1 Department of Neurobiology and Anatomy, and Children's Health Research Center,
University of Utah School of Medicine, Salt Lake City, UT 84132-3401,
USA
2 University of Ghana, Accra, Ghana, West Africa
* Author for correspondence (e-mail: schoenwolf{at}neuro.utah.edu)
Accepted 2 May 2003
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SUMMARY |
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Key words: Avian embryos, Cell migration, Gastrulation, Germ layers, Hypoblast, Ingression, Morphogenetic movements
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INTRODUCTION |
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Identification of precursor cells that ingress through the primitive
streak, and tracking of their subsequent movement to various destinations in
the developing embryo, have been the subjects of intense research. Not only do
the prospective fate maps generated from these studies suggest mechanisms
underlying cell commitment and patterning of the early vertebrate embryo, they
also provide insight into morphogenesis as a whole. The primitive-streak
origin of mesodermal precursor cells in the chick embryo has been welldefined
in previous studies (Pasteels,
1937; Spratt,
1942
; Rosenquist,
1966
; Rosenquist and DeHaan,
1966
; Nicolet,
1971
; Vakaet,
1984
; Vakaet,
1985
; Selleck and Stern,
1991
; Schoenwolf et al.,
1992
; Garcia Martinez and
Schoenwolf, 1993
;
Garcia-Martinez et al., 1993
;
Psychoyos and Stern, 1996
;
Lopez-Sanchez et al., 2001
).
For example, prospective cardiogenic cells occupy most of the rostral half of
the primitive streak at its early stages of development
(Garcia-Martinez and Schoenwolf,
1993
). By mid-primitive-streak stages, when the prospective
cardiogenic cells have undergone ingression, their position within the
primitive streak becomes occupied by ingressing prospective somitic and
lateral plate mesoderm cells (Schoenwolf
et al., 1992
; Garcia Martinez
and Schoenwolf, 1993
). Even prior to the movement of the
prospective cardiogenic cells into the primitive streak, their precise
epiblast origin is known: they reside in the epiblast lateral to the primitive
streak and caudal to the neural plate
(Lopez-Sanchez et al., 2001
).
These findings provide a cellular basis for elucidating the molecular
mechanisms involved in the morphogenesis of the cardiovascular system of the
avian embryo.
By contrast, the cellular mechanisms that underlie the formation of the
endoderm in the avian embryo have not been fully elucidated, and the existing
studies partially contradict one another. For example, several studies
reported that the endoderm receives contributions from different sources,
including the epiblast, the caudolateral region of the blastoderm and the
yolky cells derived from the germ wall (i.e. the lower layer of the area opaca
near its border with the area pellucida)
(Hunt, 1937;
Pasteels, 1937
;
Bellairs, 1953
;
Nicolet, 1971
;
Rosenquist, 1966
;
Vakaet, 1962
;
Stern and Ireland, 1981
),
whereas a study by Spratt and Haas (Spratt
and Haas, 1965
) concluded that endodermal precursors did not have
an epiblast origin. Moreover, a model based largely on timelapse observations
(Vakaet, 1970
) suggests that
three waves of cell movements occur during formation of the endoderm. First,
cells were proposed to move centrifugally (i.e. towards the center of the
blastoderm) from the area pellucida-area opaca border to give rise to the
junctional endoblast (i.e. the endoderm at the periphery of the area
pellucida). Second, cells from the region of Koller's sickle (i.e. at the
posterior marginal zone) were proposed to move rostrally to generate the
sickle endoblast [also called endoblast; see
figure 1 by Foley et al.
(Foley et al., 2000
)].
Finally, cells from the epiblast were proposed to ingress through the
primitive streak to generate the definitive endoderm. A detailed study by
Rosenquist (Rosenquist, 1972
)
is noteworthy here. He reported that the majority of cells that formed the
endodermal layer originated from the `endodermal center' within the primitive
streak, and that following ingression through the streak, they occupied an
oval area, which surrounded the rostral half of the streak. Furthermore, he
reported that these cells underwent a convergent-extension movement to
generate the definitive endoderm.
|
In the avian embryo, the spatial and temporal relationships between the
hypoblast and the developing definitive endoderm also are not fully
understood. Formation of the hypoblast layer is complex. It begins with
polyingression of cells from the epiblast
(Weinberger and Brick, 1982a;
Weinberger and Brick, 1982b
;
Penner and Brick, 1984
;
Weinberger et al., 1984
;
Stern and Canning, 1990
;
Harrisson et al., 1991
;
Lawson and Schoenwolf, 2001a
)
and is supplemented by cells moving rostrally from Koller's sickle and the
posterior marginal zone (Vakaet,
1970
; Stern and Canning,
1990
; Callebaut et al.,
1999
). By the incipient primitive streak stage of development, the
hypoblast consists of a complete layer beneath the epiblast. Its subsequent
movement to the germ cell crescent can be tracked by using hypoblast markers
such as Crescent (Pfeffer et al.,
1997
) and Goosecoid
(Izpisúa-Belmonte et al.,
1993
). Although there is evidence that when hypoblast cells are
confronted with endodermal cells in culture they tend to be displaced
peripherally, thereby surrounding the endodermal cells
(Sanders et al., 1978
), it is
unknown whether similar interactions occur in the intact embryo.
The present study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. The results demonstrate that between stages 3a/b and 4, the intra-embryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak. Second, the question of the epiblast origin of the endodermal layer was addressed by labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. The results demonstrate that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to the definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the endoderm, and combining this with in situ hybridization with a riboprobe for Crescent. The results show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells, which results in the intermingling of the two populations of cells.
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MATERIALS AND METHODS |
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Fate mapping experiments
Injections of the primitive streak
Chick embryos at stages 3a/b, 3c, 3d and 4 (n=230; n here
and subsequently refers to the number of embryos successfully labeled and
studied) were selected for injections of the primitive streak. The primitive
streak was injected with supravital fluorescent dyes at one of three levels
along its length (Fig. 1):
rostral, mid and caudal. For each level, two dyes were injected in tandem,
with the centers of the two injections being separated rostrocaudally by
125 µm: a mixture of 5-carboxytetramethylrhodamine, succinimidyl ester
(CRSE; Molecular Probes, Eugene, OR) and
1,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate
(DiI; Molecular Probes), and 5- and 6-carboxyfluorescein diacetate
succinimidyl ester (CFSE; Molecular Probes). Each injection was made across
the width of the primitive streak. Embryos were immediately examined with a
fluorescence microscope to confirm the size and site of each injection, and
they were then reincubated for periods up to 24 hours, during which they were
examined at regular intervals. At the end of culture, all embryos were fixed
in 4% paraformaldehyde in PBS, and then processed either for cryostat
sectioning or for immunocytochemistry, as described below.
Injections of the parastreak epiblast
For injections of the parastreak epiblast, embryos at stages 3a/b and 3c
(n=15) were cultured dorsal side upwards in Spratt culture
(Spratt, 1947), modified as
described by Schoenwolf (Schoenwolf,
1988
). The epiblast was injected lateral to the rostral end of the
primitive streak (Fig. 1) at a
site known to contribute cells to the somites (site bL) (see
Lopez-Sanchez et al., 2001
)
with a mixture of DiI/CRSE, and the embryos were reincubated for up to 24
hours (a subset was collected immediately after injection without reincubation
to determine the precise location and size of the injection). They were then
fixed in 4% paraformaldehyde in PBS and processed for immunocytochemistry, as
described below.
Mapping the hypoblast and definitive endoderm
For mapping the hypoblast and definitive endoderm, the primitive streaks of
embryos at stages 2-3a/b (n=18) were first injected at the
most-rostral end of the primitive streak
(Fig. 1; to mark the most
rostrally ingressing endoderm) with a mixture of DiI/CRSE and reincubated for
4 hours. They were then fixed in 4% paraformaldehyde, followed by in situ
hybridization as described below. Immunocytochemistry was subsequently carried
out, also as described below.
Immunocytochemistry
Embryos labeled with fluorescent markers were processed for whole-mount
immunocytochemistry as described previously by Patel et al.
(Patel et al., 1989), with the
following modification: the embryos were fixed with 4% paraformaldehyde in
PBS. For embryos labeled with both DiI/CRSE and CFSE, fluorescein
immunocytochemistry was carried out first as follows. The embryos were
incubated in PBT containing 3 mM levamisole for 30 minutes at room temperature
to block phosphatase activity. This was followed by treatment with the same
solution containing 5% sheep serum for a further 30 minutes. The embryos were
then incubated overnight at 4°C with alkaline phosphatase conjugated
anti-fluorescein antibody (Roche Diagnostics, Indianapolis, IN) at a dilution
of 1:600. The secondary antibody used was alkaline phosphatase conjugated
anti-digoxigenin antibody (Roche Diagnostics). For DiI/CRSE labeling, we used
anti-rhodamine (rabbit IgG polyclonal, primary; Molecular Probes) and
horseradish peroxidase-conjugated goat anti-rabbit IgG (secondary antibody;
Roche, Indianapolis). The embryos were examined and photographed as whole
mounts, and then processed for paraffin histology as described below.
In situ hybridization
In situ hybridization was carried out with Crescent RNA probe
(kindly provided by C. Stern) as described by Nieto and colleagues
(Nieto et al., 1996), except
that the proteinase K, hydrogen peroxide, and RNase A steps were omitted.
Paraffin histology
Embryos selected for histology were passed through an ascending series of
ethanol up to 100% ethanol. They were then cleared with two changes of
Histosol, infiltrated with Paraplast and embedded in fresh Paraplast.
Sectioning was carried out at 12 µm, and the sections were examined with
Hoffman modulation contrast optics.
Cryostat sectioning
Embryos selected for cryostat sectioning were passed through an ascending
series of sucrose-PBS solutions up to 30% sucrose in PBS. They were finally
embedded in OCT compound and sectioned at 30 µm. The sections were then
examined with a fluorescence microscope.
RESULTS
To determine the primitive-streak origin of cells that contribute to the
endodermal layer in embryos at stages 3a/b to stage 4, three levels along the
primitive streak (rostral, mid and caudal) were injected with vital
fluorescent dyes (Fig. 1). Two
dyes were used, a DiI/CRSE mixture and CFSE, and both dyes were injected into
each embryo but at different rostrocaudal sites about 125 µm apart,
allowing the relative displacement of prospective endodermal cells residing in
adjacent levels of the primitive streak to be followed as they migrated to new
locations in the forming endodermal layer. Below, we emphasize only the
contributions to the endodermal layer, as the mesodermal contributions have
been described in detail previously in other studies (e.g.
Garcia-Martinez et al., 1993;
Lopez-Sanchez et al.,
2001
).
Fate mapping the rostral primitive streak
After injections of the rostral level of the primitive streak at stage 3a/b
(Fig. 2A), both the
DiI/rhodamine and fluorescein labeled cells migrated rostrally where they
contributed to the rostral endoderm and mesoderm within 6 hours
(Fig. 2B,C). With further
rostral migration by 9 hours of reincubation, the labeled cells from the more
caudal injection (green fluorescence) slightly overlapped those from the more
rostral injection (red fluorescence) (Fig.
2D). After 18-24 hours of reincubation, the embryos had developed
to stages ranging from 7-8. At these stages, cells from the more rostral
injection (red fluorescence; brown after immunocytochemistry) populated most
of the intraembryonic endodermal layer, which extended from the head fold to
the caudal end of the primitive streak
(Fig. 2E,F). In addition, this
endoderm delineated a pear-shaped zone, extending the full width of the area
pellucida rostrally and tapering caudally to the width of the intraembryonic
region (Fig. 2E). By contrast,
there was minimal displacement of labeled cells from the more caudal site
[green fluorescence; purple after immunocytochemistry; the latter cells
occupied only a small region of the endodermal layer at the caudal end of the
primitive streak (Fig. 2E) and
overlap between the two populations was minimal or non-existent]. Thus, the
location of these cells was largely caudal to that of endodermal cells derived
from the more rostral site. In conclusion, the rostral tip of the primitive
streak at stage 3a/b contains cells fated to form most of the rostrocaudal
extent of the future dorsal and ventral endoderm of the gut. A similar fate
was observed when cells from the rostral tip of the primitive streak were
labeled at stage 2 (data not shown).
|
The contribution of labeled cells from the rostral level of the primitive streak (Fig. 2L) in embryos injected at stage 3d was similar to that at stage 3c (compare Fig. 2H-J with Fig. 2M-O), except that in embryos injected at stage 3d all labeled cells were restricted to the region of endoderm rostral to Hensen's node (Fig. 2O).
By stage 4, significant differences were apparent as compared to younger stages in the migration of prospective endodermal cells from the rostral level of the primitive streak (Fig. 2P) and their subsequent contribution to the developing endodermal layer. Unlike the pattern in the younger embryos described above, the migration of labeled cells at stage 4 from the most rostral level was virtually restricted to the midline (Fig. 2Q,R). Cells from this rostral level, therefore, contributed to the floor plate of the neural tube, notochord and the mid-dorsal endoderm, whereas those from the more caudal site contributed to the somites and underlying endoderm (Fig. 2S,T).
In summary, the rostral level of the primitive streak contributes to most of the rostrocaudal and mediolateral extent of the definitive endoderm. In progressively older embryos, contributions of the rostral primitive streak becomes correspondingly restricted toward the midline.
Fate mapping the mid-primitive streak
Injections of DiI/CRSE and CFSE into the mid-primitive streak level in
embryos at stage 3a/b (Fig. 3A)
resulted in the labeling of both endodermal and mesodermal cells of the
primitive streak and adjacent parts of the blastoderm by 6 hours of
reincubation (Fig. 3B,C).
Subsequently (i.e. by 9 hours), both the DiI/CRSE- and CFSE-labeled cells
within the endodermal and mesodermal layers migrated rostrolaterally,
following v-shaped streams towards the lateral margin of the area pellucida
(Fig. 3D). Between 18 and 24
hours of reincubation, the labeled prospective endodermal and mesodermal cells
at the leading edge migrated further rostrally and laterally to occupy the
cardiogenic areas of the blastoderm (i.e. the rostrolateral margins of the
area pellucida; Fig. 3E,F).
Caudally, labeled cells were found in the endoderm of the area pellucida and
the overlying mesoderm (Fig.
3E,G). Mid-primitive streak cells labeled at stages 3c and 3d
(Fig. 3H,M) followed similar
routes to populate corresponding areas of the endoderm and mesoderm
(Fig. 3I-L,N-P). Between stages
3a/b and 3d, therefore, labeled mid-primitive streak cells populated rostrally
the endoderm of the heart-forming region, the cardiogenic mesoderm, and the
lining of the pericardial cavity, whereas caudally, they populated the
endoderm of the lateral area pellucida (i.e. the future ventral gut and
extraembryonic endoderm) and its overlying mesoderm. Labeled mid-primitive
streak cells at stage 4 (Fig.
3Q) followed one of two different routes to contribute to two
different subpopulations of endodermal and mesodermal cells. Whereas the
rostromost subpopulation remained closer to the midline, eventually
contributing to the intermediate mesoderm and its underlying endoderm
(Fig. 3R,S,U), the more caudal
subpopulation of labeled cells migrated more laterally to contribute to the
lateral plate mesoderm and its underlying endoderm
(Fig. 3T,V,W).
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DISCUSSION |
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On the basis of our mapping experiments, we constructed prospective fate maps of the definitive endoderm (Fig. 7). As most of the intra-embryonic endoderm arises from the rostral end of the primitive streak as the latter forms and undergoes progression, we constructed prospective fate maps that show the rostrocaudal subdivisions of the definitive endoderm originating from the rostral end of the primitive streak (Fig. 7, to the left of the broken vertical line). In addition, as rostrocaudal position within the primitive streak ultimately translate into mediolateral position within the definitive endoderm, we constructed a prospective fate map that shows the mediolateral subdivisions of the definitive endoderm originating from the rostral, mid and caudal levels of the primitive streak (Fig. 7, to the right of the broken vertical line and above the broken horizontal line). Moreover, as mesodermal and endodermal regions arise in concert within the primitive streak and migrate in parallel after ingression, we constructed a prospective fate map comparing mesodermal and endodermal subdivisions (Fig. 7, to the right of the broken vertical line and below the broken horizontal line).
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The intermediate phase of endoderm formation
This stage of endoderm formation takes place as the primitive streak
lengthens and cells ingress through it. It is characterized by the formation
of the definitive endodermal layer of the area pellucida. The demonstration in
the present study that the primitive streak serves as a source of cells for
the definitive endodermal layer confirms the findings of several earlier
studies (Vakaet, 1970;
Nicolet, 1971
;
Rosenquist, 1971
;
Rosenquist, 1972
;
Fontaine and Le Douarin, 1977
;
Stern and Ireland, 1981
;
Garcia-Martinez et al., 1993
;
Lopez-Sanchez et al., 2001
;
Bachvarova et al., 1998
;
Foley et al., 2000
). By
following the movements of labeled cells at different levels of the primitive
streak between stages 3a/b and 3d, we demonstrated that the rostral part of
the primitive streak generates most of the intraembryonic endoderm
(Fig. 7: origins of
rostrocaudal endodermal subdivisions). This confirms the findings of
Rosenquist (Rosenquist, 1971
),
who used grafts labeled with tritiated thymidine followed by autoradiography.
Additionally, age-related differences were observed in the contribution of
labeled cells from the rostral end of the primitive streak to the definitive
endodermal layer. Whereas at stage 3a/b labeled cells from the rostral streak
populated most of the definitive endodermal layer, at stage 3d the
contribution from the rostral primitive streak was restricted only to the
region of the definitive endoderm rostral to Hensen's node. These findings,
while demonstrating that formation of the definitive endoderm is spatially and
temporally regulated, also provide insight into the nature of cell movement
taking place at the rostral end of the primitive streak during its
progression.
The late phase of endoderm formation
The late phase of endoderm formation occurs at stage 4 when the primitive
streak has attained its maximum length and Hensen's node has formed. With the
formation of Hensen's node, contributions of cells from the rostral streak
were virtually restricted to the midline, probably because Hensen's node
generates midline structures such as floor plate, notochord and dorsal midline
endoderm (as well as the ventral midline endoderm of the foregut and outflow
tract of the heart) (Kirby et al.,
2003). Cells from the rostral end of the primitive streak also
generate the prechordal plate, important for regionalizing the forebrain
(Pera and Kessel, 1997
), and
the ventral midline endoderm of the foregut, a region well situated to form a
ventral axis of the head (Kirby et al.,
2003
).
Comparison of the origin of the mesoderm and endoderm during avian
gastrulation
Previous fate mapping studies from our laboratory have revealed the
detailed origins and fates of prospective mesodermal cells in both the
primitive streak and epiblast at the full range of elongating primitive streak
stages (Garcia-Martinez and Schoenwolf,
1993; Lopez-Sanchez et al.,
2001
). Although contributions of the primitive streak and the
epiblast to the definitive endoderm were observed, they were not highlighted
in these studies. In one study
(Garcia-Martinez and Schoenwolf,
1993
), epiblast plugs of quail cells were transplanted
isochronically and homotopically to chick hosts. Quail cells were detected in
resulting chimeras by using Feulgen staining to label the quail nucleolus.
However, this method is unreliable in our hands for detecting quail cells in
highly squamous sheets of cells like the endoderm or endothelium; thus,
contributions to such layers are grossly underestimated. In the second study
(Lopez-Sanchez et al., 2001
),
also based on the transplantation of quail cells to chick hosts, an anti-quail
antibody was used to detect quail cells. This method is very effective in
detecting quail cells regardless of their histological structure. However, our
small epiblast grafts contained only a couple of hundred cells, and as the
vast majority (certainly more than three-quarters) of epiblast cells
contribute to mesoderm rather than endoderm, only small patches of endodermal
cells were detected in the published study. By contrast, with the use of dyes,
sites (especially within the primitive streak) contain reservoirs of label,
such that labeled cells are generated within the site as cells move through it
throughout the course of the experiment. Thus, many more cells are labeled,
and the contributions of such sites to the endoderm can be analyzed readily.
Although data on contributions to the endoderm are limited in our previous
studies, when their results are compared with those of the present study it is
clear that the results are consistent. Two general findings arise from these
results (Fig. 7: cross section
comparing the origin of mesoderm and endoderm from the primitive streak):
contributions to the mesoderm and endoderm occur in concert from any
particular site (i.e. primitive streak or epiblast), and contributions to the
endoderm from any particular site tend to spread more laterally than those to
the mesoderm from the same site. As reported previously, more rostral sites
within the primitive streak or epiblast tend to contribute to more medial
regions of the mesoderm, and more caudal sites tend to contribute to more
lateral regions of the mesoderm. We show here that this same relationship
holds for the endoderm, with more rostral sites tending to form more medial
regions of the definitive endoderm and more caudal sites tending to form more
lateral sites (see Fig. 7:
origins of mediolateral endodermal subdivisions).
The striking relationship in the origins of prospective mesodermal and
endodermal cells implies that signals for mesodermal patterning may be similar
to those of the underlying endodermal layer, or that both signals may occur
concurrently. Alternatively, each may be involved in the patterning of the
other. Indeed, there is evidence to suggest that interactions between endoderm
and adjacent mesodermal tissues allow the endoderm to acquire some positional
identity and morphogenetic information
(Cleaver et al., 2000;
Cleaver and Krieg, 2001
). In
addition, specification of the prechordal mesoderm may occur via rostral
endoderm-derived TGFß family signaling
(Vesque et al., 2000
).
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ACKNOWLEDGMENTS |
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