Download Extraocular dorsal signal affects the developmental fate of the optic

Develop. Growth Differ. (2005) 47, 523– 536
Extraocular dorsal signal affects the developmental fate of
the optic vesicle and patterns the optic neuroepithelium
Blackwell Publishing, Ltd.
Yuka Kagiyama,1 Nanaka Gotouda,1 Kiyo Sakagami,2 Kunio Yasuda,2 Makoto Mochii3
and Masasuke Araki1,*
1
Developmental Neurobiology Laboratory, Faculty of Science, Nara Women’s University, Nara 630-8506, Japan
Laboratory of Molecular and Developmental Biology, Nara Institute of Science and Technology, Ikoma 630-0101,
Japan and 3Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan
2
Dorsal–ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of
the eye. To address the questions of how DV specification is determined and how it affects fate determination
of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves
of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively,
when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove
the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that
surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures.
Combination cultures of collagenase-treated optic vesicles with either the dorsal or ventral part of the head
indicated that head-derived factors have an important role in the fate determination of the optic vesicle: in the
optic vesicles co-cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium,
while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in
the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles
in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and
differentiation of the neural retina and pigment epithelium.
Key words: chick embryo, cRax, Mitf, optic vesicle, organ culture.
Introduction
Development of the vertebrate eye begins with optic
vesicle formation, which develops by lateral protruding of the diencephalic neuroepithelium. The optic
vesicle subsequently invaginates to form a doublelayered optic cup, whose inner and outer layers further develop into the neural retina (NR) and the
retinal pigment epithelium (RPE), respectively (Saha
et al. 1992; Chow & Lang 2001). At the same time,
the invagination of the optic vesicle extends from the
distal to the proximal direction at the ventral part to
form the future optic fissure in the optic stalk, through
which the retinal ganglion axons pass into the optic
tectum. Thus, the early optic vesicle appears to
consist of at least two discrete dorsal and ventral
compartments that show different developmental fates.
This is also suggested by the different expression
*Author to whom all correspondence should be addressed.
Email: [email protected]
Received 14 June 2005; revised 26 August; accepted 30
August 2005.
patterns of various transcription factors (Torres et al.
1996; Schulte et al. 1999; Koshiba-Takeuchi et al.
2000). The optic vesicle is surrounded by and makes
contact with various different tissues, such as the
overlying surface ectoderm and periocular mesenchyme, and it has been assumed that the specification of the NR and RPE is influenced by signals from
the surface ectoderm and mesenchyme, respectively
(Dragomirov 1937; Lopashov 1963; Hyer et al. 1998;
Fuhrmann et al. 2000). The extraocular mesenchyme
originates from two different sources, the cephalic
neural crest cells at the dorsal region and the
precaudal mesodermal cells at the ventral region
(Johnston et al. 1979). It is not clear whether these
differently located mesenchymal tissues play different
roles in eye development (Pera & Kessel 1997).
Optic vesicle invagination begins at a point displaced
somewhat toward its ventral surface, rather than
beginning at the most lateral and distal point, and is
directed mediodorsally (Romanoff 1960; Bellairs &
Osmond 1998). Consequently, the distal and ventral
portions of the early optic vesicle are to organize the
inner layer of the optic cup, fated to develop into the
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NR, while the dorsal region will organize the outer
layer, fated to develop into the RPE. Fate mapping
of the chick optic vesicle with fluorescent dye also
reveals different developmental fates of the discrete
regions in the optic vesicle according to the dorsal–
ventral (DV) and proximal–distal directions (Dütting
& Thanos 1995). DV polarity is well understood for
its importance in eye morphogenesis, and several
transcription factors, such as Pax2 and Pax6, are
known to play an essential role in the fate determination of the optic vesicle regions (Schwartz et al.
2000; Baümer et al. 2003). The domain of Pax2
expression is complementary to that of Pax6 (Nornes
et al. 1990; Macdonald & Wilson 1996). Several signaling molecules have been implicated in the regulation of ventral optic development. The secreted
protein sonic hedgehog (Shh) plays important roles in
patterning tissues of the vertebrate embryo, including
the eye, and accumulating evidence indicates that
Shh activity is also involved in pattern formation of the
vertebrate eye (Oliver & Gruss 1997; Jean et al. 1998).
Pax2 and Pax6 expression in the optic vesicle
are regulated by Shh. Alterations in Shh activity in
zebrafish have been shown to perturb Pax6 and
Pax2 expression (Macdonald et al. 1995). Bone
morphogenetic proteins (BMP) also have profound
effects on patterning in the nervous system. BMP4,
for instance, is involved in the dorsalization of the
retina (Koshiba-Takeuchi et al. 2000). The spatial and
temporal expression patterns of Shh and BMP4 suggest a model in which ventral midline-derived Shh
and dorsally derived BMP4, either from the dorsal
midline of the neural tube or the dorsal optic cup,
play roles in establishing the DV polarity of the optic
primordium (Schulte et al. 1999; Zhang & Yang 2001;
Martí & Bovolenta 2002).
To elucidate the mechanism involved in DV polarity
formation in the developing optic vesicle, we have
previously used an embryonic transplantation technique on the chick embryo, in which the truncated
optic vesicle is transplanted inversely without changing the anterior–posterior direction (Araki et al. 2002;
Uemonsa et al. 2002). These approaches revealed
that DV polarity is established gradually between the
10- and 14-somite stages under the influence of signals derived from the midline portion of the forebrain,
and that the presumptive signals appear to be transmitted from proximal to distal regions within the optic
vesicle. Disturbance of DV polarity formation causes
the failure of optic cup formation, as well as patterning
of the NR and RPE. Accordingly, two new questions
arose: which tissue is responsible for the presumptive
dorsal or ventral signal(s), and how are they transmitted to the optic vesicle?
To answer these questions, we performed organotypic culture of the optic vesicle, either with or without
the surrounding tissues. The optic vesicle was cut into
its dorsal and ventral halves, which were cultured
separately to determine their developmental fates.
The optic vesicle was also cultured in combination
with the dorsal or ventral half of the head portion, to
examine whether the head-derived factors affect
the development of the optic vesicles. The present
results indicate that optic vesicle development is
regulated by the surrounding tissue, probably the
neural crest-derived dorsal mesenchyme. This tissue
appears to induce RPE fate in the optic vesicle,
which otherwise develops into the NR as a default
pathway. The results also indicate that the dorsal part
of the head suppresses Pax2 expression in the optic
vesicle, and that the dorsal and ventral halves of the
optic vesicle are committed to develop mainly into
the RPE and NR, respectively.
Materials and methods
Preparation of chick embryos
Fertilized eggs were incubated in a humidified atmosphere at 37.8°C. All operations were carried out
according to the procedures previously described
(Araki et al. 2002; Uemonsa et al. 2002). A small
window was made in the shell, and India ink, diluted
in Hanks’ solution, was injected into the yolk beneath
the embryos to visualize the embryonic structures
(Fig. 1). Embryos were staged according to Hamburger
and Hamilton (1951).
Preparation of optic vesicles
Embryos at given stages were placed into roundbottomed dishes filled with Hanks’ solution to wash
Fig. 1. Chick embryos at the 10-somite stage. (A) Dorsal view
showing the lateral protrusion of the optic vesicle. Bar, 300 µm.
(B) Transverse section of the proencephalon. The optic vesicle
makes contact with different types of tissues at the dorsal and
ventral regions. Arrow indicates the dorsal mesenchymal tissue.
Bar, 100 µm.
Extraocular signal and eye development
out the yolk and India ink. The embryos were then
pinned over a black silicone plate, and optic vesicles
with the covering ectoderm and mesenchyme were
truncated using a finely sharpened hand-made knife.
The surrounding tissues, like the surface ectoderm
and mesenchyme, were removed by using a fine
tungsten needle after a treatment with 0.03%
collagenase (Sigma, St Louis, MO, USA) for 60 min
at 25°C.
To obtain the dorsal and ventral halves of the whole
head portion, the head was cut transversely at the
level at the rhombomere, and then a careful lateral
incision was made exactly along the horizontal plane
that divides the dorsal from the ventral half. Particular
attention was paid to cut the optic vesicle precisely
into the dorsal and ventral halves along the horizontal
level. The dorsal or ventral halves were cultured on
a filter membrane cup (Millicell-CM, 0.4 µm, Millipore,
Bedford, MA, USA) by placing the cut surface facing
the membrane.
Fig. 2. Two
examples
of
embryonic transplantation of
the optic vesicle into the distal
wing bud. Transplantation was
performed as shown in the
schematic illustration. Optic vesicle
at the 10-somite stage was
inserted into an incision made at
the distal part of a wing bud of a
host embryo. In (A), a well developed eye is seen embedded
in the mesodermal tissue of the
wing bud. In this case, quail
optic vesicle was transplanted to
chick wing bud. At a higher
magnification numerous chicken
(host) cells are intermingled among
quail cells (C, arrowheads) in the
periocular connective tissue. In the
other case (D), a less developed
eye is found at the wing bud in
chick–chick transplantation. The
outer retinal pigment epithelium
layer appears multilayered epithelial
form with pigment granules as
shown in (E). Bar in (A) is 250 µm
and applies to (D). Bar in (B) is
40 µm, and Bar in (C) is 20 µm
and applies to (E).
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In ovo transplantation of the optic vesicle
Optic vesicles were cut from embryos at 8-, 10-, 14and 16-somite stages and placed into the wing buds
of host embryos at 3 days of incubation without
further treatment with collagenase. A small cut was
made at the distal part of the wing bud and the optic
vesicle was inserted into the limb mesenchymal
tissue (Fig. 2). The grafts were allowed to develop
further in ovo for 3–4 days until fixed for histological
preparation. In some cases, optic vesicles from quail
embryos were transplanted to the wing buds of chick
embryos to discriminate donor cells from the host.
Organotypic culture of the optic vesicle
Optic vesicles were cut from embryos at the 8-, 10-,
14-, 16- and 18-somite stages and were used for
organotypic culture. Two types of organ culture methods were used. Isolated optic vesicles were first
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laid on a filter membrane cup (Millicell-CM, pore size
0.4 µm, Millipore) and cultured with or without
embedding in a collagen gel (Cellgen I-AC, Koken,
Tokyo, Japan). They were cultured for 3 days, and
fixed for histological observation in the case of those
cultured in collagen gel. Optic vesicles cultured without collagen gel were used mainly for immunocytochemistry and in situ hybridization. The standard
medium was Dulbecco’s MEM (Nissui, Tokyo, Japan)
supplemented with sodium pyruvate (55 mg/mL),
glucose (6 mg/mL) and 8% fetal bovine serum (FBS;
Micro, Walkersville, MD, USA).
For the combination culture of the optic vesicle with
the head halves, the right optic vesicle was first cut
off, and then the head portion of embryos was divided
into two halves as described above. The optic vesicle
was laid down at the original position of the removed
optic vesicle in close contact with the head half.
Immunocytochemistry
Cultures were fixed with ice-chilled 2% paraformaldehyde in 80 mM phosphate buffer (pH 7.4) for 6 h.
They were then stained for Mitf, cRax, HNK-1 or
QCPN, either by using the indirect method or the
ABC method according to the procedure previously
described (Araki et al. 2002). Briefly, cultures were
first immersed in phosphate-buffered saline (50 mM,
pH 7.4) containing 0.2% Triton X-100 (PBS-TX) for
60 min. For the indirect method, the immunoreactive
sites were detected using a fluorescein-conjugated
secondary antibody (Alexa-488, Molecular Probes,
Eugene, OR, USA), and for the ABC method, they
were visualized by using hydrogen peroxide and
diaminobenzidine. The specificity of the antibodies
had been confirmed previously (Mochii et al. 1998;
Sakagami et al. 2003).
In situ hybridization
Plasmids containing Tbx5 and Pax2 cDNA in pBluescript SK(-) were linearized with EcoRV and EcoRI,
and transcribed in vitro with T3 RNA polymerase to
generate digoxigenin-labeled antisense and sense
RNA probes, respectively (Promega, Madison, WI,
USA). The Tbx5 cDNA was obtained from Dr. T. Ogura
(Nara Institute of Science and Technology), and the
Pax2 cDNA from Dr. H. Nakamura (Tohoku University).
Whole-mount in situ hybridization was performed
as described previously (Henrique et al. 1995). After
hybridization, embryos were incubated with 1/2000diluted alkaline phosphatase-conjugated antiDIG
antibody (Roche Diagnostics, Mannheim, Germany)
overnight, and nitroblue tetrazolium (NBT)/5-bromo-
4-chloro-3-indoyl phosphate (BCIP) staining was
employed for the detection of hybridization signals.
Embryos were photographed with a camera attached
to a stereomicroscope (Olympus, Tokyo, Japan).
Histological preparation
For histological observation, embryos were fixed with
Bouin fixative for 3–10 h depending on their size,
dehydrated in a graded ethanol series, and finally
embedded in paraffin. Serial sections of 6 µm thickness were cut in a transverse plane and stained with
hematoxylin and eosin.
Results
In ovo transplantation of the optic vesicle: Optic cup
formation and retinal pigment epithelium
differentiation
Our previous experiments suggested that diffusible
factors from the forebrain region of the embryo act on
the optic vesicles and contribute to the delineation of the
territories fated to form RPE and NR. To further define
the source of this activity, we performed a series of in ovo
transplantation and in vitro co-culture experiments.
To examine their capability of autonomic development in a foreign milieu, isolated optic vesicles were
transplanted into the wing buds of host embryos
without further treatment with collagenase. Histological
examination of the optic vesicle at the 10-somite stage
(HH stage 10) revealed that it faces dense mesenchymal tissues at the dorsal region (Fig. 1). Isolated
optic vesicles therefore contained both the epidermis
and the dorsal mesenchymal tissue.
After 3 days of in ovo culture, approximately onethird of the grafts (19 out of 60 grafts) remained at
the grafting position (the distal part of the wing
buds). Some of them had grown considerably, even
to a size similar to that of normal eyes at the corresponding stage (Fig. 2A). We further examined the
histological structures of grafts with respect to the
differentiation of the NR, RPE and the distribution of
the mesenchymal cells (Fig. 2B,C).
In all cases, a well developed lens and the inner
and outer epithelial layers of the optic cup were found.
The inner layer was usually much thicker than the
outer layer, which often showed a single or multilayered
epithelial form with pigment granules (Fig. 2C,E). In
all cases, these pigmented epithelial layers were
surrounded with mesenchymal cells, suggesting that
mesenchymal cells in the optic vesicle have an
inductive role in RPE development. The mesenchymal
cells surrounding RPE were often of the host origin,
Extraocular signal and eye development
as shown by the histological appearance of their
nuclei in the chimeric transplantation (Fig. 2C).
Organotypic culture of the optic vesicle
To examine whether in vitro culture conditions enabled isolated optic vesicles to develop autonomously,
they were maintained in vitro under two different culture conditions: gel-embedded or simply laid on filter
membrane.
In cultures of gel-embedded optic vesicles, similar
results were obtained as seen in the in ovo transplants.
After 3 days of culture, the lens, as well as the inner
and outer layer, were observed in approximately half
of the explants (29 out of 61 grafts; Fig. 3). The inner
and outer layer had no distinct difference in thickness.
Mesenchymal cells were generally poorly developed
in comparison with those of the in ovo transplants.
RPE layers were found in only a few cases (8 out of
29) and were surrounded by mesenchymal cells
(Fig. 3A,B). These observations were mostly the same
regardless of the stages (10-, 12-, 14-, 18- and 20somite stages) of the grafts.
In another culture condition, optic vesicles isolated
from 10-somite-stage embryos were laid on the membrane filter cup and cultured for 3–4 days without
being embedded in a gel. This culture method allowed
us to perform immunocytochemical staining of the
whole-mount tissues without preparing sections, and
in the following culture study we used this method
to detect Mitf and cRax immunocytochemical staining. Detection of melanin granules under a light
microscope was also a good indication of RPE
differentiation. We treated optic vesicles with collagenase to remove the ectodermal and mesenchymal
tissues, and compared the results with those obtained
in untreated optic vesicles.
Fig. 3. Gel-embedded culture of the optic vesicle removed
from embryos at the 10-somite stage. The outer layer is facing
mesenchymal tissue and contains pigment granules as shown
by arrowheads in (B). The amount of mesenchymal cells is
much less than found in the embryonic transplanted optic
vesicle. Bar (A), 100 µm; bar (B), 40 µm.
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Untreated optic vesicles, which still contained
surrounding tissues, the surface ectoderm and mesenchyme, normally became pigmented, though not
very intensely, and expressed both Mitf and cRax
(Fig. 4A–C). In contrast, collagenase-treated optic
vesicles seldom showed melanin deposition (3 out
of 22) (Fig. 4D). Mitf was not usually detected, while
cRax was always detected (15 out of 15), but less
intensely than that found in the untreated optic
vesicles (Fig. 4E,F). These observations indicate that
without the surrounding tissues the optic vesicle neuroepithelium differentiates into NR but not into RPE.
Organotypic culture of dorsal and ventral halves of
the embryonic head portion
To examine whether the dorsal and ventral parts of
the optic vesicle at these stages were already committed or determined to develop along separate fates,
we cultured dorsal and ventral halves of embryonic
heads removed from 10- to 16-somite stage embryos.
After 3 days in culture, the explants grew considerably, and the epidermis and neuroepithelial cells
intensively extended outward at the periphery of the
explants (Fig. 5A–C). The optic vesicles also extended
outward and occasionally they fused with the brain
vesicles, such as the telencephalon and diencephalon
(Fig. 5C). It was found that, in the dorsal brain culture,
melanin deposition was always seen in both sides of
the optic vesicle (Fig. 5D), while it was observed only
rarely, if at all, in the optic vesicle areas of the ventral
head culture (Fig. 5G). Cultures were fixed after
3 days in vitro to examine RPE and NR differentiation.
In the dorsal head culture, intense Mitf staining was
always found in both the right and left optic vesicles,
but only a very faint staining for cRax was observed.
In contrast, in the ventral head culture, intense
cRax staining was always observed, while Mitf
immunostaining was very faint. In cases where it was
detected, Mitf staining was always found in the posterior part of the optic vesicle. These results indicate
that the dorsal and ventral parts of the optic vesicle
are committed to differentiate mainly into the RPE and
NR, respectively, as early as at the 10-somite stage.
Taken together the above results, it was suggested
that the dorsal portion of the optic vesicle is committed to develop into RPE, and the dorsal mesenchymal tissue plays a role in fate determination.
Combination culture of the optic vesicle with the head
portion
To investigate factors involved in the fate commitment
or fate determination of NR and RPE, we cultured
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Fig. 4. Organotypic culture of
the optic vesicle. Isolated optic
vesicle was laid onto a membrane
filter cup for culture. (A,B,C)
Untreated optic vesicle. (D,E,F)
Collagenase-treated optic vesicle
in which surrounding tissues
were removed. In A, melanin
deposition can be seen (arrow).
(B,E) cRax (C,F) Mitf immunostaining. No Mitf positive nuclei
are found in cultures of
collagenase-treated optic vesicle.
Bar in (A) is 250 µm and applies
to (D). Bar in (B) is 50 µm and
applies to (C,E,F).
the isolated optic vesicle in combination with either
the dorsal or ventral half of the head portion. For this
purpose, collagenase-treated optic vesicles (without
the surrounding tissues) were placed in close contact with the dorsal or ventral half of the head at the
corresponding position of the head explant from
which the right optic vesicle had been removed in
advance (Fig. 6).
As a control, untreated optic vesicles were cultured
with the head half in a similar way, and the results
showed that both NR and RPE developed as seen
in the single culture of the untreated optic vesicle.
Neither the dorsal (Fig. 6D–F; Fig 6A–C for comparison) nor the ventral (data not shown) half of the head
significantly affected the results.
When the treated optic vesicle (without the surrounding tissues) was co-cultured with the head half,
we obtained different results depending on whether
it was co-cultured with the dorsal or ventral half. The
optic vesicle developed into both NR and RPE when
it was co-cultured with the dorsal half of the head
(Fig. 6G–I). This was confirmed by intense immunostaining for cRax and Mitf. In contrast, the treated optic
vesicle developed only into NR when co-cultured
with the ventral half of the head (Fig. 6J–L): no Mitf
immunostaining was detected, while cRax staining
increased in its intensity. These results suggest that
the dorsal area of the head portion plays an inductive
role in RPE differentiation of the optic vesicle. The
ventral area of the corresponding portion did not
have such an activity, but it enhanced NR differentiation. The effect of the dorsal head on RPE induction
in the optic vesicles was examined at different stages
of the co-cultured head portions, and dorsal head
portions at 8-, 10- and 14-somite stages showed the
same effect, while those at the 16-somite stage did
not show this effect. The statistical counting of the
number of optic vesicles with melanin deposition also
supported the results (Fig. 7).
In order to exclude the possibility that some part
of the neuroepithelium of the dorsal brain vesicle cocultured with the optic vesicle may differentiate into
RPE, we co-cultured quail optic vesicle with chicken
dorsal head. The results showed that the Mitf-expressing
domain was all stained with the QCPN antibody
(Fig. 8A–D), verifying that it was from quail optic cells
and not from chicken dorsal brain cells. Thus, it was
concluded that the differentiated RPE was derived
Extraocular signal and eye development
529
Fig. 5. Organotypic culture of
dorsal or ventral half of the optic
vesicle. (A) The embryonic brain
was cut into the dorsal and
ventral halves which were then
laid onto the membrane filter cup
and cultured for 3 days. (B)
Dorsal half on the filter cup
immediately after transferred
on the cup. (C) The same dorsal
half cultured for 3 days. Melanin
deposition can be seen at the
both sides (arrows). (B,C) are
shown at the same magnification.
(D,E,F) Dorsal half culture.
Melanin deposition can be seen
(arrows in C and D) and Mitf
expression is also found (F),
while cRax can be seldom found
(E). (G,H,I) Ventral half culture.
cRax immunostaining is usually
found in the explant (H), but Mitf
staining is rarely observed. In
case it was positively reacted, it
was found only in a small area (I).
Bar in (D) is 250 µm and applies
to (G). Bar in (E) is 50 µm and
applies to (F,H,I).
from the optic vesicle. Similarly, in quail optic vesicle
co-cultured with chicken ventral head half, the cRaxexpressing domain was stained with QCPN antibody
(Fig. 8E–H).
Pax2 and Tbx5 expression in the optic vesicle
co-cultured with the head portion
Pax2 is expressed in the optic stalk and ventral-most
region of the optic cup and is involved in the development of ventral structures of the eye (Schwartz
et al. 2000), while Tbx5 is found in the dorsal region
of the optic cup (Koshiba-Takeuchi et al. 2000; Fig. 9).
To investigate if their expression was also influenced
by signals derived from the dorsal or ventral periocular
tissues, we examined Pax2 and Tbx5 expression in
optic vesicles that were co-cultured with dorsal or
ventral head portions, respectively (Fig. 9). In the
culture of untreated optic vesicles, expression of both
Pax2 and Tbx5 was clearly detected by the in situ
hybridization technique (Fig. 9A,E). Pax2 expression
was still detected in the collagenase-treated optic
vesicles (Fig. 9B), but Tbx5 was not detected (Fig. 9F).
In the co-culture experiments, we did not observe
Pax2 expression when collagenase-treated optic
vesicles were co-cultured with the dorsal head
explant, but clear Pax2 expression was found when
co-cultured with the ventral head explant (Fig. 9C,D).
The effect of Pax2 suppression by the dorsal head
was examined at different stages. Dorsal heads
from the 18-somite stage did not suppress Pax2
expression (data not shown). In contrast, Tbx5
expression could not be seen in collagenase-treated
optic vesicles even when co-cultured either with the
dorsal or ventral head (Fig. 9G,H). These results
suggest that the dorsal signals deriving from the
dorsal part of the head induce dorsalization of the
optic vesicle as well as RPE differentiation, while
they suppress ventralization. The surrounding tissue
is required for Tbx5 gene expression, but the dorsal
head explant was not sufficient to induce Tbx5
expression.
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Fig. 6. Co-culture of the optic
vesicle with either the dorsal or
ventral half of the head. Note that
one of optic vesicle had been
removed from the head portion
before it was co-cultured with
optic vesicle graft. Four types of
combination cultures were performed as seen in the figure. (A,B,C)
Cultures of untreated optic vesicles
without additional tissue. (D,E,F)
Untreated optic vesicles were cocultured with the dorsal head
explant. Arrow in (D) indicates
melanin deposition of grafted optic
vesicle. (G,H,I) Collagenasetreated optic vesicles were cocultured with dorsal head explant.
Arrow in (G) indicates the melanin
deposition of the grafted optic
vesicle and Mitf immunostaining
was also detected in the optic
vesicle as shown in (I). (J,K,L)
Collagenase-treated optic vesicles
were co-cultured with ventral
head explant. Nither pigmentation
nor Mitf staining was seen in the
graft (J and L). Bar in (A) is
250 µm and applies to (D,G,J).
Bar in (B) is 50 µm and applies to
(C,E,F,H,I,K,L).
Topographical relationship between migrating neural
crest cells and retinal pigment epithelium
differentiation
A previous study by Fuhrmann et al. (2000) suggested
that cranial neural crest cells inhibit NR differentiation.
To clarify whether neural crest cells migrating to the
eye have any interaction with RPE (Bronner-Fraser
1996), we examined the localization of migrating neural
crest cells in cultures of dorsal or ventral halves of
the head by using HNK-1 immunocytochmisty.
In normally developing chick embryos, HNK-1 positive cells extend antero-laterally from the dorsal midline area of the future diencephalon towards the optic
vesicle. At the 10-somite stage, positive cells are distributed in the upper dorsal part of the optic vesicle,
and then migrate ventrally through the optic vesicle
in either the anterior or posterior direction (Fig. 10A–
C). No positive cells were found in the distal part of
the optic vesicle.
In the culture of the dorsal half of the head, numerous HNK-1 positive cells migrated over the explant
and further outward. Deposits of melanin granules
Fig. 7. Melanin deposition in optic vesicles which were
cultured alone, with the dorsal half head, or with the ventral half
head. Untreated optic vesicles always show melanin deposition
whether or not they were cultured with the head half. Melanin
deposition in the collagenase-treated optic vesicles is found
only when they were co-cultured with the dorsal half.
were usually observed at the optic vesicle of the dorsal half culture. Distribution of HNK-1 positive cells
obviously overlapped partially on the pigmented area,
and positive cells were often found to surround the
pigmented areas (Fig. 10D,E).
Extraocular signal and eye development
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Fig. 8. Double immunofluorescence of quail-chick chimeric culture; optic vesicles from quail embryos at 10-somite stage were cocultured with the chicken dorsal half (A,B,C,D) or with the chicken ventral half (E,F,G,H). Green fluorescence in (A) and (C) shows Mitf
immunostaining and that in (E) and (G) shows cRax staining. Red fluorescence is by QCPN antibody. Bar in (B) is 250 µm and applies
to (A,E,F). Bar in (D) is 50 µm and applies to (C,G,H).
Discussion
Morphogenesis of the vertebrate eye is regulated by
progressive interactions between the optic vesicle
neuroepithelium and the surrounding tissues, and our
previous study indicated that the dorsal and ventral
specification within the optic vesicle is crucial, not
only for optic cup formation, but also for the patterning of the neural retina and RPE (Uemonsa et al.
2002). DV specification was also suggested to be
determined by factors originating in the midline region
of the forebrain. The present study was intended to
address the question of how these factors regulate
the development of the eye anlagen. Previous studies
have shown that BMP and Shh are among the most
likely candidates, and ventrally derived Shh signals
and dorsally restricted BMP4 signals appear to act
antagonistically to regulate the growth and specification of the optic primordium (Huh et al. 1999; Zhang
& Yang 2001; Adler & Belecky-Adams 2002). The
precise mechanism involved in the DV specification
of the optic vesicle, however, is still not clear. In the
present study, we addressed this primary question
using our new organ culture method for chick embryos.
Mesenchymal tissue regulates retinal pigment
epithelium and neural retina development by directing
the retinal pigment epithelium domain
We performed a series of different cultures of the optic
vesicle: in ovo culture (embryonic transplantation),
collagen gel-embedded culture and filter membranesupported culture. We evaluated RPE differentiation
by direct histological observations, in addition to
Mitf expression. Histological observations revealed
that in ovo transplanted optic vesicles developed
similarly to the normal eye, forming an ectopic eye
on the distal tip of the wing bud. The eye had a lens,
neural retina and RPE and was surrounded by
mesenchymal tissues. This indicates that the optic
vesicle at the 10-somite stage can develop autonomously and needs no further influence from the
forebrain, but requires certain factors deriving from
mesenchymal cells. We have done careful histological observations of the grafted and cultured optic
vesicles and found that differentiating RPE always
faced mesenchymal tissues, suggesting that they
play an important role in RPE differentiation. In the
chimeric transplantation experiment, in which quail
532
Y. Kagiyama et al.
Fig. 9. In situ hybridization of Pax2 and Tbx5 expression in cultured optic vesicles. Embryos at HH stage 18 show dorsally and
ventrally located staining profiles of Tbx5 and Pax2 expression, respectively. (A,E) Untreated optic vesicles from 10-somite embryo
showed intense staining of both Pax2 and Tbx5. (B,F) In collagenase-treated optic vesicles Pax2 expression is found, though less
intense than found in untreated optic vesicle, while no Tbx5 expression could be found. (C) No Pax2 expression can be seen in
collagenase-treated optic vesicle when co-cultured with dorsal half, while it is upregulated when cultured with ventral half as shown
in (D). (G,H) Tbx5 expression can not be found in collagenase-treated optic vesicles whether they are co-cultured with dorsal half or
ventral half. Note that collagenase-treated optic vesicle did not show Tbx5 expression but displayed pigmentation (asterisk in G).
Asterisks indicate grafts of optic vesicles. Arrowheads in (D) and (G) indicate optic vesicle portions of co-cultured ventral and dorsal
heads, respectively. Bar (A), 250 µm.
Fig. 10. Neural crest cell migration
in developing chick embryos and
in cultures of the dorsal half as
shown by HNK-1 immunostaining.
(A,B,C) 8-, 10- 13-somite stages
of embryos. Positive cells became
to gradually cover the developing
optic vesicles. At 13-somite stage,
most regions of the optic cup are
covered with positive cells except
future lens-forming area and ventral
area. (D,E) Dorsal half head removed
from 10-somite stage embryo and
cultured. Arrowheads in (D) indicate
areas of melanin deposition. Arrowheads in (E) indicate the area of
melanin deposition shown at a
higher magnification. HNK-1 positive
neural crest cells appear to surround the area of melanin deposition.
Bar in (A) is 250 µm and applies
to (B,C,D). Bar (E), 100 µm.
optic vesicle was transplanted into the chick wing
bud, numerous chick mesenchymal cells surrounded
the RPE (Fig. 2C). This indicates that the mesenchymal cells need not necessarily be derived from the
periocular mesenchyme. Members of the BMP family
are expressed in the apical ectodermal ridge of
developing limbs throughout their life, as well as in
the mesenchyme (Pizette & Niswander 1999, 2001).
Since BMP4 appears to function in RPE development, the limb mesenchyme may play a role as a
source of BMP signal. It is, however, still unclear
whether, without any periocular mesenchymal tissue,
Extraocular signal and eye development
the optic vesicle can develop normally, or whether
a small amount of ocular mesenchymal cells are
needed only in the beginning of development.
Fuhrmann et al. (2000) reported that extraocular
mesenchyme, but not lateral plate mesoderm, is
required for the expression of the RPE-specific
genes. Our present data indicates that the proper
morphogenesis of the eye anlage may depend on
the extraocular mesenchyme for the initial step of
eye morphogenesis, and that once it is initialized the
non-specific mesodermal tissue can take over the
further eye development.
The significant role of the extraocular mesenchyme
was also supported by an organ-culture method.
RPE differentiated in the outer layer, making contact
with the mesenchymal tissue, while the inner layer
differentiated into NR facing the lens. This was also
evidenced by immunostaining for Mitf and cRax,
suitable markers for RPE and NR differentiation,
respectively (Mochii et al. 1998; Ohuchi et al. 1999;
Sakagami et al. 2003). When the mesenchymal tissue was removed by enzymatic treatment, no Mitf
immunostaining could be detected but cRax staining
was still found, suggesting the possibility that NR is
a default pathway of optic vesicle development.
Extraocular mesenchyme has two different sources,
the dorsally located neural crest cells and ventrally
located precaudal mesodermal cells. Although it is
difficult to clearly distinguish these two cells in the
histological sections, the former type of cells are considered to play the essential role in the patterning of
the optic vesicle. This was drawn from the culture
experiments: the collagenase-treated optic vesicle
seldom showed Mitf expression nor melanin deposition (Fig. 4), but they developed into both NR and
RPE when co-cultured with the dorsal head explant
(Fig. 6).
Developmental fates of the dorsal and ventral optic
vesicle
The dorsal and ventral parts of the optic vesicle
appear to develop somewhat differently from each
other (Romanoff 1960; Bellairs & Osmond 1998). The
distal and ventral parts are to organize the inner layer
of the optic cup and develop as NR, while the dorsalmost part is considered to organize the outer layer,
fated to become RPE. To determine the developmental
fates of the dorsal and ventral parts of the optic vesicle, we separated the head portion into its dorsal
and ventral halves by making an incisor cut at the
horizontal midline of the head, and cultured them in
an organotypic condition. The results clearly showed
that intense Mitf staining was seen in the cultured
533
dorsal part, whereas cRax staining was found only
in the ventral part, suggesting that these two regions
are specified to develop along separate fates in the
early stage of development by inductive signals from
surrounding tissues. Our recent study also supports
this notion by an experiment lesioning either the
dorsal or ventral half of the chick optic vesicle (manuscript in preparation). In contrast to the chick, Mitf
expression in the mouse starts throughout the optic
vesicle and later becomes restricted to the presumptive RPE (Bora et al. 1998). In a developing mouse
optic vesicle, Mitf and Chx10, a neuroretina-specific
marker, were initially coexpressed and later the
surface ectoderm-derived factor downregulated Mitf,
suggesting a somewhat different inductive mechanism from that of chick eye development (Nguyen &
Arnheiter 2000; Vogel-Hopker et al. 2000). Recent
studies have also implicated fibroblast growth factors
(FGF) as candidate factors released from the lens
ectoderm that pattern the distal optic vesicle (Pittack
et al. 1997; Hyer et al. 1998).
A study by Dütting and Thanos (1995) revealed the
fate mapping of the chick optic vesicle at the HH 11
stage (approximately corresponding to 13-somite
stage) by injecting DiI, and showed that the distal
sector of the dorsal part is destined to become the
dorsal-most region of the retina. This appears to be
inconsistent with the present result that positive staining for cRax was rarely found in the organ-cultured
dorsal half of the head portion (Fig. 5). Although
results obtained by a fate-mapping study have a different meaning from those by explant culture experiments, several possibilities can be considered for
that inconsistency: isolation and culturing of the dorsal head explant may alter the geographic relations
among the neuroepithelium, mesenchymal tissue and
surface ectoderm, and this will affect the developmental fate of the dorsal optic vesicle. Alternatively,
the optic vesicle may be patterned by diffusible factors emanating from the dorsal and ventral poles of
the vesicle (or the surrounding tissue at these poles)
and that separation of the vesicle into dorsal and
ventral halves depletes the dorsal explant from the
ventrally derived signal and vice versa. The expression pattern of transcription factors such as Pax2,
cVax, and Tbx5 in the optic cup indicates that the
optic cup can be divided into three sectors in relation
to the DV direction (Schulte et al. 1999; Peter & Cepko
2002). The medial part is devoid of the expression
of these genes, and may not be rigidly committed
as early as at the stage of the optic vesicle. Their
fates may be influenced by the reciprocal signals
from the dorsal and ventral parts of the optic vesicle,
as mentioned above.
534
Y. Kagiyama et al.
Dorsal head-derived factor regulates dorso-ventral
specialization
We hypothesized factors that may be derived from
the dorsal part of the head and play a role in RPE
determination. This was examined in the present
study by co-culturing the optic vesicle with the dorsal
head (Fig. 6). The optic vesicle was treated to remove
the surrounding tissues and was juxtaposed in vitro
with the dorsal head. It was shown that this treatment
induced Mitf expression in the optic vesicle that otherwise could not be found. The ventral head did not
have this effect on Mitf but it apparently upregulated
cRax expression to some extent. These results clearly
indicate the existence of a dorsal factor(s) in the
dorsal head that upregulates Mitf expression. Interestingly, this dorsal factor also suppressed Pax2
expression in the optic vesicle. Pax2 plays an important role in the development of the ventral structure
of the eye, such as the optic stalk and ventral fissure
(Torres et al. 1996; Schwartz et al. 2000). Pax2
appears to repress the expression of Otx2, a gene
that has a crucial role for the determination of the
presumptive RPE territory (Martinez-Morales et al. 2001;
Bovolenta et al. 1997). Pax2 and Pax6 seem to cooperate to establish the boundary between the optic
stalk and optic cup by reciprocally controlling their
expression levels (Schwartz et al. 2000). In the present
study, Pax2 expression was not affected by the
removal of the surrounding tissues, but was repressed by co-culturing with the dorsal head (Fig. 9).
Thus, the dorsal factors appear to have two effects
on the optic vesicle: induction of Mitf expression and
suppression of Pax2, leading the optic vesicle to be
dorsalized. In contrast, the ventral head appears not
to have a crucial role in the development of the NR
and RPE, although it enhanced cRax expression in
addition to Pax2 expression to a certain extent.
With respect to dorsalizing factors, it has been
reported that BMP play crucial roles in the regional
morphogenesis of the dorsal forebrain in mouse
embryos by regulating specific gene expression,
cell proliferation and local cell death, and BMP4 is
expressed in the dorsal region of the optic cup in
the mouse embryo (Furuta et al. 1997). It has also
been reported that when mouse BMP4 is misexpressed in the ventral half of the optic cup in chick
embryos, round eyes are formed with the expansion
of Tbx5 expression in the ventral half, and that
expression of Vax and Pax2 is repressed (KoshibaTakeuchi et al. 2000). When BMP4-soaked beads
were implanted into the head region at HH stage 11/
12 of the chick embryo, the resulting optic tissue was
entirely pigmented with a complete absence of neural
retinal tissue (Hyer et al. 2003). The present culture
study indicates that the surrounding tissue is essential for Tbx5 expression in the optic vesicle and the
dorsal head explant can not compensate for the
tissue. The previous in ovo explant culture study
showed that in explants taken from embyros earlier
than the 10-somite stage Tbx5 expression could not
be seen, while Pax2 was expressed. Explants taken
from embryos at the 14-somite stage normally
expressed Tbx5 (Uemonsa et al. 2002). These results
suggest that Tbx5 expression is specified between
the 10- and 14-somite stages under the influences
coming from both the surrounding (mesenchymal)
tissues and brain portions. It is also suggested that
Tbx5 does not directly suppress Pax2 expression,
since loss of Tbx5 expression did not cause expansion of Pax2 positive areas as shown presently and
in the previous study.
The previous work by Fuhrmann et al. (2000) indicated that the extraocular mesenchymal cells inhibited the expression of the NR-specific transcription
factor, Chx10, and that these cells may originate from
the cranial neural crest. From our present observations that HNK-1 positive cells were always gathering
at the area with pigmented granules (Fig. 10), we can
also suppose that the neural crest-derived mesenchymal cells, densely distributed at the dorsal part
of the optic vesicle, must direct the optic vesicle to
develop into RPE. Neural crest cell migration to the
optic vesicle still continues up to later stages (18somite stage), and these migrating cells may reinforce
the fate of the dorsal optic vesicle. In early Xenopus
embryos, BMP-2,4 are expressed in the head neural
crest cells (Fainsod et al. 1994; Clement et al. 1995).
BMP-regulated Smad proteins, intracellular signaling
molecules specific for BMP, are also localized in the
neural crest-derived ocular mesenchymal cells (Kurata
et al. 2001). These studies indicate a possibility that
ocular neural crest cells may be the source of BMP
signals.
Tissue interaction between the neuroepithelium and
the surface ectoderm may also bring about a separation into a discrete expression domain, and FGF
signaling plays an important role in this process
(Hyer et al. 1998; Nguyen & Arnheiter 2000; VogelHopker et al. 2000; Zhao et al. 2001). Conditional
deletion of Pax6 expression in the surface ectoderm
by using a Pax6 surface ectoderm-specific Cre mouse
indicated that the developing lens is required for the
proper organization of the NR and RPE (Ashery-Padan
et al. 2000). Hyer et al. (2003) reported that surgical
removal of the pre-lens ectoderm of the 11-somitestage embryo resulted in a persistent optic vesicle
that initiated NR differentiation but failed to undergo
Extraocular signal and eye development
invagination. Such an ectoderm-less optic vesicle
still showed patterned development of RPE and NR,
indicating that the mesenchymal tissue has the
primary function in RPE differentiation, as shown in
the present study. It is plausible to consider that the
surface ectoderm that covers the optic vesicle also
plays some role in the DV patterning of the eye
primordium, but this must be examined in future
experiments.
Acknowledgements
We are sincerely grateful to Dr Dorothea Schulte for
valuable discussion and critical reading of the
manuscript. This work was supported in part by a
Grant-in-Aid and Special Coordination Funds for Brain
Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan to M. Araki.
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