Download Ventral diencephalic cells divide eye field - Development

5533
Development 126, 5533-5546 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV4225
Anterior movement of ventral diencephalic precursors separates the
primordial eye field in the neural plate and requires cyclops
Zoltán M. Varga, Jeremy Wegner and Monte Westerfield
Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
*Author for correspondence (e-mail: [email protected])
Accepted 22 September; published on WWW 24 November 1999
SUMMARY
A currently favored hypothesis postulates that a single field
of cells in the neural plate forms bilateral retinas. To learn
how retinal precursors segregate, we followed individual
labeled neural plate cells in zebrafish. In the late gastrula,
a single field of odd-paired-like-expressing cells contributed
to both retinas, bordered posteriorly by diencephalic
precursors expressing mariposa. Median mariposaexpressing cells moved anteriorly, separating the eyes, and
formed ventral anterior diencephalon, the presumptive
hypothalamus. In cyclops mutants, corresponding cells
failed to move anteriorly, a ventral diencephalon never
formed, and the eyes remained fused. Ablation of the region
containing these cells induced cyclopia in wild types. Our
results indicate that movement of a median subpopulation
of diencephalic precursors separates retinal precursors into
left and right eyes. Wild-type cyclops gene function is
required for these morphogenetic movements.
INTRODUCTION
midline. Movement of axial mesendodermal cells and the
subsequent elongation of the neural plate midline have been
proposed as playing a role in separation of the eyes (Woo and
Fraser, 1995; Heisenberg and Nüsslein-Volhard, 1997; Marlow
et al., 1998). In contrast, other fate map studies indicated that
cells in the anterior midline of the neural plate do not contribute
to the retina, but to diencephalic brain regions between the eyes
(Couly and Le Douarin, 1988; Eagleson and Harris, 1990;
Eagleson et al., 1995; Li et al., 1997) consistent with the
hypothesis that two fields of neural plate cells give rise to the
retinas. Based on this second set of observations, an alternate
interpretation has been proposed. Under the influence of the
underlying prechordal plate, median neural plate cells down
regulate expression of genes characteristic of retinal precursors
and instead contribute to the ventral diencephalon (Pera and
Kessel, 1997; Li et al., 1997). This model predicts that in the
absence of prechordal plate signaling, median neural plate cells
would continue to develop as retinal precursors resulting in a
single retina fused across the midline. It is unclear, however,
how this model can account for the previous fate map studies
that indicated that median cells contribute to the retinas
(Ballard, 1973; Jacobson and Hirose, 1978; Hirose and
Jacobson, 1979; Woo and Fraser, 1995).
To distinguish between these apparently contradictory
hypotheses and to understand the morphogenesis of the
diencephalon and the eyes, we fate mapped the anterior neural
plate of zebrafish embryos at high resolution, measuring the
movements of labeled cells relative to morphological
landmarks and patterns of gene expression. We found a single
field of retinal precursor cells that express the odd-paired-like
During development, vertebrate embryos normally form two,
bilateral eyes. The retinas are derived from the anterior neural
plate. Under the influence of various genetic (Cohen, 1989;
Hatta et al., 1991; Muenke et al., 1994; Schier et al., 1996;
Solnica-Krezel et al., 1996; Ming and Muenke, 1998) and
environmental factors (reviewed by Adelmann, 1936a; Roach
et al., 1975; Blader and Strähle, 1998), however, a single,
median, cyclopic eye can form. Such observations have led to
two hypotheses to explain the developmental origin of bilateral
eyes. The first proposes that two fields of retinal precursor cells
in the neural plate, separated by medial diencephalic
precursors, independently produce the two eyes (Meckel, 1826;
Spemann, 1904, 1912; reviewed by Adelmann, 1936a,b). This
hypothesis predicts that during abnormal development, the two
fields of retinal precursors fuse to form a cyclopic eye. The
second hypothesis postulates a single field of retinal precursor
cells that separates into left and right eyes (Huschke, 1832;
Stockard, 1913; LePlat, 1919; Adelmann, 1929a,b,c; reviewed
by Adelmann, 1936a,b), leaving diencephalic precursors
medially. Failed separation of the single primordial neural plate
eye field would result in cyclopia.
Fate map analyses of the gastrula and anterior neural plate
in several species have indicated the presence of a single
median field of retinal precursors (Ballard, 1973; Jacobson and
Hirose, 1978; Hirose and Jacobson, 1979; Woo and Fraser,
1995). Cells labeled in the medial part of the anterior neural
plate can contribute progeny to either or both eyes, suggesting
that the field of retinal precursor cells extends across the
Key words: Fate map, Anterior neural plate, Cyclopia,
Holoprosencephaly, Zebrafish, Danio rerio, Gene expression,
Morphogenesis, odd-paired-like (opl)
5534 Z. M. Varga, J. Wegner and M. Westerfield
gene (opl, Grinblat et al., 1998) at the end of gastrulation. This
field extends across the midline and all cells within the field
contribute to the eyes. Diencephalic precursor cells express the
forkhead gene, mariposa (mar, Moens et al., 1996; also called
forkhead 3, Odenthal and Nüsslein-Volhard, 1998), and are
located posterior to the opl-expressing eye field. Median marexpressing cells move anteriorly along the midline, separating
the retinal precursors into left and right eyes and forming the
ventral diencephalon. In embryos mutant for Cyclops (Hatta et
al., 1991), a nodal-related member of the Transforming Growth
Factor-β superfamily of signaling molecules (Rebagliati et al.,
1998; Sampath et al., 1998), these posteriorly located cells
express opl instead of mar and fail to move anteriorly. Thus,
the eye field fails to separate and a single cyclopic eye forms.
Ablation of mar-expressing median ventral diencephalic
precursors and underlying prechordal plate from wild-type
embryos also prevents separation of the eye field and produces
cyclopia. Our results suggest that movement of diencephalic
precursors anteriorly along the midline is required to separate
the primordial eye field into left and right eyes and that this
morphogenetic movement requires cyclops gene function.
MATERIALS AND METHODS
Wild-type (AB), two cyclops alleles, cycb16 (Talbot et al., 1998) and
cycb426, and maternal-zygotic one-eyed-pinhead (MZoep; Gritsman et
al., 1999) mutant embryos were obtained from zebrafish (Danio rerio)
lines maintained with standard procedures (Westerfield, 1995).
Similar results were obtained with both cyclops alleles. Embryos were
staged by hours post fertilization (h) and by standard staging criteria
(Kimmel et al., 1995).
Accuracy of the fate map
To obtain a high-resolution fate map at the neural plate stage, several
parameters were controlled. We selected embryos of similar size
(690±5 µm mean diameter ±s.d. measured at the equator), and
developmental stage (100% epiboly) and we corrected for shrinkage
during in situ hybridization, which averaged 11-12% as measured at
the circumference of the embryonic equator. These corrections
allowed us to achieve a resolution of 1-4 cell diameters indicated by
the deviation bars in our fate map.
Intracellular labeling of single cells
Micropipettes were pulled (Sutter P2000) and filled with a mixture of
2.5% rhodamine dextran and 2.5% fluorescein dextran (both 3 kDa;
Molecular Probes) in 0.2 M KCl. Embryos were mounted in normal
Ringer’s solution and methyl cellulose (Westerfield, 1995), oriented
with their prospective heads up and tailbuds down and viewed with a
10× objective and a 10×10 grid reticule (KR406; Zeiss/Tanji) in the
ocular (10×) of a fixed stage microscope (Zeiss Standard). Single cells
were injected with current by overcompensating for the electrode
capacitance with the amplifier (Getting Instruments Inc.). The
embryos were monitored again 10 minutes after injection for
successful labeling.
Single cells were labeled in embryos derived by crossing cyc/+
heterozygotes. The genotypes were determined in fixed embryos by
RNA in situ hybridization at 80% epiboly and tailbud stage using the
absence of the mar protrusion as a scoring criterion. At 24h, the
morphology indicated the genotype.
Whole-mount RNA in situ hybridization and lineage tracer
detection
Embryos were fixed and hybridized with one or two mRNA probes
(Hauptmann and Gerster, 1994). After double in situ hybridization,
digoxigenin-UTP and fluorescein-UTP (Boehringer) labeled probes
and the fluorescein-conjugated dextran were detected sequentially
with anti-digoxigenin-alkaline phosphatase and anti-fluoresceinalkaline phosphatase Fab fragments (Boehringer Mannheim). This
allowed us to detect the fluorescein-labeled mRNA probe and
fluorescein-dextran-injected cells at the same time. Fast Red
(Boehringer, Sigma) was used as the substrate for the alkaline
phosphatase color reaction to detect one mRNA probe and the lineage
tracer and NBT/BCIP (Boehringer Mannheim) was used to detect the
other mRNA probe.
Generation of the fate map
To analyze the lineages of cells relative to gene expression domains
and morphological landmarks as precisely as possible, we performed
Fig. 1. The anterior borders of the neural plate, the
prechordal plate and the opl expression domain
colocalize at 80% epiboly. (A-C) Dorsal views, anterior
to the top, 80% epiboly, white arrowheads indicate
midline; (A-B), Nomarski optics. (A) Dorsal view of live
embryo focused at the level of the ectoderm. The
anterior border of the neural plate is apparent as a
thickening of the ectoderm (black arrowhead). (B) A
deeper focal plane view of the embryo pictured in A.
Cells with filopodia (white arrows) are apparently
migrating at the anterior edge (black arrowhead) of the
axial mesendoderm. (C) An ectodermal cell was labeled
with lineage tracer (arrow, red cell) superficial to the
anterior edge of the migrating axial mesendoderm (black
arrowhead), and the embryo was immediately fixed and
hybridized with RNA probes for opl (black expression
domain) and otx2 (red expression domain). This
demonstrated that at 80% epiboly, the anterior edges of
the axial mesendoderm, the neural plate, and the opl
expression domain coincide. (D) Distance between the
anterior edge of the axial mesendoderm and the anterior
edge of the neural plate between the 80% epiboly and 1-somite stages. The distance between these two morphological landmarks can be used
for precise staging of the embryos as the axial mesendoderm advances relative to the anterior edge of the neural plate and the tailbud. 80%
epiboly, n=9; tailbud stage, n=14; 1-somite stage, n=10. Distance = average ± s.d. in µm. 1 Box, the length of a box in the 10×10 reticule used
for the fate map (see also Fig. 2B). Time scale is approximately linear with developmental time. Scale bars, 50 µm (A-B), 200 µm (C).
Ventral diencephalic cells divide eye field 5535
double in situ hybridizations with probes for otx2, opl, and mar on 1015 size-selected and carefully staged embryos. We viewed each
embryo with a 10×10 reticule in the eyepiece of the microscope and
sketched the dimensions of its expression domains on a 10×10 grid
that we used as a coordinate system. We defined the intersection of the
anterior-posterior axis and the anterior edge of the neural plate (opl
expression domain) as the origin of our coordinate system (✱, Figs 2B,
4). The average gene expression domains of opl (n=14), otx2 (n=14),
and mar (n=10) were calculated by measuring the distance along the
anterior-posterior axis from the origin and the lateral distance from the
axial midline. This allowed us to predict the locations of cells inside
or outside these gene expression domains, using the reticule and
morphological landmarks in live, unlabeled embryos.
We always injected pairs of embryos, side by side, in matching
locations of the neural plate using embryonic morphology and the
10×10 grid reticule as landmarks for orientation. One of the embryos
was allowed to develop until 24h to analyze the size and location of
its labeled progeny; the other embryo was fixed immediately after
injection and processed for RNA in situ hybridization and
Fig. 2. Gene expression domains subdivide the anterior neural plate.
Dorsal views anterior to the top; (A,B,D) tailbud stage, (C) 1-somite
stage; (A) The opl (black) expression domain is contained within the
otx2 (red) domain as shown by fluorescence microscopy. The white
arrowhead points to the indentation in the opl expression domain.
(B) 10-15 embryos were doubly labeled with mRNA probes for otx2
and opl or mar. Gene expression borders were recorded on 10×10
grids representing the reticule in the eyepiece of the microscope. The
distances from the midline and the origin (✱) were measured in
individual embryos and average values and standard deviations
calculated. Such analysis produced averaged gene expression
domains for otx2 (red), opl (blue) and mar (green). The averaged
outline of the polster is indicated in black. Error bars indicate
standard deviations. (C) opl (red) and pax6 (black) occupy
overlapping domains in the anterior neural plate. The posterior
portion of the pax6 expression domain overlaps the lateral arms of
the mar expression domain (not shown). Cells farther anterior
express pax6 less intensely and also express opl. A white arrowhead
indicates the region corresponding to the mar protrusion and the
indentation in the posterior border of the opl expression domain.
(D) Cells of the prechordal plate and median neural plate express the
cyclops gene (black). The site of the mar protrusion (white
arrowhead) is not distinguished by cyclops gene expression pattern.
opl expression is shown in red. Scale bar, 100 µm.
visualization of the injected lineage tracer. Thus, 50% of all injected
embryos were fixed immediately (control group). The other 50% were
transferred to embryo medium containing 1% Penicillin/Streptomycin
and allowed to develop until 24h (experimental group). The data
obtained from the experimental group were used for the fate map only
if all injected cells in the control group were located in the intended
region of the expression domain. If a single lineage tracer labeled cell
was located outside the region to be fate mapped during a given
experiment, the data obtained from the experimental group were
discarded. This approach is a modification of previous methods
(Melby et al., 1996; Müller et al., 1996).
The movement of lineage tracer-labeled cells was analyzed relative
to morphological landmarks including the anterior edge of the axial
mesendoderm and the anterior edge of the neural plate in dorsal and
side views. In addition, the anterior movement of the axial
mesendoderm was analyzed at the end of gastrulation and during early
somitogenesis stages in side views relative to the tailbud. Gene
Fig. 3. Opl and mar share a border of gene expression at tailbud
stage that is perturbed in cyclops and MZoep mMutant embryos.
Dorsal views; anterior to the top; insets in A and B, sagittal sections
with dorsal to the top and anterior to the left. (A) Wild-type embryo
labeled with mRNA probes for opl (red) and mar (black). The
anterior protrusion of the mar expression domain fits into the
indentation at the posterior border of the opl expression domain
(arrowhead). The inset shows that the protrusion of mar-expressing
cells is wedge-shaped beneath the posterior part of the opl domain.
(B) cyclops mutant embryo labeled with mRNA probes for opl (red)
and mar (black). The indentation in the opl expression domain and
the protrusion in the mar domain are absent (arrowhead). Inset, no
wedge shaped protrusion is visible; instead, a vertical border forms
between the mar and opl expression domains. (C,E) Wild-type
embryos labeled with mRNA probes for opl (C) and mar (E). The
arrowheads point to the indentation at the posterior border of the opl
expression domain (C) and to the anterior protrusion in the mar
expression domain (E). (D,F) MZoep mutant embryos labeled with
mRNA probes for opl (D) and mar (F). The indentation in the opl
expression domain (D) and the protrusion in the mar domain (F) are
absent (arrowheads). The dorsal axis is shortened in MZoep mutant
embryos. Scale bar, 100 µm (A-F and insets).
5536 Z. M. Varga, J. Wegner and M. Westerfield
expression patterns were used in addition to morphological landmarks
to judge cell shifts in the neural plate.
Labeling of dying cells
To detect cells undergoing apoptosis, we labeled wild-type and
cyclops mutant embryos using Terminal-UTP-Nick-End-Labeling
(TUNEL) as described by Whitlock and Westerfield (1998).
We labeled live embryos with Acridine Orange (AO; Sigma) to
detect dying cells (Furutani-Seiki, et al., 1996) and cells labeled with
lineage tracer simultaneously. Embryos were incubated for an hour in
approximately 5 ml of embryo medium (Westerfield, 1995) containing
1 µl of saturated AO stock solution. The embryos were then washed
in embryo medium, mounted in methyl cellulose, and viewed with the
confocal microscope (Zeiss). Fluorescent images were collected and
reconstructed using Voxel View and Voxel Math (Vital Images) and
Photoshop 4.0 (Adobe).
Tissue ablations
Pieces of median neural ectoderm and underlying axial mesendoderm
of wild-type embryos were removed at the tailbud stage using an
eyebrow hair (similar to Adelmann, 1929a,b; Woo and Fraser, 1997).
For this operation, wild-type embryos at the tailbud stage were
mounted dorsal side up in normal Ringer’s solution and methyl
cellulose (Westerfield, 1995) and were maintained in normal Ringer’s
solution after the operation until 24h. Rectangular blocks of tissue
were removed, thus ablating the neural plate region of the mar
protrusion and the underlying prechordal plate. In 9 sham operations,
the tissue was replaced into its original location. Five types of
ablations were performed, removing various regions to define the field
of cells required for normal separation of the eyes (Ablation types IV, Table 2).
50% of the embryos in each experiment were fixed immediately
and analyzed by RNA in situ hybridization with probes for mar and
cyc. The other 50% were allowed to develop to 30h and were analyzed
for cyclopia. The results were used only if all ablations, in embryos
fixed immediately after operation, removed the intended region. In
addition, we examined all operated embryos immediately after the
ablation to ascertain the location and extent of the ablated tissue using
the microscope’s grid reticule and the fate map.
RESULTS
To understand how the eyes and the diencephalon form, we
measured the movements of cells that give rise to these
structures, labeling them in the anterior neural plate by
intracellular injection of vital dyes. We localized individual
cells as precisely as possible in the neural plate on the basis of
their positions relative to visible morphological landmarks in
the live embryo and to patterns of gene expression visualized
by RNA in situ hybridization after fixation.
Axial mesendoderm migrates anteriorly relative to
gene expression domains in the overlying neural
plate
We used the relative positions of the anterior edges of the
neural plate and underlying axial mesendoderm as landmarks
for localizing cells in live embryos. The anterior axial
mesendoderm can be subdivided into an anterior part that
forms the polster and later gives rise to the hatching gland
(Kimmel et al., 1990) and a posterior part that we term
prechordal plate. At 80% epiboly, the neural plate became
distinguishable from non-neural ectoderm in live embryos as a
slight thickening of the dorsal epithelium (Fig. 1A; Papan and
Campos-Ortega, 1994). Although this thickening defined an
apparent edge of the neural plate, we found by fate mapping
that some more peripheral cells later contributed to the
telencephalon and the placodes (Whitlock and Westerfield,
1998; data not shown). At this stage, we found the anteriormost cells of the axial mesendoderm one cell layer deeper than
and at the same anterior-posterior position (±5 µm s.d.; n=9
embryos) as the anterior edge of the neural plate (Fig. 1A,B).
These most anterior mesendodermal cells often displayed
filopodia and migrated anteriorly (Fig. 1B).
To correlate gene expression domains in fixed embryos with
visible landmarks in live embryos, we injected lineage tracer
into single ectodermal cells one cell layer superficial to the
anterior edge of the axial mesendoderm in live embryos at
various developmental stages and fixed the embryos
immediately. We processed them for detection of lineage tracer
(Fig. 1C, arrow) and gene expression by RNA in situ
hybridization. At 80% epiboly, we found that the anterior edge
of the neural plate coincided with the anterior-most border of
the opl expression domain (Fig. 1C). Thus, at 80% epiboly, the
anterior edge of the migrating axial mesendoderm, the anterior
edge of the neural plate, and the anterior border of the opl
expression domain co-localize. At later stages, the anterior
edge of the opl expression domain continued to correlate with
the visible anterior edge of the neural plate while the
underlying polster and prechordal plate migrated farther
anteriorly (Fig. 1D). This movement of axial mesendoderm
was also apparent by measuring cell positions relative to the
tailbud. In contrast, the anterior edge of the neural plate did not
shift relative to the tailbud during this period. Thus, the
distance between the anteriorly advancing mesendodermal
cells and the anterior edge of the neural plate provided an
accurate and reproducible measure of developmental stages
between 80% epiboly and 1-somite stage (Fig. 1D).
The wild-type neural plate is divided by gene
expression domains
We characterized the gene expression domains of opl, otx2, and
mar in the anterior neural pate. In the late gastrula, a large field
of prospective head ectodermal and mesendodermal cells
expressed otx2 (Li et al., 1994; Mori et al., 1994) and
ectodermal cells in a central patch within this domain also
expressed opl (Figs 1C, 2A). The opl expression domain had
an indentation at the midline of its posterior border (Fig. 2A,B).
To correlate cell positions in the live embryo with gene
expression patterns, we first measured the average shape and
size of each gene expression domain. We selected staged
embryos of uniform size and fixed and hybridized them
simultaneously with RNA probes for opl and otx2 (Fig. 2A),
opl and mar, or otx2 and mar. We used otx2 as a reference to
calculate and compare the locations of opl and mar expression
domains and sketched the expression domains on a 10×10 grid
corresponding to the eyepiece reticule in the microscope. In
each embryo, we measured the distance to the border of each
gene expression domain from the intersection of the dorsal
midline with the anterior border of the opl expression domain.
We could also localize this point in live embryos, because it
overlies the center of the polster (Fig. 2B, ✱). The calculated
standard deviations from the average positions of the gene
expression borders (Fig. 2B) indicate both the accuracy of the
fate map and the limits of our resolution in predicting the
Ventral diencephalic cells divide eye field 5537
locations of cells in live unlabeled embryos relative to
expression domains. The average diameter of neural plate cells
at the tailbud stage was 11 µm (±2.2 µm, s.d.; n=23 cells) in
live embryos. Thus, each box in the reticule corresponded to
about four to six cells at this stage. These results suggest that
we were able to predict the position of the anterior border of
opl expression in live embryos with an accuracy of ±9 µm, or
approximately 1 cell diameter, whereas in more posterior
regions, our accuracy was limited to 2-3 cell diameters (±21
µm).
Other gene expression patterns subdivided the anterior
neural plate. By the tailbud stage, for example, the mar
expression domain consisted of ectodermal cells at the midline
and three transverse stripes (Odenthal and Nüsslein-Volhard,
1998). By labeling with probes for both opl and mar, we found
that the most anterior mar stripe borders the posterior edge of
the opl expression domain (dark green in Fig. 2B). The anterior
median protrusion of the mar expression domain extends into
the indentation in the posterior opl expression domain (Figs
2A, 3A). Sagittal sections revealed that the mar-expressing
cells form a wedge ventral to the opl-expressing cells (Fig. 3A,
inset).
The anterior-most portion of the pax6 gene expression
domain (Püschel et al., 1992a; Amirthalingam et al., 1995)
overlaps the opl expression domain, including the indentation
at the midline of the opl domain and the protrusion of the mar
domain (Fig. 2C, arrowhead). The more strongly expressing
posterior portion of the pax6 gene expression domain overlaps
the anterior most transverse stripe of the mar expression
domain (data not shown). pax6 gene expression appeared at the
1-somite stage, considerably later than opl, mar, and otx2.
By the end of gastrulation, the prechordal plate cells and the
ventral-most cells of the entire medial neuraxis also expressed
the cyclops gene (Fig. 2D; Rebagliati et al., 1998; Sampath et
al., 1998). Similarly, we found shh expression in prechordal
plate cells as well as in the ventral midline of the neural plate
(similar to Dale et al., 1997 in chick and rat; Müller et al., 1999;
data not shown). The anterior-most cells of the median
neurectoderm that expressed shh were located at the level of
the mar protrusion, approximately 200 µm posterior to the
anterior edge of the neural plate (data not shown).
The cyclops and one-eyed-pinhead mutations alter
gene expression domains in the neural plate
To understand how patterning of the neural plate relates to
separation of the eyes, we examined gene expression patterns
in cyclops (Hatta et al., 1991) and one-eyed-pinhead mutant
embryos (Schier et al., 1996; Solnica-Krezel, 1996) that lack
the ventral diencephalon and form a single retina fused across
the midline.
In embryos mutant for cyclops, a nodal-related gene
(Rebagliati et al., 1998; Sampath et al., 1998), both the
indentation of the opl expression domain and the protrusion of
the mar expression domain were missing (Fig. 3B). Cells
expressed opl in the region that corresponded to the mar
protrusion in wild-type embryos. In sagittal sections, the
anterior border of the mar expression domain was
perpendicular to the axis (Fig. 3B, inset) rather than wedge
shaped as in wild-type embryos (Fig. 3A, inset).
To examine further the role of the nodal-related signaling
pathway, we analyzed the expression of opl and mar in the
neural plate of maternal-zygotic one-eyed pinhead (MZoep;
Gritsman et al., 1999) mutant embryos. A recent study showed
that MZoep mutant embryos have a phenotype like double
mutants for the nodal-related genes cyclops and squint
(Feldman et al., 1998) suggesting that oep is an essential
extracellular cofactor required for nodal-related signaling
(Gritsman et al., 1999). Consistent with this interpretation, we
observed even more severe changes in neural plate patterning
in MZoep than in cyclops and oep mutant embryos: the opl
expression domain displayed a posterior extension (Fig. 3D)
and the mar expression domain an indentation (Fig. 3F). This
indicated that extension of and signaling from the axial
mesendoderm is a prerequisite for normal neural plate gene
expression.
Bilateral retinas form by separation of a single field
of precursor cells
To determine the fates of cells that express opl, mar or otx2 in
the anterior neural plate, we localized single cells and labeled
them with lineage tracer, using the reticule and morphological
landmarks as points of orientation in live embryos. We
analyzed cell fate at 24h. After injection at 80% epiboly, we
found that essentially all cells within the predicted opl
expression domain later gave rise to retinal cells (Fig. 4A,B).
Some of these precursors crossed the midline and contributed
to the eye that formed on the contralateral side. At tailbud
stage, cells in the opl expression domain still predominantly
gave rise to retinal cells but were now more restricted to the
ipsilateral side (Fig. 4C,D). The majority of cells near the
midline gave rise to cells later located on both sides, as
previously reported in more posterior regions of the CNS
(Kimmel et al., 1994; Papan and Campos-Ortega, 1994). At the
anterior and lateral borders of the opl expression domain, we
occasionally found telencephalic precursors (not shown here),
and at the lateral posterior border of the domain, we found
dorsal diencephalic precursors. These non-retinal precursors
were all within 3 cell diameters of the predicted opl expression
domain border and, hence, were within the limits of our ability
to predict the location of the border. The presence of retinal
precursors throughout the opl domain, the absence of other cell
fates in the midline of the opl domain, and the bilateral
distribution of retinal cells derived from single precursors
together suggest that the opl expression domain marks a single
field of retinal precursors in the neural plate at tailbud stage.
Injected precursors in the predicted anterior mar expression
domain at 80% epiboly and tailbud stages gave rise to
diencephalic cells at 24h. Cells located within the anterior
protrusion of the mar expression domain contributed to the
ventral diencephalon, the prospective hypothalamus (Fig. 4AD, green). Thus, median cells located posterior to the retinal
field in the neural plate were found anterior to and between the
eyes at 24h. Cells from more posterior and lateral regions of
the predicted transverse stripe of mar expression at the tailbud
stage contributed to dorsal and more posterior regions of the
24h diencephalon (Fig. 4C,D, yellow).
In a dorsal view of the neural plate, retinal and diencephalic
cell fates seemed to overlap in the anterior part of the mar
protrusion (Fig. 4C). We found, however, that labeled
superficial cells contributed to retina, whereas deeper cells
gave rise exclusively to diencephalon. This correlated well with
the wedge shape of the mar protrusion (Fig. 3A, inset). Thus,
5538 Z. M. Varga, J. Wegner and M. Westerfield
Fig. 4.
Ventral diencephalic cells divide eye field 5539
Fig. 4. Median cells in the mar protrusion of late gastrula stage
embryos form ventral anterior diencephalon in wild-type but not
cyclops mutant embryos. A,C,E, Dorsal views, anterior to the top. A,
80% epiboly; C,E, tailbud stage,. Gene expression domain borders
are shown as solid lines; red, otx2; blue, opl; green, mar. Black
indicates the outlines of axial mesendoderm (A) and the polster
underlying the neural plate (C,E, ✱ indicates the origin from which
we measured the positions of the gene expression borders.
(B,D,F) 24h; (B) frontal view; (D,F) side views of head, anterior to
the left, dorsal to the top. (A,B) The fate map at 80% epiboly shows
that cells in the median region of the opl expression domain in the
neural plate contribute to the retina. (A) Positions of injected cells in
the neural plate are shown as red, green and blue dots. (B) Later
positions of the same cells at 24h. Precursors that gave rise to retinal
cells in the left eye are shown in red, those that gave rise to cells in
the right eye are shown in blue, diencephalic precursors (green) are
located posterior to the opl expression domain in the neural plate.
(C,D) Retinal and diencephalic precursor cells occupy distinct
positions in the neural plate. Numbers indicate positions of cells in
the neural plate (C) and the same cells or their daughters at 24h (D).
Usually, the daughter cells of a single precursor were located close to
each other (several dots under a number); exceptions are shown by
two dots at different positions with the same number. Neural plate
cells located in the opl expression domain (C, red) constitute a single
field of retinal precursor cells (D, red). Other cell fates, except optic
stalk (D, blue), are not intermingled at the midline (C, blue). Neural
plate cells that contribute to the telencephalon (not shown here) are
located peripherally, at the border between the opl and otx2
expression domain. Cells injected in the anterior protrusion of the
mar expression domain (C, green) contribute to the ventral
diencephalon (D, green) which is later located between and anterior
to the eyes. Precursors located in the lateral and posterior portions of
the mar expression domain (C, yellow) give rise to dorsal
diencephalon and pretectal regions (D, yellow). Only a few cells
(three; C, shown as red/green) labeled near the border between the
opl and mar expression domains gave rise to both retinal (D, red) and
diencephalic (D, green) cells. (E,F) The cyclops mutation alters gene
expression patterns and cell movements. In cyclops mutants, the
posterior indentation in the opl expression domain and the anterior
protrusion of the mar expression domain are absent (E). Cells
(E, green) injected in this region of the neural plate failed to move
anteriorly but remained in the diencephalon posterior or dorsal to the
eyes (F, green). The eyes remained fused at the anterior midline.
Scale bars, 100 µm.
there was no significant overlap of opl and mar expression in
the protrusion; deep cells in the mar protrusion contributed to
diencephalon, whereas overlying retinal precursors expressed
opl. These results suggest that in the midline at the site of the
mar protrusion, retinal and diencephalic cell fates are divided
at the border between the opl and mar expression domains.
Subsequently, diencephalic precursors in the protrusion of the
mar expression domain shift anteriorly, separating the single
opl-expressing retinal field into the two bilateral eyes.
The cyclops mutation blocks the anterior shift of
diencephalic precursor cells
To understand how these inferred cell movements relate to the
separation of the eyes, we examined the fates of cells in cyclops
mutant embryos.
We injected single cells in the predicted opl expression
domain of cyclops mutant embryos and found they gave rise to
retinal cells except in the region that corresponded to the mar
protrusion in wild-type embryos (n=11). Cells in this region
Fig. 5. Wild-type cyclops gene function is required for anterior
movement of ventral diencephalic precursors. (A-D) Dorsal views,
anterior to the top. (E,F) Side views, anterior to the left, dorsal to the
top. e, eye; mhb, mid-hindbrain boundary. (A,B) Single cells in
corresponding regions of the neural plate of wild-type
(A, arrowhead) and cyclops mutant embryos (B, arrowhead) were
injected with rhodamine dextran. The first group of embryos (n=9
wild type; n=3 cyclops) was immediately fixed and labeled for mar
expression by mRNA in situ hybridization. (C,D) The second group
of embryos (n=7 wild type; 2 cyclops) was fixed 2 hours later and
labeled for mar expression. (C) By this time the injected cell
(arrowhead) had shifted anteriorly, and had stopped expressing mar
in wild-type embryos. (D) In cyclops mutant embryos, the injected
cell failed to move anteriorly and remained near the anterior border
of the mar expression domain. (E,F) The third group (n=6 wild type;
n=3 cyclops) was fixed 4 hours after injection. These embryos were
labeled (black) to detect the expression of pax2 in the prospective
optic stalk region, emx1 in the prospective telencephalon, eng3 in the
prospective midbrain hindbrain border and krx20 in the prospective
hindbrain. In wild-type embryos (E), the injected cell (red,
arrowhead) was found anterior and ventral to the eye primordia. The
ventral diencephalon had formed. (F) In cyclops mutants, the injected
cell failed to move forward and remained posterior to the eye
(arrowhead). cyclops mutants lacked ventral diencephalon. Scale bar,
200 µm.
expressed opl aberrantly (Fig. 4E) and gave rise to dorsal
diencephalic cells located posterior and dorsal to the cyclopic
eye (Fig. 4F). cyclops mutants failed to form a ventral
diencephalon (Hatta et al., 1991), the later fate of cells in the
mar protrusion. Thus, the lack of ventral diencephalon in
cyclops mutant embryos correlates with the absence of the
protrusion of the mar expression domain that marks the
prospective ventral anterior diencephalic area of the neural
plate in wild-type embryos. These findings suggested that the
two retinas remain fused in cyclops mutants because
diencephalic precursors fail to shift forward along the midline
as they do in wild-type embryos.
To test this hypothesis that cell movements are affected in
cyclops mutant embryos, we labeled cells, at 80% epiboly, in
the mar protrusion in wild-type embryos and in the
corresponding region of the neural plate in cyclops mutant
embryos and assayed their positions later. Some of the embryos
(n=9 wild type; n=3 cyclops) were fixed immediately after cell
labeling and processed for mar expression and detection of the
5540 Z. M. Varga, J. Wegner and M. Westerfield
Table 1. Cell proliferation is uniform in the anterior neural plate
Location of injected cell at
tailbud stage
opl domain
mar protrusion
mar lateral arms
Location of progeny
at 24h
n
Av. clone size
s.d.
Eye
Vent. diencephalon
Dors./post. diencephalon
50
30
22
3.8
3.0
3.3
1.5
1.6
1.7
Sizes of clones in the eyes and diencephalon at 24h derived from single cells labeled with rhodamine dextran at the tailbud stage. Location refers to the
positions of cells at the time of injection (tailbud) or of the progeny (24h). n, number of preparations; Av., average number of labeled cells present at 24h; s.d.,
standard deviation.
lineage tracer (Fig. 5A,B) to ensure that we labeled cells in the
correct region of the neural plate. We fixed a second group (n=7
wild types; n=2 cyclops) 2 hours later at the tailbud stage (Fig.
5C,D), and a third group (n=6 wild types; n=3 cyclops) at the
6-somite stage (Fig. 5E,F). The first and second groups were
labeled for the lineage tracer and by RNA in situ hybridization
with a probe for mar. The third group was labeled for the
lineage tracer and by RNA in situ hybridization with probes
for emx1 (Morita et al., 1995), pax2 (Püschel et al., 1992b),
krx-20 (Oxtoby and Jowett, 1993) and eng-3 (Ekker et al.,
1992). By tailbud stage in wild-type embryos, the cells injected
at 80% epiboly had shifted anteriorly relative to the mar
protrusion and no longer expressed mar (Fig. 5C) or did so
only at very low levels. Injected cells lateral to the mar
protrusion remained at the same anterior-posterior position
(data not shown). These results suggest that cells in the mar
protrusion shift rapidly past cells that are more lateral into a
region anterior to the mar protrusion and ventral to the eye
field. In cyclops mutants, on the other hand, injected cells in
this region, corresponding to the mar protrusion of wild-type
embryos remained in the same position (Fig. 5D), even though
the underlying mesendoderm migrated normally relative to the
neural plate (n=23). By the 6-somite stage, the cells in the
protrusion of wild-type embryos that were injected at 80%
epiboly had shifted to the anterior ventral part of the
prospective diencephalon in the region close the optic stalk and
the polster (Fig. 5E). In cyclops mutant embryos, injected cells
failed to shift anteriorly, remained in the midline of the neural
keel posterior to the eyes, and contributed to prospective
posterior diencephalon (Fig. 5F). These results showed that
cells in the anterior protrusion of the mar expression domain
shift anteriorly with respect to the edge of the neural plate and
the mar gene expression domain, separate the eye field, and
form the ventral anterior diencephalon.
Cells that fail to shift in cyclops mutants die
In cyclops mutant embryos, cells failed to move anteriorly and
remained in the posterior diencephalon. Although cyclops
mutants lack a ventral anterior diencephalon, they do not have
an obviously expanded posterior or dorsal diencephalon. Thus,
it was unclear what happened to the cells that failed to move
anteriorly in cyclops mutants.
To examine the possibility that these misplaced cyclops
mutant cells die, we used TUNEL as an indicator of cell death
(Gavrieli et al., 1992). At 24h there were only a few dying cells
in the forebrain or the retinas of wild-type embryos (Fig. 6A;
n=20). We observed significant cell death only in the lens and
to a lesser extent in the surface ectoderm. In cyclops mutant
embryos, however, cell death was prominent posterior and
dorsal to the eye (Fig. 6B; n=7), the region of the diencephalon
occupied by the cells that failed to shift (Fig. 4B). We also
observed TUNEL labeled cells in the lens, retina, and surface
ectoderm of cyclops mutants. To learn whether the cells that
fail to shift in cyclops mutants die, we labeled neural plate cells
with lineage tracer anterior to the mar expression domain in
the region that corresponded to the mar protrusion of wild-type
embryos. We waited until 24h, and then doubly labeled with
Acridine Orange to identify dying cells in live embryos
(Furutani-Seiki, et al., 1996). We found many doubly labeled
cells (Fig. 6C; n=5 embryos). These results suggest that in
cyclops mutants, cells in the region of the neural plate that
corresponds to the mar protrusion of wild types express opl
instead of mar, fail to shift anteriorly, and die.
Clone size is uniform across the anterior neural
plate
In addition to cell movements, cell proliferation could also
potentially contribute to the separation of the eyes. Cells near
the midline could divide at a higher rate generating new cells
that would push the slower dividing more lateral cells apart. To
calculate proliferation rates, we measured the sizes of clones
generated by precursor cells in various regions of the neural
plate. We found no obvious differences in the sizes of clones
generated by labeled precursors (Table 1), which indicates that
differential cell proliferation probably does not play a
significant role in separation of the eyes. Our results are also
consistent with cell proliferation studies in the rat neural plate
(Tuckett and Morriss-Kay, 1985).
Ventral diencephalic precursors are required at the
end of gastrulation to form bilateral eyes
The cyclopic eye and the absence of ventral diencephalon in
cyclops mutant embryos, together with our observations of cell
movements, suggested that during normal development, the
anterior shift of cells from the mar expression domain is
required to separate the eyes and form the prospective ventral
anterior diencephalon. We tested this hypothesis by ablating
the mar protrusion in wild-type embryos at tailbud stage.
Adelmann (1929a,b) had previously shown that removal of
median anterior neural plate can induce cyclopia. To define
which cells are required, we removed median pieces of tissue
from the anterior neural plate at tailbud stage, ablating both
ectoderm and the underlying mesendoderm in various regions.
The lesions ‘healed’ rapidly; within 10-15 minutes after
ablation, neighboring cells filled in the ablated region. Sham
ablations, in which the tissue was removed and then placed
back into its original location produced no visible
developmental changes.
We ablated several different regions of the neural plate
(Ablation types I-V, Table 2) but only one type of ablation
Ventral diencephalic cells divide eye field 5541
reliably produced cyclopia in wild-type embryos. In these
operations (Type I) we removed all cells from the mar
protrusion, some median cells from the opl expression domain,
and underlying prechordal plate cells (Fig. 7B-D; Table 2).
Median cells posterior to the protrusion that normally
contributed to posterior ventral diencephalic regions (Fig. 4C)
were also removed. A portion of the anterior-most prechordal
plate, the posterior third of the prechordal plate, and the
anterior portion of the median opl expression domain in the
neuroectoderm were left intact. At 30h, we fixed the embryos
and labeled them for sonic hedgehog (shh) expression to
visualize the ventral diencephalon (Krauss et al., 1993). In 12
out of 15 preparations, this type of ablation produced cyclopic
embryos with a single retina fused across the midline. These
embryos also lacked the ventral diencephalon as indicated by
the absence of ventral diencephalic shh expression (Fig. 7H).
We compared normal (Fig. 7E) and operated (Fig. 7F)
embryos at the 12-somite stage. In normal embryos, we found
that the eye primordia were nearly completely separated by the
ventral diencephalon, although they were still connected by the
optic stalk (Fig. 7E). In contrast, after ablation of the mar
protrusion and flanking regions (Type I ablation), embryos
maintained a single field of cells continuous across the midline
(Fig. 7F) and later formed fused eyes (Fig. 7H) closely
resembling the morphology of cyclops mutant embryos at
equivalent developmental stages (data not shown).
In contrast, type II ablations (n=16) that removed the
anterior half of the prechordal plate and the overlying median
ectoderm resulted in cyclopia only in 31% of the cases (n=5;
Table 2). A few cells expressed shh in the posterior
diencephalon at 30h, consistent with the anterior movement of
some remaining diencephalic precursors along the midline
from regions posterior to the lesion. Embryos with type II
ablations formed extremely small eyes probably because such
a large portion of the retinal field was ablated.
In type III ablations, which involved removing the anterior
portion of the mar protrusion, only one embryo out of a total
of 5 was cyclopic. The other 4 had reduced ventral diencephalic
shh expression domains at 30h, consistent with the inferred
movement of some cells, located initially posterior to the
ablation in the protrusion of the mar expression domain,
forward into the ventral diencephalic region.
Other types of ablations, such as Type IV that removed the
mar protrusion incompletely, and Type V (Fig. 7A,G) that
removed mesendoderm and a piece of median anterior retinal
field, failed to produce cyclopia (Table 2).
DISCUSSION
A single eye field
Studies in a variety of species have indicated that the bilateral
eyes of vertebrates arise from a single field of cells in the
anterior neural plate (Adelmann, 1929a,b,c, 1936a,b; Ballard,
1973; Jacobson and Hirose, 1978; Woo and Fraser, 1995). Our
fate map of the zebrafish late gastrula supports this view. We
labeled cells in specific locations within the anterior neural
plate using morphology and gene expression patterns as
landmarks. We found a large, single field of eye precursor cells
within the opl expression domain. All the cells within this
domain later contributed to the retinas, we found no other cell
fates intermingled at the midline. Many labeled cells located
medially within the opl domain crossed the midline and some
divided to give rise to progeny that contributed to both eyes.
Thus, the classic theory of Huschke (1832) is firmly supported
by modern observations.
Our analysis provides new insights into how cells in the
single field of retinal precursors segregate into the bilateral
eyes. Several lines of evidence support the hypothesis that
anterior movement of diencephalic precursor cells splits the
retinal field into the left and right eye primordia. First, by fate
mapping, we showed that the retinas derive from a single field
of opl-expressing cells in the anterior neural plate (Fig. 4).
Second, cell labeling by intracellular injection suggested that
cells located posterior to the eye field in the median part of the
mar expression domain, shift anteriorly along the midline of
the neural plate (Figs 5, 8). Later, these cells form the ventral
diencephalon that lies between and anterior to the eyes (Fig.
4). Third, in cyclops mutant embryos, the cells corresponding
to the mar protrusion fail to shift anteriorly, the eye field
remains fused across the midline, and a single cyclopic retina
forms (Fig. 5). Fourth, removal of the median mar-expressing
cells and the directly underlying portion of the prechordal plate
from wild-type embryos blocks the anterior shift of cells into
the opl domain and the eye field remains fused across the
midline resulting in cyclopia (Fig. 7).
Vertebrate forebrain fate maps and cell movements
in the neural plate
Previous fate map studies (Ballard, 1973; Jacobson and Hirose,
1978; Hirose and Jacobson, 1979; Woo and Fraser, 1995) and
our results show that the eyes are derived from a single field
of cells in the neural plate and that all cells within this domain
contribute to the eyes. These results seem to contradict the
interpretation of recent gene expression studies that indicate
neural plate cells near the midline of the retinal precursor field
down regulate expression of ‘retinal’ genes and contribute to
the diencephalon (Li et al., 1997). Our results provide an
explanation for this seeming disagreement. Until the end of
gastrulation, retinal precursor cells occupy a single field that
extends across the midline. All cells within this field contribute
to the eyes and medial cells are free to cross the midline. This
field probably corresponds to the region fate mapped in the
previous studies (Ballard, 1973; Jacobson and Hirose, 1978;
Hirose and Jacobson, 1979; Woo and Fraser, 1995) that showed
the presence of a single eye field. During late gastrula and early
segmentation stages (80% epiboly to 5-somite stage in
zebrafish), diencephalic precursor cells move anteriorly along
the ventral midline, separating the retinal field later (5- to 12somite stages) into bilateral, left and right eye primordia. Thus,
the disappearance of retinal precursors and retinal gene
expression from the midline is most likely due to lateral
movement or displacement of retinal precursor cells rather than
to a change in their patterns of gene expression and inferred
fate from eye to diencephalon.
We analyzed cell position and inferred movements relative
to a variety of visible landmarks and gene expression patterns
(opl, mar and otx2) in the neural plate. pax6, the eyeless gene
in Drosophila and the small eye gene in mice, is expressed by
retinal precursor cells, and is required for normal eye
development (Quiring et al., 1994; Halder et al., 1995). In
mouse and zebrafish embryos, a variety of other CNS regions
5542 Z. M. Varga, J. Wegner and M. Westerfield
Fig. 6. Cells that fail to migrate
in cyclops mutant embryos
die. (A,B) bright field;
(C) superimposed confocal and
bright-field image; side views,
anterior to left, dorsal to the top,
24h. (A) TUNEL labeling of wildtype embryo. Cell death is
observed only in the surface
ectoderm and the lens (out of
focus). No cell death is apparent in
forebrain or midbrain. Similar
results were obtained in 19 other
embryos. (B) TUNEL labeling
reveals dying cells dorsal and
posterior to the eye of cyclops
mutant embryos (n=7).
(C) Embryos (n=5) doubly labeled
with lineage tracer (rhodamine
dextran, red) and Acridine
Orange (AO, green) showed that
diencephalic precursors failed to
move forward to separate the
single retinal field. Lineage tracer
injected cells remained
posterior / dorsal to the eyes and
died in dorsal diencephalic and
pretectal regions (yellow, doubly labeled with AO and lineage tracer).
A few hours later all rhodamine labeled cells were doubly labeled
with AO (not shown). In wild-type embryos, lineage tracer labeled
cells were unlabeled by AO (n=12). Prospective brain regions:
t, telencephalon; vd, ventral diencephalon; d, dorsal/posterior
diencephalon; e, eye; m, midbrain. Scale bar, 100 µm.
Table 2. Ablation of median cells of the mar protrusion
induces cyclopia
Ablation
type
n
% Cyclopic
I
15
80
II
16
31
III
5
20
IV
9
0
V
7
0
Ablation Type refers to the locations of ablated regions as indicated in the
diagram on the right; n, number of preparations; % cyclopic, number of
preparations showing cyclopia at 24h divided by n. Expression domains of
otx2 (red), opl (blue), mar (green) and cyc (shaded area) are indicated at
tailbud stage. The ablations are represented as boxes. Ablation Type I, black;
Type II, dark blue; Type III, light blue; Type IV, green; Type V, red. n, number
of preparations; dorsal view, anterior to the top.
also express pax6 (Püschel et al., 1992a; Kammandel et al.,
1999) and our results indicate that in the zebrafish neural plate
both retinal and diencephalic precursors express pax6 (Fig.
2C). pax6 expression, thus, might not be a suitable marker for
following the fates of retinal precursors (Li et al., 1997). In
contrast, our fate map analysis shows that at tailbud stage the
border between the opl and mar gene expression domains in
the neural plate of zebrafish provides an accurate and
Fig. 7. Diencephalic precursors are required for separation of the
single eye field. (A-F) Dorsal views, anterior to the top;
(A-D) tailbud stage. (A,B) Diagram showing gene expression
domains: otx2, red; opl, blue; mar, green; cyclops, grey. (A) The
Type V ablation is marked by the red rectangle. Type V ablations,
like unoperated and sham ablations never produced cyclopia. (B) The
Type I ablation at neural plate stage is marked by the black rectangle.
This type of ablation removed the entire mar protrusion and flanking
regions. (C) Embryo with Type I ablation, fixed immediately after
operation and hybridized in situ with probes for hgg-1 (red, polster;
Thisse et al., 1994) and mar (black) mRNAs. (D) Embryo with Type
I ablation, fixed immediately after operation and hybridized in situ
with a probe for cyclops mRNA. (E,F) 12-somite stage.
(E) Unoperated embryo. Retinal precursors had separated in
unoperated embryos by this stage (n=15), and only the optic stalk
(arrowhead) connected the eye primordia. Sham operated embryos
(n=9) are indistinguishable from unoperated siblings (not shown).
(F) Cells in the protrusion of the mar expression domain were
ablated at neural plate stage (Type I as in A-C). A single retinal field
persisted in operated embryos (12-somite stage, n=15), which closely
resembled the morphology of cyclops mutant embryos at the same
developmental stage (not shown). (G,H) 30h, side views, anterior to
the left, dorsal to the top. (G) shh expression marks ventral
diencephalon in sham (not shown) and unoperated embryos
(arrowhead). (H) Operated embryos (Type I ablation; 10 out of 13
embryos) lack a ventral diencephalon, as indicated by the absence of
sonic hedgehog (black) expressing cells (arrowhead) at 30h, and are
completely cyclopic. Prospective brain regions: t, telencephalon;
e, primordial eye (field); vd, ventral diencephalon; d, dorsal/posterior
diencephalon; m, midbrain; mhb, mid-hindbrain boundary. Scale
bars, 100 µm.
convenient subdivision of prospective retinal and diencephalic
brain regions and that retinal precursors express opl.
Our observations indicate that the shift of mar-expressing
cells from the area of the protrusion is probably due to an
anterior movement of ventral diencephalic precursor cells
relative to the retinal field, the anterior edge of the neural plate,
Ventral diencephalic cells divide eye field 5543
groups of cells. Our results resolve this ambiguity by showing
directly the anterior movement of labeled diencephalic
precursors relative to many landmarks at tailbud stage.
Fig. 8. Proposed model for the separation of a single retinal field by
median posterior cells. Ventral diencephalic precursors (blue) move
anterior (blue arrow) and ventral to the retinal field (red) and form
the primordium of the hypothalamus. In late neural plate stages, they
occupy a position ventral to the retinal field and dorsal to the axial
mesendoderm (green). Retinal precursors move laterally (red arrows)
to form the bilateral eyes. Telencephalic cell fates at the anterolateral
periphery of the neural plate are not shown here. Anterior to the
lower left, posterior to upper right, dorsal to the top.
the anterior edge of the mesendoderm, and the tailbud. We
found no apparent posterior movement of the retinal field
relative to these landmarks.
We suggest that patterning and morphogenesis of the
anterior neural plate may be similar in different vertebrates.
Studies that indicated a single eye field were carried out before
or by the end of gastrulation (Huschke, 1832; Stockard, 1913;
LePlat, 1919; Adelmann, 1929a,b,c; reviewed in Adelmann,
1936a,b; Ballard, 1973; Jacobson and Hirose, 1978; Hirose and
Jacobson, 1979; Woo and Fraser, 1995), whereas studies that
indicated two morphogenetic fields of retinal precursors were
obtained later during segmentation stages (Spemann, 1912;
Couly and Le Douarin, 1988; Eagleson and Harris, 1990;
Eagleson et al., 1995; Li et al., 1997). Our results predict that
anterior movement of ventral diencephalic precursors occurs
between these two developmental times. Therefore, similar cell
movements that separate a single eye field may be expected in
other vertebrates. This notion is further supported by the recent
observation of anterior movement of chick ventral diencephalic
precursors (Dale et al., 1999).
Our results clarify the interpretation of previous studies in
wild-type and mutant zebrafish that suggested a role for cell
movements in separation of the eye field (Woo and Fraser,
1995; Heisenberg and Nüsslein-Volhard, 1997; Marlow et al.,
1998). The previous zebrafish fate map indicated a large
overlap between diencephalic and retinal precursors, leaving
open the possibility of two retinal fields in the neural plate
(Woo and Fraser, 1995). Other studies suggested an anterior
movement of ventral midline neural plate cells using shh gene
expression as a marker (Heisenberg and Nüsslein-Volhard,
1997; Marlow et al., 1998). Gene expression, however, can
change rapidly in specific cell populations during development
and is therefore inadequate to predict cell movements and cell
fates. Moreover, prechordal plate cells express shh and are
located anterior and ventral to shh-expressing ventral midline
cells in the neural plate, obscuring the identification of these
two different cell types. Thus, changes in the shh expression
pattern could reflect shifts in axial mesendodermal cells,
extension movements of the neural plate midline, combinations
of these two movements, or changes in gene expression in
Ventral diencephalic precursors are required at the
end of gastrulation to form bilateral eyes
We found that removal of a median stripe of neuroectodermal
and underlying mesendodermal cells from wild-type embryos,
including the protrusion of the mar expression domain,
mimicked the cyclops mutation (Type I; Table 2) even though
only a subset of the cyclops-expressing cells were removed.
Ablation of cells in more anterior or posterior regions did not
produce cyclopia. Moreover, although neighboring cells
quickly filled the wound, these cells were unable to split the
eye field. Surprisingly, removal of nearly all median cells in
the eye domain (Type V, Table 2) still resulted in bilateral,
although smaller eyes. Together, these results indicate that
median cells in a particular anterior-posterior region of the
cyclops expression domain are required for normal forebrain
and eye development, consistent with an early specification of
cells in this region. However, because we were unable to
separate the neuroectoderm from the mesendoderm, we do not
know whether one or both of these tissues are required.
Function of Hh and cyclops signaling
Several inductive events are believed to pattern the neural plate:
signals from an anterior organizer (Houart et al., 1998), planar
signals from the blastoderm margin (Woo and Fraser, 1997)
and vertical signals from the axial mesendoderm (Kessler and
Melton, 1994; Kelly and Melton, 1995; Dale et al., 1997, 1999;
Foley et al., 1997; Pera and Kessel, 1997). In the anterior neural
plate, vertical signals such as BMP7 and Shh emanate from the
prechordal plate and ventral diencephalon and are required in
vitro for specification of forebrain cells (Dale et al., 1997,
1999). Prechordal plate and the ventral neural midline also
express Cyclops but the relationships between Cyclops and Hh
signaling are not well understood (Rebagliati et al., 1998;
Sampath et al., 1998).
Hh emanating from the prechordal plate may be insufficient
to regulate normal pax gene expression and differentiation of
optic stalk and retinal cells (Macdonald et al., 1995). Initially,
anterior movement of ventral diencephalic precursors may be
required to separate retinal precursors at the midline. Ventral
diencephalic precursors express additional Hh which may be
needed to elevate Hh signaling in the anterior midline to a level
required for maintenance of normal pax gene expression in the
anterior neural plate. Cyclopia in our ablation experiments can
then be explained by the failure of median neural plate cells to
move anteriorly, independently of Hh expression and extension
movements in the prechordal plate. This interpretation is
supported by explant studies of deep neural cells in Xenopus
that were shown to converge and extend autonomously (Elul
et al., 1997), although, studies in other vertebrates, including
mouse and human, indicate some role for Hh signaling in
cyclopia (Chiang et al., 1996; reviewed in Ming and Muenke,
1998). Thus, Hh alone may be insufficient for formation of the
ventral diencephalon and separation of the eyes. Cyclops
dependent movement of ventral diencephalic precursors may
also be required.
We found that convergent extension movements of the
prechordal plate are unaffected in cyclops mutant embryos.
5544 Z. M. Varga, J. Wegner and M. Westerfield
cyclops gene function appears to be required along the entire
ventral midline of the neuroectoderm. Mutation of the cyclops
gene removes the floorplate (Hatta et al., 1991) and perturbs
the forebrain (Hatta et al., 1994). The eyes are variably fused
at the midline, the ventral diencephalon is completely missing,
and the thickness of the forebrain is reduced to about half of
normal. Apparently, the eyes remain connected by optic stalk
precursors, which are thought to differentiate into retina later
(Hatta et al., 1994; Macdonald et al., 1995). Analysis of
genetic mosaics, obtained by transplanting wild-type cells
into cyclops mutant embryos, suggested that most of the
forebrain deficiency in cyclops mutants is non-autonomous;
transplantation of very few wild-type cells can rescue the
mutant phenotype apparently completely (Hatta et al., 1994).
Cells in the embryonic shield, the prechordal plate, and the
ventral CNS midline express Cyclops (Sampath et al., 1998;
Fig. 2D). The spatiotemporal expression pattern, the rescue
data, and the cell-nonautonomous inducing activities led to the
hypothesis that nodal-related gene function is required early at
the onset of gastrulation for the specification of floorplate,
ventral diencephalon, and mesendoderm (Sampath et al.,
1998). The inferred structure of this protein is also consistent
with Cyclops’ cell-nonautonomous function, providing a
potential mechanism for cell signaling from the prechordal
plate to the overlying midline of the anterior neural plate.
We found that like axial mesendodermal cells, ventral
diencephalic precursor cells move anteriorly; however, unlike
the mesendodermal cells, this movement of neural plate cells
requires cyclops gene function. The presence of filopodia
suggests that anterior movement of the axial mesendoderm is
an active migration. It remains unclear, however, whether the
movement of ventral diencephalic precursors is similarly active
or whether it results from oriented cell divisions along the
neural plate midline (Concha and Adams, 1998), and/or from
mediolateral intercalation (Elul et al., 1997) that leads to
extension. Although we found that cell division rates in the
neural plate midline were equivalent to those in lateral
positions (Table 1), cell divisions oriented along the
anteroposterior axis could play a role in the extension
movements of the neural epithelium during gastrulation
(Concha and Adams, 1998).
The role of cyclops function in these movements is unclear.
Studies of chick and rat have implicated signals emanating
from the prechordal plate in ventral forebrain cell specification
(Dale et al., 1997, 1999; Foley et al., 1997). Thus, prechordal
plate expression of Cyclops may be involved in specification
of median neural plate cells. In this scenario, Cyclops would
act indirectly in the separation of the eyes. Once specified in
the shield, median neural plate cells could undergo convergent
extension autonomously, as in Xenopus (Keller et al., 1992;
Elul et al., 1997), no longer requiring Cyclops signaling from
the prechordal plate at a later stage. However, this
interpretation may be inconsistent with mosaic studies
suggesting that most of the forebrain deficiency in cyclops
mutants can be rescued by transplantation of very few wildtype cells into the midline of the mutant forebrain (Hatta et al.,
1994). Alternatively, Cyclops could act directly to induce
movement of ventral diencephalic precursors. The expression
of Cyclops in the prechordal plate is consistent with this
possibility. However, Cyclops is expressed along much of
the ventral axis, whereas the movement of only ventral
diencephalic precursors seems affected in cyclops mutant
embryos. Thus, in this scenario, these cells would be specified
to respond specifically to Cyclops signaling. Our results do not
distinguish between these possibilities.
cyclops may be required for cell survival. We found that the
anterior median cells that fail to move anteriorly in cyclops
mutant embryos later die. These cells remain posterior to the
eyes and are thus located in inappropriate regions of the
diencephalon. The reason for their death is unclear. Their
abnormal morphogenesis and position in the developing
forebrain suggest they are mis-specified and may thus execute
an internal program of cell death. On the other hand, however,
cyclops mutant cells differentiate normally and apparently
survive in the diencephalon in the presence of wild-type cells
that are transplanted at blastula stages into mutant hosts (Hatta
et al., 1994) suggesting that at least some mutant cells can
survive in a supportive environment. This suggests that in the
absence of cyclops gene function, the cells that normally
populate the anterior diencephalon may not receive trophic
support required for their survival. Such trophic support could
come directly from the underlying cyclops-expressing cells of
the prechordal plate, which are abnormal in mutants, or from
cells located in the anterior neural plate region into which the
mutant cells fail to move (Houart et al., 1998).
Our results suggest a new function for Cyclops signaling. In
normal embryos, median neural plate cells in the region of the
mar protrusion move anteriorly forming the ventral anterior
diencephalon and separating the eye field into left and right
eyes. If these cells are surgically removed, the ventral anterior
diencephalon fails to form and the eyes remain fused.
Similarly, in the absence of cyclops gene function, these cells
fail to shift anteriorly, the ventral anterior diencephalon fails to
form, and the eyes are cyclopic. We suggest that a function of
Cyclops signaling is to induce the morphogenetic movements
of neural plate cells that are required for formation of the
ventral diencephalon and eyes.
We wish to thank Y. Grinblat and H. Sive for providing the opl
mRNA probe, Y. Yan and J. H. Postlethwait for providing the mar
probe, C. and B. Thisse for providing the hgg-1 probe, M. Halpern
and M. Rebagliati for providing cyclops mRNA probes and A. Schier
for providing MZoep mutant embryos. We thank R. Reyes and T.
Maynard for their advice with the TUNEL labeling technique. We are
grateful for critical reading of the manuscript by J. Eisen and C.
Kimmel. We also thank K. Whitlock for many critical discussions and
useful suggestions, A. O’Shea for her help breeding the fish lines, and
the staff of the Oregon zebrafish facility for their excellent care of
the fish. This work was supported by NIH HD 22486, AR45575
and the W. M. Keck and M. J. Murdoc Foundations, and by a
Boehringer Ingelheim Fond Postdoctoral Stipend and a Deutsche
Forschungsgemeinschaft research stipend to Z. V.
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