Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
* Author for correspondence (e-mail: heisenberg{at}mpi-cbg.de)
Accepted 23 December 2004
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SUMMARY |
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Here we show that at the onset of zebrafish gastrulation, mesendodermal progenitors in dorsal/axial regions of the germ ring internalize by single cell delamination. Once internalized, mesendodermal progenitors upregulate E-Cadherin (Cadherin 1) expression, become increasingly motile and eventually migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. When E-Cadherin function is compromised, mesendodermal progenitors still internalize, but, with gastrulation proceeding, fail to elongate and efficiently migrate along the epiblast, whereas epiblast cells themselves exhibit reduced radial cell intercalation movements. This indicates that cadherin-mediated cell-cell adhesion is needed within the forming shield for both epiblast cell intercalation, and mesendodermal progenitor cell elongation and migration during zebrafish gastrulation.
Our data provide insight into the cellular mechanisms underlying mesendodermal progenitor cell internalization and subsequent migration during zebrafish gastrulation, and the role of cadherin-mediated cell-cell adhesion in these processes.
Key words: E-Cadherin, Shield formation, Cell migration, Gastrulation, Zebrafish
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Introduction |
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Insight into the cellular mechanisms that regulate vertebrate germ layer
formation at the onset of gastrulation comes primarily from studies in the
amphibian Xenopus (for a review, see
Winklbauer et al., 1996).
Here, mesodermal and endodermal progenitor cells involute as a coherent sheet
of cells at the blastopore lip. The involuting mesodermal and endodermal cell
layers fold back onto the non-involuting sheet of ectodermal progenitors and
move along this layer towards the animal pole of the gastrula. The layer of
ectodermal progenitors is thought to facilitate and direct the migration of
mesodermal progenitors by secreting both extracellular matrix proteins, such
as Fibronectin, and guidance signals, such as Platelet-Derived Growth Factors
(PDGFs) (Ataliotis et al.,
1995
; Davidson et al.,
2002
; Nagel et al.,
2004
).
Much less is known about the cellular rearrangements underlying the
internalization and migration of mesendodermal progenitor cells at the onset
of zebrafish gastrulation (for a review, see
Kane and Adams, 2002). In
zebrafish, the first mesendodermal progenitors are induced at the margin of
the blastoderm when the blastoderm starts to spread over the yolk cell (dome
stage) (for reviews, see Kimelman and
Schier, 2002
; Warga and
Stainier, 2002
). When the blastoderm covers about half of the yolk
cell (50% epiboly), the germ ring forms as a local thickening at the margin of
the blastoderm. Germ ring formation is accompanied by convergence movements of
blastodermal cells, leading to a compaction of cells at the dorsal side of the
germ ring, where the embryonic organizer or `shield' forms
(Warga and Kane, 2003
). This
is also the time, when mesendodermal progenitors within the germ ring begin to
internalize by moving first to the margin of the blastoderm, then downwards in
direction of the yolk sac and eventually migrating back towards the animal
pole of the gastrula (Warga and Kimmel,
1990
).
How mesendodermal progenitor cells internalize in zebrafish is still not
fully understood. Two possible modes of mesendodermal cell internalization
have been proposed (Stern and Ingham,
1992; Trinkaus,
1996
): (1) `involution', describing the inward movement of
mesendodermal progenitors cells as a cohesive sheet of cells; and (2)
`ingression', the internalization of single mesendodermal progenitors cells as
they undergo an epithelial to mesenchymal transition. Experiments in which
single mesendodermal progenitors were transplanted into places of the gastrula
where no other mesendodermal progenitors can be found have shown that these
cells ingress in a cell-autonomous manner, suggesting that single-cell
ingression is the primary mode for mesendodermal cell internalization
(Carmany-Rampey and Schier,
2001
; David and Rosa,
2001
). Similarly, experiments demonstrating that mesendodermal
progenitors in outer cell layers of the shield at the onset of gastrulation
intermingle with ectodermal progenitors have been interpreted as evidence for
mesendodermal progenitors undergoing single-cell ingression rather than
involution (Shih and Fraser,
1995
). By contrast, studies in which the movements of cells within
the forming shield were analyzed have demonstrated that mesendodermal
progenitor cells at the germ ring margin involute as a continuous stream of
cells (D'Amico and Cooper,
1997
; D'Amico and Cooper,
2001
). It is likely that these contradictory views are due to the
fact that the morphogenetic movements within the germ ring have not yet been
looked at in sufficient resolution to be able to unambiguously distinguish
between these two different modes of cell internalization.
The molecular pathways that control tissue morphogenesis at the onset of
vertebrate gastrulation have only begun to be unraveled. The zebrafish
Nodal-related genes Squint and Cyclops are required for germ layer and shield
formation at the onset of gastrulation
(Feldman et al., 2000;
Feldman et al., 1998
;
Gritsman et al., 1999
).
Furthermore, inactivation of the Nodal antagonists Lefty 1 and Lefty 2 both
during zebrafish and Xenopus gastrulation causes deregulation of
Nodal signaling, expansion of mesendoderm and loss of ectoderm
(Branford and Yost, 2002
;
Feldman et al., 2002
). The
expansion of mesoderm after Lefty 1/Lefty 2 inactivation is accompanied by an
extended period of mesendodermal cell internalization, and a failure of
epiboly movements in zebrafish (Feldman et
al., 2002
) and exogastrulation in Xenopus
(Branford and Yost, 2002
).
Similarly, activation of Nodal signaling in single cells transplanted into the
blastoderm margin of maternal zygotic one eyed pinhead
(mz-oep) mutants that are defective in receiving Nodal signals, or,
alternatively, into the animal pole of wild-type embryos where endogenous
Nodal signaling is low, causes these cells to autonomously express
mesendodermal marker genes and to undergo cell internalization movements
(Carmany-Rampey and Schier,
2001
; David and Rosa,
2001
). This indicates that Nodal signals are required and
sufficient to induce both mesendodermal cell fate and the morphogenetic cell
behaviors underlying mesendodermal cell internalization during vertebrate
gastrulation.
The regulation of cell adhesion has also been shown to be required for germ
layer formation at the onset of vertebrate gastrulation. Cadherins form a
large group of cell-cell adhesion molecules regulating tissue morphogenesis in
a variety of developmental processes
(Tepass et al., 2000). During
Xenopus and zebrafish gastrulation, the over or underexpression of
paraxial protocadherin (papc) leads to defects in
convergence and extension of the forming embryonic body axis
(Hukriede et al., 2003
;
Kim et al., 1998
;
Yamamoto et al., 1998
).
Furthermore, when XB/U- or EP/C-Cadherin function is blocked at the onset of
Xenopus gastrulation, both mesodermal progenitor cell involution and
migration, as well as germ layer separation, are affected
(Kuhl et al., 1996
;
Lee and Gumbiner, 1995
;
Wacker et al., 2000
). Finally,
inactivating E-Cadherin in Xenopus and zebrafish embryos causes
variable defects before and during gastrulation, including impaired prechordal
plate formation and migration in zebrafish and ectodermal lesions in
Xenopus (Babb and Marrs,
2004
; Levine et al.,
1994
). While these results provide clear macroscopic evidence for
an involvement of Cadherins in tissue morphogenesis during vertebrate
gastrulation, their function in vivo on a cellular level is much less
understood.
In this study we have addressed two main questions. (1) What are the cellular mechanisms that underlie mesendodermal progenitor cell internalization and subsequent migration within dorsal/axial regions of the germ ring during early stages of zebrafish gastrulation? (2) How is Cadherin-mediated cell-cell adhesion involved in these processes? We provide evidence that axial mesendodermal progenitors delaminate as single cells and then migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. We further show that E-Cadherin is needed both in mesendodermal progenitors for elongation and efficient migration along the epiblast, and in epiblast cells for undergoing radial cell intercalation movements.
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Materials and methods |
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mRNA, morpholino and fluorescein-dextran injections
For mRNA synthesis the following constructs were used: pCS2-YFP,
pCS2-GAP43-YFP and pCS2-Ndr2 (Rebagliati
et al., 1998). mRNA was synthesized using the Ambion mMessage
mMachine Kit. For mRNA overexpression, 120 pg of YFP, 40 pg of
GAP43-YFP and 100 pg of ndr2 mRNA was injected into
one-cell-stage embryos. Time-lapse movies were recorded from embryos injected
with either 120 pg of YFP and 40 pg of membrane
bound-(GAP43-YFP) (see Movie 2 in supplementary material) or,
alternatively, a combination of 40 pg of GAP43-YFP and 125 pl of 0.5%
high molecular weight fluorescein-dextran (see Movies 1, 3 and 4 in
supplementary material). For e-cadherin morpholino (MO) injections, 8
ng of a MO targeted against the 5' end of the e-cadherin cDNA
(Babb and Marrs, 2004
) was
injected into one-cell-stage embryos. The specificity and efficiency of this
MO has been tested previously (Babb and
Marrs, 2004
). We determined the efficiency of our own MO
injections by measuring the total amount of E-Cadherin present in uninjected
wild-type (control) and e-cadherin MO (8 ng)-injected embryos at
shield and bud stage through western blotting (see Fig. S3 in supplementary
material).
Embryo sectioning and immunostaining
Embryos at shield stage were fixed in 4% paraformaldehyde (PFA) at 4°C
overnight, dehydrated and embedded in paraffin wax. For immunohystochemistry
10 µm sections were taken. Samples were hydrated and blocked with serum for
1 hour and incubated overnight in a humidity chamber with the following
primary antibodies: E-Cadherin, 1:745; ß-Catenin, 1:100, Sigma;
pan-Cadherin, 1:100, Sigma. The primary antibodies were visualized by using a
FITC-coupled secondary antibody (Molecular Probes, 1:2000).
Electron microscopy
Embryos were fixed in 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M
phosphate buffer overnight. They were postfixed in 1% osmium tetroxide
(Science Services, Munich) for 1 hour, dehydrated through a graded series of
ethanol and infiltrated in Embed-812 resin (Science Services, Munich)
overnight. The samples were cured for 48 hours at 65°C. Ultrathin sections
(70 nm thick) were cut on an Ultracut microtome (Leica Microsystems, Vienna).
The samples were viewed in a Morgagni EM (FEI, Eindhoven).
In situ hybridisation
Whole-mount in situ hybridisation was performed as previously described
(Barth and Wilson, 1995). For
gsc, ntl, fkd3 and gata5 in situ hybridisation, antisense RNA probes
were synthesized from partial sequences of the respective cDNA.
Embryo mounting, confocal imaging and image analysis
Embryos at shield stage were manually dechorionated and mounted in 0.8%
low-melting point agarose in E3. For confocal time-lapse imaging, we used a
Nikon TE 300 microscope and a BioRad Radiance 2000 Multiphoton Confocal
Microscope with a 60x water immersion lens. For data aquisition, we used
the LaserSharp 2000 program version 1.4 on a Windows NT-based PC. Images were
taken by scanning a 207x207 µm area with 50 lines per second. The
images were analyzed using Volocity (Improvision, UK) for cell movements and
morphology, and ImageJ to measure the relative intensity of immunostaining in
fixed tissue sections. The relative intensity of immunostaining for the
antibodies used in this study (E-Cadherin, ß-Catenin, pan-Cadherin) was
determined by comparing the plot profiles of the plasma membrane staining
between ectodermal and mesendodermal progenitors using ImageJ. For cell
movement traces, we defined the cell nucleus as the center of the cell. We
either determined the movement tracks of cells within the frame of our movies
(blue tracks in Figs 2,
5), or, alternatively, within
the germ ring margin itself (red tracks in Figs
2,
5). For the latter, we
determined the position of the cell relative to the margins of the germ ring
along the x-axis (animal-vegetal extent of the germ ring) and
y-axis (`inner-outer' extent of the germ ring); for a schematic
diagram of how the margins were defined see Fig. S1 in supplementary material.
For analysis of significance between two mean values, Student's
t-test was chosen, based on an unequal variance between the two mean
values.
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Cell transplantations
Cells from donors fluorescently labeled with Rhodamine- or
Fluorescein-dextran (Molecular Probes) and expressing membrane-bound
(GAP43-) YFP mRNA were transplanted into unlabeled hosts, as
described (Reim and Brand,
2002), and monitored using a dissecting scope (Nikon) or a
Multiphoton Confocal Microscope (BioRad).
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Results |
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The germ ring starts to form when the blastoderm has spread about halfway
over the yolk cell (50% epiboly), and becomes apparent by an accumulation of
cells at the margin of the blastoderm all around the circumference of the
blastula (Warga and Kimmel,
1990). This accumulation is triggered by cells close to the margin
of the blastoderm slowing down their movement towards the vegetal pole of the
blastula (epiboly movements) and, instead, moving as a continuous stream of
cells over the margin down towards the yolk cell, which causes the blastoderm
to `fold-in' at its margin; examples for the `folding-in' of the blastoderm
margin in dorsal/axial regions of the forming germ ring are shown in
Fig. 2A-F (cells 1-3); for a
complete picture of all cell tracks see Fig. S2A and Movie 1 in supplementary
material).
As soon as a recognizable germ ring has formed, single mesendodermal (prechordal plate) progenitor cells within dorsal/axial domains of the germ ring segregate or `delaminate' from their neighboring cells and become increasingly motile, eventually giving rise to a distinct hypoblast (mesendodermal) cell layer positioned between the yolk cell and overlying epiblast (ectodermal) cell layer (Movie 2 in supplementary material). Prechordal plate progenitor delamination is predominantly seen for cells at the tip of the germ ring directly adjacent to the yolk cell (as an example of a delaminating cell within the shield see cell 5 in Fig. 2G-I,K, and for a complete picture of all cell tracks see Fig. S2B in supplementary material). Notably, all of the prechordal plate progenitor cells - out of the 70 epiblast cells we analyzed in dorsal/axial of the germ ring - delaminated not more than 4-5 cell diameters away from the tip of the germ ring; this indicates that prechordal plate progenitor cell internalization is restricted to the marginal-most region of the germ ring. Moreover, prechordal plate progenitors positioned at the tip of the germ ring synchronously undergo delamination with direct neighbors showing similar cell behaviors (for examples of neighboring cells delaminating see the red tracks in Fig. S2B in supplementary material).
Once prechordal plate progenitors have delaminated, they move up towards the overlying epiblast (ectodermal germ layer), intercalate between each other at the forming interface between epiblast and hypoblast (mesendodermal germ layer), and eventually migrate along the epiblast in the direction of the animal pole of the gastrula (as an example for such a cell within the shield, see cell 6 in Fig. 2G-I,L; for a complete picture of all cell tracks, see Fig. S2B in supplementary material; and for a quantification of the rate of radial cell intercalations see Table 1). At the same time as the germ ring forms and prechordal plate progenitors begin to delaminate, ectodermal progenitors further away from the germ ring margin move from the deeper layers of the epiblast up towards the surface of the epiblast, `radially' intercalate in between more superficially located cells, and move together with the germ ring margin towards the vegetal pole of the gastrula (as examples for such cells within the shield, see cells 1 and 4 in Fig. 2A-D,G-J; for a complete picture of all cell tracks, see Fig. S2A,B; and for a quantification of the rate of radial cell intercalations, see Table 1).
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In order to visualize the function of E-Cadherin in controlling cellular movements at the onset of gastrulation, we took high-resolution two-photon confocal time-lapse movies of the germ ring margin at the region of the shield in e-cadherin MO-injected embryos during early stages of gastrulation [6-8.5 hours post fertilization (hpf)]. The analysis of these movies showed that blastodermal cells further away from the blastoderm margin of e-cadherin MO-injected embryos exhibit reduced movements towards the surface of the blastoderm (reduced radial intercalation) and, together with the germ ring, in direction of the vegetal pole of the gastrula (reduced epiboly), when compared with uninjected wild-type cells during all stages analyzed (Table 1; see cells 1, 2 and 4 in Fig. 5A-E,G-J; see also Movies 3 and 4 in supplementary material; for a complete picture of all cell tracks, see Fig. S2C,D). By contrast, mesendodermal progenitors from e-cadherin MO-injected embryos appear to normally delaminate and move away from the germ ring margin but, at the same time, fail to efficiently move up towards the overlying epiblast and to intercalate between each other at the forming interface of epiblast and hypoblast (Table 1; as an example, see cell 3 in Fig. 5A-C,F; see also Movie 3 and see Fig. S2C). In addition, when gastrulation has proceeded and a border has formed between the epiblast and hypoblast (7.5 hpf), mesendodermal cell migration along this border is reduced when compared with uninjected wild-type cells (Table 1; as examples see cells 5 and 6 in Fig. 5G-I,K,L; see also Movie 4 and Fig. S2D in supplementary material). This indicates that E-Cadherin function is required for radial intercalations of ectodermal and radial intercalation/anterior migration of mesendodermal progenitors within the shield during gastrulation.
To investigate whether these cell movement defects in e-cadherin
morphant embryos are due to defective cell-cell adhesion in ectodermal and
mesendodermal cells, we established primary cultures of ectodermal and
mesendodermal progenitor cells and monitored the cell aggregation behavior
throughout the first few hours in culture. Ectodermal cells were taken from
maternal-zygotic one-eyed-pinhead mutant embryos (mz-oep),
which lack most of their mesendodermal progenitors
(Gritsman et al., 1999),
whereas mesendodermal cells were taken from wild-type embryos overexpressing
the mesendoderm-inducing Nodal signal Cyclops (Cyc)
(Montero et al., 2003
;
Rebagliati et al., 1998
). We
confirmed the difference in cell fate between these two populations of cells
by detecting strong expression of the anterior axial mesendodermal (prechordal
plate) marker gene gsc in cyc-injected mesendodermal, but
not mz-oep ectodermal, cells (data not shown). In both ectodermal and
mesendodermal cell cultures, cells rapidly assemble into distinct clusters of
cells consisting of 20-100 cells (Fig.
6A,C,G,I). When E-Cadherin is inactivated in ectodermal or
mesendodermal cells, they still bind to each other but are unable to cluster
as efficiently as their wild-type counterparts
(Fig. 6B,D,H,J). This
difference in the ability to cluster between wild-type and e-cadherin
morphant cells becomes particularly obvious when wild-type and morphant cells
are co-cultured and wild-type cells form tight clusters that are surrounded by
loosely associated cells from e-cadherin MO injected embryos
(Fig. 6E,F,K,L and Movies 5,
6). Taken together, these data indicate that E-Cadherin is required for
cell-cell adhesion in gastrulating ectodermal and mesendodermal cells.
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To determine whether the defect in cell-cell adhesion of ectodermal and
mesendodermal cells in the absence of E-Cadherin leads to alterations of their
cellular morphology that might correspond to their cell movement phenotypes,
we recorded and analyzed the shape of anterior mesendodermal (prechordal
plate) and overlying ectodermal (epiblast) cells from wild-type and
e-cadherin MO-injected embryos at early stages of gastrulation. At
these early stages, the identity of prechordal plate progenitors appears to be
unchanged in e-cadherin morphant embryos
(Babb and Marrs, 2004).
Although the overall appearance of epiblast cells looks indistinguishable
between wild-type and e-cadherin MO-injected embryos (for details on
cellular processes, see below), cells at the leading edge of the prechordal
plate, close to the border between epiblast and hypoblast, are less elongated
in e-cadherin MO-injected embryos than in wild-type siblings
(circularity of wild-type cells was 0.69±0.17 compared with
0.82±0.12 in morphant cells; P<0.05;
Fig. 7A,B). These results
suggest that E-Cadherin is needed for the elongation of cells at the leading
edge of the prechordal plate close to the border between epiblast and
hypoblast, a morphological feature previously shown to correlate with the
migratory activity of prechordal plate cells at the onset of zebrafish
gastrulation (Montero et al.,
2003
; Ulrich et al.,
2003
).
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The reduced cell elongation and migration of prechordal plate cells at the border between epiblast and hypoblast in e-cadherin MO-injected embryos might be a consequence of more general defects in the formation of the interface between these tissues. We, therefore, analyzed specifically the border region between the epiblast and hypoblast within the shield at the onset of gastrulation (65% epiboly) in wild-type and e-cadherin morphant embryos. In e-cadherin MO-injected embryos, the border between epiblast and hypoblast within the shield is, although present and recognizable, morphologically less distinct when compared with uninjected control embryos (compare Movie 2 with Movies 3 and 4) suggesting that E-Cadherin controls the apposition of these layers. This assumption is further supported by the observation that the dense layer of cellular processes found in between the epiblast and hypoblast layers in wild-type embryos (see also Fig. 3) is strongly reduced in e-cadherin MO-injected embryos, as detected both in confocal sections of this region (Fig. 8A,B) and in electron microscopy images of cells at the border between these tissues (Fig. 8C,D). By contrast, although the border epiblast and hypoblast was often indistinct in e-cadherin morphant embryos, we did not observe cells crossing this border, neither from the epiblast nor from the hypoblast (data not shown), nor is the differential sorting behavior of ectodermal and mesendodermal cells in culture affected when E-Cadherin function is diminished (Fig. 6M-O). This indicates that E-Cadherin is required for cell elongation and process formation at the border between epiblast and hypoblast, but is not needed to restrict the movement of cells between the forming germ layers.
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Discussion |
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Cellular mechanisms underlying germ ring formation, mesendodermal cell internalization and migration
Zebrafish gastrulation starts when the blastoderm covers about half of the
yolk cell (50% epiboly) and the germ ring emerges as a local thickening at the
margin of the blastoderm all around the circumference of the blastula
(Warga and Kimmel, 1990). We
show that the germ ring in zebrafish forms by cells at the blastoderm margin
transiently slowing down their movement towards the vegetal pole of the
blastula (epiboly movements) and moving over the margin down towards the yolk
cell where they accumulate. This movement resembles the `folding-in' of a
sheet of cells at its margin and shares some similarities with the involution
movements of the mesodermal and endodermal germ layers at the onset of
Xenopus gastrulation (for a review, see
Winklbauer et al., 1996
).
However, in contrast to Xenopus involution, germ ring formation in
zebrafish occurs before the first mesendodermal progenitors delaminate and
appears to be at least partially independent of mesendodermal cell fate
inducing signals such as Nodals (J.-A.M. and C.-P.H., unpublished). This
suggests that the formation of the germ ring and the germ layers in zebrafish
are separable events that involve different sets of cell fate inducing and
morphogenetic signals.
Although the molecular pathways that trigger germ ring formation at the
onset of zebrafish gastrulation have yet to be elucidated, much more is known
about the signals responsible for mesendodermal cell fate induction and
progenitor cell internalization. The Nodal-related genes cyclops and
squint are required and sufficient to induce mesendodermal cell fate
(Feldman et al., 2000;
Feldman et al., 1998
;
Gritsman et al., 1999
), and to
trigger the morphogenetic processes leading to mesendodermal progenitor cell
internalization (Branford and Yost,
2002
; Feldman et al.,
2002
). Furthermore, the observation that Nodal signaling can
induce cell internalization in a cell-autonomous way, has been interpreted as
if single-cell ingression would be the predominant way by which zebrafish
mesendodermal progenitors internalize
(Carmany-Rampey and Schier,
2001
; David and Rosa,
2001
). In this study, we found that single mesendodermal
(prechordal plate) progenitors delaminate at the margin of the germ ring close
to the yolk cell, supporting the notion that mesendodermal progenitors ingress
as single cells. Importantly, we did not observe ingression of cells further
away from the germ ring margin (greater than four cell diameters) suggesting
that ingression is restricted to a marginal region within the germ ring.
We also see - predominantly in paraxial regions of the germ ring - a continuous flow of cells originating within the blastoderm further away from the germ ring margin, and moving over the margin towards the place where mesendodermal progenitors delaminate (J.-A.M. and C.-P.H., unpublished). This might create the impression that delaminating mesendodermal progenitors are part of a continuous `involuting' sheet of cells, although cells within the germ ring are clearly not organized into sheets, neither in the epiblast nor in the hypoblast, and certainly not between epiblast and hypoblast. This indicates that single-cell ingression or `delamination' at the margin of the germ ring, but not involution of a continuous sheet of cells, is the primary way by which mesendodermal progenitors internalize during zebrafish gastrulation.
Once internalized, mesendodermal (prechordal plate) progenitors move towards the interface between epiblast and hypoblast, and migrate along this interface in the direction of the animal pole. The epiblast, therefore, forms the substrate on which internalized prechordal plate progenitors migrate. As cells within the epiblast undergo radial cell intercalations that move these cells towards the vegetal pole of the gastrula, the prechordal plate progenitors migrate on a layer of cells that moves in the opposite direction to their own movement. In order to generate some net movement towards the animal pole, prechordal plate progenitors have therefore not only to reach the animal pole, but also to counteract the movement of the epiblast cell layer in the opposite direction.
The role of E-Cadherin-mediated cell adhesion for the formation of the germ layers
E-Cadherin has previously been shown to control tissue integrity and
morphogenesis during vertebrate gastrulation
(Babb and Marrs, 2004;
Levine et al., 1994
). In
Xenopus, blocking E-Cadherin function using an anti-E-Cadherin
antibody leads to defects in the integrity of the ectoderm during epiboly
(Levine et al., 1994
).
Furthermore, `knocking down' E-Cadherin translation in zebrafish results in
variable defects before and during gastrulation, including delayed epiboly,
reduced convergent extension of the body axis and impaired advancement of the
prechordal plate towards the animal pole of the gastrula
(Babb and Marrs, 2004
). The
results of our study provide insight into the cellular mechanisms by which
E-Cadherin controls these different processes. We show that the epiboly defect
in e-cadherin morphant embryos is most likely due to reduced radial
intercalation movements of ectodermal progenitors in these embryos. Similarly,
we present evidence that reduced cell elongation, process formation and
anterior migration of prechordal plate progenitors is the cause for the
posterior displacement of the prechordal plate previously observed in these
embryos (Babb and Marrs, 2004
).
As E-Cadherin function has not yet been directly associated with any of these
morphogenetic processes, either during zebrafish or Xenopus
gastrulation, this is the first demonstration of such a function of E-Cadherin
at early stages of gastrulation.
Internalizing prechordal plate progenitors in zebrafish have recently been
suggested to undergo an epithelial to mesenchymal transformation (EMT), which
is triggered by the nuclear localization of Snail, a transcriptional repressor
of e-cadherin expression
(Yamashita et al., 2004). We
were therefore surprised to see that prechordal plate progenitors in our study
upregulated rather than downregulated E-Cadherin expression. The most likely
explanations for this discrepancy are that either the downregulation of
E-Cadherin is not a suitable readout for EMT in zebrafish, or, alternatively,
that mesendodermal cells are actually not undergoing EMT. Supporting the
latter notion that mesendodermal progenitors are not undergoing a `classical'
form of EMT are our own observations that neither epiblast cells nor hypoblast
cells exhibit clear epithelial features such as an apical-basolateral
polarity. Instead, both cell types move within their tissues, show dynamic
changes in their cellular morphology and form multiple cellular extensions
(Ulrich et al., 2003
). The
only obvious difference between epiblast and hypoblast cells is that hypoblast
cells, in particular in paraxial regions of the germ ring, are more loosely
associated and show a more prominent elongation in the direction of their
individual cell migration. It therefore is quite likely that internalizing
mesendodermal progenitor cells, rather than undergoing EMT, primarily change
their general state of adhesiveness (e.g. through the upregulation of
E-Cadherin expression), which allows them then to delaminate from the epiblast
and take on a more mesenchymal appearance.
The differential expression of E-Cadherin between epiblast (ectodermal) and
hypoblast (mesendodermal) progenitor cells might not only determine the
different states in cellular motility between these two cell types, but might
also allow mesendodermal progenitor cells to delaminate and remain separated
from the pool of ectodermal progenitors. Evidence for a role of Cadherins in
germ layer separation comes primarily from studies in Xenopus
demonstrating that XB/U- and EP/C-Cadherin activity is required for the
separation of the ectodermal and mesodermal germ layers at the onset of
gastrulation (Wacker et al.,
2000). By contrast, we did not observe any such defects in
zebrafish embryos lacking E-Cadherin activity. The most likely explanation for
this is that the function of E-Cadherin in germ layer separation is redundant
to the function of other yet unidentified Cadherins expressed in the same
territory. Future studies will have to address which other Cadherins are
expressed within the zebrafish germ ring and how they functionally interact in
regulating germ layer formation and separation at the onset of
gastrulation.
Conclusions
With this study we provide the first detailed analysis of the cellular
rearrangements underlying germ ring formation and mesendodermal progenitor
cell internalization at the onset of zebrafish gastrulation. From this
analysis we conclude: (1) that mesendodermal progenitor cells segregate or
`delaminate' as single cells within dorsal/axial regions of the germ ring
margin; (2) that delaminated mesendodermal progenitors migrate along the
overlying layer of non-internalizing ectodermal progenitors away from the germ
ring towards the animal pole of the gastrula; and (3) that Cadherin-mediated
cell-cell adhesion controls cell movements within the germ layers, and
mesendodermal progenitor cell elongation and process formation at the
interface between the germ layers. These observations provide a solid starting
point from where to further analyze the cellular and molecular mechanisms
underlying germ layer formation during zebrafish gastrulation.
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ACKNOWLEDGMENTS |
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