Download Formation of the dorsal marginal zone in Xenopus laevis analyzed

Developmental Biology 305 (2007) 161 – 171
www.elsevier.com/locate/ydbio
Formation of the dorsal marginal zone in Xenopus laevis analyzed by
time-lapse microscopic magnetic resonance imaging
Cyrus Papan ⁎, Benoit Boulat, S. Sendhil Velan 1 , Scott E. Fraser, Russell E. Jacobs
Beckman Institute, California Institute of Technology, 1200 California Blvd., Pasadena, CA 90124, USA
Received for publication 11 December 2006; revised 16 January 2007; accepted 5 February 2007
Available online 13 February 2007
Abstract
The dorsal marginal zone (DMZ) of the amphibian embryo is a key embryonic region involved in body axis organization and neural induction.
Using time-lapse microscopic magnetic resonance imaging (MRI), we follow the pregastrula movements that lead to the formation of the DMZ of
the stage 10 Xenopus embryo. 2D and 3D MRI time-lapse series reveal that pregastrular movements change the tissue architecture of the DMZ at
earlier stages and in a different fashion than previously appreciated. Beginning at stage 9, epiboly of the animal cap moves tissue into the dorsal
but not into the ventral marginal zone, resulting in an asymmetry between the dorsal and the ventral sides. Time-lapse imaging of labeled
blastomeres shows that the animal cap tissue moves into the superficial DMZ overlying the deeper mesendoderm of the DMZ. The shearing of
superficial tissue over the deeper mesendoderm creates the radial/vertical arrangement of ectoderm outside of mesendoderm within the DMZ,
which is independent of involution and prior to the formation of the dorsal blastoporal lip. This tilting of the DMZ is distinct from, but occurs
synchronously with, the vegetal rotation of the vegetal cell mass [R., Winklbauer, M., Schürfeld (1999). “Vegetal rotation, a new gastrulation
movement involved in the internalization of the mesoderm and endoderm in Xenopus.” Development. 126, 3703–3713.]. We present a revised
model of gastrulation movements in Xenopus laevis.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Developmental biology; Xenopus laevis; Gastrula; Spemann organizer; Longitudinal study; MRI; Contrast media
Introduction
The dorsal marginal zone (DMZ) is a key embryonic tissue
region of the early gastrula stage Xenopus embryo because it
houses the Spemann–Mangold organizer (Spemann and
Mangold, 2001). It contains precursors of all three germ layers
(Keller, 1975, 1976); thus, knowledge of the cell movements
and the timing of the cell interactions are important for
understanding inductive processes among the cells within the
DMZ (Nieuwkoop, 1999; Nieuwkoop and Koster, 1995).
In the current view, Xenopus gastrulation begins with the
appearance of a dorso-vegetal pigment line at stage 10, marking
⁎ Corresponding author. Institute of Bioengineering and Nanotechnology, 31
Biopolis Way, The Nanos 04-01, Singapore, 138669, Singapore. Fax: +65 6478
9080.
E-mail address: [email protected] (C. Papan).
1
West Virginia University, Department of Radiology and Center for
Advanced Imaging, One Medical Center Drive HSC South, PO Box 9236,
Morgantown, WV 26506-9236, USA.
0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2007.02.005
the site of the future blastopore lip (Keller and Davidson, 2004;
Nieuwkoop and Faber, 1994; Shih and Keller, 1994). Following
this, mesendodermal precursors are thought to be brought into
vertical contact beneath the overlying ectoderm by means of
two types of morphogenetic movements: involution (Keller,
1975; Vogt, 1929) and vegetal rotation (Winklbauer and
Schürfeld, 1999), both of which are thought to begin at stage
10 (Poznanski and Keller, 1997; Shih and Keller, 1994;
Winklbauer and Schürfeld, 1999).
However, histological examination makes it likely that the
first morphogenetic movements occur prior to stage 10, and
internal tissue rearrangements before this stage have been
described (Bauer et al., 1994; Brachet, 1935; Hausen and
Riebesell, 1991; Holtfreter, 1943; Keller, 1978; Nieuwkoop
and Florschütz, 1950; Schechtman, 1934; Vodicka and
Gerhart, 1995; Vogt, 1929). Nevertheless, the true nature,
timing and extent of these motions, and their relationship to the
outward signs of gastrulation, have remained uncertain. Due to
the optical opacity of amphibian embryos, hypotheses about
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internal tissue movements have relied on the observation of live
tissue explants, on interpretations of tissue movements from
fixed material or on extrapolations from the externally visible
cell movements. Thus, the morphogenetic tissue movements at
blastula and early gastrula stages and their significance for
tissue interrelationships have not been convincingly defined.
Here we use 2D and 3D time-lapse microscopic magnetic
resonance imaging (MRI) to trace the pregastrular tissue
movements that result in the formation of the DMZ at stage
10. We find that the tissue movements leading to the formation of
the DMZ begin by stage 9. Pregastrula epiboly moves tissue
from the animal cap into the DMZ, but not into the ventral
marginal zone, explaining the well-known dorso-ventral tissue
asymmetry in the marginal zone of the stage 10 Xenopus embryo
(Hausen and Riebesell, 1991; Nieuwkoop and Florschütz,
1950). Time-lapse tracing of B1- and C1-blastomeres labeled
with MRI contrast agent confirms this process and reveals that
animal cap cells (e.g. descendants of the B1-blastomere) form
the superficial DMZ by moving outside mesendoderm cells
(e.g. the descendants of the C1-blastomere). Thus, beginning at
stage 9, pregastrula epiboly transforms the planar/horizontal
tissue juxtaposition into radial/vertical tissue juxtaposition prior
to the formation of the dorsal blastoporal lip. The outcome is
the same as proposed to result from an internal involution
movement; however, this apposition is not brought about by
involution (the in-folding of invaginating tissue back onto an
external cell layer).
Time-lapse analysis furthermore reveals that the tissue
movements associated with vegetal rotation (Winklbauer and
Schürfeld, 1999) also begin at stage 9, much earlier than
previously appreciated. These vegetal rotation motions bring a
portion of the blastocoel floor into contact with the animal cap in
the upper (anterior) deep marginal zone. Epiboly and vegetal
rotation occur synchronously, so that by the onset of gastrulation, the vegetal cell mass has come into contact with the animal
cap by two different paths. Based on these observations we
present a schematic model of the pregastrula morphogenetic
movements and DMZ formation.
Materials and methods
Embryo handling, mounting, imaging and image processing
Embryos were handled, mounted and imaged as described previously
(Papan et al., 2006, 2007). If embryos were to be injected with dye, they were
de-jellied at the 4- to 8-cell stage by incubating them in 2% cystein pH 8.0 for 4–
5 min and rinsed with Rearing medium. For 3D imaging, the Rearing medium
Fig. 1. 2D time series showing how animal cap tissue moves into the DMZ but not the VMZ during late blastula. Embryonic stages (Nieuwkoop and Faber, 1994) are
given at the upper left. The elapsed time is shown at the lower right in hour:minute relative to the first scan shown. “Hist” shows an optical image of a stage 10.25
embryo for comparison. Images are sagittal, the dorsal side is to the right and the animal pole is up. A color look-up table (shown at the right of the figure) was applied,
with the animal cap (AC) rendered in blue and the vegetal cell mass (veg) in yellow. The blastocoel (bc) is white. The black horizontal lines indicate the position of the
animal cap boundaries at stage 8. Placing the same line on the subsequent images indicates the original position of the boundary. White lines indicate the moving front
of the animal cap tissue in the subsequent stages. For confirmation of the stage, the inset figure in 10.25 shows the blastopore morphology of the embryo immediately
after the scan. In “Hist”, the white dotted lines indicate the boundary between the region of smaller cells of the marginal zone and the larger cells of the vegetal cell
mass. Arrowhead = dorsal blastoporal lip. Scale bar = 200 μm.
C. Papan et al. / Developmental Biology 305 (2007) 161–171
was supplemented with Feridex (Berlex, Seattle, WA) at a 1:40 dilution to
suppress the water signal. No morphological differences were observed in
comparison to sibling embryos developing in a Petri dish. To slow development,
scanning was done at 15 °C. At this temperature, 24 h are required to reach stage
12, yielding sufficient temporal resolution for 3D time series to follow
development. For confocal microscopy, embryos were fixed in MEMFA and
sagittally bisected with a razor blade in order to image just beneath the cut face.
Embryos were cleared and mounted in benzyl alcohol–benzyl benzoate (Sive et
al., 1998) in depression slides. Z-stacks of confocal images of fixed and cleared
embryos were acquired using a Zeiss LSM 510 Axiovert using a C-Apochromat
10× 0.45 W objective lens with pinhole size of ca 2 Airy units at 0.7× scan zoom,
which allows imaging the whole embryo. MR and fluorescence images were
processed using Amira software (Mercury Computer Systems, Inc.). Animal cap
tissue movement was measured using the measuring tool of the Amira software.
For this, a two-color look-up table was applied to the images in a way that
sharply distinguishes the animal and the vegetal tissue (see Fig. 1). The animal
cap advancement between each five scans was measured at the lowest point of
the animal cap-vegetal cell mass boundary as a linear distance.
Contrast agent and fluorescent dye injection
A polymeric gadolinium-based T1 contrast agent (P717 (Corot et al., 1997),
provided by Guerbet Research, France) was supplied as an aqueous NaCl
containing solution. To reduce the amount of sodium, P717 solution was
desalted by diluting 10-times with reverse-osmosis-treated H2O and the volume
then reduced to the original volume with a Microcon YM-10 centrifugal filter
(Millipore, Billerica, MA). This procedure was carried out twice. Desalted P717
was supplemented with 25 mg/ml TexasRed Dextran 10 kDa (Molecular Probes,
Oregon, USA).
Injection capillaries were fabricated from quartz tubing O.D. 1 mm, I.D.
0.75 mm (Sutter Instrument Co, Novato, USA) with a Sutter P-2000
microelectrode puller. Contrast agents and fluorescent dyes were backfilled
into the capillaries and intracellularly injected into individual blastomeres using
a PLI-100 microinjector (Harvard Apparatus, Holliston, USA). Quantification of
the injected amount was done by expelling dye into mineral oil on a
haemocytometer before injections, then determining drop volumes. Single cell
labeling was confirmed under a dissecting stereomicroscope equipped with
fluorescence epi-illumination (Leica).
Results
Pregastrula epiboly moves animal cap tissue into the
superficial dorsal marginal zone
With the aid of intrinsic tissue specific contrast (Jacobs et al.,
2003; Lee et al., 2007; Papan et al., 2007; Sehy et al., 2001), we
can follow the morphogenetic movements of the animal cap and
the vegetal cell mass in time series of developing embryos. Fig.
1 (see also supplementary Movie 1) shows a time-lapse
sequence of pregastrula movements. At stage 8 (Fig. 1) the
embryo is radially symmetric. No difference in tissue
architecture between the dorsal and the ventral marginal zone
is apparent, in agreement with histological observations
(Hausen and Riebesell, 1991). A black line on the dorsal side
and on the ventral side indicates the boundary between the bluelabeled animal cap tissue and the yellow-labeled vegetal cell
mass. Using this line as a landmark, the vegetal-ward
advancement of the animal cap can be observed in subsequent
time-lapse frames (Fig. 1, stages 9–10.25). By stage 9, the
dorsal side of the animal cap has just begun to advance vegetalward (white line in Fig. 1 stage 9). No advancement is observed
on the ventral side. By stage 9.5, the dorsal side of the animal
cap has further advanced but still no advancement is evident on
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the ventral side. By stage 10, the animal cap has advanced about
220 μm on the dorsal side (white line in Fig. 1 stage 10), while
no movement can be seen ventrally. The dorsal blastoporal lip
becomes visible as a slight indentation (indicated by the black
arrowhead). By stage 10.25, the animal cap has advanced about
290 μm from its original location, and now the ventral side too
has begun to advance (ventral white line in Fig. 1 stage 10.25).
The blastoporal lip is prominently visible (black arrowhead),
but no invagination has occurred yet. For staging confirmation,
the inset Figure at stage 10.25 shows an optical image of the
embryo taken immediately after the MRI scan.
The animal cap tissue moves into the superficial DMZ
outside of the Cleft of Brachet (Brachet, 1935). Though the cleft
is not directly visible in the MR images, intrinsic MRI tissue
contrast and the well-known timing and location of its
appearance (Hausen and Riebesell, 1991) allow defining the
cleft between the mesendodermal and ectodermal germ layers,
separating the superficial from the deep DMZ (white dashed
line; stage 10.25). The asymmetry seen in the MR image of the
stage 10.25 embryo correlates with the well-known histological
appearance of a comparable stage embryo (Fig. 1 Hist; see also
plate 18 in Hausen and Riebesell, 1991), where a domain of
smaller cells is present in the DMZ, but not in the ventral
marginal zone (VMZ).
Timing of dorsal vs. ventral pregastrula epiboly
The timing of the dorsal vs. the ventral pregastrula epiboly
movement was found to be variable. In one embryo, the dorsal
and the ventral side appeared to start moving at the same time
and speed, while in three other embryos, the onset of the ventral
movement lagged that of the dorsal movement by five, ten and
twenty scans respectively. In two further embryos, both sides
began moving at the same time, but the dorsal side moved faster
compared to the ventral side.
To quantify the difference of the pregastrula movement, we
measured the advancement of the animal cap at the dorsal
side and the ventral side in six embryos. The measurements
are summarized in Fig. 2. The diagram shows the traveled
Fig. 2. Pregastrula epiboly movement of the animal cap tissue, measured in six
embryos. Abscissa: scan number (6:44 min/scan); Ordinate: distance in
micrometers. Open circles: dorsal side of the animal cap; solid circles: ventral
side of the animal cap. A 3rd order polynomial regression was used to fit the
data. Dashed graph: dorsal; solid graph: ventral.
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distance of the dorsal and ventral animal cap boundary as a
function of the scan number. Image sequences were aligned
with respect to the first sign of the upward movement of the
mesendodermal mantle to provide a time frame independent
of the animal cap movement. The polynomial regression lines
indicate the average pregastrula epiboly movement of the
dorsal and ventral side of the animal cap. It can be seen that,
on average, the dorsal side begins to move about 10 scans
(corresponding to 1 h 7 min) earlier compared to the ventral
side.
Time-lapse tracing of labeled blastomeres shows pregastrula
apposition between the animal cap and the deep DMZ
To define in more detail the spatial and temporal changes of
the tissue relationships brought about by pregastrula movement,
we traced the morphogenetic movements of B1- and C1blastomeres labeled at stage 6 with contrast agent through late
blastula in time-lapse experiments.
B1 descendants move into the superficial DMZ
Fig. 3 (see also Supplementary Movie 2) shows selected
images from a 2D (Figs. 3A–D) and a 3D time series (Figs. 3E–
H) of two different embryos with a labeled B1-blastomere.
Seven such embryos were analyzed. At stage 9 (Figs. 3A and
E), the B1-clones are located mostly above the equator of the
embryo. At stage 9.5 (Figs. 3B and F), the clones have begun to
move vegetally; they continue to extend in the vegetal direction
at stage 10 (Figs. 3D and G). By stage 10.25 (Figs. 3D and H),
the clones have extended vegetally by about 200 μm and almost
reach the blastoporal lip (indicated with white arrowheads in
Figs. 3D and H). Both of the depicted clones are now located
within the superficial DMZ outside of the Cleft of Brachet
(indicated with a black dashed line in Figs. 3D and H). Thus,
these time-lapse images show that pregastrula morphogenetic
movement translocates cells from above the equator of the stage
9 embryo into the superficial DMZ of the stage 10 embryo prior
to the onset of involution movements.
Fig. 3. 2D (A–D) and 3D (E–H) time series showing how labeled B1-blastomere descendants move into the superficial dorsal marginal zone during late blastula
stages. In panels E–H, the front part of the embryo is digitally cut away to facilitate visualization of internal structures. bc = blastocoel; veg = vegetal cell mass;
asterisks = labeled cell clone; black dashed line: Cleft of Brachet. Embryonic stages (Nieuwkoop and Faber, 1994) are shown at the bottom of each panel. Arrowheads
point to the dorsal blastoporal lip. Images are sagittal with dorsal to the right and the animal pole up. The medium surrounding the lower embryo is black because the
Feridex added to the medium makes the water appear dark in MRI. Scale bar = 200 μm.
C. Papan et al. / Developmental Biology 305 (2007) 161–171
C1 descendants move into the deep and superficial DMZ
Fig. 4 (see also supplementary Movie 3) shows selected
images from a 2D (Figs. 4A–D) and a 3D time series (Figs.
4E–H) of two different embryos with a labeled C1-blastomere.
Nine C1-labeled embryos were analyzed. At stage 9 (Figs. 4A
and E), the C1-clones are located at or slightly below the
embryo's equator. The C1-clones span the entire thickness of
the DMZ from the blastocoel floor to the embryo surface. By
stage 9.5 (Figs. 4B and F), the superficial parts of the clones
have moved vegetal-ward. This movement has become more
apparent at stage 10 (Figs. 4C and G), when pregastrula
epiboly has begun to move unlabeled tissue from the animal
cap into the superficial DMZ (white arrows). In contrast, the
deeper parts of the clones have not moved vegetal-ward to the
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same extent; thus, a superficial layer of unlabeled tissue (white
arrows in Fig. 4) comes to lie above the labeled deep tissue. By
stage 10.25 (Figs. 4D and H), pregastrula epiboly movements
have moved the outer part of the clones further vegetal-ward
into the lower superficial DMZ; more unlabeled tissue has
moved into the superficial DMZ superficial to the labeled deep
DMZ. Thus, two distinct clone subsets can be distinguished: a
large part in the deep DMZ inside the Cleft of Brachet
(indicated with a black dashed line), and a second part in the
lower superficial DMZ outside of the Cleft of Brachet.
Tissue relationship examined by optical microscopy
To confirm the observations made using MRI, we compared
the stage 10 configurations of the B1- and the C1-clones using
Fig. 4. 2D (A–D) and 3D (E–H) time series showing how labeled C1-blastomere descendants move into the deep and the superficial dorsal marginal zone during late
blastula stages. Images are sagittal with dorsal to the right and the animal pole up. In panels E–H, the front part of the embryo is digitally cut away to facilitate
visualization of internal structures. bc = blastocoel; veg = vegetal cell mass; asterisk = labeled cell clone; black dashed lines: Cleft of Brachet. The white dashed lines in
panels E–H outline the blastocoel. White arrowheads point to the dorsal blastoporal lip. In 3D images, the blastoporal lip is not apparent, so the arrowhead indicates its
approximate location. Embryonic stages (Nieuwkoop and Faber, 1994) are shown at the bottom of each panel. White arrows indicate the unlabeled animal cap tissue
that moves into the superficial DMZ overlaying the labeled C1 descendants of the deep DMZ. Black arrowhead: C1-clone lining of the blastocoel floor; dotted white
line: blastocoel floor lining of the VCM as defined by the intrinsic image contrast (see text). Scale bar = 200 μm.
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confocal microscopy of embryos in which both blastomeres had
been injected with different fluorophores at the 32-cell stage in
the same embryo. While this does not allow the tracking of the
developmental history of the labeled cells over time, it has the
advantage of showing directly the complementary positions of
the two neighboring labeled sets of clonal descendants at a
particular time point in development.
Fig. 5 shows two examples of stage 10 embryos in which
the B1-blastomere was labeled with fluorescein and the C1blastomere with Texas red. The configuration of the dyelabeled descendants agrees with the observations made by the
time-lapse sequences: the B1-clone is mostly located in the
superficial marginal zone and the C1-clone makes a large
contribution to the deep DMZ, underlying the B1-derived
tissue in the superficial marginal zone. The C1-clone also
contributes to the superficial DMZ just above the blastoporal
lip. Note that the boundary between the clones roughly
coincides with the Cleft of Brachet (indicated by dashed lines),
which, due to the volume rendering technique, is not well
defined in the images. In Fig. 5B, it is evident that cells of both
clones straddle this boundary. For the case of the B1-clone, this
means that most cells are located in the outer presumptive
ectoderm, while some clonally related sibling cells are located
within the mesendoderm.
Interrelation of blastocoel floor expansion and DMZ
formation
Beginning at the onset of gastrulation, the blastocoel floor
(BCF) expands (Winklbauer and Schürfeld, 1999), and cells
labeled in the center of the blastocoel floor at stage 9 have been
found to have moved toward the dorsal side of the embryo by
stage 10.5 (Jones et al., 1999). We directly analyze the BCF
expansion process from time-lapse sequences in further detail
and relate it to the animal cap tissue as defined by labeled B1and C1-blastomeres.
Timing of BCF expansion
The timing of the blastocoel floor expansion can best be
observed in a 2D time series as shown in Figs. 4A–D. The
intrinsic contrast defines the edge of the blastocoel floor, which
is indicated with a white dotted line. At stage 9 (Fig. 4A), the
BCF is separated from unlabeled animal cap tissue by the
labeled tissue of the C1-clone, which is exposed to the blastocoele (black arrowhead in Fig. 4A). As the blastocoel floor
expands, the amount of labeled C1-tissue lining the blastocoel
becomes successively reduced (Figs. 4B and C). At stage 10
(Fig. 4C), the clone retains only limited contact with the
blastocoel floor (black arrowheads in Fig. 4C). By stage 10.25
(Fig. 4D), as the blastocoel floor has maximally expanded, the
labeled cells have disappeared from the blastocoel lining
because the BCF has overgrown the C1-clone. In this way, the
BCF has moved into the upper deep marginal zone beneath the
Cleft of Brachet, where it has now come into contact with
unlabeled tissue from the animal cap. These images show clearly
that the BCF expansion begins earlier than previously thought
(at around stage 9) and cells of the BCF already contribute to the
deep DMZ by the onset of gastrulation at stage 10.
Note that the blastocoel floor overgrows the labeled clone
cells because the whole BCF is elevating and thus raising above
the original deep marginal zone (compare the level of the BCF
in Figs. 4A–D and Supplemental Movie 3). However, at the
same time the labeled clone cells destined to populate the deep
marginal zone are moving downwards only little. The cells
destined to form the outer marginal zone are moving down to a
greater extend and in this way overlaying the deep marginal
zone cells as explained above. This simultaneous movement can
best be understood in the movie sequence of the C1-labeled
embryo (Supplemental Movie 3).
Site of the vegetal cell mass—animal cap apposition
Fig. 5. 3D volume renderings of confocal image stacks of two stage 10
embryos showing the spatial relationship at the onset of gastrulation between
a fluorescein-labeled B1-clone (rendered in green) and a Texas-red-labeled
C1-clone (rendered in orange/red). Bc: blastocoel; Veg: vegetal cell mass;
asterisk: site of the dorsal blastoporal lip; white arrowheads: deep portion of the
C1-clone underlying the more superficial B1 cells (black arrowheads); white
arrow: region of intermingling between the C1- and the B1-clones; dashed line:
Cleft of Brachet. The Cleft of Brachet is not directly apparent in the images
because the figures are volume renderings of an approximately 300-μm-thick
image stack and the lower signal intensities of the unlabeled tissue set
transparent. Scale bar = 200 μm.
The site of the apposition between the rim of the vegetal cell
mass (VCM) and the animal cap (AC) was determined from
time-lapse sequences of embryos in which a B1-blastomere had
been labeled. Fig. 6 shows a 2D time series (Figs. 6A and B) and
a 3D time series (Figs. 6C and D) of two such labeled embryos.
At stage 9 (Figs. 6A and C), the B1-clones are situated in the
animal cap in both embryos. In the first embryo (Fig. 6A), the
labeled clone cells are seen just above the embryo's equator,
reaching 300 μm into the animal cap. In the second embryo
(Fig. 6C), the labeled cells reach about 200 μm higher toward
the animal pole. At stage 10 (Figs. 6B and D), pregastrula
epiboly has shifted the clones into the DMZ and the blastocoel
floor has expanded. In the first embryo, the clone has
completely moved into the superficial DMZ and the edge of
C. Papan et al. / Developmental Biology 305 (2007) 161–171
167
Fig. 6. Formation of the contact between the animal cap and the vegetal cell mass during late blastula. (A–B) 2D time series; (C–D) 3D time series with the front part of
the embryo digitally cut away to facilitate visualization of internal features. Veg: vegetal cell mass; bc: blastocoel. A dashed line outlines the blastocoel in panels C and
D. White arrowheads: blastoporal lip. Embryonic stages (Nieuwkoop and Faber, 1994) are shown in the upper left of each panel. In both embryos, a B1-blastomere was
labeled with contrast agent at the 32-cell stage, but occupies different regions in the embryo due to variability in the early blastomere cleavage patterns. The clones are
visible as a bright region in the animal cap at the dorsal side. The regions, which will contact each other, are indicated with an asterisk and the direction of movement is
indicated with white arrows. At stage 9 (A, C), both clones are located in the lower animal cap. By stage 10 (B, D), morphogenetic movements have brought these
tissue regions in close proximity to each other: In panel B, the upper limit of the clone is now on the level of the blastocoel floor, and in panel D, one half of the clone
has moved into the superficial DMZ. At the same time, the blastocoel floor has expanded, now contacting the animal cap. Scale bar = 200 μm.
the blastocoel floor is in apposition to the anterior/animal limit
of the B1-clone (Fig. 6B). In the second embryo, only the
posterior half of the labeled clone has moved into the DMZ,
reflecting the initially more animal location of the clone, so that
the edge of the blastocoel floor is contacting the middle of the
clone (Fig. 6D). The contact site is indicated by asterisks. These
images directly show that the edge of the blastocoel floor comes
into contact with the animal cap by stage 10; by this stage, the
vegetal cell mass has moved into the upper/anterior deep DMZ
where it contacts the animal cap approximately at the boundary
between the A-tier and the B-tier blastomeres.
(DMZ) and Spemann organizer (Spemann and Mangold, 2001),
could not be adequately described. Using time-lapse microscopic MRI, we were able to eliminate the problem of tissue
opacity and observe the process of the DMZ formation noninvasively in live embryos. Because a single embryo is tracked
over time, time-lapse MRI also overcomes the problem of
embryo-to-embryo variability (Ewald et al., 2004; Masho,
1988), which makes the analysis of internal movements from
time-course data difficult. From our studies, we found that
pregastrula epiboly begins during late blastula around stage 9
and changes the structure of the dorsal marginal zone of the
stage 10 embryo (Fig. 1).
Discussion
Pregastrula epiboly leads to marginal zone asymmetry
Epiboly begins during late blastula
Histological examinations have suggested that the first
morphogenetic movements occur prior to stage 10 Xenopus
gastrula embryos (Bauer et al., 1994; Brachet, 1935; Hausen
and Riebesell, 1991; Holtfreter, 1943; Keller, 1978; Nieuwkoop
and Florschütz, 1950; Schechtman, 1934; Vodicka and Gerhart,
1995; Vogt, 1929). However, due to the opacity of the
amphibian embryos, the precise nature of these movements,
and their role for the formation of the dorsal marginal zone
From histological preparations, the tissue structure of the
early Xenopus embryo is well known: at early-to-mid blastula
stages, the embryo is rotationally symmetric, with smaller cells
in the animal cap and larger cells in the vegetal cell mass. By
stage 10, the internal structure the embryo has become
asymmetric; the dorsal marginal zone is populated with smaller
cells reaching deep into the embryo, while the ventral marginal
zone is not (Hausen and Riebesell, 1991; Nieuwkoop and
Florschütz, 1950).
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Measurements of the pregastrula epiboly in six embryos
(Fig. 2) showed some variability between the early pregastrula
movements of the dorsal and ventral side of the embryo. In
some cases, the dorsal side begun to move earlier, while in other
cases both sides begun to move at the same time but faster on
the dorsal side. In one case, both sides moved at the same time
and speed. The average measurement from six embryos showed
that pregastrula epiboly begins about 1:07 h earlier on the dorsal
side than on the ventral side. From this, we suggest that the
pregastrula epiboly movement establishes the dorso-ventral
asymmetry of the marginal zone seen in histological images.
Pregastrula epiboly movements resulting in dorso-ventral
asymmetry of the embryo may be widespread among lower
vertebrates, as a similar morphogenetic pattern is found in the
zebrafish where the blastoderm advances from 30% to 50%
epiboly prior to the onset of gastrulation (Kimmel et al., 1995).
As a result, the rotationally symmetric blastula-stage zebrafish
embryo becomes bilaterally symmetric well before the onset of
the involution/ingression movements (Schmitz and CamposOrtega, 1994).
Planar-to-vertical tissue rotation within the DMZ
To investigate the tissue rearrangements in more detail, we
studied the pregastrula morphogenetic movements in the DMZ
with the aid of cell labeling (Figs. 3 and 4). At stage 9, the
descendants on the B1- and the C1-blastomeres are in locations
reminiscent of their locations at the time of labeling at stage 6.
The B1-clone is located above or animal-ward of the C1-clone
in a planar (also termed horizontal) juxtaposition. With the onset
of the pregastrula epiboly movements, the B1-derived tissue
moves into the DMZ outside of the Cleft of Brachet. Given that
the C1 descendants in the deep DMZ remain in position, the
downward-moving B1-tissue comes to overlie this C1-derived
internal tissue. Thus, the tissue within the DMZ folds from a
planar/horizontal into a radial/vertical juxtaposition, bringing
non-involuting presumptive ectodermal tissue from the B-tier
blastomere in vertical/radial contact with the deeper mesendodermal tissue of the C-tier blastomere before the onset of
gastrulation and without involution.
This shearing movement effectively generates an apposition
similar to the result of an involution movement. An internal
involution movement within the deep DMZ tissue characterizing the onset of gastrulation in Xenopus has been described
previously by Nieuwkoop and Florschütz (1950). Based on
their fixed histological sections the authors describe a rolling up
of dorsal internal material around an internal lip at the beginning
of gastrulation. Our study confirms the anatomy they report but
shows that this apposition is not brought about by the
movements they envisaged; instead, it results from the surface
layer moving downward over the internal tissue. Therefore, this
process should not be termed internal involution.
Strong shifting of C1-derived tissue from a marginal/animal
toward the dorso-vegetal position has been suggested previously. In a fate-mapping analysis for the Spemann organizer
precursors (Bauer et al., 1994), the C1-blastomere was reported
to rotate all the way from the equatorial region into the vegetal
cell mass below the limit of the dorsal blastoporal lip. Our
observations correct this notion by showing that there is no such
strong displacement of the C1-blastomere (Vodicka and
Gerhart, 1995).
Spatial relation between neural and mesodermal precursors at
the onset of gastrulation
The time-lapse study of the B1-blastomere (Fig. 3) shows
that tissue from this blastomere moves into the superficial DMZ
and comes to overlie the deep tissue that originates from the C1blastomere. Fate mapping studies (Keller, 1975, 1976; Vodicka
and Gerhart, 1995) show that the descendants of the B1blastomere at stage 10 (Fig. 3) contribute to the non-involuting
dorsal marginal zone (NIDMZ), and thus will give rise to
neuroectoderm. To clarify these relationships, Fig. 7 shows an
overlay of an embryo in which a C1-blastomere was labeled
with the surface fate map of the stage 10+ embryo (after Keller,
1975, modified). Our studies demonstrate that the stage 10+
presumptive neuroectoderm is underlain by C1-derived tissue,
which previous studies have shown to possess neural-inducing
capability (Jones et al., 1999; Lupo et al., 2002; Nieuwkoop and
Koster, 1995). Thus, the pregastrula epiboly movement brings
presumptive neuroectoderm into vertical contact with inductive
mesendodermal precursors during late blastula, independent of
the involution/invagination movements of gastrulation.
A vertical relation between presumptive prechordal mesoderm and neuroectoderm in the Xenopus embryo at the onset of
gastrulation was observed by (Nieuwkoop and Koster, 1995).
Our study agrees with their original notion; however, the
authors explain this relationship as occurring through an
Fig. 7. Overlay of a sagittally oriented 3D NMR image of a stage 10.25 embryo
with a stage 10+ surface fate map after Keller (modified) (Keller, 1975, 1976).
The front half of the 3D NMR image has been digitally cut away to facilitate
the visualization of the inner structures. The regions above the double line
(white arrowhead) are presumptive ectoderm, and regions below the double
line are presumptive mesendoderm. AN = anterior neuroectoderm; MN = midneuroectoderm; PN = posterior neuroectoderm; NIDMZ = non-involuting dorsal
marginal zone; IDMZ = involuting dorsal marginal zone. The black dashed line
indicates the Cleft of Brachet, which is forming at this developmental stage.
White arrowhead = dorsal blastoporal lip. Scale bar: 200 μm.
C. Papan et al. / Developmental Biology 305 (2007) 161–171
internal involution movement beginning at stage 10. Our data
show that this interaction is brought about by simultaneous
pregastrula epiboly and blastocoel floor expansion, beginning at
stage 9.
The role of blastocoel floor expansion in DMZ formation
The onset of the blastocoel floor expansion (Winklbauer and
Schürfeld, 1999) has been reported to begin at the time of the
pigment line appearance at stage 10. Our direct time-lapse
observations show that blastocoel floor expansion on the dorsal
side is already beginning at stage 9, so that a considerable part
of the apposition between the vegetal cell mass and the animal
cap is already established by stage 10 (Figs. 4A–D). As the BCF
expands, it overgrows the cells previously at the edge of the
BCF (the labeled C1 descendants) coming to lay animal-ward of
them. In this way, the anterior subset of the deep tissue of the
DMZ (inside the Cleft of Brachet) is composed of descendants
of the D-tier blastomeres, while its posterior subset is composed
of C-tier descendants. It should be noted that the ventral side of
the BCF does not significantly extend until the beginning of the
ventral epiboly movement (data not shown).
The site of apposition between the blastocoel floor and the
animal cap at stage 10 is located approximately at the boundary
between the A- and the B-tier descendants. The coincident
motions of BCF expansion and epiboly move tissue vegetal
mass and from the animal cap into the DMZ. This brings
169
presumptive anterior neuroectoderm into contact with the
expanding margin of the blastocoel floor by stage 10. This is
in agreement with observations by Koide et al. (2002), who
showed that as early as stage 10.25, the leading edge of
gastrulation has reached the anterior neuroectoderm in the
animal cap.
Model of DMZ formation in Xenopus laevis
Based on time-lapse MRI data, we present a model for the
pregastrula tissue movements leading to the formation of the
DMZ of a stage 10 embryo (Fig. 8). At stage 9, morphogenetic
movements begin to transform the dorsal marginal zone (Fig.
7A). Epiboly moves tissue from the animal cap vegetal-ward;
the internal tissue from the C-region does not move to the same
extent, as evident by the tilting of the interface between the Bregion (orange in Fig. 8) and the C-region (blue in Fig. 8). The
shearing motion of the B-descendants over the C-descendants
transforms the original animal–vegetal boundary between
regions into an exterior–interior boundary, which approximately coincides with the Cleft of Brachet. At the same time, the
blastocoel floor expands. Cells in a more medial location in the
BCF overgrow the cells at the rim; by the onset of gastrulation at
stage 10, they have come in direct contact with the animal cap
cells. In this way, the deep DMZ underneath the non-involuting
marginal zone is composed of cells from D-tier blastomeres
(anterior) and from C-tier blastomeres (posterior).
Fig. 8. Semi-schematic model depicting the morphogenetic movements and the resulting changes in tissue relationships during late blastula stages. The B-blastomere
region is colored in orange and the C-blastomere region is colored in blue. Embryonic stages are shown below the figures. For details see text. Bc: blastocoel; Veg:
vegetal cell mass. Arrowheads: dorsal blastoporal lip; IMZ: involuting marginal zone; NIMZ non-involuting marginal zone; dashed line: Cleft of Brachet. Scale
bar = 200 μm.
170
C. Papan et al. / Developmental Biology 305 (2007) 161–171
Note that in our model, the boundary between the B- and the
C-region coincides with the future Cleft of Brachet (dashed
lines in Fig. 8). In embryos in which the B1-blastomere
occupies a more vegetal position at stage 6 (due to the
variability in early cleavage patterns; Masho, 1988, 1990; Wetts
and Fraser, 1989), some of the B1 descendants would be found
on the inside of the Cleft of Brachet (see Fig. 5B). This can
explain why B-tier blastomeres, while giving rise to a large
fraction of anterior neural tissue (retina, brain), often give rise to
a significant populations of anterior mesodermal and endodermal tissue (Bauer et al., 1994; Dale and Slack, 1987). In an
involution-based gastrulation model, this separation of fates
would be problematic, as it must involve long-range cell
migrations, which seem inconsistent with cell lineage studies
(Wetts and Fraser, 1989). For example, the mesodermal
descendants of the B1-blastomere would need to migrate
toward the blastopore lip, involute and then move all the way to
the animal pole (a distance of roughly 1 mm) in 6–8 h, while
sibling cells with a neural fate would remain on the embryo's
surface and move little. Our in vivo MRI observations offer a
much simpler scenario: cleavages of the B1-blastomere separate
it into deeper (anterior mesoderm) and superficial (neuroectoderm) daughters, which become separated by later shearing
motions and mitoses. Because the B1 descendants fated to
become anterior mesoderm are already in place in the deeper
regions, there is no need for the long-range migrations required
of the involution models.
The rotation of the boundary between the B-descendants and
the C-descendants from a horizontal/planar into a vertical/radial
configuration resembles an “internal involution”. However, in a
true involution movement, the internalizing tissue folds back
over the external cell layer, distinct from the motions we
document of the external tissue moving over the deeper tissue
layers.
Acknowledgments
We thank Vladimir Korzh, Rachel S. Kraut and Rudolf
Winklbauer for suggestions to improve the manuscript, Michael
J. Tyszka and Seth Ruffins for technical discussion and Andrey
Demyanenko for building the NMR coil. We thank Dr. Claire
Corot (Guerbet Research, France) for the generous gift of the
P717 contrast agent. This work was supported by the NIH Grant
number HD25390. C.P. was in part supported by the Deutsche
Forschungsgemeinschaft, Grant number PA 562/1-1.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.ydbio.2007.02.005.
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