Development 120, 2879-2889 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
2879
A fate map of the epiblast of the early chick embryo
Yohko Hatada* and Claudio D. Stern*
Department of Human Anatomy, South Parks Road, Oxford OX1 3QX, UK
*Present address: Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, 701 West 168th Street, New York, NY
10032, USA
SUMMARY
We have used carbocyanine dyes (DiI and DiO) to generate
fate maps for the epiblast layer of the chick embryo
between stage X and the early primitive streak stage (stages
2-3).
The overall distribution of presumptive cell types in
these maps is similar to that described for other laboratory
species (zebrafish, frog, mouse). Our maps also reveal
certain patterns of movement for these presumptive areas.
Most areas converge towards the midline and then move
anteriorly along it. Interestingly, however, some presump-
tive tissue types do not take part in these predominant
movements, but behave in a different way, even if enclosed
within an area that does undergo medial convergence and
anterior movement. The apparently independent
behaviour of certain cell populations suggests that at least
some presumptive cell types within the epiblast are already
specified at preprimitive streak stages.
INTRODUCTION
Giladi and Kochav (1976; in Roman numbers for preprimitive streak
stages) stage-X and Hamburger and Hamilton (1951; in Arabic
numerals for later stages) stage-3. Embryos were explanted in Pannett
and Compton (1924) saline by the technique of New (1955), with
modifications (Stern and Ireland, 1981). Following marking with DiI
and/or DiO (see below), the embryos were incubated for 24-36 hours
at 38°C in a humid atmosphere.
The early stages of development in amniotes are particularly
important subjects to study because it is at this time that the
early body plan is laid down. However, there are no detailed
fate maps available for these stages in any amniote. In the chick
embryo, which is probably the best studied, one reason was the
absence of a suitable staging system for the preprimitive streak
embryo until 1976, when Eyal-Giladi and Kochav produced
their detailed stage table. Most of the chick fate maps that have
been published were produced before this staging system
became available.
Now that both an accurate staging system and a better
method for mapping (carbocyanine dyes) exist, we have used
them to construct a detailed fate map for the preprimitive streak
and early primitive streak chick embryo. The results reveal an
orderly pattern of cell movements, consistent with previous
observations of gross morphogenetic movements in the early
blastoderm. The present results also reveal considerable
overlap between different prospective areas, as has recently
been described for the mouse (Lawson et al., 1991), rather than
sharp dividing lines between different territories, as suggested
by some older publications. Less expected was the finding that
certain presumptive areas appear to move independently of
others, even against the predominant gross pattern of
movements.
Key words: chick embryo, primitive streak, epiblast, DiI, DiO,
carbocyanine dyes, mesoderm induction, gastrulation
MATERIALS AND METHODS
Fate mapping with carbocyanine dyes, DiI and DiO
The carbocyanine dyes 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (Molecular Probes, Inc.) (DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate (Molecular Probes Inc. Di-OC18-(3)) (DiO) were used for labelling, following methods previously
described (see Stern, 1990; Selleck and Stern, 1991). These intensely
fluorescent dyes are lipophilic, become incorporated into cell
membranes and are not transferred between cells (Honig and Hume,
1989; Serbedzija et al., 1990; Wetts and Fraser, 1989).
Briefly, DiI or DiO was first dissolved at 0.5% (w/v) in absolute
ethanol and this diluted 1:9 with 0.3 M sucrose in distilled water at
40°C (see Serbedzija et al., 1990; Selleck and Stern, 1991). The dye
was applied to the desired region using gentle air pressure, using
micropipettes pulled from 50 µl capillaires (Sigma) with an Ealing
vertical electrode puller. A small group of cells (5-30) could thus be
labelled.
A total of 1326 injections were made into 726 embryos (600
embryos were labelled with both dyes), applied to different positions
(see Table 1). Embryos were not used for subsequent analysis if after
incubation: (i) they had developed abnormally; (ii) most of the
embryo contained label; or (iii) dye was seen as fine granular material
over the surface of the tissues. 358 of the embryos developed normally
to stages 11-13. A total of 332 injections, in 200 embryos, was used
for the final analysis.
Embryo techniques
Fertile hens’ eggs (obtained from Coppocks Poultry Farm, Carterton)
were incubated at 38°C for 0-8 hours to obtain embryos between Eyal-
Staging of embryos and standardization of position of
labelled cells
To use a more accurate staging system than those published, a quan-
2880 Y. Hatada and C. Stern
Table 1. Number of embryos used and number of
injections performed
Total number of embryos marked
Number developing normally to stages 11-13
Number of embryos used for analysis
Total number of injection sites used to construct fate maps
726
358
200
332
titative method was used (Fig. 1), in which the position of the anterior
border of the hypoblast sheet was used as a reference for staging. An
eyepiece graticule divided into 100 units was used. Using a zoom in
the dissecting microscope (Nikon SMZ-2T), the visible size of the
area pellucida was adjusted to fit exactly into the 100 eyepiece units,
with position ‘0’ corresponding to the posterior edge of Koller’s
sickle. Using our scale, the width of the sickle varies between 5 and
12 units. The position of the anterior edge of the hypoblast at the
midline was recorded, as was the site of injection. A similar method
was used to record the position of the site of injection relative to the
width of the area pellucida. In this way, a grid was constructed from
which the positions of injection sites were mapped.
According to this method, in stage XI embryos, the anterior border
of the hypoblast sheet had reached a position of less than 30; stage
XII was ascribed to a hypoblast sheet extending to position 30-69;
stage XIII corresponds to a position in the range 70-100. Stage XIV
embryos were those in which a cellular bridge visible posterior to
Koller’s sickle (see Eyal-Giladi and Kochav, 1976) and/or the sickle
itself has become V-shaped. Embryos with a primitive streak were
used up to the stage at which the tip of the streak had reached no
further than the 55 position.
This quantitative method also allowed us to assess retrospectively
the original position of marked cells in those cases where the orientation of the embryo had not been assessed correctly at the time of
marking. In total, the orientation had been assessed correctly in
323/358 (90%) embryos. It is interesting that even stage 2 embryos,
which already have developed a primitive streak, sometimes (3/111)
changed the orientation of the embryonic axis during subsequent
culture. Because of this, we analyzed particularly carefully each of
the 35 embryos in which the orientation had shifted from the original:
in none of these did the fate of the labelled cells fall outside the boundaries of that presumptive region as determined from the remaining
323 embryos.
Fixation and histology
After incubation, the embryos were fixed in PBS (pH 7.0) containing
0.25% glutaraldehyde and 4% formaldehyde for at least 1 hour and
stored in this fixative. All embryos were mounted in cavity slides and
examined in toto with an epifluorescence microscope (Olympus
Vanox-T, with 200 W high-pressure mercury lamp). DiI was visualized using 547 nm (green) peak excitation, where it emits at 571 nm
(red); DiO was excited at 484 nm (blue) and observed by its emission
at 507 nm (green). In a few cases, labelled embryos were also observed
using a BioRad MRC500 confocal laser scanning microscope.
In addition to examination of the fixed embryos as whole mounts,
some embryos were processed histologically to confirm the localization of the labelled cells. For this, the fluorescence had first to be photoconverted to an insoluble product by photooxidation of 3,3′diaminobenzidine (DAB) exactly as described previously (Stern,
1990; Selleck and Stern, 1991). After this, embryos were dehydrated
in a series of alcohols, embedded in fibrowax, sectioned at 10 µm,
mounted on glass slides and dewaxed in xylene before being mounted
in DePeX for bright-field observation.
RESULTS
Spatial and temporal resolution of the maps
The results obtained (based on a pattern of 873 contributions
Fig. 1. Diagram summarising the method used to determine the
position of marked regions in preprimitive streak stage embryos. The
diameters of the area pellucida along the midline and at right angles
to this (left lateral to right lateral) were each divided into 100 units.
The position of the site of dye injection was recorded. The same
scale was used to measure, in each embryo, the positions of the
anterior border of the hypoblast sheet (at stages X+ to XIV) and of
the anterior tip of the primitive streak (stages 2-3). These
measurements provide an additional, quantitative assessment of
developmental stage.
to different tissues from 332 injections analysed), are shown in
Figs 2-3, and specific examples shown in Figs 4-5.
We decided to standardize the positions of the injections
using a scale of orthogonal coordinates, adjusted to 100 units
along each the anteroposterior axis and the left-right axis, as
described in Materials and Methods. To standardize the
temporal aspect of the maps, we used the position of the
anterior border of the hypoblast at stages X-XIII and the
anterior tip of the primitive streak at stages XIV-3 (Fig. 1). In
this way, a continuous scale was obtained, spanning these
stages. For the summary maps shown in Figs 2 and 6, however,
embryos were grouped by the stage criteria of Eyal-Giladi and
Kochav (1976).
The following description summarises the main results
emerging from detailed analysis of the data, classified
according to each cell type containing labelled cells.
Gut endoderm
111 injections contributed labelled cells to the gut (Figs 2A,
4D,I). The gut endoderm differs from most other presumptive
areas in that it is quite compact at all stages studied. At stage
X, no presumptive gut cells are found in the midline except for
a very small component at the posterior end of the embryo;
they form a wing shape, hinged at the posterior midline (Fig.
2A). In general, this presumptive area converges towards the
midline and moves anteriorly at the stages studied. The anterior
movement seems to occur in two distinct stages (Fig. 3): a first
one, at stages X-XI, when the anterior border of the prospective gut tissue at the midline moves at the same rate as the
anterior border of the hypoblast. The second stage occurs at
stages XIII-XIV, with the midline component of the prospec-
Fate map of chick epiblast 2881
tive gut region reaching the centre of the blastoderm at stage
XIV. Between stages XII-XIII, there is no apparent anterior
movement at the midline. The most lateral cells contributing
to the gut converge towards the midline up to stage XIV. In
general, our impression is that injections into the midline of
the presumptive gut region produced labelled cells in only the
dorsal part of the gut at stages 11-13; more lateral regions of
the blastoderm contribute to other, lateral/ventral regions of the
gut (data not shown).
Prechordal plate
Only a few (20) injections contributed cells to this region (Fig.
5A). For this reason, it is difficult to draw general conclusions
about its movement. From the data obtained, it can be seen that
this presumptive population tends to be restricted to the
midline of the embryo at all stages studied (Fig. 2B). When
comparing the three stages that contributed more than one data
point (XI, XII, 2-3), a tendency for this area to move anteriorly during development can be seen.
Chordamesoderm (notochord/head process)
93 injections contributed labelled cells to the chordamesoderm
(Figs 2C, 4I,K). Like the presumptive gut, this region is wingshaped at stage X, hinged at the posterior midline; the
movements of the two regions are also similar and occur in two
distinct stages (Fig. 3). At stages 2-3, the presumptive chordamesodermal territory is wider than that of the gut at the same
stage (Figs 2C,6).
Somite mesoderm
49 injections contributed to the somitic mesoderm (Figs 2D,
4G,H,K, 5C): 40 to the medial halves (Figs 2D, 4K, 5C) and
18 to the lateral halves (Fig. 2D). Nine of the injections (two
at stage X, three at stage XI, two at stage XIII and two at stage
2-3) contributed labelled cells to both halves (Fig. 4H). At
stage X, the lateral somite territory is contained completely
within that of the medial somite, but the overlap gradually
diminishes. The separation appears to be brought about mainly
by the medially directed movement of the medial-half somite
population, whilst the lateral cells remain at their original
position. The presumptive medial-half-somite cells, in
addition, show a tendency to move anteriorly at these stages.
Intermediate mesoderm
10 injections gave rise to labelled cells in the intermediate
mesoderm (Figs 2E, 4G,H). Their positions are sparsely distributed over many regions of the blastoderm and it is therefore
difficult to draw definite conclusions about their movement.
Heart
38 injections contributed to the heart (Figs 2F, 4D,H). From
stage XII to stages 2-3, there is relatively little change in the
position or size of the presumptive heart territory.
Lateral plate mesoderm
Cells contributing to the lateral plates (88 injections) are
widely spread around the surface of the blastoderm, but still
forming wing-shaped territories hinged about the posterior
midline (Figs 2G, 4G,J,L, 5D). There is little indication of
movement before formation of the primitive streak.
Neuroectoderm
211 injections contributed cells to neural tissues (Figs 2I,J,
4A-C,E, 5B), of which 65 were restricted to the forebrain,
anterior tip of the neuroepithelium (prospective olfactory
region) or optic lobes (Figs 2J, 4A-C, 5B). The neuroectoderm
territory, like the prospective lateral plate mesoderm, is widely
distributed over the surface of the blastoderm at all stages
studied and shows no particular movement, except for anterior
movement of its posterior border from stage XIV, leading to
absence of this presumptive cell type from the posterior-lateral
region. The presumptive olfactory and optic regions form a
discrete sub-region of the neuroectodermal territory, which
gradually becomes localized at the midline. showing anteriorly directed movement from stage XI (Fig. 3). Several injections produced descendants in neural tissues and in lateral
plate mesoderm, and the two labelled regions were continuous at the posterior end of the embryo, level with the regressing Hensen’s node at stage 11-13. A similar phenomenon has
been observed in the urodele Triturus pyrrhogaster (Hama,
1978).
Surface ectoderm and extraembryonic tissues
These tissue types are considered together for three reasons:
(1) because at early stages and in posterior regions the
boundary between amnion and surface ectoderm is poorly
defined, (2) because it was sometimes difficult to distinguish
unambiguously the precise layer containing the labelled cells
and (3) because many of the injections contributed to both
tissues. 244 injections contributed to these regions (Figs 2H,
4A,C). In general, cells contributing to surface ectoderm and
to extraembryonic tissues (amnion, yolk sac and its stalk, etc.)
are very widely distributed over the entire surface of the
prestreak blastoderm. However, at stage XIV, there is a large
lateral/posterior region devoid of cells with these fates (Fig.
2H).
Shifting patterns in the mesoderm
Six injections (one at stage XI, one at stage XII, four at stages
2-3; in Fig. 4G) gave rise to labelled progeny. In these cases,
labelled cells were found laterally in anterior regions, and more
medially in more posterior parts of the embryo (as described
by Hama, 1978 for Triturus pyrrhogaster). In the majority of
cases, the most anterior cells were located in the lateral
mesoderm and gradually shifted via the intermediate
mesoderm to the lateral halves of the somites at more posterior
positions.
Extent of contribution to different levels of the
anteroposterior axis
In general, many injections contributed progeny to large
regions of the anteroposterior axis of the embryo. We are
therefore unable to ascribe particular regions of the anteroposterior axis to specific areas in the early stages of development.
This is consistent with the findings of Selleck and Stern (1991)
who reported that even progeny derived from some single
labelled cells spans extended regions of the embryonic axis.
However, one exception is the sensory areas associated with
the forebrain, where some injections only contributed progeny
to this region.
2882 Y. Hatada and C. Stern
Incidence of progeny crossing the
midline
We also analyzed the distribution of descendants of labelled cells in terms of whether or
not they crossed the midline. The results of this
analysis are shown in Table 2. In general,
labelled progeny tend to be restricted to one
side of the embryo. When this analysis is
extended by comparing the distribution in
different tissue types, it is seen that the descendants of presumptive somite cells do not follow
this rule: labelled progeny tend frequently to
cross the midline (χ2, 1 d.f. = 7.06; P<0.01).
We also compared the frequency with which
progeny crossed the midline in terms of the
stage at which the injection had been done. No
significant differences were found.
DISCUSSION
Assessment of the technique used
Fate maps of the early embryo have been constructed by many authors, both before the
formation of the primitive streak (Kopsch,
1926; Gräper, 1929; Wetzel, 1929; Kopsch,
1934; Pasteels, 1937; Malan, 1953; Vakaet,
1970, 1984) and after (e.g. Rosenquist 1966;
Nicolet, 1971). These maps were produced
either by local killing, or marking using spots
of water-soluble dyes applied through the
vitelline membrane in ovo, or using carbon
particles. However, these studies all suffer
from technical defects. Transplantation of
marked cells might have disturbed the spatial,
and perhaps temporal, organization of the
tissues to be mapped, while carbon or carmine
particles may not always follow the cells in
their movements. In addition to the disadvantages of these methods, the classical maps
suffer from the problem that they were
produced before a reliable staging system was
available for early stages of development.
Recently, both some new marking methods
(the carbocyanine dyes, DiI and DiO; see
Honig and Hume, 1989; Wetts and Fraser,
1989; Serbedzija et al., 1990; Stern, 1990;
Selleck and Stern, 1991) and a new staging
table (Eyal-Giladi and Kochav, 1976) have
been described, allowing the labelling of small
groups of cells at precisely controlled stages
of the very early chick blastoderm without
using transplantation or particulate markers.
We opted for a quantitative method for
assessing the position of sites of injection and
for staging the embryos, so that data obtained
from different embryos can be compared. The
positions of injection sites were measured with
respect to a pair of orthogonal axes (anteroposterior and mediolateral, each divided into
Fate map of chick epiblast 2883
100). By this method, size variation along each of these axes
of the area pellucida was eliminated by scaling.
To avoid complications introduced by possible left/right
asymmetry of the embryo (c.f. Strehlow and Gilbert, 1993), we
Fig. 2. Diagrams showing the data obtained from cell marking
experiments. Each point represents one injection in one embryo,
made at the stage shown (X-2/3), which contributed to a given cell
type (see below). Thus, each group of 6 diagrams (A, B, etc.)
represents the contributions to one tissue type according to the stage
at which labelling was performed; intermediate stages between
labelling and fixation were not analyzed. Each diagram represents
the right half of the area pellucida (seen from the ventral side of the
embryo). (A) Contributions to the definitive (gut) endoderm.
(B) Contributions to the prechordal plate. (C) Positions of cells
contributing to the chordamesoderm (notochord and head process).
(D) Locations of somite progenitors; v, medial halves of the
somites; ¶, lateral halves. (E) Intermediate mesoderm (prospective
pro- and mesonephros). (F) Heart. (G) Lateral plate mesoderm.
(H) Surface ectoderm and extraembryonic membranes.
(I) Neuroectoderm (excluding sensory placodes and optic lobes).
(J) Sensory organs: olfactory and optic evaginations.
decided to collect data from the left half of the blastoderm; the
degree of left/right asymmetry remains to be assessed in future
studies. It is worth mentioning, however, that injections to the
left side often gave rise to labelled cells that extended to the
contralateral side of the embryo, as found by
Gallera and Nicolet (1969) and Rosenquist
(1966) in chick and Lawson et al. (1991) in
mouse.
In some cases, the embryos were observed
under fluorescence immediately after injection,
to confirm the position and size of the label.
After incubation, embryos were again
observed by fluorescence and the germ layers
containing labelled cells discriminated by
focussing through a high numerical aperture
objective (10×, NA=0.40 and 20×, NA=0.65).
If there was doubt concerning which tissues
contained labelled progeny, the embryos were
photooxidised and examined in histological
sections.
It is worth pointing out that these fate maps
were constructed by labelling groups of cells
rather than single cells. Therefore, when an
injection produces progeny in more than one
tissue, we cannot formally distinguish between
two possibilities: (a) each presumptive area
contains a mixture of cells with different presumptive fates and (b) each area contains
common progenitors for these fates.
Comparison with other vertebrate
species
In general, the distribution of different
prospective cell types in the early chick
embryo agrees broadly with findings made in
other vertebrate species (zebrafish, amphibian,
mouse). There are a few minor differences,
however. Among them, in the chick, the region
contributing to mesodermal cell types appears
broader than in the mouse (Lawson et al.,
1991). Also, compared to the mouse and
urodele fate maps (see Lawson et al., 1991 for
summaries of both), the region of cells contributing to the notochord in the chick is also
broader. In contrast, the region contributing to
gut endoderm appears marginally broader in the mouse than in
the chick.
Convergence and anterior movement start long
before primitive streak formation
Our maps show that specific prospective areas change shapes
and positions during development. From these changes, the
gross pattern of movements can be extrapolated. Overall, the
predominant movements for most areas between stages X and
3 are convergence towards the midline and anterior
movement along the midline, in the posterior half of the area
pellucida. Different presumptive tissues undergo these
movements at different times. For example, ‘axial’ tissues such
as the chordal mesendoderm undergo convergence at stages XXI and appear to move anteriorly in two steps, at stages X-XI
and XIII-XIV (Fig. 3). The presumptive prechordal plate is
2884 Y. Hatada and C. Stern
already at the midline at stage X-XI. The posterior-medial parts
of the prospective neuroectoderm and of the notochord territories appear to move anteriorly together with the tip of the
primitive streak.
This overall pattern, convergence towards the midline and
anterior migration, has been described previously for early
stages of the chick (Patterson, 1909; Graeper, 1929; Wetzel,
1929; Pasteels, 1937; Spratt, 1946; Vakaet 1960, 1970, 1985)
and other vertebrates, such as teleosts (Oppenheimer, 1936;
Pasteels, 1936; Ballard, 1973, 1981, 1982, Trinkaus and Fink,
1992), amphibians (Nakamura, 1938; Pasteels, 1940;
Nieuwkoop and Sutasurya, 1979) and reptiles (Pasteels, 1937).
In the mouse, Lawson et al. (1991) describe a similar convergence (“anisotropic” spread between members of a clone, orientated towards the forming primitive streak). Thus, the
“Polonnaise” movements that have been described in the literature can be seen as an overall trend in the epiblast. However,
when individual prospective areas are followed, it is found that
many depart from this general pattern.
The pattern of movements explains the shapes of
prospective regions
After stage XIII, but before the appearance of the primitive
streak, the anterior border of the presumptive olfactory placode
and optic lobe, notochord, medial somite and prechordal plate
areas reach the centre of the area pellucida. This observation
agrees with Spratt’s (1946) careful study of epiblast
movements. He describes a convergence of the whole epiblast
to the posteromedial part, which could be likened to a fan,
hinged at the centre of the area pellucida and closing before
the primitive streak appears. The centre of the area pellucida
does not appear to move until later (stage 3), during primitive
streak elongation.
After stage XIV areas such as the heart and neuroectoderm
(Figs 2F,I, 6) appear ‘T’-shaped, but are not all coincident.
Vakaet’s (1984) study of cell movement seems best to explain
how this shape arises from the patterns seen at earlier stages.
He showed not only medial convergence towards the posterior
streak, but also anteromedially directed movement just in front
of the elongating streak at stage 2. Our study suggests that this
movement may start even before stage XIV.
Cell mixing is not random
Classical fate maps of the chick embryo were drawn with the
different presumptive territories separated by sharp borders
(Patterson 1909; Graeper, 1929; Wetzel, 1929; Pasteels, 1937;
Rosenquist 1966; Vakaet, 1984, 1985; Nicolet, 1971).
However, more refined techniques have now become available.
These are starting to indicate that, in general, such sharp
borders do not exist. For example, in the frog, Wetts and Fraser
Fig. 3. Graphs showing the change in the position (Y-axis), with
increasing developmental stage (X-axis), of different presumptive
cell populations that had been labelled at the midline of the area
pellucida. The position of the anterior border of the hypoblast sheet
is shown as an interrupted line on the left of each graph, and the
anterior tip of the primitive streak as a dashed line on the right.
These graphs are derived from data included in Fig. 2.
Fig. 4. Some examples of the results obtained. (A) After labelling
cells at position [x=10, y=50] in a stage 2 embryo with DiI (red),
labelled cells are seen in the surface ectoderm and amnion, seen here
from the ventral side of the embryo. (B) DiO (green) was applied to
cells at position [10,40] of a stage XI embryo; their descendants are
found in the forebrain, including the olfactory region. (C) Optic
lobes and diencephalon. At a different focal plane, labelled cells
were also found in the ectodermal covering of the diencephalic and
mesencephalic regions. (D) DiO-labelled cells derived from an
injection at position [25,30] of a stage XII embryo contribute to the
midline of the foregut and in the heart. The cells in the heart have the
characteristic spindle-like shape of cardiac myocytes. (E) Neural
tube and migrating neural crest cells derived from an injection at
position [15,15] at stage XIV. (F) The endothelial lining of the
embryonic blood vessels contain DiI-labelled cells, descended from
progenitors labelled at position [30,70] at stage XII. (G) DiI-labelled
descendants (from an injection at position [20,40] at stage XIII) in
the somites and DiO-labelled cells (from dye applied to position
[40,40]) in the intermediate and lateral plate mesoderm. In the latter
two tissues, the labelled cells are found at a more lateral position
anteriorly and more medially in more posterior regions. (H) The
somites, intermediate mesoderm and heart contain DiI-labelled cells,
derived from progenitors at position [25,30] of a stage XII embryo.
(I) Notochord and midline of the gut endoderm, arising from an
injection at stage XI in position [10,50]. (J) Unilaterally distributed
cells in the lateral plate, from cells labelled at position [20,50] in a
stage XIII embryo. (K) DiI (red)-labelled cells are found in the
notochord and DiO (green)-labelled cells in the medial halves of the
somites. (L) Bilateral distribution of labelled cells in the lateral
plates, after injection into position [30,60] of a stage 2 embryo. Scale
bars, 500 µm in I,J; 200 µm in A,B,L; 100 µm for the remaining
photographs.
Fate map of chick epiblast 2885
(1989) report that the descendants of labelled ectoderm cells
become mixed, albeit slowly, a process that continues throughout early development. During gastrulation, some of this
mixing is driven by cell intercalation (Shih and Keller,
1992a,b; Keller et al. 1992), as is also seen in teleosts (Trinkaus
and Fink 1992). In the French frog, Pleurodeles waltl, Delarue
and Boucaut (1992) have demonstrated finer differences in the
extent of cell mixing between deep and superficial circumblastoporal cells: dorsally, deep and superficial cells intermix
more extensively than ventrally. In the zebrafish (Kimmel and
Law, 1985a,b; Kimmel and Warga, 1988; Warga and Kimmel,
1990; Ho, 1992) and preimplantation (Winkel and Pedersen,
1988) and postimplantation mouse (Lawson et al., 1991);
however, there is much more extensive intermixing of cells,
resulting in some indeterminacy in the patterns of descendants
derived from single, identified cells. Lawson et al. (1991) summarised this with particular clarity, by concluding that “morphogenetic movements occur in the presence of extensive,
although not indiscriminate, cell mixing in the epiblast, and
that descendants of a single progenitor may be spread widely,
and also be present in different germ layers” (p. 905).
The present experiments reveal two, apparently contradictory, trends. First, the finding that certain presumptive cell
types in the epiblast undergo rather different movements to
2886 Y. Hatada and C. Stern
Fig. 5. Transverse sections through specimens like those shown in Figs 4-5, after photo-oxidation of DiI-labelled cells. (A) Section at the level
of the prechordal plate. (B) Section through the optic region, showing labelled cells in one of the optic evaginations. (C) Labelled cells located
in the medial and dorsal part of a somite. (D) The anterior tip of the segmental plate of a stage 11 embryo, showing labelled cells restricted to
the lateral plate. Scale bars, 25 µm in A,B; 50 µm in C,D.
those of other presumptive regions suggests that there is indeed
extensive cell mixing. Second, the finding that descendants of
a small group of cells, labelled by a single dye injection, tend
to contribute only to a few, more or less adjacent tissue types,
suggests that this mixing is not as extensive. This is the case
in most regions except the posterior midline, where many presumptive cell populations come together and overlap. One way
to reconcile these observations is to suggest that cell mixing is
fairly widespread, affecting large areas of the epiblast, but that
it is not random and that cells do not wander to very distant
sites (c.f. Lawson et al., 1991: “not indiscriminate”; see
above).
Some cell types may become specified early during
chick development
Interestingly, certain presumptive cell types move in characteristic ways, sometimes against the prevailing currents. For
example, the prospective lateral plate mesoderm territory does
Table 2. Laterality of distribution of labelled descendants
Tissue type
Neural
Olfactory region, optic lobes
Somites
Intermediate mesoderm
Lateral plate
Extraembryonic tissues and
surface ectoderm
Total
Unilateral
distribution
Bilateral
distribution
Total
15 (79)
10 (83)
13 (54)
4
16 (70)
38 (84)
4 (21)
2 (17)
11 (46)
0
7 (30)
7 (16)
19
12
24
4
23
45
96 (76)
31 (14)
127
Number of labelled cells found unilaterally or bilaterally after a single
lateral injection in the area pellucida, classified according to various
presumptive tissue types. These tissue types exclude the notochord, heart and
gut, where this type of analysis is not meaningful. The table includes all
embryos in which the required details had been recorded (n=127). The
numbers in brackets are % for the tissue type being considered.
not appear to change position between stages X and 3, even
though this territory overlaps with others, which do move as
described above from stage X. Likewise, the presumptive optic
lobe and olfactory areas seem to move differently from other
surrounding cell fates. This could be taken to indicate that
some prospective cell types are already specified at very early
stages of development, perhaps as early as stage X, although
we cannot formally rule out the possibility that morphogenetic
cell movements are not well coordinated at these early stages
of development. The exact degree of such specification for
different cell types may vary between different vertebrate
classes, giving rise to the apparent discrepancies in the findings
of different authors in terms of the degree of cell mixing for
different species.
Induction may be involved in the specification of
prospective gut cells
Slack (1991) has argued that a comparison between fate maps
and specification maps is of great value in identifying those
regions that require cell interactions (‘induction’) to define the
fates of the cells contained in them. In addition to the present
study, fate maps of the early embryo before the formation of
the primitive streak were published by Kopsch (1926), Gräper
(1929), Wetzel (1929), Kopsch (1934), Pasteels (1937), Malan
(1953) and Vakaet (1970, 1984, 1985). ‘Specification maps’
for the unincubated blastoderm were produced by Hoadley
(1926a,b,c,d, 1927), Olivo (1928a,b), Murray and Selby
(1930), Waddington (1933, 1935), Butler (1935), Dalton
(1935), Rudnick (1932, 1935, 1938a,b, 1944, 1948, 1961),
Hunt (1937), Spratt (1940, 1942, 1947) and Rawles (1943).
These were generated by culturing pieces of blastoderm on the
chorioallantoic membrane of a host embryo, in a plasma clot
or on the surface of another early blastoderm. Unfortunately,
these specification maps are very crude because no good
staging system was available at the time, because the pieces
Fate map of chick epiblast 2887
isolated are often very large and often comprised more than
one germ layer.
In addition, many reviewers (e.g. Pasteels, 1945; Romanoff,
1960; Nicolet, 1971; Balinsky, 1975) have failed to distinguish
between fate maps and specification maps and have amalgamated results from the literature to construct composites that
probably have little value. The maps produced by Butler
(1935) and by Rudnick (1948) stand out from others
because they appear to have been constructed more
carefully by attempts to separate the layers. In the former
case the description of the early embryos is clear enough
to allow us to conclude that it refers to embryos at around
stage XII.
We have therefore compared our results with those of
Butler (1935) and Rudnick (1948) (Fig. 6) for stages XII
and 2-3, respectively. Several areas, like neuroectoderm,
eye, notochord and heart are in equivalent positions in our
fate maps and in the published specification maps. The
only territory that does differ is the presumptive gut. This
is smaller in our fate map than in Butler’s specification
map. When parts of the epiblast (including area opaca)
are isolated and cultured, they become gut irrespective of
their original positions. However, in normal development, only the posterior one third of the epiblast becomes
gut. This indicates that cell interactions are required after
stage XII for restricting the gut territory, implying that
one or more inductive interactions are involved in the
early development of the gut.
Cell- or region-specific markers and the origin
of early mesendodermal cells
Staining with monoclonal antibody HNK-1 reveals a
mosaic, salt-and-pepper pattern in chick embryos at
stages XII-XIII (Canning and Stern, 1988). At stages XII2, staining is graded in the posterior-to-anterior and
medial-to-lateral directions, with the primitive streak containing more immunoreactive cells than more remote
regions of epiblast. By immunogold labelling and
ablation experiments, Stern and Canning (1990) showed
that the HNK-1-positive cells at stages XII-XIII are precursors of the mesendoderm of the early primitive streak.
In agreement with these patterns, our fate maps, as well
as those of previous authors, show that the posterior
epiblast and that surrounding the streak contribute more
cells to the mesendoderm than anterior and lateral
regions.
The finding that the epiblast contains a mixture of
HNK-1-positive and -negative cells, together with the
results of immunogold lineage analysis, led to the suggestion (Stern and Canning, 1990) that precursors of the
early mesendoderm are mixed with other cells in this
tissue before appearance of the primitive streak. Our fate
maps cannot provide an independent test of this because
the method used labels groups of cells rather than single
ones. Future analysis of the descendants of single epiblast
cells will be required to provide further insights into this
question.
Several authors have described restricted expression of
other markers at early stages of chick development (stage
X-3). For example the carbohydrates FC10.2 (Loveless et
al. 1990) and (NAc-lac)n (Thorpe et.al. 1988), the
cytoskeletal proteins vimentin and cytokeratin (Page, 1989)
and the homeobox gene goosecoid (Izpisúa-Belmonte et al.
1993) are expressed in certain subsets of cells in the early
embryo. In general the investigators concluded, however, that
these are markers specific for cell states such as ingression,
movement or ‘organizing’ properties rather than for particular
fates.
FATE
FATE
stage X
FATE
stage XIV
Prechordal Plate
Notochord
Medial Somite
SPECIFICATION
stage XI-XIII
FATE
SPECIFICATION
stage 2-3
Lateral Somite
Heart
Lateral Plate
Olfactory Region, Optic Lobe
Neuroectoderm
Gut
Fig. 6. Summary fate maps at different stages of development: X, XI-XIII,
XIV and 2-3. Each line is made to enclose all of the positions contributing to
each prospective cell type. In cases where there were too few data to allow
us to draw a line with confidence, dashed lines are used. For stages XI-XIII
and 2-3, the right half of the diagram shows the specification maps produced
by other authors for these stages, for comparison. Only those cell types for
which there is specification information available (Butler, 1935; Rudnick,
1948) are shown. The preprimitive streak specification map is based on
Butler (1935); the map for primitive streak stage embryos is based on
Rudnick (1948). In these specification maps, the frequency of each cell type
is represented as the density of symbols.
2888 Y. Hatada and C. Stern
Origin of Hensen’s node and of the ‘organizer’
property
Hensen’s node, the ‘organizer’ of the amniote embryo, has
been shown by many studies to contain several distinct cell
types: gut endoderm, prechordal plate, notochord/head
process, the medial halves of the somite mesoderm and floor
plate of the neural tube (e.g. Rosenquist, 1966; Selleck and
Stern, 1991; Schoenwolf, 1992; Schoenwolf et al., 1992).
Which cell populations of the early embryo give rise to the
node? Although we have not analyzed the movements of each
region in detail at intermediate stages, our data are consistent
with the view that regions containing various presumptive cell
types later found in the node may come together at the posterior
midline very early, at about stage XII. After this, but still
before primitive streak formation (about stage XIV), they
move together to a position close to the centre of the blastoderm, where Hensen’s node will eventually form (Spratt,
1946).
In a recent study, Izpisúa-Belmonte et al. (1993) showed that
the homeobox gene goosecoid, a marker for organizer cells, is
first expressed in a small population of cells in the middle layer
of the prestreak stage embryo, associated with Koller’s sickle.
Fate mapping with DiI reveals them to be precursors of some
of the cells of Hensen’s node, which also express this gene.
However, the same study demonstrated that these early
goosecoid-expressing cells have the ability to induce others
also to express the gene, and the node itself contains more cells
than can be accounted for from the early expressing population. Therefore, the node appears to be derived from at least
two distinct populations of cells: one group, found in the
middle layer around Koller’s sickle from stage X, and others,
in the epiblast.
Our fate maps show the locations of the territories occupied
by cell types derived from the node in the epiblast between
stages X and XIV. In the future, it will be interesting to investigate the onset of inducing ability: is it associated with any
particular prospective cell type, or does it require a particular
combination of cell types?
This study was funded by a Wellcome Trust Prize Studentship to
YH and by grants from the Human Frontier Science Program (held
jointly with Drs E. M. De Robertis and P. Gruss) and the Wellcome
Trust to C. D. S. We are grateful to Dr A. Stoker for allowing access
to his computer, to Dr T. Cunnane for his generous help with printing
of computer graphics in colour, and to Mr. G.J. Carlson for skilfull
technical assistance. We are also grateful to Drs Kirstie Lawson,
Julian Lewis and Jonathan Slack for their helpful comments on the
manuscript.
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(Accepted 15 July 1994)