1 Department of Biology and Biological Imaging Center, California Institute of
Technology, Pasadena, CA 91125, USA
2 Department of Molecular and Cell Biology, University of California, Berkeley,
CA 94720, USA
3 Department of Molecular, Cell and Developmental Biology and Institute for
Cellular and Molecular Biology, University of Texas, Austin, TX 78712,
USA
Author for correspondence (e-mail:
wallingford{at}mail.utexas.edu)
Accepted 21 October 2004
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SUMMARY |
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Key words: Gastrulation, Dishevelled, Morphogenesis, Xenopus
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Introduction |
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Tissue isolation experiments have permitted the individual events to be
studied in detail, and have identified the autonomous morphogenetic processes
that drive amphibian gastrulation. For example, isolated head mesoderm moves
directionally, mimicking its advance across the intact blastocoel roof
(Davidson et al., 2002;
Nagel et al., 2004
;
Winklbauer et al., 1992
).
Isolated vegetal mass (endoderm) undergoes vegetal rotation movements, much
like the early motions in the embryo that expand the blastocoel floor and move
mesendoderm into contact with the overlying ectoderm
(Winklbauer and Schurfeld,
1999
). Finally, explanted axial mesoderm engages in convergent
extension, narrowing and lengthening to adopt the elongate morphology
appropriate for the forming body axis
(Holtfreter, 1944
;
Keller and Danilchik, 1988
;
Schectman, 1942
;
Wilson and Keller, 1991
). Such
explant experiments have been central to our understanding of the cell
behaviors driving each of these morphogenetic processes. For example,
time-lapse imaging of cells in the Xenopus dorsal marginal zone
revealed that convergent extension is driven by mediolateral cell
intercalation; detailed imaging experiments demonstrated that the
intercalation is accomplished by cell protrusions that are stabilized and
polarized mediolaterally (Shih and Keller,
1992a
; Shih and Keller,
1992b
; Wallingford et al.,
2000
; Wilson and Keller,
1991
). By comparison, much less is understood about the
inter-relationships between these different autonomous processes, and about
their coordination, in the embryo, to accomplish the end goals of
gastrulation.
We sought to study the mechanistic connection between different tissue movements during gastrulation in intact embryos. The internal events of gastrulation in Xenopus remain poorly understood, largely because of the large size and opaque nature of the early embryo. To better define these internal events, we produced high-resolution 3D digital datasets of fixed frog gastrulae; to examine their coordination in detail, we developed a series of morphometric measurements to simultaneously assess the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To understand how the diverse morphogenetic engines work together to accomplish gastrulation, we applied these tools to wild-type and experimentally manipulated embryos of various gastrula stages.
Members of the planar cell polarity (PCP) pathway, including Dishevelled,
are among the most well-studied genes involved in gastrulation in
Xenopus (reviewed by Wallingford
et al., 2002). In contrast to the patterning defects that result
from perturbing the canonical Wnt signaling pathway, the disruption of genes
in the Wnt/PCP pathway results in a failure of convergent extension
(Darken et al., 2002
;
Deardorff et al., 1998
;
Goto and Keller, 2002
;
Medina et al., 2000
;
Moon et al., 1993
;
Park and Moon, 2002
;
Rothbächer et al., 2000
;
Sokol, 1996
;
Takeuchi et al., 2003
).
Moreover, time-lapse imaging has revealed that cells lacking Xenopus
Dishevelled (Xdsh) signaling fail to stabilize and polarize the lamellipodia
that drive cell intercalation during convergent extension
(Wallingford et al., 2000
).
Disruption of PCP signaling has been shown to result in defective blastopore
closure (Sokol, 1996
).
However, the effect of disrupted Xdsh signaling on other events of
gastrulation remains poorly defined.
We show that mesendoderm internalization proceeds very effectively in the nearly complete absence of convergent extension and blastopore closure in embryos with disrupted Xdsh signaling. On the contrary, the failure of blastopore closure in embryos lacking Xdsh was tightly correlated with a failure of convergent extension. In addition, we found that blastopore closure required Xdsh signaling not only in the dorsal marginal zone, but also in the ventral/lateral marginal zone. We also found that archenteron elongation was independent of Xdsh signaling during the second half of gastrulation. In embryos lacking Xdsh function, nearly half of the length of the late gastrula-stage archenteron was generated in the absence of both convergent extension and blastopore closure.
Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, namely convergent extension and blastopore closure. Moreover, the data reveal a surprising degree of dissociability between these processes and mesendoderm internalization and archenteron formation. This loose relationship between morphogenetic processes may have allowed for the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species.
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Materials and methods |
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Surface-imaging microscopy
Embryos were fixed in Bouin's fixative and imaged as described
(Ewald et al., 2002).
Three-dimensional renderings were performed in ResView 3.1 (Resolution
Corporation; now Microscience Group, Corte Madera, CA). Images in
Fig. 1G-I were processed using
Unsharp Mask (75%, radius 5, threshold 0) in Adobe Photoshop 6.0. Surface
rendering (Fig. 12) was
performed with Amira 3.1 (TGS, San Diego, CA).
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Embryonic center and radius estimates
Assuming that the embryo is essentially spherical allows a geometrically
consistent estimate of surface areas from their 2D projection in plane-field
micrographs. The embryonic sphere radius and center can be estimated from a
circle approximation to the embryo boundary in the 2D image. The center
(x0,y0) and radius (a) of the circle approximation were
calculated from three manually-defined points on the embryo boundary in the
plane-field image. Applying the spherical embryo approximation, the elevation
(z) of any point (x,y) within the embryo boundary can be estimated using:
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Natural spline tracing of blastopore boundary and surface area ratio calculations
Blastopore and Xnot domain boundaries were defined by fitting a
closed natural spline to a set of manually selected points in the 2D image.
Typically between 5 and 15 points were used to define the spline that was then
approximated by a polygon with more than 100 vertices. The true surface area
enclosed by the natural spline boundary was calculated by re-projection of the
2D polygon spline approximation to the 3D embryonic sphere surface. The area
enclosed by this spherical polygon was determined using a discrete
line-integral approach implemented by MatLab. The ratio of the blastopore
surface area to the surface area of the embryonic hemisphere was calculated
using the radius estimate, a.
Xnot length/width calculations
The length and width of the Xnot domain
(von Dassow et al., 1993) were
determined by re-projection of the polygon spline approximation to the surface
of the embryonic sphere, rotation of the long axis of the domain to the
equator followed by determination of the maximum azimuth and elevation extents
of the domain. The long axis of the domain was defined as the great circle on
the embryo surface passing through the re-projection of two points manually
defined in the 2D image. Calculations presented divide the maximum length of
the domain by the median width. Ratios calculating median, maximum and mean
width were compared. and gave the same statistical results.
Archenteron and mesendoderm progress calculations
Images of mid-sagittally cleaved embryos were analyzed by manually defining
a boundary circle for the embryo as described above. Lines were then defined
from the center of the boundary circle to each of four points: the dorsal lip
of the blastopore (DBPL), the anterior limit of the Xnot domain, the anterior
limit of the archenteron, and the anterior limit of the mesendoderm. All angle
differences were calculated between the dorsal lip of the blastopore
(Fig. 2D), except for the
progress of mesendoderm migration, which was calculated as the angle from the
vegetal pole to the leading edge of the mesendoderm. Archenteron extension was
calculated as the angle from the DBPL to the anterior limit of the
archenteron. Anterior archenteron expansion was defined as the length of the
archenteron, in degrees, from the anterior limit of the Xnot domain to the
anterior limit of the archenteron. Archenteron inflation was quantified by
manually defining the outline of the embryo and of the archenteron (using
natural splines) on mid-sagittal surface. Progress in inflation was calculated
as the ratio of the area of the archenteron to the area of the embryo.
|
Optical flow analysis of time-lapse movies
Motion of embryonic surface features was assessed by optical flow analysis
(Barron et al., 1994), which
estimates the velocity field within an image from frame-to-frame changes in
local intensity. Movies were screened to identify heavily pigmented embryos
that provide appropriate image contrast for optical flow calculations. Optical
flow estimates were generated using the Lucas and Kanade algorithm
(Lucas and Kanade, 1981
),
implemented by the FlowJ plugin
(http://bij.isi.uu.nl/flowj.htm)
(Abramoff et al., 2000
) for
ImageJ
(http://rsb.info.nih.gov/ij/).
The resulting flow fields for selected frames within the time-lapse movies
were visualized using DC format spot noise also implemented by the FlowJ
plugin. In these images color is used to code the direction of flow. All
images depict dorsal to the right.
Statistics
To evaluate blastopore phenotypes within a day of experiments, blastopore
areas from different conditions were statistically evaluated with a
Kruskal-Wallis non-parametric ANOVA, with a 5% significance level for Dunn's
multiple comparison post-test, with comparisons between all conditions. Where
individual comparisons are made in the text between two conditions (e.g.
control versus ventral), the significance values reported are always from
ANOVAs calculated, as above, for all conditions performed in that experiment.
All calculations were performed using GraphPad Prism version 4.0a for
Macintosh (GraphPad Software, San Diego, CA). Embryo images presented are
representative of the mean (black bar in image), except in
Fig. 8, where the images
correspond to the final time point.
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Results |
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Beyond providing an excellent platform for observing the detailed morphology of the gastrula, the digital nature of this normal series enabled rapid evaluation of different perspectives within the same embryo (see Movie 1 in supplementary material). Surprisingly, these datasets revealed that the degree of blastopore closure was only loosely correlated with either mesendodermal mantle movement or archenteron elongation (Fig. 1G-I). At late gastrula stages, the blastopore appeared to be similar in embryos with radically different progress in archenteron elongation, archenteron inflation, mesendoderm migration and blastocoel displacement (Fig. 1G-I). Conversely, we also identified embryos with near identical archenterons and vastly different blastopores (data not shown). These observations prompted us to develop a set of morphometric measurements to allow us to investigate the apparent dissociability of internal and external gastrulation events.
Quantifying the events of gastrulation and the role of Xdsh
To quantify gastrulation events, we used obvious landmarks within the
embryo, and quantified the events of gastrulation with ratios and angles that
eliminate the confounding influence of natural variations in embryo size from
clutch to clutch.
We measured progress in blastopore closure by calculating the surface area
occupied by the blastopore and dividing it by the area of the vegetal
hemisphere of the embryo to generate a blastopore surface area/embryo surface
area ratio (Fig. 2A) (see also
Nakatsuji, 1974). Surface
areas were inferred from the 2D projected areas by reprojection to the
estimated spherical embryo surface in 3D, as described in Materials and
methods (Fig. 2C); 0.0
indicates complete closure and 1 indicates a blastopore covering the
hemisphere.
To assess the extent of convergent extension of dorsal tissues, we
processed embryos for in situ hybridization to the Xnot transcript,
which marks the dorsal midline tissues: notochord, floorplate and hypochord
(von Dassow et al., 1993). We
then measured the length-to-width ratio (LWR) of this expression domain, as it
appears on the surface of the embryo (Fig.
2B,C).
Quantifying convergent extension in intact embryos is difficult because the
marginal zone folds in upon itself during gastrulation. However, in a large
normal series, our values for Xnot LWR increased as gastrulation
advanced, and the increasing values for Xnot LWR showed a very significant
(P<0.001) correlation to decreasing values for blastopore closure
(see Fig. 3D, below),
indicating the coordinate progress of these two morphogenetic events. To
further test the validity of our metric, we also measured the progress in
convergent extension by measuring the length of the Xnot domain, in
degrees, as viewed on the mid-sagittal plane
(Fig. 2D); this alternate
formulation resulted in identical P values for all comparisons illustrated in
Fig. 3. Finally, the normal
progress of convergent extension, as gauged by Xnot LWR, was severely
inhibited by expression of Xdd1 (Fig.
3A,B), which is known to block convergent extension in dorsal
marginal zone explants (Sokol,
1996; Wallingford et al.,
2000
). Together, these data suggest that the LWR of the Xnot
domain provides an effective gauge of the progress of convergent extension in
intact embryos.
|
We next sought to understand the consequences of defective Xdsh signaling on other aspects of gastrulation. To achieve this, we imaged dorsally injected embryos using SIM. In these digital datasets, we observed open blastopores, but we were intrigued by the consistent presence of excess material immediately inside the blastopore lip (Fig. 4). In control embryos viewed in sagittal section, the marginal zone appears as a sheet that smoothly curves animalward from the blastopore (Fig. 4A). By contrast, in Xdd1-injected embryos, the marginal zone appears as a rounded ridge at the blastopore lip that gradually thins away from the lip (Fig. 4B, red arrowhead). Taking advantage of our SIM digital datasets, we evaluated the radial extent of internalized material in Xdd1-injected embryos. The ridge was still observed after rotating our digital datasets 30° in either direction from mid-sagittal (Fig. 4b', red arrowhead), suggesting that internalization of marginal zone material had occurred in at least a 60° arc on the dorsal side of this Xdd1-injected embryo.
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Because blastopore closure is commonly thought to be tightly coupled to mesendoderm internalization, we studied gastrulation of normal and Xdd1-injected embryos in time-lapse movies. In control embryos, the involution of marginal zone material began dorsally and proceeded to the lateral and then ventral lips of the blastopore (see Movie 2 in supplementary material). This movement is represented in Fig. 5 by optical flow diagrams. In this diagram, the colors indicate the direction of movement (see index; Fig. 5C) and the length of the lines corresponds to the speed of movement. Material moving from the surface of the embryo to the interior, at the blastopore lip, is indicated by regions of opposed flow (white boxes in Fig. 5a'). In control embryos, the internalization of surface material from the marginal zone was always accompanied by a coordinate internalization of yolk plug material under the lip of the blastopore (Fig. 5a'; Movie 2). The internalization of yolk plug cells also began dorsally and propagated ventrally (Fig. 5a'; Movie 2).
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Blastopore closure requires Xdsh function in both ventral/lateral and dorsal marginal zone tissues
The finding that both marginal zone and yolk plug internalization proceeds
in embryos with disrupted Xdsh signaling suggested that the mechanisms of
internalization and blastopore closure are not tightly coupled. Because we
observed that Xdsh is required for blastopore closure, we next sought to
better define this requirement. We tested the regional requirements for Xdsh
activity in blastopore closure by performing targeted injections of Xdd1 into
the two dorsal, the two ventral, or all four cells (circumferential) at the
four-cell stage. These injections produced a graded series in severity, with
increasing effect when injected in the ventral, dorsal, or all four cells.
Xdd1 had the greatest effect when expressed circumferentially, but all three
experimental conditions were significantly different statistically from
control embryos (Fig. 6,
P<0.001). The ventral requirement is apparent at the mid-gastrula
stages, indicating an early role for Xdsh in the ventral/lateral marginal
zone.
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Archenteron elongation requires Xdsh signaling during early gastrulation but not during late gastrulation
To assess the role of Xdsh signaling in archenteron development, we
measured the degrees of arc between the dorsal lip of the blastopore and the
anterior limit of the archenteron (Fig.
2D) (see also Moore,
1946). Archenteron inflation was also estimated as the 2D area of
the archenteron divided by the area of the embryo, both as viewed in
mid-sagittally cleaved embryos (Fig.
2E). We tested the requirement for Xdsh in archenteron formation
through ventral, dorsal and circumferential injections of Xdd1 (as above).
When embryos were fixed and analyzed at mid-gastrula stages, both dorsally and
circumferentially injected embryos displayed a significant defect in early
archenteron elongation compared with controls
(Fig. 9A; P<0.001;
means=79° control, 19° dorsal, 18° circumferential).
|
In light of the requirement for Xdsh in both dorsal and ventral/lateral tissues for blastopore closure, discussed above (Fig. 6), we tested for differences in archenteron elongation based on regionally targeted ventral, dorsal and circumferential injections. Embryos were fixed at both mid- and late-gastrula stages, and in all cases the ventrally injected embryos were statistically indistinguishable from the controls (not shown), and the dorsally injected embryos were statistically indistinguishable from the circumferentially injected embryos (Fig. 9A,B). From these data, we conclude that, in contrast to blastopore closure, PCP signaling is required only in dorsal tissues for archenteron elongation.
During late gastrula stages, the anterior portion of the archenteron
expands rapidly (Keller, 1975;
Keller, 1981
). We measured
this anterior expansion as the degrees of arc between the anterior limit of
the Xnot domain and the anterior end of the archenteron
(Fig. 2D). We observed no
significant difference in the amount of anterior archenteron between control
and Xdd1-injected embryos (Fig.
9C), further supporting the independence of archenteron elongation
from convergence and extension during the second half of gastrulation
(Fig. 9B).
To assess later archenteron development, we injected embryos with Xdd1 in the two dorsal cells, and cultured embryos to tailbud stages; SIM was used to generate 3D images of the whole embryos. These embryos had profound defects in their dorsal axes, but had relatively normal archenteron lengths, extending all the way to the head of the embryos (Fig. 10).
|
Interestingly, we saw no correlation between archenteron elongation and archenteron inflation in normal or Xdd1-injected embryos (Fig. 11B). For a given length of archenteron, a huge spread of values for inflation can be found. Also, we observed that the circumferentially injected embryos have less inflation (P<0.01) than do those injected dorsally (Fig. 11A). As dorsally injected embryos are not significantly different from circumferentially injected embryos in archenteron length (Fig. 9B), these findings support the idea of dissociability between archenteron elongation and inflation.
|
To determine whether or not such cavities could connect the blastocoel to the archenteron, we used the high-resolution 3D datasets obtained by SIM to generate volume renderings of cavities within a late gastrulae frog embryo. These volume renderings revealed connections linking such cavities to both blastocoel and archenteron (Fig. 12D,E; see Movie 8 in supplementary material).
Mesendoderm extension appears to be independent of Xdsh signaling
The mesendodermal mantle moves animal-ward along the roof of the blastocoel
in a hemispherical wave (Davidson et al.,
2002). Although it can be difficult to assay the exact position of
the leading edge of the mesendoderm, we measured the progress of mesendoderm
extension in degrees of arc (Fig.
2D). At mid-gastrula stages, we observed only a very modest
difference in mesendoderm progress in dorsally injected Xdd1 embryos
(P<0.05; control, 173°; dorsal, 161.5°). In
circumferentially or ventrally injected embryos, we saw no significant
difference (P>0.05; control 173°; ventral, 179°;
circumferential 163.2°). By the end of gastrulation, the mesendodermal
mantle had closed at the animal pole in all injected embryos. This result
suggests that Xdd1 does not generally inhibit cell motile behaviors, and is
consistent with the normal development of anterior structures in Xdd1-injected
embryos (Sokol, 1996
).
Dissociability of morphogenetic processes during Xenopus gastrulation
Examination of SIM datasets (Fig.
1G-I) indicated that blastopore size is a poor indicator of the
internal progress of gastrulation. To quantify the normal variability in
gastrulation, we plotted a normal series of embryos such that each embryo is a
single point plotted for both archenteron progress and blastopore progress
(Fig. 13A). As a reference, we
plotted the Niewkoop and Faber normal stages on the same graph, with the width
of the circle corresponding to the approximate predicted range of variability
(10° of arc) (Nieuwkoop and Faber,
1994). We observed a much higher degree of variability than had
been previously described; for example, for a given blastopore size, embryos
can have almost 100° of arc difference in the extent of archenteron
elongation (Fig. 13). For a
single given archenteron length, blastopore size can indicate stage 10.5
through stage 12 (Fig.
13).
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Discussion |
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Convergent extension, marginal zone internalization and blastopore closure
One current model of blastopore closure suggests that convergent extension
movements, which begin on the dorsal side of the marginal zone and propagate
ventrally, generate arcs of hoop stress that are shortened progressively as
the behaviors propagate (Keller et al.,
2003). This progressive arc-shortening is thought to progressively
constrict the blastopore, thereby internalizing the marginal zone
(Keller et al., 2003
;
Keller and Jansa, 1992
;
Shih and Keller, 1992b
;
Shih and Keller, 1994
).
Alternately, it has been suggested that blastopore closure and marginal zone
internalization may be separable
(Phillips, 1984
), and
internalization of at least ventrolateral material in the absence of dorsal
convergent extension has been previously shown in microsurgical experiments
(Schectman, 1942
). Indeed,
more recent studies indicate that much internalization occurs through the
combined action of vegetal rotation movements and the active internalization
of individual cells (Ibrahim and
Winklbauer, 2001
; Winklbauer
and Schurfeld, 1999
).
Because defective blastopore closure was consistently correlated with
defective convergent extension in our Xdd1-injected embryos
(Fig. 3), our data support the
progressive arc-shortening model of blastopore closure. Furthermore, the
presence of compact, internalized marginal zone material in embryos with
defective blastopore closure (Fig.
4) is consistent with the notion that it is convergent extension
of post-involution material that drives the vegetalward movement of the
blastopore lip (Keller, 1984;
Keller et al., 1985
). However,
blastopore closure can be accomplished without convergent extension in
UV-irradiated Xenopus embryos
(Cooke, 1985
;
Gerhart et al., 1989
;
Malacinski et al., 1977
). In
these cases, other tissue movements, such as convergent thickening
(Keller and Danilchik, 1988
),
are thought to drive blastopore closure. This fact leaves open the possibility
that Xdsh is also required for the other processes that contribute to
blastopore closure in Xenopus. Further study will be necessary to
discern the role of Xdsh in these other processes.
In addition, the ability of Xdd1-injected embryos to reduce their
blastopore area to some degree (Figs
7,
8) implies that other,
Xdsh-independent cellular mechanisms contribute to blastopore closure as well.
One such mechanism could be the evacuation of marginal zone cells from the
blastopore lip and insertion of these cells into the lining of the cleft of
Brachet (Ibrahim and Winklbauer,
2001). This model also helps to explain the observed separability
of marginal zone and yolk plug internalization from blastopore closure. We
consistently observe robust, internalization of both marginal zone and yolk
plug tissue during gastrulation in embryos in which the blastopore is not
closing (see Movies 2-7 in supplementary material,
Fig. 5).
Together, our data suggest that vegetal rotation and active involution are the primary motors of mesoderm internalization throughout the gastrula embryo, with convergent extension contributing only a small amount to internalization. Conversely, we argue that post-involution convergent extension is the primary motor driving the closure of the blastopore, with active involution contributing relatively little. Further studies into the molecular and cellular mechanisms driving these events of gastrulation will be required for a clear understanding to emerge.
An important role for Xdsh in the ventrolateral marginal zone during blastopore closure
Although often ignored, cells of the lateral and to a lesser degree
the ventrolateral marginal zone do engage in modest convergent
extension during the second half of gastrulation
(Keller and Danilchik, 1988).
Our data indicate that these ventrolateral cells make an important
contribution to blastopore closure in an Xdsh-dependent manner (Figs
6,
7,
8). This contribution of
ventrolateral cells to blastopore closure may help to explain the ability of
embryos ventralized by UV irradiation to close their blastopore
(Cooke, 1985
;
Gerhart et al., 1989
;
Malacinski et al., 1977
).
Although the cells of the ventrolateral marginal zone undergo only limited
convergent extension, they may also contribute mechanically to events driven
on the dorsal side. Ventral marginal zone cells make stable, if not polarized,
contacts with neighboring cells throughout gastrulation
(Reintsch and Hausen, 2001).
Stable attachments such as these may contribute to the establishment of
tension around the circumference of the marginal zone. In fact, relaxation of
such tension lines by microsurgical manipulation perturbs gastrulation and
results in abnormally open blastopores
(Beloussov et al., 1975
;
Beloussov et al., 1990
).
Furthermore, it has been shown that relaxation of the circumferential tension
in the marginal zone can reduce the efficacy of cell intercalations
(Beloussov et al., 1990
;
Beloussov et al., 2000
).
Consistent with a role for tissue tension in blastopore closure, the affected
side of the blastopore in Xdd1-injected embryos appears to be visibly less
taut at mid-gastrulation (Fig.
6, red arrowheads).
As we have previously shown that Xdsh is required to maintain normal
lamellipodial stability (Wallingford et
al., 2000), we propose that, when Xdd1 is expressed ventrally, it
disrupts the stability of cellular protrusions that are important for
maintaining tension within the ventral marginal zone, even at early gastrula
stages when this tissue is not engaged in convergent extension, explaining the
observed deficit in ventrally-injected embryos at mid-gastrula stages
(Fig. 8). Later in
gastrulation, the failure of these lamellipodia to polarize correctly will
block ventrolateral convergent extension, further disrupting blastopore
closure.
Finally, the ventral marginal zone undergoes convergent thickening during
late gastrulation (Keller and Danilchik,
1988), as mentioned above. The cellular basis of convergent
thickening is poorly understood, but it is clearly possible that this process
could involve polarized cell behaviors that require intact Xdsh signaling.
Xdsh, convergent extension and archenteron elongation
Convergent extension of the marginal zone produces a dramatic amount of
force (Moore et al., 1995),
and it is tempting to speculate that most aspects of anteroposterior axis
elongation during gastrulation are driven by this tissue movement. However,
the relationship between convergent extension and archenteron formation has
not, to our knowledge, been previously examined by direct experiment, and
evidence can be found to the contrary. For example, it has been shown that in
hybrids of Rana species in which convergent extension is defective,
archenteron elongation can proceed (Gregg,
1957
; Gregg and Klein,
1955
; Moore,
1946
). Likewise, hydrolytic sulfatase disrupts convergent
extension potently, and whereas archenteron inflation is severely affected,
archenteron elongation is only mildly reduced
(Wallingford et al.,
1997
).
We show here that interfering with Xdsh signaling by Xdd1 overexpression in
the dorsal tissues produced a significant defect in archenteron elongation by
the mid-gastrula stage (Fig. 9)
that is correlated to a defect in convergent extension
(Fig. 14). However, during the
second half of gastrulation, the archenteron elongates even in embryos that
lack additional dorsal convergent extension
(Fig. 9). Our data demonstrate
that anterior archenteron expansion is Xdsh independent
(Fig. 9C). Moreover, the data
suggest that most of the elongation of the archenteron during late gastrula
stages can be accounted for by anterior expansion and does not require
convergent extension. This result implies that at least two mechanisms are
required independently to elongate the archenteron. Two distinct phases of
archenteron elongation have also been noted in echinoderms
(Kominami and Takata, 2000).
The mechanisms contributing to archenteron elongation in the second half of
gastrulation remain to be determined, although several candidates exist.
The apical surfaces of bottle cells at the blastopore lip re-spread at the
end of gastrulation (Keller,
1975), and manual removal of bottle cells results in a truncation
of the archenteron, suggesting that re-spreading of these cells contributes to
archenteron length (Keller,
1981
). Likewise, disruption of epiboly by overexpression of
Xoom mRNA (Hasegawa and
Kinoshita, 2000
) has much more severe effects on archenteron
elongation than does the blockage of convergent extension with Xdd1 reported
here.
We suggest that convergent extension and epiboly drive distinct aspects of
archenteron elongation. If this is the case, then molecular perturbations that
disrupt both epiboly and convergent extension should have very severe effects
on archenteron formation. This is in fact the case, as disruption of
fibronectin function results in defects in both epiboly and convergent
extension, and in these embryos no archenteron ever forms
(Marsden and DeSimone, 2001;
Marsden and DeSimone, 2003
).
Disruption of Wee1 signaling also disrupts convergent extension, and to a
lesser degree epiboly, and again results in a severe failure of archenteron
formation (Murakami et al.,
2004
). These very severe archenteron defects resulting from these
molecular disruptions are likely to represent the sum of defects in multiple
motors contributing to archenteron elongation.
To highlight the potential contribution of epiboly to archenteron
formation, it is informative to compare Xenopus gastrulation with
that in the sturgeon, where the cell behaviors driving distinct tissue
movements are similar, but where the different tissue movements are used in
different ways (Bolker, 1994).
Sturgeon gastrulation begins with robust epiboly commensurate with archenteron
elongation, only later does convergent extension begin in earnest
(Bolker, 1993a
;
Bolker, 1993b
), suggesting that
epiboly can contribute to the formation of an archenteron in the absence of
convergent extension.
In order to elongate the archenteron, the vegetal-ward movement of
superficial tissue driven by epiboly would need to work in concert with
animal-ward movement of internal tissue. So, another likely contributor to
archenteron elongation is active movement of vegetal yolk cells by vegetal
rotation (Winklbauer and Schurfeld,
1999). Active, animal-ward movements continue in the yolk cells
until late gastrula stages (Ibrahim and
Winklbauer, 2001
), and could drive archenteron elongation.
Alternatively, intercalation of archenteron cells around the circumference of
the forming gut tube could produce convergent-extension movements of the
archenteron epithelium and thereby elongate it. Such a mechanism elongates
tubes in echinoderm archenterons and in Drosophila hindgut tubes
(Ettensohn, 1999
;
Lengyel and Iwaki, 2002
).
Archenteron inflation
Our data suggest that the inflation and the elongation of the archenteron
are separable morphogenetic events (Figs
9,
13). The mechanisms
specifically governing archenteron inflation remain obscure, but it is likely
that bottle cell respreading contributes to both archenteron inflation and
elongation (Keller, 1975;
Keller, 1981
). Moreover, our
3D datasets demonstrate that archenteron inflation could be driven by the
transfer of fluid from the blastocoel to the archenteron
(Fig. 12) (see also
Adolph, 1967
;
Humphrey, 1960
;
Tuft, 1957
;
Tuft, 1965
;
Zotin, 1965
).
It is interesting that embryos deficient in convergent extension of dorsal
tissues form relatively normal-looking archenterons by the tailbud stages
(Fig. 10). Beginning during
neurulation, the archenteron will eject fluid and shrink
(Brown, 1941;
Zotin, 1965
), and the entire
archenteron will eventually disappear before the onset of formation of the
definitive gut (Chalmers and Slack,
2000
). It has been suggested that the archenteron is a crucial
organ for both fluid balance and for respiration
(Adolph, 1967
;
Brown, 1941
), but it is also
possible that the archenteron is in fact most important as a morphogenetic
device.
There is much evidence to suggest that fluid transfer in embryos can
contribute to morphogenesis (Desmond and
Jacobson, 1977). For example, the blastocoel is necessary as a
space into which the internalized material can move
(Eakin, 1939
), and the rapid
transfer of fluid from blastocoel to archenteron may be a necessary
consequence of this invasion of material. Indeed, dye experiments in sturgeon,
Xenopus and Ambystoma have indicated that fluid is
transferred from blastocoel to archenteron during gastrulation
(Humphrey, 1960
;
Tuft, 1957
;
Zotin, 1965
).
Defects in fluid transfer may explain some of the phenotypes observed in
embryos lacking Xdsh function. Gastrulation movements, in particular the
constriction of the blastopore, have been suggested to exert hydrostatic
pressure on the blastocoel, forcing fluid into the archenteron
(Zotin, 1965). So, by
disrupting the progress of the blastopore lip, Xdd1 could indirectly reduce
the pressure on the blastocoel. Indeed, we often observed a failure of
blastocoel shrinkage in Xdd1-injected embryos
(Fig. 4B,
Fig. 11A). In turn, defects in
the constriction of the blastopore would then also secondarily influence
archenteron inflation, which may explain the different effects of Xdd1
injection on archenteron inflation and elongation (Figs
9,
11).
Evolutionary implications of the loose coupling of morphogenetic processes during gastrulation
There is tremendous variety in the overall sequences and patterns of
morphogenetic processes during gastrulation in amphibians
(Chipman et al., 1999;
del Pino, 1996
;
Minsuk and Keller, 1996
;
Purcell and Keller, 1993
;
Shook et al., 2002
;
Shook et al., 2004
). For
example, although the marginal zone of salamander embryos undergoes convergent
extension by mediolateral intercalation, this convergent extension does not
drive blastopore closure; it is instead the subduction of surface mesoderm
that forms arcs of hoop stress and drives blastopore closure in that species
(Shi et al., 1987
;
Shook et al., 2002
). Likewise
embryos of Gastrotheca close their blastopores prior to the onset of
convergent extension (del Pino,
1996
). Moreover, gastrulation in the frog Hyperolius
puncticulatus more closely resembles that of the sturgeon, with a
near-completion of epiboly occurring prior to the onset of internalization at
the blastopore, or of axis elongation
(Bolker, 1993a
;
Chipman et al., 1999
). In
addition, the yolk-rich gastrulae of this frog species form very small
blastocoels and also very small, slit-like archenterons
(Chipman et al., 1999
).
The Xenopus archenteron can elongate significantly during late gastrulation in the nearly complete absence of elongation of the axial mesoderm by convergent extension (Figs 9, 14), and the marginal zone and yolk plug material can be internalized in the absence of blastopore closure (Figs 4, 5). These findings suggest that each of the morphogenetic programs that run in parallel during gastrulation can act to a large degree independently of one another, yet can still function together as a whole.
Changes in gastrulation mechanisms could play an important role in the
diversification of egg size and life history strategies in Amphibia (see
Arendt and Nubler-Jung, 1999).
As such, the observed dissociability of morphogenetic events during
gastrulation may be evolutionarily significant. If the different aspects of
frog gastrulation were more tightly coupled, then it would be relatively
difficult to change one element without severe consequences on other
processes. If this were the case, one would expect that relatively similar
gastrulation processes would be observed in different amphibian species.
Because the Amphibia display a wide diversity of patterns of gastrulation, we
suggest that the lack of coupling between morphogenetic processes observed in
Xenopus gastrulation reflects allowances for variability in the
timing and the relative contributions of different events in gastrulation.
This variability may in turn have allowed for evolutionary innovations that
led to the diverse gastrulation patterns observed in amphibians.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/24/6195/DC1
* Present address: Department of Anatomy, University of California, San
Francisco, CA 94143, USA
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