Download Self-Organization of Vertebrate Mesoderm Based on Simple

DEVELOPMENTAL DYNAMICS 231:576 –581, 2004
RESEARCH ARTICLE
Self-Organization of Vertebrate Mesoderm Based
on Simple Boundary Conditions
Jeremy B.A. Green,1* Isabel Dominguez,1,† and Lance A. Davidson2
Embryonic development requires cell movements whose coordination is robust and reproducible. A dramatic
example is the primary body axis of vertebrates: despite perturbation, cells in prospective axial tissue coordinate their
movements to make an elongated body axis. The spatial cues coordinating these movements are not known. We
show here that cells deprived of preexisting spatial cues by physical dissociation and reaggregation nonetheless
organize themselves into an axis. Activin-induced cells that are reaggregated into a flat disc initially round up into a
ball before elongating perpendicular to the disc. Manipulations of the geometry of the disc and immunofluorescence
micrography reveal that the edge of the disc provides a circumferential alignment zone. This finding indicates that
physical boundaries provide alignment cues and that circumferential “hoop stress” drives the axial extrusion in a
manner resembling late-involuting mesoderm of Xenopus and archenteron elongation in other deuterostome species
such as sea urchins. Thus, a population of cells finds its own midline based on the form of the population’s boundaries
using an edge-aligning mechanism. This process provides a remarkably simple organizing principle that contributes
to the reliability of embryonic development as a whole. Developmental Dynamics 231:576 –581, 2004.
© 2004 Wiley-Liss, Inc.
Key words: Xenopus; gastrulation; convergent extension; hoop stress; polarity; vegetal alignment zone; activin
Received 28 May 2004; Revised 15 June 2004; Accepted 15 June 2004
INTRODUCTION
Embryonic development is evolved
to be robust and reproducible
(Kirschner and Gerhart, 1998). This
finding is often expressed in terms of
redundancy in regulatory networks,
but it applies just as significantly to
cell movements. A dramatic example is the primary body axis of chordates and vertebrates in particular:
despite perturbation, cells in prospective axial tissue coordinate their
movements to make an elongated
body axis (Keller et al., 1985; Munro
and Odell, 2002). Although the cell
movements involved in this morpho-
genesis have been described in exquisite detail, the nature of the cues
that coordinate these movements
and provide the necessary robustness is not known.
The vertebrate embryo transforms
itself during development from a
one-layered structure into a threelayered one and from a round
shape into an elongated one by
means of the well-studied orchestration of cellular movements and behaviors collectively known as gastrulation. The best known of the cell
behaviors is mediolateral intercalation in which mediolaterally elon-
gated cells crawl between one another to drive narrowing and
elongation of axial mesoderm
(Keller, 2002; Wallingford et al.,
2002). A fundamental but unanswered question is where the directional information that initiates and
coordinates the orientation of these
movements comes from. The initial
randomly oriented protrusive activity
of mesodermal cells first becomes
oriented at the intersection of the
future notochord-somite boundary
(NSB) and the vegetal alignment
zone (VAZ; Domingo and Keller,
1995; Lane and Keller, 1997). There is
The Supplementary Material referred to in this article can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat
1
Department of Cancer Biology, Dana Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts
2
Departments of Biology and Cell Biology, University of Virginia, Charlottesville, Virginia
Grant sponsor: National Institutes of Health; Grant numbers: RO1-HD36345; R01-HD26402; R01-HD25594/R01-HD036426.
†
Dr. Dominguez’s present address is Hematology-Oncology, Boston Medical Center, Boston, MA 02118.
*Correspondence to: Jeremy B.A. Green, Department of Cancer Biology, Dana Farber Cancer Institute and Department of Genetics,
Harvard Medical School, Boston, MA 02115. E-mail: [email protected]
DOI 10.1002/dvdy.20163
Published online 16 September 2004 in Wiley InterScience (www.interscience.wiley.com).
© 2004 Wiley-Liss, Inc.
XENOPUS CELL AGGREGATE SELF-ORGANIZATION 577
Fig. 2. a: Frames of a time-lapse video recording showing side and top views of the same
elongating reaggregate at successive times shown. Arrows (center) show upward elongation (black arrow) and direction of toppling (white arrow). b: The shape of the initial disc
of cells determines subsequent elongation: a disc of reaggregating cells with a “peninsula” (arrowed, left) rounds up but then elongates both on its main axis and on a branch
at the site of the “peninsula” (arrowheads and three-headed arrows, right). Scale bar ⫽ 250
␮m in a,b.
Fig. 1. Aggregates of activin-treated cells
elongate in vitro. a–f: Elongation is dosedependent (treated with 0, 0.25, 0.5, 1.0, 2.0,
and 4.0 units/ml activin, respectively). g,aⴕ–
gⴕ: Elongation is evident at neurula stages
(g, control embryo, stage 18) and is more
pronounced by tail bud stage (aggregates
a⬘–f⬘, control embryo, g⬘, stage 24). h: Aggregates contain notochord (MZ15 antibody staining). Scale bars ⫽ 300 ␮m in a
(applies in a– g⬘), 100 ␮m in h.
no evidence for a midline signaling
center that would orient and attract
these movements (Keller et al.,
2000). Despite the beauty of the observations and the models that describe this process, they do not adequately explain why notochordless
ultraviolet (UV) -ventralized embryos
can still undergo gastrulation (Gerhart and Keller, 1986), and, therefore, how cell intercalation becomes oriented in the absence of a
NSB. Furthermore, the absence of
evidence for a midline signaling
center does not constitute evidence
of its absence, and a formal test of
this model has been lacking.
Many aspects of axial convergent
extension, including migratory and
protrusive cell behaviors characteristic of endogenous mesoderm, can
be mimicked by growth factor treatment, especially activin treatment,
of animal pole explants (animal
caps; Symes and Smith, 1987). Elongation of animal cap explants has
now become a commonplace assay for convergent extension, in part
because the assay is relatively
straightforward and provides great
manipulative freedom (Green, 1999;
Yao and Kessler, 2000). Convergent
extension of animal caps also requires coordinated cell orientation.
Both mesodermal explants and
animal cap explants contain potentially orienting information derived
from the zygote. For mesodermal explants, cellular orientation clearly
Fig. 3. Notching of the aggregate disc periphery inhibits elongation. a,b: Smaller excised piece and larger remaining aggregate (a) show much less coherent
elongation that uncut induced aggregate
(b), although a small elongated peninsula
is sometimes seen (compare with Fig. 2b).
c: Control uninduced aggregate.
derives from that of the zygote, because all the relevant tissue arrangements are explanted intact. For ani-
578 GREEN ET AL.
mal cap explants, it has been shown
that they are highly asymmetrical
with respect to inducibility of dorsal–
anterior- and ventral–posterior-derived halves (Sokol and Melton,
1991) and, thus, clearly contain potentially orienting information. It is
plausible, therefore, that coherent
convergent extension requires the
orienting influence of something laid
down earlier in development. The alternative hypothesis is that coherent
convergent extension is generated
autonomously and autocatalytically
with minimal polarity information
from prior events.
RESULTS
We examined the gastrulation behaviors of inner layer animal cap
cells that had been dissociated and
reaggregated to abolish preexisting
polarity information. Unlike intact
mesodermal explants and explanted animal caps, such cells contain no global intrinsic orientation
deriving from the zygote. They are,
thus, a good test of the autonomy of
the cell orientation process. In previous work, we dissociated cells from
animal cap explants by incubation
in calcium-free medium and then
treated them with the protein
growth factor activin to make multiple mesodermal tissue types (Green
et al., 1990). At the right dose, aggregates consist almost entirely of
notochord, but surprisingly failed to
elongate (Green et al., 1992). We
had thought that this finding was because they lacked directional cues.
However, in previous work, rapid reaggregation was achieved by lowspeed centrifugation of cell suspensions (Green et al., 1990). This
centrifugation step abolishes elongation ability (J.B.A. Green and A.
De Jesus-Escobar, data not shown),
perhaps because hydrostatic pressure during centrifugation disrupts
microtubules that are essential for
orientation of intercalation behavior
(Lane and Keller, 1997). Convergent
extension was observed when, instead of centrifuging the cells, they
were allowed to reaggregate spontaneously by pipetting into a pile in
medium containing calcium ions.
Uninduced cells simply rounded up
into a ball, but cells treated with
0.5–2
units/ml
activin
initially
rounded up into a ball then reorganized themselves into an elongated
shape (Fig. 1a). Thus, a population of
cells deprived of all preexisting directional cues from the egg or zygote was able to reconstitute directional cues to organize a coherent
axis. When allowed to differentiate,
such axes contained notochord arranged cylindrically around the aggregate’s long axis (Fig. 1b).
To understand the mechanism of
elongation, we made time-lapse
video recordings (see Supplementary
Material, which can be found at
http://www.interscience.wiley.com/
jpages/1058-8388/suppmat). In the
initial stages, cells rapidly aggregated
into a flat disc or pancake shape (Fig.
2a). This finding presumably represents simple maximization of cell– cell
contact dominated by the initial condition that the cells are sitting on a flat
surface. With time, these disc aggregates “rounded up” into a spherical
shape, regardless of activin induction
and with little influence of the initial
disc shape, whether circular, ellipsoid,
or irregular. This spherical shape would
be predicted by a simple maximization of cell– cell contact. However, after this stage, control and activininduced aggregates immediately
diverged. Control aggregates remained spherical, whereas aggregates
of induced cells elongated upward,
perpendicular to the plastic or agarose-coated surface of the dish. Eventually, the now-elongated cell mass
would topple over but continue elongating (Fig. 2a). The elongation and its
upward orientation were highly reliable,
occurring in 100% of cases.
Simple consideration of the asymmetries of the aggregate led to the
conclusion that cells at the periphery of the disc stage are likely to
experience the most asymmetrical
environment and might, perhaps,
provide the leading cue for the coordination of subsequent convergent extension. By altering the geometry of the cell aggregates at the
disc stage, we were able to determine its significance for subsequent
cell movements. First, we allowed
the disc to form and then cut a
notch or “cake slice” from it. This
strategy dramatically inhibited convergent extension (Fig. 3), suggest-
ing that the disc periphery is indeed
an important component of the
elongation mechanism. We also
made video recordings of aggregates with small peninsulas (Fig.
2b and Supplementary Material).
Like the other aggregates, these
rounded up into spheres, with no obvious appendage at the site of the
preceding peninsula. However,
upon elongation, a secondary axis
appeared at the site of the peninsula (Fig. 2b), showing the significance of the disc stage periphery
and its significance at the disc rather
than the subsequent ball stage.
We examined cell shape in aggregates by fixing and immunofluorescently staining for ␤-catenin protein
to reveal cell outlines. Confocal sections showed that control aggregates had small lumens and contained cells with no signs of
elongation (Fig. 4). Elongating aggregates, by contrast, contained
many elongated cells arranged circumferentially around the geometrical center of the long axes of the
aggregates (Fig. 4). The elongating
cells were all aligned underneath a
layer or two of nonelongated cells.
The arrangement of cells was very
reminiscent of that in posterior lateinvoluted mesoderm of normal tadpoles (Keller et al., 1989; Hausen and
Riebesell, 1991).
DISCUSSION
Our results prove that vertebrate axial cells—which may or may not be a
homogeneous population— do not
need global spatial cues from the
egg or sperm to organize an axis.
Instead, a population of such cells
regenerates an axis based on the
form of the population’s boundaries.
This finding strengthens the hypothesis proposed by Keller and colleagues, that the shape of dorsal axial mesoderm is determined by tissue
boundaries and the center of mass,
not by some preexisting midline signal (Keller et al., 2000). Although sophisticated cell intercalation and bipolarity behaviors underlie this
mechanism, it nonetheless provides
a remarkably simple organizing principle whereby these behaviors are
coordinated macroscopically.
Our data also suggest a mecha-
XENOPUS CELL AGGREGATE SELF-ORGANIZATION 579
Fig. 4. Cells within aggregate axis are circumferentially elongated. a– c: A montage of
confocal sections collected through a bisected aggregate of deep ectoderm cells form
cavities (a) and exhibit isodiametric cell shapes near the cavity (b) and far from the cavity
(c). d: In contrast, a montage of confocal sections collected transverse to the axis of an
activin-induced aggregate produces circumferentially elongated cells. e,f: The outer-most
cell layer is not aligned, but alignment is strong two to three cells from the surface. Scale
bars ⫽ 200 ␮m in a, 50 ␮m in b (applies to b,c,e,f), 120 ␮m in d.
nism whereby the global orientation
of convergent extension can be triggered with minimal (indeed, inevitable) spatial cues (Fig. 5). We observed that the superficial cells in the
aggregates are not elongated and
do not appear to adhere well to one
another (Fig. 4). Therefore, if cells
have a tendency to bipolarity and
exert tensile force along their bipolar
axis, this force will be random in the
middle of an aggregate (Fig. 5a) but
must be circumferential near its
edge, where cells lack outward tension-generating neighbors (Fig. 5b).
If there is positive feedback between tensile force and bipolarity,
as demonstrated, for example, in bipolar fibroblasts cultured in collagen
(Stopak and Harris, 1982), cells at the
periphery will increasingly tend to
align their long axes with each other
circumferentially. Coherent alignment in response to an edge is observed in other systems (Elsdale and
Wasoff, 1976; Bard, 1990). Circumferentially aligned cells will initially link
up to create short arcs of aligned
cells, eventually joining into hoops of
tension running in a great circle
within the aggregate (Fig. 5c). As
these hoops contract they will extrude deep cells into an axis as circumferential intercalation shrinks the
perimeter. Thus, a local cell behavior, namely tension-stimulated tensile
bipolarity, would propagate by local mechanical interactions to
achieve a macroscopic organization and to orient tissue shape
change on a large scale.
Importantly,
this
mechanism
would also apply at any boundary
between a bipolar cell population
and another for which its adhesiveness or tensile resistance is weak. In
the aggregate, this could arise at
the surface layer because superficial cells are a different cell type
(due to signals within the aggregate) and/or because they are
physically poorly anchored. In the
embryo, the edge alignment mechanism would apply to the boundary
between prospective endoderm
and mesoderm—in other words, at
the VAZ. Selective adhesion between germ layer cells is a well-established cell sorting behavior
(Townes and Holtfreter, 1955) and is
particularly evident between marginal zone and vegetal cells of Xenopus (Turner et al., 1989). The localization of the Xenopus VAZ has not
been established precisely with respect to cell fate, but it is certainly in
the vicinity of the junction between
marginal chordamesodermal cells
and more vegetal endodermal or
mesendodermal cells.
An alternative a priori model of
aggregate elongation could have
included radial contraction mediated by radially elongated cells. Radial elongation does occur in vivo: it
accompanies radial intercalation
movements that cause spreading
and thinning (i.e., epiboly) in the embryonic ectoderm (Keller, 1980;
Marsden and DeSimone, 2001) and
may play a role in axial extension
after neural tube closure (Davidson
and Keller, 1999). We did not see any
indications of radial elongation, ruling out this mode of tissue extension.
The cellular morphology of the aggregates and the timing of elongation resemble that of late involuting
mesoderm in Xenopus (see, for example, plate 22A of Hausen and
Riebesell [1991]). It is also reminiscent
of the radially symmetrical gastrulation of other species such as sea urchin, in which circumferential intercalation extends all the way around
580 GREEN ET AL.
posed in flies (Tree et al., 2002). In
that model, as in ours, relatively simple and subtle global cues are reinforced at the cellular level to align
cell orientation. This type of mechanism for global and local polarization is evidently extraordinarily robust
and resistant to radical perturbation.
EXPERIMENTAL PROCEDURES
Fig. 5. A model for cell alignment at cell population boundaries. a: In the middle of an
aggregate (a), cells may elongate transiently but lack cues to coordinate the competing
orientations of their long axes. b: At the periphery of a disc, the outer ends of radially
elongated cells (brown) encounter medium or loose surface cells (lighter yellow) and the
elongation is not sustainable. c: Circumferentially elongated cells (green) can link up and
form a peripheral alignment zone.
the elongating archenteron (Hardin
and Cheng, 1986; Hardin, 1989).
Both of these are clear examples of
a hoop-stress mechanism (Hardin
and Cheng, 1986; Keller and Hardin,
1987). A hoop-stress mechanism
may serve to extrude the axis of UVventralized frog embryos and zebrafish mutants such as somitabun
(Mullins et al., 1996; Hammerschmidt
and Mullins, 2002) and ultimately be
responsible for fatal rupture in some
of those mutant embryos. A similar
hoop-stress mechanism may be important for blastopore closure.
The cellular morphology of the aggregates is indistinguishable from
that of the central notochord region
of the embryo undergoing dorsal
convergent extension. In the latter,
cells are generally bipolar and intercalate along their axis to shorten the
tissue dimension in that direction.
Only the notochord–somite boundary interrupts the propagation of
arcs of alignment by anchoring cells
at their lateral ends, in effect, limiting
contraction to a segment of a hoop.
More posteriorly, these arcs extend
all the way around the blastopore. It
therefore seems likely that hoopstress-driven axial extrusion differs
from dorsal convergent extension
only in its circumferential extent.
Reaggregating activin-induced
cells provides an accessible model
system in which controlled geometry
may enhance our ability to dissect
the cellular and subcellular mechanisms of gastrulation morphogenesis.
For example, this system could be
used to analyze the gradual propagation of cellular alignment (which is
seen in vivo) and to distinguish mechanical force from a graded external cue as the driver. The absence of
a relatively impervious epithelial
layer may also facilitate experiments
with soluble factors added to the
medium. Adding inhibitors, such as
those for gap junctions, mitosis, transcription, and translation, at different
times could be used to establish
whether, when, and how particular
programs of cell behavior are initiated. The morphogenetic effects of
target protein depletion by antisense morpholino oligonucleotides
of relevant proteins can also be analyzed in great detail. Likely interesting proteins would be those involved
in planar cell polarity (PCP), because not only are several PCP-associated proteins implicated in convergent extension, but also the
locally autocatalytic aspects of this
model are reminiscent of a PCP
propagation model recently pro-
Xenopus embryos and cells were
obtained their animal caps excised
in calcium- and magnesium-free
medium as previously described
(Green and Smith, 1990; Green et
al., 1992). Xenopus activin B was
made in baculovirus (materials were
the gift of Dr. Doug Melton, Harvard
University). For time-lapse recording,
cells were pipetted next to a small 45
degree mirror (Edmund Optics, Inc.,
Barrington, NJ) mounted at approximately 60 degrees to the horizontal
by use of a piece of modeling clay
inside the Petri dish (Supplementary
Fig. S1). Activin or control medium
was used to fill the dish to the brim
and a lid placed on top to prevent
evaporation and rippling of the liquid surface. Images were captured
by using a Leica MZ stereo zoom microscope, a Q-Imaging Retiga digital CCD camera and Openlab (Improvision) software.
For fluorescent immunostaining of
notochord, aggregates were fixed
in buffered formaldehyde (MEMFA),
embedded in acrylamide, frozen,
cryostat-sectioned, and stained as
previously described (Green et al.,
1992). For immunostaining of ␤-catenin, aggregates were fixed in icecold Dent’s (80% methanol 20% dimethyl sulfoxide; Dent et al., 1989)
and rehydrated and incubated
overnight with rabbit polyclonal
against ␤-catenin (Sigma; C-2206).
After incubation with a rhodamineconjugated secondary antibody
(Jackson ImmunoResearch Laboratories; West Grove, PA) and washing,
whole aggregates were cut with a
scalpel transverse to the direction of
elongation and the fragments dehydrated in ethanol and mounted in
clearing medium (2:1 benzyl benzoate: benzyl alcohol). Cleared samples were mounted and sealed in
shallow wells formed by nylon washers (Small Parts, Inc.; Miami Lakes, FL)
XENOPUS CELL AGGREGATE SELF-ORGANIZATION 581
glued in place by finger nail polish
(Sally Hanson “Hard as Nails,” clear;
Farmingdale, NY). Serial optical sections were collected by using either
a ⫻20 (dry lens; n.a. 0.70) or a ⫻60
(oil immersion; n.a. 1.40) and a confocal laser scan head (Nikon
PCM2000; Melville, NY) mounted on
an inverse compound microscope
(Nikon). The eight-bit image stacks
were transferred to a computer and
analyzed with image processing
software (ImageJ; Wayne Rasband
NIH). Single image projections were
created using the “brightest-point”
algorithm.
NOTE ADDED IN PROOF
While this article was in press, Winklbauer and colleagues published
data consistent with our findings,
showing that an activin-inducible
cell-type boundary corresponding
to the VAZ aligns convergent cell
movements (Ninomiya et al. [2004]
Nature 430:364 –367).
ACKNOWLEDGMENTS
We thank José DeJesus for assistance with Figure 1h; Doug Melton
for activin baculovirus constructs;
and Sergei Sokol, Olga Ossipova,
Karen Symes, and Nick Hirsch for
comments on the manuscript. J.B.A.
Green was funded by the National
Institutes of Health.
REFERENCES
Bard JBL. 1990. Morphogenesis: the cellular and molecular processes of developmental
anatomy.
Cambridge:
Cambridge University Press. p 145–157.
Davidson LA, Keller RE. 1999. Neural tube
closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergentextension.Development126:4547–
4556.
Dent JA, Polson AG, Klymkowsky MW.
1989. A whole-mount immunocytochemical analysis of the expression of
the intermediate filament protein vimentin in Xenopus. Development 105:
61–74.
Domingo C, Keller R. 1995. Induction of
notochord cell intercalation behavior
and differentiation by progressive signals in the gastrula of Xenopus laevis.
Development 121:3311–3321.
Elsdale TR, Wasoff FL. 1976. Fibroblast cultures and dermatoglyphics: the topology of two planar patterns. Wilhelm
Rouxs Arch Dev Biol 180.
Gerhart J, Keller R. 1986. Region-specific
cell activities in amphibian gastrulation. Annu Rev Cell Biol 2:201–229.
Green JBA. 1999. The animal cap assay.
Methods Mol Biol 127:1–13.
Green JBA, Smith JC. 1990. Graded
Changes in dose of a Xenopus activin
A homologue elicit stepwise transitions
in embryonic cell fate. Nature 347:391–
394.
Green JBA, Howes G, Symes K, Cooke J,
Smith JC. 1990. The biological effects of
XTC-MIF: quantitative comparison with
Xenopus bFGF. Development 108:173–
183.
Green JBA, New HV, Smith JC. 1992. Responses of embryonic Xenopus cells to
activin and FGF are separated by multiple dose thresholds and correspond
to distinct axes of the mesoderm. Cell
71:731–739.
Hammerschmidt M, Mullins MC. 2002.
Dorsoventral patterning in the zebrafish: bone morphogenetic proteins
and beyond. Results Probl Cell Differ
40:72–95.
Hardin J. 1989. Local shifts in position and
polarized motility drive cell rearrangement during sea urchin gastrulation.
Dev Biol 136:430 –445.
Hardin JD, Cheng LY. 1986. The mechanisms and mechanics of archenteron
elongation during Sea Urchin gastrulation. Dev Biol 115:490 –501.
Hausen P, Riebesell M. 1991. The early
development of Xenopus laevis: an atlas of the histology. Berlin: Springer-Verlag.
Keller RE. 1980. The cellular basis of epiboly: an SEM study of deep-cell rearrangement during gastrulation in Xenopus laevis. J Embryol Exp Morphol 60:
201–234.
Keller R. 2002. Shaping the vertebrate
body plan by polarized embryonic cell
movements. Science 298:1950 –1954.
Keller R, Hardin J. 1987. Cell behaviour
during active cell rearrangement: evidence and speculations. J Cell Sci
Suppl 8:369 –393.
Keller RE, Danilchik M, Gimlich R, Shih J.
1985. The function and mechanism of
convergent extension during gastrulation of Xenopus laevis. J Embryol Exp
Morphol 89:185–209.
Keller R, Cooper MS, Danilchik M, Tibbetts
P, Wilson PA. 1989. Cell intercalation
during notochord development in Xenopus laevis. J Exp Zool 251:134 –154.
Keller R, Davidson L, Edlund A, Elul T, Ezin
M, Shook D, Skoglund P. 2000. Mecha-
nisms of convergence and extension
by cell intercalation. Philos Trans R Soc
Lond B Biol Sci 355:897–922.
Kirschner M, Gerhart J. 1998. Evolvability.
Proc Natl Acad Sci U S A 95:8420 –8427.
Lane MC, Keller R. 1997. Microtubule disruption reveals that Spemann’s organizer is subdivided into two domains by
the vegetal alignment zone. Development 124:895–906.
Marsden M, DeSimone DW. 2001. Regulation of cell polarity, radial intercalation and epiboly in Xenopus: novel
roles for integrin and fibronectin. Development 128:3635–3647.
Mullins MC, Hammerschmidt M, Kane DA,
Odenthal J, Brand M, van Eeden FJ,
Furutani-Seiki M, Granato M, Haffter P,
Heisenberg CP, Jiang YJ, Kelsh RN, Nusslein-Volhard C. 1996. Genes establishing dorsoventral pattern formation in
the zebrafish embryo: the ventral specifying genes. Development 123:81–93.
Munro EM, Odell G. 2002. Morphogenetic pattern formation during ascidian notochord formation is regulative
and highly robust. Development 129:1–
12.
Ninomiya H, Elinson RP, Winklbauer R. 2004.
Antero-posterior tissue polarity links mesoderm convergent extension to axial
patterning. Nature 430:364–367.
Sokol S, Melton DA. 1991. Pre-existent
pattern in Xenopus animal pole cells
revealed by induction with activin. Nature 351:409 –411.
Stopak D, Harris AK. 1982. Connective tissue morphogenesis by fibroblast traction. I. Tissue culture observations. Dev
Biol 90:383–398.
Symes K, Smith JC. 1987. Gastrulation
movements provide an early marker of
mesoderm induction in Xenopus laevis.
Development 101:339 –349.
Townes PL, Holtfreter J. 1955. Directed
movements and selective adhesion of
embryonic amphibian cells. J Exp Zool
128:53–120.
Tree DR, Ma D, Axelrod JD. 2002. A threetiered mechanism for regulation of planar cell polarity. Semin Cell Dev Biol
13:217–224.
Turner A, Snape AM, Wylie CC, Heasman
J. 1989. Regional identity is established
before gastrulation in the Xenopus embryo. J Exp Zool 251:245–252.
Wallingford JB, Fraser SE, Harland RM.
2002. Convergent extension: the molecular control of polarized cell movement during embryonic development.
Dev Cell 2:695–706.
Yao J, Kessler DS. 2000. Mesoderm induction in Xenopus. Oocyte expression system and animal cap assay. Methods
Mol Biol 137:169 –178.