations had indeed killed the ventronasal cells by locating pyknotic cells
using DIC optics.
Drug treatment
Dechorionated TL embryos were treated with cyclopamine or vehicle (DMSO or
methanol), fixed and stained with ath5, ptc1 or ptc2
riboprobes. Cyclopamine was obtained from Toronto Research Chemicals and by
the generous gift of Dr J. K. Chen (Johns Hopkins/Stanford). We tested dosages
between 100-400 µM, but for all experiments shown and quantified here we
used cyclopamine at 200 µM, which has been shown by real-time RT-PCR
analysis to be optimal for blocking all Hh dependent transcription
(Wolff et al., 2003).
Quantification of wave position in syu and cyclopamine experiments
The position of the ath5 or RGC wave front was determined by
examining stained embryos (labeled with either the ath5 riboprobe or
with zn5/anti-GFP antibodies) on a Leica MZ-FLIII dissecting microscope or the
Zeiss compound microscope using 10 x or 20 x objectives. Each
animal was scored as belonging to one of four categories: (1) wave front in
ventronasal retina; (2) wave front in central retina; (3) wave front in
temporal retina; or (4) wave over (ath5 expression only in the
ciliary margin or RGCs evenly filling the GCL). Because vehicle-treated
animals from cyclopamine experiments (both 13 and 25 hpf groups) were
indistinguishable from untreated wild-type syu siblings, we pooled
the data from these groups into a single `wild-type' category.
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Results |
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Fig. 1 shows both the
ath5 and RGC differentiation waves, revealed either by staining for
ath5 mRNA (Fig. 1A-C);
staining for the zn5 antigen (Trevarrow et
al., 1990), a specific RGC marker
(Fig. 1G-L); or by expression
of an ath5:GFP transgene (Masai
et al., 2003
) that faithfully recapitulates the spread of
ath5 mRNA across the retina (Fig.
1D-F; compare with Fig.
1A-C). The ath5 wave starts in the ventronasal retina
(Fig. 1A,D) with an initial
cluster of cells known as the ventronasal patch
(Hu and Easter, 1999
), and
spreads from there to fill the rest of the nasal retina and then a small patch
of the central retina (Fig.
1B,E). Next the wave begins spreading in a central-to-peripheral
manner, filling increasingly more peripheral regions of dorsal and temporal
retina. Ventrotemporal retina is the last to express ath5 and to make
RGCs (Fig. 1C,F)
(Hu and Easter, 1999
).
The sequential-induction model predicts that spread of the ath5
wave should require RGC-derived signals. To test this hypothesis, we used the
zebrafish lakritz (lak) mutant, in which a null mutation in
the ath5 gene causes complete elimination of RGCs
(Kay et al., 2001). We found
that the spread of ath5 expression was normal in lak mutants
(Fig. 2 and data not shown;
n>10 mutants and >20 wild-type siblings for each time point).
Thus, neither RGCs nor the ath5 gene itself are essential for driving
the ath5 wave.
RGCs form without sequential induction by pre-existing RGCs
We next tested whether signals from earlier-born RGCs are essential for
driving the RGC differentiation wave. Because the lak/ath5 gene acts
cell-autonomously in RGC specification
(Fig. 3D-H), we were able to
test this prediction by generating lak/wild-type chimeras in which a
small number of wild-type cells were situated in an otherwise mutant (and thus
RGC-free) retina (see model, Fig.
3C). We generated chimeras at the 1000-cell stage
(Ho and Kane, 1990) and
assayed RGC differentiation at 55 hpf using the zn5 antibody. Regardless of
the host's genotype, retinal clones derived from lak mutant donors
never gave rise to zn5+ RGCs (n>50 clones;
Fig. 3G,H), while clones from
wild-type donors always did. Of the wild-type-into-lak chimeras
(n=9 eyes with multiple clones per eye), we found three retinae with
central or temporal clones that were well isolated from both the optic stalk
and other RGC-containing wild-type clones. Contrary to the
sequential-induction model's predictions, the isolated clones gave rise to
RGCs in all three cases (Fig.
3E,F and data not shown). Indeed, clones in the temporal retina
could produce RGCs even when the rest of the retina was completely devoid of
RGCs (Fig. 3F). Thus, RGC
formation in later-differentiating retinal regions does not depend on prior
RGC formation in earlier-differentiating regions.
Laser ablation of the ventronasal patch does not block the ath5 wave
We next asked whether the ath5-expressing retinoblasts themselves
might signal to neighboring retinoblasts, inducing them to express
ath5 (Masai et al.,
2000). If this version of the sequential-induction model is
correct, then removal of the ventronasal patch before ath5 expression
should prevent relay of ath5-inducing signals, thereby blocking the
wave. To test this prediction, we laser-ablated the neuroblasts of ventronasal
retina at times ranging from 22 somites to 26 hpf. To guide the ablations we
used a transgenic line that expresses GFP in all retinal neuroblasts
(Pax6-DF4:mGFPs220)
(Kay et al., 2004
).
Photobleaching of GFP protein allowed us to precisely delineate the retinal
region targeted in each larva (Fig.
4A-B).
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We next tested whether elimination of the ventronasal patch would affect spread of ath5 expression through the rest of the retina. Ablations were performed as before, but this time we assayed for ath5 mRNA expression at 33 hpf. In ablated retinas, the ventronasal portion of the ath5 domain was absent, indicating successful removal of the ventronasal patch. However, outside the laser-targeted region, ath5 expression was similar to that seen in unablated retinas (Fig. 4G-H; see Fig. S1 in the supplementary material). In all treated animals (n=27 at 24-26 hpf; n=12 at 22-28 somites), the extent of ath5 wave progression into the central retina was the same in both the ablated and unablated eyes. The ath5 wave evidently can skip over the ablated region, and begin instead in more dorsal regions of the nasal retina, in the central retina, or even in the temporal retina, depending on the size of the ablated domain (see Fig. S1 in the supplementary material). This finding indicates that, if ath5+ cells do in fact generate signals that induce ath5 expression in neighboring retinoblasts, such signals are not necessary for propagating the ath5 wave across the retina.
Retinal signals are not required for ath5 expression
The results of our experiments so far suggested that cell-cell signaling is
not as essential for triggering ath5 expression as had previously
been assumed. We therefore wondered whether retinoblasts would express
ath5 even when removed entirely from the normal retinal signaling
milieu. To test this possibility, we devised a method for transplanting
retinoblasts from a labeled donor retina into non-retinal tissues of an
unlabeled host (note that this experiment is quite different from the
lak/wild-type blastula transplants; see Materials and methods). If
retinal signals are required to trigger ath5 expression,
heterotopically transplanted retinoblasts should fail to express
ath5. Donor cells (labeled with RDA and carrying the
ath5:GFP transgene) were removed from the temporal retina at various
times between the 20-somite stage and 29 hpf. This latest time point is still
5 hours before the first temporal retinal cell expresses mRNA for the
ath5:GFP transgene (data not shown). Donor cells were placed in the
telencephalic or mesencephalic ventricular space of a wild-type,
non-transgenic host brain; some transplants were also placed in the head
mesenchyme. By 50 hpf, grafted cells in both the ventricular space
(Fig. 5A-B) and the head
mesenchyme (not shown) expressed GFP. Some of these cells had clearly
differentiated into RGC-like neurons, as they possessed long axons tipped by
growth cones (Fig. 5B). To
ensure that the transplants were done before the onset of ath5
expression, we used donors at the 20-24-somite stage. RT-PCR experiments
showed that ath5 is not yet expressed at 24 somites (not shown).
Three of the seven grafts from 20-24-somite donors expressed ath5:GFP
by 50 hpf. These results demonstrate that retinoblasts can express
ath5, and possibly even assume the RGC fate, even when stripped out
of the retinal neuroepithelium and placed in a variety of ectopic
locations.
We next tested whether the transplanted retinoblasts could undergo neuronal
differentiation. We found that heterotopically grafted retinoblasts in the
brain, brain ventricles or head mesenchyme, as well as homotopically grafted
cells in the retina, could differentiate as both RGCs and photoreceptors, as
judged by expression of cell-type-specific markers (see Figs S2, S3 in the
supplementary material). Not every grafted cell was able to differentiate
successfully. For example, only 15-35% of cells transplanted to the host
retinal ganglion cell layer (GCL) successfully expressed RGC markers by 72 hpf
(see Fig. S2 in the supplementary material). This observation suggests that
certain cell-cell signals, perhaps those provided by being part of an intact
neuroepithelium, may be permissive for the activation of neurogenesis and/or
for differentiation. Nevertheless, intra-retinal signals do not appear
essential for activation or execution of the retinal neurogenic program, as
both ath5 expression and cell-type-specific marker expression can
occur in the absence of such signals.
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We first took nasal retinoblasts from RDA-labeled ath5:GFP
carriers at 24-26 hpf and placed them into the temporal region of a host
ath5:GFP retina. GFP expression was then monitored in live fish at
regular intervals (generally 1-2 hours), until well after the wave was
complete (58 hpf). We found that these nasal-derived cells in temporal retina
either began expressing ath5 while the host wave was still confined
to nasal retina (Fig. 5C,D;
31%; n=4/13), or failed to express ath5 at all by 58 hpf
(n=9/13). None of the grafted nasal cells expressed ath5 on
a temporal schedule. This result supports the notion that intrinsic factors,
not signals from the advancing wave front, determine the timing of
ath5 expression. Although only 30% of nasal-into-temporal grafts
succeeded in expressing ath5:GFP by 58 hpf, this rate of
differentiation was typical for grafts into the GCL that were made without
regard to the retinal sector from which the cells were taken from or where
they were placed (see Fig. S2 in the supplementary material). It is therefore
unlikely that the temporal environment inhibited nasal donor cells from
differentiating.
We next asked whether relative timing differences would be maintained even in the absence of retinal signals. Using RDA-labeled ath5:GFP carriers as donors, retinoblasts were removed from either the central or temporal retina at 26-29 hpf and transplanted into the head of wild-type (non-transgenic) hosts. This time point was several hours prior to the onset of transgene mRNA expression in the central or temporal retina (not shown). Each donor gave rise to two hosts, one carrying the donor's central retinal cells and one carrying its temporal cells. These live hosts were then checked periodically for ath5:GFP expression in the grafted cells. In each pair of hosts, the one carrying the central graft always expressed ath5:GFP first. In fact, all the central grafts had begun expressing ath5 before the first temporal graft did so (Fig. 5B; n=5 central, n=5 temporal). Only one graft failed to express ath5 at all (n=11 hosts). Thus, progenitors do not require retinal signals after 26 hpf (27°C) to maintain the neurogenic timing conferred by their original retinal position. These experiments suggest that the spatiotemporal pattern of the RGC differentiation wave might arise because retinoblasts are intrinsically programmed, before the wave, to begin neurogenesis at different times.
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We first examined ath5 expression in the shh null mutant
syut4 (Schauerte et
al., 1998). We found that the spatial pattern of the ath5
wave was unaffected in these mutants, but the timing of wave progression was
dramatically altered (Figs 6,
7). Initiation of the wave in
ventronasal retina was normal at 30 hpf
(Fig. 6A,E; Fig. 7A). Subsequently, the
ath5 wave spread to the central and temporal retina as in wild type,
but it did so on a delayed schedule. For example, whereas the wild-type wave
reached the temporal retina at 41 hpf, in syu mutants the
ath5 wave was confined to the central retina at 41 hpf
(Fig. 6C,G;
Fig. 7C). The wave did not
reach the temporal retina until 50 hpf in the mutants, indicating that
temporal retinoblasts were delayed in expressing ath5 by almost 10
hours (Fig. 6D,H;
Fig. 7D). Loss of shh
function thus did not block the ath5 wave, but it did have a
substantial effect on the time of neurogenic activation.
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To determine whether the altered timing of the ath5 wave also changes the timing of RGC differentiation, we analyzed the expression of two different RGC markers following 13 and 25 hpf cyclopamine treatment. Indeed, we found that treatment at 13 hpf, but not 25 hpf, delays expression of RGC-specific markers (Fig. 9). Thus, while loss of Hh signaling during the ath5 wave has no effect on ath5 expression or RGC differentiation, blockade of early, midline-derived Hh signals causes a delay in both the ath5 and RGC differentiation waves. This finding raises the possibility that axial patterning mechanisms, such as midline Hh signaling, might be involved in creating spatial differences in the timing of neurogenic activation.
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Discussion |
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Setting the neurogenic timer: the role of midline-derived Sonic hedgehog
We show that central retinoblasts are already competent to express
ath5 independently of signals from ventronasal retina by 22 somites
(20 hpf at 28.5°C), and temporal retinoblasts are independent of
retinal signals by 20 somites (
19 hpf at 28.5°C). When the retina is
removed at 18 somites and the nasal and temporal halves are cultured
separately, the temporal explant is delayed or fails to express ath5
(Masai et al., 2000
). This
result, together with ours, may indicate that an important intra-retinal
signaling event occurs between the 18- and 20- somite stages (a time window of
about 1 hour). Alternatively, explanting the retina may remove a source of
extra-retinal signals, such as Shh, that are required for timely
differentiation of temporal retinoblasts. Regardless of the precise cellular
mechanism through which the timing of ath5 expression is set, our
results are significant for showing that the time window during which signals
act to influence neurogenic activation is substantially earlier than
previously suspected.
If retinoblasts have an intrinsic tendency to activate neurogenesis at a
particular time, there must be some mechanism that establishes the neurogenic
timing for each cell. What is this mechanism, and when does it act? We find
that retinal location is a key variable, implying that an asymmetric spatial
signal might set the neurogenic timer in order to impart location-specific
timing information. A good candidate for such a signal is Shh derived from the
ventral midline of the diencephalon. Previously, midline Hh signals were
reported to be required for ath5 expression
(Stenkamp and Frey, 2003).
Here we extend this important finding by showing that Shh acts between 13 and
25 hpf not as an absolute prerequisite for ath5 expression, but
rather to ensure timely expression of ath5 during the wave, hours
later. Before 25 hpf, shh and its relative, tiggy-winkle
hedgehog (twhh), are expressed in the diencephalic ventral
midline but not in the retina (Ekker et
al., 1995
). In fact, shh expression is not detectable in
the retina before the ath5 wave has already reached the temporal
retina (Masai et al., 2005
).
These combined results suggest that the midline source of Hh signals, in
addition to patterning the DV axis of the eye
(Amato et al., 2004
;
McLaughlin et al., 2003
), also
seems to have a role in patterning the timing of retinal neurogenesis.
In a recent study, Masai et al. (Masai
et al., 2005) narrowed the time window of Hh action even further
by using forskolin, a potent activator of protein kinase A (PKA), which in
turn antagonizes Hh signaling. In keeping with our results, these authors show
that the ath5 wave can only be blocked when forskolin treatment
occurs before, but not during, wave progression (21-25 hpf at 28.5°C)
(Masai et al., 2005
). Because
forskolin causes a more severe ath5 phenotype than either cyclopamine
or various genetic manipulations that block Hh signal transduction, it appears
that multiple signaling pathways impinge on PKA to regulate the subsequent
timing of ath5 expression.
Evidence against a role for retinal Hh signaling in ath5 expression
Loss of early Hh signaling delays both the ath5 and RGC
differentiation waves. By contrast, retina-derived Hh appears not to be
required for ath5 expression or RGC differentiation. In
Xenopus, chick and mouse, reducing retinal Hh signaling has effects
on RGC genesis similar to those we describe
(Dakubo et al., 2003;
Perron et al., 2003
;
Zhang and Yang, 2001
). By
contrast, it had previously been reported that retinal Hh signaling is
essential for RGC formation in zebrafish
(Neumann and Nüsslein-Volhard,
2000
), a finding that we could not confirm. It is not clear why
the results of the two studies differ. In both experiments, cyclopamine was
used to block Hh signaling specifically during the period of RGC genesis
(starting at
25 hpf). We show, using ptc1/2 staining on larvae
treated in parallel with those tested for RGC formation, that our drug was
effective at eliminating Hh signaling. To control for drug quality, we
obtained cyclopamine from two independent sources and dissolved it in two
different solvents each permutation of the experiment gave identical
results.
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Integration of intrinsic and extrinsic factors during retinal neurogenesis
To date, there have been few attempts to determine how specific neuroblast
populations integrate cell-intrinsic and cell-extrinsic information for
neurogenic activation. For oligodendrocyte precursors, it has been shown that
an intrinsic timer operates, which is licensed by permissive extrinsic signals
(Durand and Raff, 2000).
Retinoblasts may use a similar cell-intrinsic mechanism to decide when to
differentiate (Cayouette et al.,
2003
). While this work in cell culture, together with our
experiments in vivo, highlights the importance of cell-intrinsic factors, RGC
neurogenesis is certainly not completely cell-autonomous. First, as in
oligodendrocyte precursors, there may be licensing signals the fact
that the majority of transplanted retinoblasts fail to differentiate in our
experiments implies the existence of such signals. Second, cell-cell signals
may affect steps of RGC differentiation downstream of the cell-intrinsic
trigger that activates the neurogenic program (and thus presumably downstream
of ath5 expression).
Third, cell-cell signaling may be necessary to counteract the effects of
the intrinsic timer. Our results reveal the existence of an intrinsic tendency
toward neurogenesis, but there must be some signal that opposes this tendency
otherwise no retinoblasts would be reserved to make later-born cell
types. Similarly, to ensure that the correct number of progenitors become
RGCs, there may also be signals that promote neurogenesis over and above the
basal level provided by the intrinsic timer. There is strong evidence that
Notch, perhaps in concert with other signals, plays both these roles, acting
in one context to promote cell cycle exit during the RGC wave
(Ohnuma et al., 2002), and in
another context to terminate RGC genesis behind the wave front
(Silva et al., 2003
). However,
Notch probably accomplishes both these roles without affecting the
spatiotemporal pattern of differentiation
(Ohnuma et al., 2002
;
Scheer et al., 2001
;
Silva et al., 2003
). Thus, it
appears that extrinsic signals balance but do not fundamentally alter the
intrinsic program underlying RGC genesis.
Conserved and divergent mechanisms of retinal neurogenesis in insects and vertebrates
In the developing Drosophila retina, a progressive cell-cell
signaling loop, centered on the ato and hh genes, initiates
neurogenesis (Kumar, 2001).
The discovery that close homologs of these genes, ath5 and
shh, play a role in differentiation of the first-born neurons of the
vertebrate retina led to the hypothesis that the mechanisms for initiating
retinal neurogenesis are conserved between flies and vertebrates (reviewed by
Kumar, 2001
). Despite the
appeal of this hypothesis, there have been few attempts to test whether
neurogenesis spreads across the vertebrate retina by a fly-like sequential
mechanism. In one such study, the peripheral portion of a chick retinal
explant, dissected away from the RGC wave front and cultured separately, was
still competent to generate RGCs, implying that differentiation does not
require a progressive, wave-like mechanism
(McCabe et al., 1999
).
However, a similar experiment in zebrafish yielded the opposite result
(Masai et al., 2000
). Although
there are experimental design differences that might explain the differing
results of these two studies, neither has settled the question of whether
sequential induction triggers retinal neurogenesis in vertebrates. Here we
have devised in-vivo tests of the fly-inspired sequential-induction model, and
we have found very little evidence to support it. Our findings indicate that
while hh and ato family genes may have a broadly conserved
function in RGC genesis, the cellular context in which they operate differs
significantly between vertebrates and insects.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/11/2573/DC1
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