1 Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254,
USA
2 Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY,
UK
* Present address: Developmental Genetics Program and Department of Cell
Biology, Skirball Institute of Biomolecular Medicine, New York University
School of Medicine, New York, NY 10016, USA
Present address: Department of Anatomy, University of Cambridge, Downing
Street, Cambridge CB2 3DY, UK
Author for correspondence (e-mail:
rja46{at}cam.ac.uk)
Accepted 18 November 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Gradients, Morphogenesis, no tail, Mediolateral intercalation behavior, Notochord, Convergence, Extension, Gastrulation, Epiboly, Zebrafish
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our current view of the cellular behaviors that drive these tissue-level
shape changes comes primarily from a series of studies of explanted
Xenopus dorsal mesoderm (Shih and
Keller, 1992a; Shih and
Keller, 1992b
; Keller et al.,
2000
). Molecular and genetic studies (including studies in
zebrafish and other species) have enriched our current understanding
(Solnica-Krezel, 1999
;
Tada and Concha, 2001
;
Wallingford and Harland, 2001
;
Myers et al., 2002a
;
Myers et al., 2002b
). The
explant studies in Xenopus show that dorsal mesodermal converges and
extends largely by cell rearrangement within the tissue, and without
dependence upon an external substrate
(Shih and Keller, 1992a
).
Furthermore, from this work has evolved the concept of a single but complex
cellular behavior, termed mediolateral intercalation behavior (MIB), which
underlies the rearrangements (Fig.
1). The MIB hypothesis is elegant because a single
force-generating cellular machine, distributed across a field of cells,
produces both convergence and extension, both narrowing and elongation of the
field. By the MIB hypothesis, as applied particularly to the domain of
notochord-forming cells within dorsal mesoderm, motile and adhesive cells
become polarized along one particular axis, the mediolateral (ML) axis. The
polarity may depend on, and be coordinated within the field, by a noncanonical
Wnt signaling planar polarity pathway (Choi
and Han, 2002
; Heisenberg et
al., 2000
). The cells take on a bipolar shape, elongating along
the ML axis. This process requires that the individual cells all correctly
orient actin-based cytoskeletal machinery that mediates motility, and perhaps
also orient associated adhesion complexes on their plasma membranes
(Montell, 1999
;
Zalik et al., 1999
). Localized
release of intracellular Ca2+, via connexin 43 channels
(Essner et al., 1996
), and
activation of Rho GTPases may be crucial in such polarized cellular
morphogenesis (Choi and Han,
2002
; Hall and Nobes,
2000
; Smith et al.,
2000
). The cells intercalate mediolaterally. To accomplish this,
they all protrude filopodial processes both medially and laterally that extend
between immediate cellular neighbors, and new cell-cell contacts are made,
perhaps involving adhesion molecules of the cadherin/protocadherin superfamily
localized to their filopodial tips (Keller
et al., 2000
). The newly contacting cells then contract their
processes. They exert traction upon one another and pull together. Such
events, which occur across the entire field of cells, narrow the field
(convergence). The resulting intercalations push previously neighboring cells
apart along the AP axis, lengthening the field along this axis
(extension).
|
Previous work suggests that MIB may also underlie convergence and extension
in the zebrafish gastrula (Concha and
Adams, 1998; Heisenberg et
al., 2000
; Kimmel et al.,
1994
; Solnica-Krezel et al.,
1996
; Warga and Kimmel,
1990
; Myers et al.,
2002a
; Myers et al.,
2002b
). The cells disperse along the AP axis by intercalating with
neighbors to form a discontinuous AP string, as expected if MIB mediates
extension (Kimmel and Warga,
1986
; Kimmel et al.,
1994
). However, it is not known to what extent the phenomenon is
coupled to convergence as the MIB hypothesis predicts it should be.
Additionally, other more coherent cellular flows, e.g. migrations that do not
involve cellular intercalations, might also occur to increase the AP length of
the developing notochord.
To test the MIB model in zebrafish, we used a confocal microscope to image
and make time-lapse recordings of the dorsal mesoderm in dye-labeled intact
wild-type (WT) and no tail (ntl) mutant embryos
(Cooper et al., 1999b) (N. S.
Glickman, PhD Thesis, University of Oregon, 2000). ntl is the
zebrafish homolog of the mouse T-box gene Brachyury
(Schulte-Merker et al., 1994
).
Our recordings begin during gastrulation, shortly after mesodermal
internalization at the blastoderm margin, and we followed the movements of
most of the cells in the field of view during convergence and extension. The
method differs significantly from most previous studies in that confocal
imaging, 4D recording and cell-by-cell analysis of these records allows us to
keep track of, and quantify, the behaviors (e.g. movement velocities and
intercalation behaviors) of a substantial fraction of the dorsal mesodermal
cells rather than just a small sample. We use this method to measure rate
constants for convergence and extension, providing for meaningful comparison
between tissues or embryos.
We find that the rate of convergence within the notochord domain is comparable with that in the somite, whereas extension is several-fold higher in the notochord than in the somite, suggesting that the cells are reorganizing differently in the two domains. Quantitative features of convergence and extension and local cellular reorganizations within notochord domain are as expected from the MIB hypothesis. Our data suggest furthermore that for the WT notochord domain, MIB quantitatively accounts for the observed morphogenesis. We show that ntl is a key regulator of convergence of the notochord domain; in ntl mutants, convergence is severely disrupted, and our analyses suggest that MIB is initiated but cannot be completed. Surprisingly, in the near absence of early convergence in ntl mutants, the dorsal mesoderm can still extend. Hence, extension does not require MIB. We discuss the meaning of these finding for understanding mesodermal morphogenesis and its regulation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Time-lapse production and analysis
Dechorionated embryos (3.3-3.5 hpf)
(Kimmel et al., 1995) were
incubated in embryo rearing medium plus 10 mM HEPES buffer (pH 7.2)
(Cooper et al., 1999a
;
Cooper et al., 1999b
;
Westerfield, 1995
). Embryos
were stained in 100 µM BODIPY FL C5-Cer/C5-DMB-Cer
(N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)
sphingosine (in 1% DMSO), here after referred to as BODIPY ceramide (Molecular
Probes) (Cooper et al., 1999a
;
Cooper et al., 1999b
).
Embryos were mounted in a viewing chamber
(Westerfield, 1995) in 3%
methylcellulose and 0.3% agar, dorsal side upwards. We examined seven
wild-type embryos and four ntl mutant embryos. Images were taken with
a Zeiss 310 upright confocal microscope with a Kr/Ar laser. We imaged 12 focal
planes, at 3 µm intervals for each time point, set 2 minutes apart. The 4D
confocal images were compiled into a 4D movie for playing in a modified
version of NIH image. Cell locations over time were collected using a modified
version of NIH Image (Wasband, NIH) customized to animate 3D time-lapse image
series. Each cell was traced over time by manually marking its approximate
geometric center in three dimensions through each time frame, recording all
cell divisions and final cell fate from its location. All data produced in
this way were further analyzed by routines written in the analysis environment
IDL (Research Systems Inc, CO). Final presentations were rendered by code
generated for the ray-tracer POVRay (POVray).
The precise records of cell positions over time allow us to generate metrics that describe the patterns of motion of morphogenesis. The raw cell tracks were rotated in space to align the AP axis vertically. AP location and velocities can then be expressed in terms of y coordinate and velocities calculated as dy/dt. Similarly, mediolateral movements proceed in x with velocities of dx/dt. Cell velocity and speed were calculated as changes in location about the time frame of interest e.g. dx/dt at time t is calculated from the displacement x[t+I] x[t-I] for a short period I, usually ±8 minutes. The area of the entire tracked field and the tracked axial domain was measured by drawing a convex hull around all the tracked cells. The width of the field of tracked cells is the mean width of the lateral edges of the hull. The 2D density of the tracked field was approximated by dividing the area contained by the convex hull by the number of cells within the box. This measure is approximate and assumes cells are uniformly tracked through time, to reduce possible errors the relative change in density rather than absolute measures were used in these analyses.
In order to analyze average local cell rearrangement, we calculated for each cell in turn the relative location and movement of each other cell in its vicinity. Then a smoothed map of all accumulated relative cell movements was used to generate a vector field to visualize the mean local tissue shape change during a short epoch.
Specific cell rearrangement motifs can be looked for in the cell tracks by
calculating a connectivity matrix that describes adjacency of neighboring
cells based upon their geometry in space (2D or 3D). Neighbor changes will
reflect, for example, intercalation events because as two cells move towards
each other mediolaterally, they become new neighbors and displace their
anterior and posterior neighbors. The analysis of neighbor changes are limited
to the wild-type and mutant notochord/axial domains, where the optics are best
and we have most completely tracked the cell populations. We calculated the
immediate cell neighbors of all traced cells for each time frame, from a
complete 3D Delaunay triangulation. The program Qhull
(Barber et al., 1996) was built
as a library callable from within IDL for this purpose. Each vertex
corresponds to the location of a cell. The algorithm assumes that the local
field has been completely tracked, that cells are compact in shape and are in
close contact with their neighbors as is evident from inspection of
the field (see below, Fig. 3A).
To exclude erroneous apparent neighbor changes between non-contiguous cells, a
conservative maximum cell spacing of 18 µm was used. Any cells separated by
an edge longer than this threshold were not considered to be neighbors for
this analysis. (In practice, the patterns of change seen in connections were
relatively insensitive to variation in this threshold, despite significant
changes in the total number of permitted connections.) Changes in neighbor
connections over time were detected from the changes in connectivity in the
triangulation. Changes due to cell divisions, cells moving into and out of the
traced field, and transient make-break events were excluded from the analysis.
Hand-checking a set of such identified intercalations, in the original 4D
record set, showed the accuracy of the computer-assisted method to be at least
90%. The angle at which cells make and break connections reflects the angle of
the interaction event. The distributions of angles were tested for uniformity
using a Watson U2 test and any samples found to be non-random
(P<0.05) were tested for their mean axial alignment. Variations in
alignment over time were visualized by a moving sample window through the
duration of the experiment.
|
|
|
|
|
|
![]() |
For the purposes of comparison between experiments, an equivalent single rate constant equal to the mean rate over that interval can be used. The same calculations were made for an extension rate constant kE, but in this case, the change in the AP position (dy/dt) of each cell was calculated.
We made a simulation to show how an idealized 2D field of cells would reorganize if converging with a linear velocity gradient in space and extending with an equal gradient of opposite sign (thus preserving cell density), see Fig. 2. A hexagonal array of cells was incrementally constricted and stretched to demonstrate the resulting affect. Globally, the field narrowed exponentially and extended exponentially, as expected. Individual cell reorganization relative to adjacent neighbors was constant and reiterated across the field.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During MIB cells reorganize, as illustrated by close-up views near the field center (*) at the beginning (Fig. 2E) and end (Fig. 2F) of the same sequence. A single cell (*) retains several of its original neighbors (numbered for identification) but changes others. Cells that start out a rank apart along the ML axis become new neighbors (e.g. cells 2 and 6). Conversely, cells that were neighbors along the AP axis become separated by a rank (e.g. cells 1 and 10). This local reorganization is uniform across the field.
Strikingly, however, movement velocities of the cells are not uniform because of the incremental nature of MIB. The cell at the center (*; which we shall see represents a point along the midline of the embryo) does not move. Cell 8 moves inward a single rank, and cell 9, located three times as far away along the ML axis, moves inward three ranks (Fig. 2E,F), moving three times faster because it is pulled by its own intercalations and also pulled by inward-moving more medial cells. `Converging' cells move inwards with a velocity proportional to their distance from the midline, such that a linear gradient of velocity with position is present across the field (Fig. 2H). This position dependence on the rate of movement can also be seen by examining the paths the cells take during a given time interval (Fig. 2G). The paths of the cells at more lateral positions are longer than the paths of cells close to the center, reflecting their rate of travel.
Similarly, along the AP axis the rate of outwards movement depends on AP position. Cell 11 is three times farther from the center than cell #1 and it moves three times faster (Fig. 2E,F). Again, the relationship between cellular position in the field and velocity is linear (Fig. 2I). The slopes of the lines in Fig. 2H,I give convenient measures (rate constants) for convergence and extension (kc and kE, respectively). The convergence rate constant is negative, reflecting inward movement, and the extension rate constant is positive, reflecting outward movement. Their absolute values are equal.
The MIB hypothesis thus predicts linear gradients of velocity with position. Furthermore, it follows from the same arguments that the shape changes of the field should not be constant with time. As the field narrows during convergence, only smaller ML positional values remain (the larger ones are lost as the population moves closer the midline) and only lower velocities should be observed. Hence, the rate of change in field width should decrease with time. As the field lengthens during extension new, larger AP positional values are established and velocities are correspondingly higher. Hence, the rate of change in field length should increase with time. The kinetics are exponential in both cases (Fig. 2J,I), and the exponential curves have the same slopes as the gradients described above: kc and kE.
A perhaps surprising feature of MIB is that a large change in shape of the cellular field is produced by relatively small local cellular rearrangements. This is another prediction of the hypothesis.
Tissue domains and cellular flows
Convergence and extension in embryos has usually been measured by tracking
the movements of one or more labeled cells or small cell groups toward the
dorsal midline, and along the AP axis. The above analyses show clearly that
this method is unreliable when used to make interpretations about the movement
of the entire field of cells. Even where convergence and extension are uniform
across the field and during time, the ML and AP cellular velocities are not
uniform with time, and depend on cell position within the field. Hence, we
used a labeling and recording method
(Cooper et al., 1999a;
Cooper et al., 1999b
), and
analytical tools (Concha and Adams,
1998
) (see Materials and Methods) that allowed us to keep track of
all or most of the cells being recorded, and we tracked the field of
developing cells as fully as we could. Fig.
3 illustrates the results for a wild-type embryo. Cell outlines
are apparent in the time series of original recordings (A), and the series of
records with respect to both time and depth (z-axis sectioning with
the confocal microscope) intervals were close enough to one another to permit
us to follow unambiguously individual cells in the field of view, no matter
how the cells moved, and during cell divisions. The field is approximately
centered on the dorsal midline, and the particular z-level
illustrated in Fig. 3A is at an
intermediate depth through the internalized prospective mesoderm (or
hypoblast) at the center of the field. The blastoderm margin is evident at
first (Fig. 3A1), and then,
because of epiboly (Kimmel et al.,
1995
; Solnica-Krezel et al.,
1996
), it moves off towards the vegetal pole (downwards in the
figure), out of the field being recorded. Brachet's cleft
(Kimmel et al., 1995
), the
boundary between ectoderm and mesoderm, is evident (arrows in
Fig. 3A2).
Animating a recording such as used for
Fig. 3A (see Movie 2 at
http://dev.biologists.org/supplemental/)
reveals that the overall cellular flows are as expected of MIB
(Fig. 2G). Movement pathways of
lateral cells are predominantly ML, whereas those of medial cells are
predominantly AP (see Myers et al.,
2002b). The animation also allows visualization of another
predicted feature of MIB: the position-dependent rates of cell movement
(Fig. 2H,I). Lateral cells move
towards the midline faster than cells already close to the midline. Cells near
the top and bottom of the field move faster along the AP axis than those near
the middle of the field.
Within the mesoderm at 9.3 hpf, boundaries between the prospective notochord and presomitic mesoderm become clearly visible (notochord-somite or axial-paraxial boundaries; arrowheads, Fig. 3A3). By animating the recordings, and playing Movies 2 and 3 backwards (see http://dev.biologists.org/supplemental/), one can see that these boundaries arise in a patchy (or piecemeal) way; they can be vaguely recognized in Fig. 3A2 (8.8 hpf), farther apart than at later stages, thus revealing that the notochord domain is converging. However, the boundaries are not at all visible at the initial time point (7.3 hpf, midgastrula period). At the last time point, 4 hours later, the notochord-somite boundaries have straightened out and come together to enclose a notochord domain that is now only two or three cells wide (Fig. 3A4). The boundaries between the most anterior somites are also just becoming visible (arrows, Fig. 3A4).
Once such landmarks become visible, we could assign cells particular fates (e.g. position of a cell within the area demarcated by the notochord boundaries defines a notochord cell). Fig. 3B shows a time series from the same records, where we represent tracked cells as spheres color-coded according to these eventual fates. We find, in this and in other wild-type embryos similarly analyzed, that the notochord domain is coherent from outset of the recording; i.e. the domain of prospective notochord cells is spatially separate from the somitic domains (and also from overlying floorplate-forming cells, Fig. 3C), before overt boundaries arise. During convergence, the notochord domain narrows from 10-14 cells across at the time our recording begins to two or three cells across. This kind of decrease is expected from sustained MIB.
Differential movements can be detected between regions adopting different fates. For example, we observe slipping apart or shear between cells the notochord domain and the immediately overlying floor plate-forming cells in the prospective neural plate. As is evident in Fig. 3C, the notochord and floor plate-forming cells start out at the same AP position but after undergoing marked convergence and extension, the final position of the floor plate-forming cells is posterior relative to the notochord cells. Tracking other cells in the epiblast (i.e. in the prospective neural plate outside of the medial floor plate domain; data not shown) reveals that this shearing is not unique to the notochord and floorplate, but is a general feature of epiblast cellular movement relative to that in the internalized mesoderm of the hypoblast. We can account for the shear by considering the combined morphogenetic movements of epiboly and internalization of cells by ingression at the blastoderm margin (see Discussion).
The notochord domain also shears with the neighboring somite domains
(Fig. 3D), as previously
reported at later stages in zebrafish by Devoto et al.
(Devoto et al., 1996). In this
case, as we describe next, we can account for the shearing by a difference in
the rate in which the two domains extend. The presentation in
Fig. 3D reveals that extension
of the notochord domain is dramatically greater than extension of the somite
mesoderm. The same difference is also present in Xenopus
(Keller et al., 1989
;
Keller et al., 2000
).
Rates of convergence and extension
We quantitatively analyzed the kinetics of the cellular flows in our
records to explore further the similarities and differences in convergence and
extension in the notochord and somite domains. We observed that during
gastrulation and continuing into the early segmentation period of development,
the width of the notochord domain decreases with the kinetics of exponential
decay (Fig. 4A), as predicted
by the MIB hypothesis (Fig.
2J). This finding suggests that if MIB, or a similar mechanism
underlies convergence, cellular interactions (including rates of ML
intercalations) remain approximately constant during the recording period.
|
Plotting ML velocities of all the cells tracked in the field as a function
of their ML position, at any time point, reveals the linear gradient
underlying convergence (Fig.
4B) predicted by the model
(Fig. 2H). As shown in
Fig. 4B, the data points for
the velocities of cells in left and right somite domains (red) fall on the
same line as those for cells in the notochord domain (green). This finding
shows that the rates of convergence in the three domains are similar.
Calculating the rates separately for each of the domains and integrating (or
averaging: see Materials and Methods) across all time points confirms this
conclusion; kc=-0.0066, -0.0065 and -0.0057 for the notochord, and
left and right somite domains, respectively. Evidently, the rate of
convergence is slightly higher in the notochord domain (8% higher that
the average of the somite domains in this example), a conclusion substantiated
by similar estimates in a second wild-type embryo. Convergence in the
notochord domain estimated by this method is in excellent agreement with that
measured directly from its decreasing width with time
(Fig. 4A; kc=-0.0064
compared with -0.0066).
Kinetic analysis also confirms the relatively higher extension of the notochord domain when compared with the somite domain, noted above (Fig. 3D). Cells in the notochord domain have much higher AP velocities than somite domain cells beginning at the same AP position. The relationship between AP velocity and AP position, within either the notochord or somite domain, is linear (Fig. 4C). As we see from Fig. 4C, two separate lines describe the separate rates of extension of the two domains; the slope (kE) is substantially higher for the notochord. Over all time points, extension in the notochord domain is about three times higher than in the somites in this embryo; kE=+0.0064 for the notochord domain compared with +0.0023 for average of both somite domains.
The rates of convergence and extension are expected to be equal if MIB is pushing cells apart exclusively along the AP axis (as in the model in Fig. 2) and if no factors, other than MIB, are contributing to convergence and extension. In this example, as predicted by this extreme model, convergence of the notochord domain almost exactly equals extension (|kc|=0.0066 and |kE|=0.0064; 3% difference). In a second wild-type embryo examined in this way, |kc| is 35% higher than |kE|. By contrast, extension is much lower than predicted by the model in the somite domain, occurring, as we have seen at about one third of the predicted rate. An explanation that we favor for why convergence and extension are more in balance in the notochord domain than in the somite domain is that MIB is driving convergence at nearly the same rate in the two domains, but that during MIB, cells are rearranging themselves differently in the two domains (see Discussion).
The overall results of our kinetic analyses in wild-type embryos are in accordance with the MIB hypothesis. In particular, the kinetics of cell movement are approximately as predicted. Thus, MIB may account for all (or most) of the cellular movements within the wild-type notochord domain. To test this conclusion more rigorously, we ask if the velocity gradients underlying the cell movements, presumably generated by MIB, can account for the all observed convergence and extension of the notochord domain. We do this by using the values of the convergence and extension rate constants to subtract the cell movement due to MIB. If MIB accounts for all cell movement, then this procedure should subtract all motion (other than random noise) from the field of cells. This is essentially what we observe, as illustrated for the notochord domain in Fig. 5. The purple lines indicate the observed locations and movements of the notochord cells at 7.3 hpf (Fig. 5A) and 4 hours later (Fig. 5B). Convergence and extension of the tissue are clearly observed. However, after removing the calculated components of the cell movements resulting from MIB, little convergence and extension is observed after 4 hours (yellow lines in Fig. 5). The field has approximately the same shape at the end as at the beginning. This analysis strongly suggests that we are not missing some major factor contributing to the change in shape of the field.
Failure of early convergence but not extension in the no
tail mutant axial dorsal mesoderm
By the MIB model, active convergence drives extension in the dorsal
mesoderm, and our analyses in the wild-type notochord and somite domains (see
also below for cell neighbor analyses in the notochord) are consistent with
this hypothesis. We reasoned that if convergence were abolished in the
notochord domain, then so too should extension.
We used ntl mutants to carry out this experiment. The ntl
gene encodes a T-box transcription factor (the zebrafish ortholog of mouse
Brachyury) (Schulte-Merker et
al., 1994) that is broadly expressed in nascent mesoderm and then
maintained specifically in the gastrulating axial mesoderm that develops as
notochord (Schulte-Merker et al.,
1994
). In ntl loss-of-function mutants, notochord
development fails and gene expression analysis suggests that, within the
ntl- axial domain, convergence fails during gastrulation
(Melby et al., 1997
).
Recordings made in the ntl mutant begin the same way as in the
wild type. Cells ingress at the blastoderm margin in the early gastrula, and
we made our recordings of the internalized dorsal mutant `mesodermal' layer
present during gastrulation. However, as gastrulation continues and the
notochord-somite boundary becomes visible in the wild type, no such boundary
is at first apparent in the mutant (Fig.
6A,E). Later, by the one-somite stage, a pair of boundaries
appears in the mutant, encompassing an `axial' domain only slighter broader
than the wild-type notochord domain at this stage
(Fig. 6B,F). We could use these
boundaries in the mutant to score our tracked mesoderm-derived cells as
`axial' cells (i.e. cells enclosed by the boundaries) versus somitic cells
(cells outside of the boundaries). The axial/somite boundaries appear not to
enclose a mutant notochord domain, however, but to enclose the ventral part of
the primordium of the spinal cord (termed the neural keel at this stage)
(Papan and Campos-Ortega,
1994). We infer this from the cellular morphology at early somite
stages: in the wild-type notochord domain, the cells become highly organized.
They take on a wedge-shape and are significantly elongated along the ML axis
[Fig. 6C,D (parts 4 and 5)]. At
a more dorsal focal plane in the wild type, just before coming to the floor
plate one encounters the notochord-floor plate boundary, a flat, thin zone
diffusely labeled with BODIPY-ceramide, where cell outlines are difficult to
discern (Fig. 6D, part 3).
Dorsal to this boundary the cells of the floor plate region are, at the
three-somite stage, disorganized and irregular in shape
(Fig. 6D, parts 1 and 2). In
the ntl mutants at the same stage, the cellular morphology at any
focal plane is irregular (Fig.
6F), like that of the wild-type floor plate. Furthermore, a
notochord-floor plate boundary cannot be discerned.
The width of the axial domain in ntl mutants, as judged from the positions of the axial-somite boundaries, becomes very similar to that of the wild-type notochord domain, clearly revealing that the mutant axial domain has undergone convergence by the three-somite stage. In the wild-type embryo, as we have seen, the narrowing occurs during much of the gastrulation and early segmentation periods. By contrast, convergence of the axial domain in the ntl mutant is at first nearly absent. For example, the field of tracked mutant cells does not appreciably narrow during the 1.5 h interval during gastrulation shown as cell pathways in Fig. 7A, and by computed width of the field in Fig. 8A. Then, at postgastrula stages, just preceding the appearance of the boundaries surrounding the mutant axial domain, convergence of the mutant axial domain dramatically picks up (Fig. 8A; time points after about 9.5 hpf).
|
Notably, at same stages when convergence of the mutant axial field is severely perturbed, extension is occurring at a high rate. The field of tracked cells is lengthening quite prominently in Fig. 7A, and Fig. 8B shows that in this example, extension of the mutant axial domain is substantially higher than the wild-type notochord domain during the gastrula period. Without substantial convergence, seeing any extension at all during this period was unexpected. In the wild type, because of the close balance between convergence and extension, the cross-sectional (or planar) area of the notochord remains nearly unchanged (Fig. 8C; first a 20% increase then a 20% decrease over the recorded period). In the mutant, with marked extension but not convergence, the computed area of the domain expands markedly, about threefold, implying that an active process of extension or stretching is taking place (Fig. 8C).
A possible explanation for observing extension in the mutant comes from
understanding that ntl- midline axial `mesoderm' joins the
ventral neural tube, and expresses the floor plate fate
(Amacher et al., 2002;
Halpern et al., 1993
). We
could suppose that, in the mutant, the mutant axial cells leave the axial
mesodermal domain and move into the overlying epiblast-derived floor plate.
These cells would then undergo rounds of cellular intercalation within the
epiblast. Intercalations of axial mesodermal cells within the epiblast of
ntl mutants may contribute to the observed extension of the axial
domain. To examine this hypothesis, we tracked some epiblast cells overlying
the midline axial domain in a ntl mutant. We observe that, as in the
wild type, the two sets of cells do not mix
(Fig. 7B). Hence, `ectopic'
intercalations between midline hypoblast and epiblast do not appear to account
for the observed extension. Rather, during extension in the mutant, the cells
seem to thin out, forming a layer one-cell thick
(Fig. 7B,C). As the cells thin
out, they cover a greater area, resulting in extension of the axial domain.
The overall density of cells in the ntl mutant axial domain
decreases, while the density of cells in the wild-type notochord increases
(Fig. 8D). The observed
decrease in cell density in ntl mutants also helps explain the
increase in area, and extension, of the axial mesoderm
(Fig. 8C).
MIB appears to initiate, but not to continue correctly in no
tail mutant axial domain
Our analyses reveal cellular correlates of reduced convergence of
ntl mutant axial domain cells during gastrulation. ML movements are
present in wild-type embryos, but most sustained movements in mutants are AP
(vertical versus horizontal tracks, Fig.
7A). In mutants, cells appear to mix abnormally between the axial
domain and somite-forming domains (Fig.
7A; red and green interspersed in ntl-).
Wild-type cells show only limited mixing with medial and lateral neighbors
during convergence and extension (Fig.
9A). Of course, we expect limited mixing because constrained cell
mixing is the basis of convergence and extension by MIB. However, ML mixing is
prominent in ntl mutants (Fig.
9A), a circumstance where convergence is greatly reduced and
mesoderm thins out. Cells in the wild type move towards the dorsal midline, as
expected during MIB, but movement with respect to the midline is scrambled in
ntl mutants (Fig.
9B).
These data show that the ntl mutant cells intermix far more extensively than do wild-type cells, but do not reveal how the intercalations are oriented. To address this, we examined cell neighbor exchanges. In cellular fields undergoing intercalations, new neighbor pairs will be generated and old ones lost in a reciprocal fashion. By the MIB model, neighbor gains should be oriented mediolaterally, and neighbor losses should be oriented anteroposteriorly (Fig. 2E,F; Fig. 10A). We detect large numbers of both neighbor gains and losses in the axial domains of wild-type and ntl- embryos, and in the wild type they are strongly oriented as predicted (Fig. 10B). Surprisingly, in ntl mutants, neighbor changes are still made with a significant but broadened tendency to align as in MIB (Fig. 10B). A most interesting change underlies the broadening. Analysis of the time course of the neighbor changing events (Fig. 10C) shows that the wild-type axial domain is able to maintain oriented intercalation activity nearly throughout the recording period. However, in the ntl mutant, only pulses of ordered neighbor gains or losses occur, interspersed with generally longer periods of disorganization.
|
This disorderly behavior suggests that MIB can be initiated but not sustained in ntl mutants, and prompted us to examine how cells are actually moving with respect to their local neighbors over both short and long time periods. We have shown that in a cellular field uniformly undergoing MIB, ML cell movement is towards the field center and AP movement is away from the field center, with velocity gradients accompanying both. If local cellular reorganizations are in fact uniform and if we now hold constant the position of any cell in the field, we expect ML neighbors to move towards that cell and AP neighbors to move away. Strikingly, we observe these predicted local correlates of both convergence and extension in the notochord domain of wild-type embryo (Fig. 11A). The ML inward movements are comparable in magnitude to the AP outward movements, reflecting the close balance of convergence and extension. Relative velocity gradients are evident (from the arrow lengths) along both axes, mirroring the global velocity gradients across the field. Furthermore, as shown by comparing the series of panels in Fig. 11A, the wild-type embryo is able to maintain these highly oriented local behaviors, i.e. the pattern is unchanging over significant time courses.
However, we see this same pattern only occasionally in the ntl mutant (Fig. 11B). Behavior is wild type-like at the first time point selected (first panel). Afterwards, whereas the AP movements tend to remain outward from the reference cell, the ML movements are scrambled, first inward (first panel), then outward (last panel in Fig. 11B). Disorganized local ML convergence of cells toward one another underlies the global disruption in convergence of the axial field.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our studies also reveal complexity in the behaviors that underlie
convergence and extension. The notochord domain and adjacent domains of
somite-forming cells converge at a similar rate, but they extend at different
rates. In mutants that lack function of a key mesodermal patterning gene (the
T-box gene no tail)
(Schulte-Merker et al., 1994),
convergence of the notochord domain is substantially blocked during
gastrulation, but unexpectedly, the domain extends rapidly. A likely
explanation for these findings, that we explore below, is that in addition to
MIB, other morphogenetic force-generating processes are shaping the dorsal
mesoderm during this crucial period of early development.
Mediolateral intercalation behavior quantitatively accounts for
dorsal mesodermal convergence and extension
A striking finding of our study is that linear relationships, gradients,
between cellular movement velocities and cellular position underlie both
convergence and extension. These relationships arise because of the
incremental and additive nature of ML intercalation events across the field:
during convergence, cells located laterally will be displaced inward towards
the center of the field (the dorsal midline) not only by their own
intercalations with their own neighbors, but because they are connected by a
chain of adhesions to more medial cells, and cells all along the chain are
undergoing MIB. The resulting velocity gradient along the chain will be
linear, as we observed, if the local cellular behavior along the chain (i.e.
MIB) is, on average, uniform. Exactly the same kind of explanation works to
explain the AP gradient underlying extension.
These simple relationships between velocity and position are extremely useful in practice. If we plot cellular velocity versus cellular position, from our recordings of the cell movement pathways, the slopes of the lines are straightforward to determine. The slopes serve as rate constants (kc, kE) that reveal how rapidly the shape of the field is changing. It follows from the linear relationships that if MIB is constant over time, then the kinetics of the shape change are exponential (first order), as we confirmed experimentally.
From our estimate of kc=-0.006 min-1, halving of the
width of the notochord domain occurs within 2 hours. The notochord domain is
about 20 cells wide in the early gastrula (as determined from expression of
the Not gene floating head at 6 hpf) (see
Melby et al., 1996;
Talbot et al., 1995
). If the
rate of convergence is constant, the domain will narrow to a domain only about
a single cell wide during about 8 hours.
From our data, we propose that MIB is not only a prominent feature in
dorsal mesoderm, but that this behavior quantitatively accounts for the shape
changes of the field. We performed a severe test of this proposition for the
notochord domain: From our measured velocities and locations of each cell in
the field, we determined the overall values of kc and kE
for the domain at each time point in our recording. Then, we computationally
subtracted from the movement pathways of each cell those components (the ML
and AP relocations) of the movements that were accounted for by the MIB. As
predicted, the resulting movement pathways, with these components absent,
exhibited no apparent trends. The cells jitter about in a possibly random
fashion, and there was no prominent change in the shape of the field. Hence,
MIB appears to be the single cellular morphogenetic behavior rearranging cells
and shaping the dorsal mesoderm, during the mid- and late gastrula period.
This finding is in marked contrast to descriptions of teleost dorsal
mesodermal morphogenesis in literature from a century ago (e.g.
Morgan, 1895). At that time,
internalized dorsal mesodermal cells were proposed to migrate, with cell
intercalations playing no role at all. By that model there would be no
gradients, the migrating cells would all be moving with the same velocities.
We observed mesodermal migrations without gradients on the ventral side of the
zebrafish gastrula (data not shown) (Myers
et al., 2002a
). We observed no such cell migration in the dorsal
trunk-forming mesoderm of the embryo.
Notochord and somite
We show that in the wild-type embryo, convergence of the notochord-forming
mesoderm occurs at about the same rate as the adjacent somitic mesoderm. This
finding suggests that during gastrulation, all of the trunk dorsal mesoderm is
behaving as a single unit with respect to axis narrowing, i.e. there appears
to be mechanical continuity across the entire field. This is an extremely
interesting result because the field becomes subdivided during the same period
by prominent notochord-somite boundaries. From cell lineage analyses we know
that from the onset of gastrulation, within the dorsal mesoderm, the domain of
prospective notochord cells is already a lineage compartment separate from the
somitic domains (Kimmel and Warga,
1986; Melby et al.,
1996
). Our recordings also show that the domains are spatially
separate during and after gastrulation: even as intercalations occur within
the notochord and somite domains, they apparently do not occur across the
notochord-somite boundaries, or between the two domains before the boundaries
become recognizable. Moreover, by the stage our recordings begin, the
notochord and somite domains are already expressing different patterns of
developmental regulatory genes; genes encoding transcription factors,
signaling molecules and adhesion molecules
(Kodjabachian et al., 1999
).
Hence, it would be naive to imagine that the molecular basis of convergence of
dorsal mesoderm is uniform.
Whereas the rates of convergence are similar for the notochord and somite domains, extension is much higher in the notochord domain, more than three times higher than in the somite for the wild-type example shown in Fig. 4C. If MIB is driving convergence at the same rate in both domains, then why are the rates of extension different? One possibility is that the cellular populations are rearranging differently during MIB. In the notochord, convergence and extension are in relatively close balance. We understand this to mean that essentially all of the ML intercalations are pushing cells apart along the AP axis, as in the model shown in Fig. 2. Hence the lengthening of the axis is proportional to its narrowing, and the area of the domain examined along the plane of the intercalations is preserved (Fig. 8A-C). However, in the wild-type somite domain, the intercalations might thicken the tissue as well as lengthen it; i.e. the ML intercalations would push some cells apart along the dorsoventral axis. Our data are consistent with this possibility (e.g. Fig. 7C), but our recording procedures were not optimized to quantify the thickening in the somitic domains. Whether the difference we observe is due to somitic thickening or something else, these findings reveal complexity of MIB, suggesting convergence and extension are separately regulated.
Conserved cellular basis of notochord convergence and extension
Mediolateral cell intercalations underlie convergence and extension of the
notochord in Xenopus (Keller et
al., 1989) in a way similar to what we observe in zebrafish. By
midgastrula stages, the notochord-forming cells have a distinct bipolar shape
and exhibit protrusive activities at their tips
(Keller et al., 1989
;
Keller et al., 1992
). Then
they become wedge-shaped as the notochord narrows to a domain only one or two
cells wide. During early morphogenesis, convergence of the Xenopus
notochord is at least roughly balanced by extension
(Keller et al., 1989
).
Convergence and extension also occurs in the somitic mesoderm in
Xenopus (Keller et al.,
2000
). Extension is more rapid in the notochord, and shearing
between the notochord and somite is observed. Intercalating somitic cells that
do not contribute to extension may thicken the somites dorsoventrally (see
Keller et al., 2000
). Finally,
disruption of the no tail ortholog in Xenopus, Xbra,
disrupts convergence (Conlon and Smith,
1999
).
In each of these features, the convergence and extension behavior and regulation appear similar in zebrafish and Xenopus, suggesting that the basic cellular machinery of MIB has been highly conserved between the two species.
One difference between zebrafish and Xenopus is that in zebrafish
the yolk syncytial layer (YSL) provides a substrate for the mesendoderm,
rather than a blastocoele roof present in amphibians. YSL nuclei undergo
convergence and extension, and their movements are similar to those of the
overlying mesendodermal cells (D'Amico and
Cooper, 2001). This observation raises the possibility that the
zebrafish cell movements are, at least in part, passively imposed by the YSL.
However, passive convergence and extension of the zebrafish notochord-forming
cells seem quite unlikely. Examined in detail, the blastoderm cell movements
generally occur faster than the YSL nuclear movements
(D'Amico and Cooper, 2001
),
arguing that convergence and extension are autonomous in the YSL and cellular
blastoderm.
How might loss of no tail function disrupt convergence?
As expected from previous studies of gene expression of ntl
mutants (Melby et al., 1997),
we see a prominent defect in convergence of the gastrula-stage axial mesoderm
essentially the field does not narrow
(Fig. 8A). The defect appears
largely limited to the axial mesoderm and is transient, observed only during
the gastrula period. Later, after epiboly is completed, convergence of the
domain in the mutant begins at a rapid rate, and the domain width becomes
similar to that of the WT notochord by the three-somite stage. It may not be
coincidental that the time when convergence initiates in ntl mutants
is when we see morphological boundaries enveloping the axial domain together
with the ventral neural keel (including the floor plate) that undergoes
pronounced convergence and extension. Notably, wild-type floor-plate cells do
not express ntl at these stages: its axial expression is limited to
the notochord. Hence, the ntl- cells, as they join the
floor plate, may acquire the floor plate, ntl-independent, program of
regulation of morphogenesis.
How does ntl regulate notochord morphogenesis, such that lack of
function produces such a major defect? A candidate implicated in
Xenopus is Xwnt11 (Tada
and Smith, 2001). This gene is a direct transcriptional target of
the ntl ortholog Xbra. Dominant-negative constructs of
Xwnt11 (Tada and Smith,
2000
), its receptor Frizzled-8
(Wallingford et al., 2001b
),
or a key downstream target Dishevelled/Xdsh
(Wallingford et al., 2000
) all
block convergence in Xenopus, apparently acting via a non-canonical
planar polarity Wnt/Ca2+ pathway
(Choi and Han, 2002
;
Tada and Concha, 2001
;
Wallingford et al., 2001a
).
Mutational analyses involving silberblick/wnt11
(Heisenberg et al., 2000
)
clearly reveals a role in early morphogenesis of the Xwnt11 ortholog
in zebrafish. However, key aspects of Wnt11 regulation differ between the two
species. slb/wnt11 is first expressed in the early blastoderm margin,
and this expression domain is independent of ntl
(Makita et al., 1998
). Midline
ntl-dependent expression becomes strong only later, reaching highest
levels at the 5- to 12-somite stages. Hence slb/wnt11 is an unlikely
candidate for mediating ntl-dependent MIB in the early axial mesoderm
in zebrafish.
FGFs are attractive alternative candidates. FGFs are regulated by T-box
genes, and are strongly implicated in the control of mesodermal morphogenesis,
convergence and extension in particular. FGFs act via downstream targets
including ephrins (Chong et al.,
2000), and, like Wnt11, intracellular Ca2+ release
(Nutt et al., 2001
). Again
there are key regulatory changes between Xenopus and zebrafish: in
Xenopus the key Xbra-dependent FGF in axial mesoderm is
eFGF, but in zebrafish other FGFs, including FGF8 and FGF3, play at least part
of the role of this gene in dorsal mesodermal morphogenesis during
gastrulation (B. Draper, personal communication).
Current understanding is that both noncanonical Wnt signaling and FGF signaling are regulating cell polarity as would be required for the cells to orient MIB properly. Interestingly, our analyses indicate that the major defect in ntl mutants may not be loss of cell polarity: We find that cells in the mutant can apparently initiate correctly oriented MIB. They frequently move towards their neighbors (Fig. 11), and make and break contacts with their neighbors (Fig. 10) in the same orientation as in the wild type. However, although many ML contacts are made in the mutant, the cells appear unable to exert the necessary tension to pull together rather, they frequently slip apart. This proposal explains the ML cellular intermixing, as well as the randomized ML movement of cells with respect to the midline (Fig. 8) and one another (Fig. 11).
Loss of the tension necessary to pull the tissues together may reflect a
requirement for an adhesion molecule missing in ntl mutants. Studies
of the paraxially expressed T-box gene spadetail (spt;
tbx16 Zebrafish Information Network)
(Griffin et al., 1998) provide
a clue about what might be missing from the ntl-
convergence machinery. spt function is required for proper paraxial
cell behaviors (Ho and Kane,
1990
; Kimmel et al.,
1989
) and is hierarchically upstream of a protocadherin that is
critical for proper mesodermal cell-cell adhesion during gastrulation
(Kim et al., 1998
;
Yamamoto et al., 1998
). Lack
of a corresponding adhesion molecule in the ntl- axial
domain (Kuroda et al., 2002
)
might underlie the defects we observe.
How can extension work when convergence does not?
Dorsal mesoderm efficiently extends in ntl mutants. It extends
without significant convergence in the axial domain, clearly revealing that a
mechanism other than MIB can drive extension in this domain. Furthermore,
somitic mesoderm in the mutant also appears to extend more rapidly in the wild
type, where, as discussed above, convergence normally seems to be driving
thickening as well as extension.
The result comes as a surprise because there was no hint from our data, or from the literature, that anything other than MIB drives dorsal mesodermal extension. Finding that extension occurs in the mutant without convergence may well be providing a clue about how normal development works. For example, we do not understand what normally orients the AP pushing apart of cells during MIB such that convergence and extension are normally in balance in the notochord domain. The convergence-independent extension in the mutant may be uncovering something about this mechanism.
We examined specifically whether abnormal radial intercalation between axial hypoblast and epiblast might account for the observed extension in the mutant, and observed that it did not (Fig. 7B). However, radial intercalations are very likely to be occurring within (not between) these cell layers, particularly in the dorsal mesoderm. We infer this because by late in gastrulation in ntl mutants (before convergence picks up in the mutant), both axial and paraxial mesoderm have thinned to a layer only a cell or two thick (Fig. 7C). During the same period, the cellular density of the mutant axial domain is substantially decreasing (Fig. 8D). Rather than packing together as in the wild-type notochord (which eventually becomes very cell-dense; Fig. 8D), the cells of the mutant axial domain are spreading apart predominantly in the AP direction, and this spreading accounts for the extension of the domain.
The cellular spreading observed in ntl mutants suggests that
blastoderm epiboly could be driving extension in the mutant. Epiboly occurs
simultaneously with convergence during the stages we studied. During epiboly,
blastoderm cells intercalate radially
(Kimmel and Law, 1985;
Wilson et al., 1993
) and
spread apart along the AP axis (Concha and
Adams, 1998
; Warga and Kimmel,
1990
). Solnica-Krezel et al.
(Solnica-Krezel et al., 1996
)
previously suggested that epiboly contributes to extension in volcano
mutants, and epiboly is underlain by an AP velocity gradient (R. J. A., D.
Faruque and M. L. Concha, unpublished). We emphasize that our proposals for
epiboly driving extension in the ntl mutant does not in any way argue
against MIB as the key mechanism in the wild type. Indeed, in other mutants
with disrupted convergence such as knypek and trilobite, or
the knypek-trilobite double mutants, extension is also disrupted,
even though epiboly is intact (Myers et
al., 2002a
). These genes function in the Wnt planar polarity
pathway (Myers et al., 2002b
)
and the results, combined with our findings with ntl, might be
revealing that Wnt signaling drives extension that is closely coupled to
convergence, but that ntl- disrupts this relationship
leaving other, parallel, mechanisms able to extend the axis. Furthermore, in
epiboly mutants (Kane et al.,
1996
) or in embryos treated with a teratogen
(Bauman and Sanders, 1984
),
both convergence and extension of the notochord can occur in the absence of
epiboly, the opposite of the findings with the Wnt signaling mutants. Hence,
it is likely that MIB normally functions redundantly with other cellular
mechanisms to produce extension of the dorsal mesoderm. This proposition might
be examined with new genetic analyses, e.g. in double mutants in which
epiboly, Wnt signaling and ntl function are disrupted in combination.
With the approach and quantitative methods we have developed for this study,
we can now account for the individual reorganizations of populations of cells
and distinguish between competing models for the mechanism of morphogenesis
and its control.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amacher, S. L., Draper, B. D., Summers, B. and Kimmel, C. B.
(2002). The zebrafish T-box genes no tail and
spadetail are required for development of trunk and tail mesoderm and
medial floor plate. Development
129,3311
-3323.
Barber, C. B., Dobkin, D. P. and Huhdanpaa, H. T. (1996). The Quickhull algorithm for convex hulls. ACM Trans. Math. Software 22,469 -483.[CrossRef]
Bauman, M. and Sanders, K. (1984). Bipartite axiation follows incomplete epiboly in zebrafish embryos treated with chemical teratogens. J. Exp. Zool. 230,363 -376.[Medline]
Blagden, C., Currie, P., Ingham, P. and Hughes, S.
(1997). Notochord induction of zebrafish slow muscle mediated by
Sonic hedgehog. Genes Dev.
11,2163
-2175.
Carmany-Rampey, A. and Schier, A. F. (2001). Single-cell internalization during zebrafish gastrulation. Curr. Biol. 11,1261 -1265.[CrossRef][Medline]
Choi, S. C. and Han, J. K. (2002). Xenopus Cdc42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway. Dev. Biol. 244,342 -357.[CrossRef][Medline]
Chong, L. D., Park, E. K., Latimer, E., Friesel, R. and Daar, I.
O. (2000). Fibroblast growth factor receptor-mediated rescue
of x-ephrin B1-induced cell dissociation in Xenopus embryos.
Mol. Cell Biol. 20,724
-734.
Concha, M. L. and Adams, R. J. (1998). Oriented
cell divisions and cellular morphogenesis in the zebrafish gastrula and
neurula: a time-lapse analysis. Development
125,983
-994.
Conlon, F. L. and Smith, J. C. (1999). Interference with brachyury function inhibits convergent extension, causes apoptosis, and reveals separate requirements in the FGF and activin signaling pathways. Dev. Biol. 213,85 -100.[CrossRef][Medline]
Cooper, M. S., D'Amico, L. A. and Henry, C. A. (1999a). Analyzing morphogenetic cell behaviors in vitally stained zebrafish embryos. Meth. Mol. Biol. 122,185 -204.[Medline]
Cooper, M. S., D'Amico, L. A. and Henry, C. A. (1999b). Confocal microscopic analysis of morphogenetic movements. Methods Cell Biol 59,179 -204.[Medline]
D'Amico, L. A. and Cooper, M. S. (2001). Morphogenetic domains in the yolk syncytial layer of axiating zebrafish embryos. Dev. Dyn. 222,611 -624.[CrossRef][Medline]
Devoto, S. H., Melancon, E., Eisen, J. S. and Westerfield,
M. (1996). Identification of separate slow and fast muscle
precursor cells in vivo, prior to somite formation.
Development 122,3371
-3380.
Essner, J. J., Laing, J. G., Beyer, E. C., Johnson, R. G. and Hackett, P. B., Jr (1996). Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos. Dev. Biol. 177,449 -462.[CrossRef][Medline]
Griffin, K. J., Amacher, S. L., Kimmel, C. B. and Kimelman,
D. (1998). Molecular identification of spadetail:
regulation of zebrafish trunk and tail mesoderm formation by T-box genes.
Development 125,3379
-3388.
Hall, A. and Nobes, C. D. (2000). Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355,965 -970.[CrossRef][Medline]
Halpern, M. E., Ho, R. K., Walker, C. and Kimmel, C. B. (1993). Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell 75,99 -111.[Medline]
Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C. and Wilson, S. W. (2000). Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76-81.[CrossRef][Medline]
Ho, R. K. and Kane, D. A. (1990). Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors. Nature 348,728 -730.[CrossRef][Medline]
Kane, D. A., Hammerschmidt, M., Mullins, M. C., Maischein, H.
M., Brand, M., van Eeden, F. J. M., Furutani-Seiki, M., Granato, M., Haffter,
P., Heisenberg, C. P. et al. (1996). The zebrafish epiboly
mutants. Development
123, 47-55.
Keller, R. and Winklbauer, R. (1992). Cellular basis of amphibian gastrulation. Curr. Top. Dev. Biol. 27, 39-89.[Medline]
Keller, R., Cooper, M. S., Danilchik, M., Tibbetts, P. and Wilson, P. A. (1989). Cell intercalation during notochord development in Xenopus laevis. J. Exp. Zool. 251,134 -154.[Medline]
Keller, R., Shih, J. and Domingo, C. (1992). The patterning and functioning of protrusive activity during convergence and extension of the Xenopus organizer. Development Suppl. 81-91.
Keller, R., Davidson, L., Edlund, A., Elul, T., Ezin, M., Shook, D. and Skoglund, P. (2000). Mechanisms of convergence and extension by cell intercalation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355,897 -922.[CrossRef][Medline]
Kim, S. H., Park, H. C., Yeo, S. Y., Hong, S. K., Choi, J. W., Kim, C. H., Weinstein, B. M. and Huh, T. L. (1998). Characterization of two frizzled8 homologues expressed in the embryonic shield and prechordal plate of zebrafish embryos. Mech. Dev. 78,193 -198.[CrossRef][Medline]
Kimmel, C. B. and Law, R. D. (1985). Cell lineage of zebrafish blastomeres. III. Clonal analysis of the blastula and gastrula stages. Dev. Biol. 108,94 -101.[Medline]
Kimmel, C. B. and Warga, R. (1986). Tissue specific cell lineages originate in the gastrula of the zebrafish. Science 231,365 -368.
Kimmel, C. B., Kane, D. A., Walker, C., Warga, R. M. and Rothman, M. B. (1989). A mutation that changes cell movement and cell fate in the zebrafish embryo. Nature 337,358 -362.[CrossRef][Medline]
Kimmel, C. B., Warga, R. M. and Kane, D. A.
(1994). Cell cycles, clonal strings, and the origin of the
zebrafish central nervous system. Development
120,265
-276.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Kodjabachian, L., Dawid, I. B. and Toyama, R. (1999). Gastrulation in zebrafish: what mutants teach us. Dev. Biol. 213,231 -245.[CrossRef][Medline]
Kuroda, H., Inui, M., Sugimoto, K., Hayata, T. and Asashima, M. (2002). Axial protocadherin is a mediator of prenotochord cell sorting in Xenopus. Dev. Biol. 244,267 -277.[CrossRef][Medline]
Makita, R., Mizuno, T., Koshida, S., Kuroiwa, A. and Takeda, H. (1998). Zebrafish wnt11: pattern and regulation of the expression by the yolk cell and No tail activity. Mech. Dev. 71,165 -176.[CrossRef][Medline]
Melby, A., Kimelman, D. and Kimmel, C. (1997). Spatial regulation of floating head expression in the developing notochord. Dev. Dyn. 209,156 -165.[CrossRef][Medline]
Melby, A. E., Warga, R. M. and Kimmel, C. B.
(1996). Specification of cell fates at the dorsal margin of the
zebrafish gastrula. Development
122,2225
-2237.
Montell, D. J. (1999). The genetics of cell
migration in Drosophila melanogaster and Caenorhabditis elegans
development. Development
126,3035
-3046.
Morgan, T. H. (1895). The formation of the fish embryo. J. Morphol. 10,419 -472.
Munro, E. M. and Odell, G. (2002a).
Morphogenetic pattern formation during ascidian notochord formation is
regulative and highly robust. Development
129, 1-12.
Munro, E. M. and Odell, G. M. (2002b).
Polarized basolateral cell motility underlies invagination and convergent
extension of the ascidian notochord. Development
129, 13-24.
Myers, D. C., Sepich, D. S. and Solnica-Krezel, L. (2002a). Bmp activity gradient regulates convergent extension during zebrafish gastrulation. Dev. Biol. 243, 81-98.[CrossRef][Medline]
Myers, D. C., Sepich, D. S. and Solnica-Krezel, L. (2002b). Convergence and extension in vertebrate gastrulae: cell movements according to or in search of identity? Trends Genet. 18,433 -488.[CrossRef][Medline]
Nutt, S. L., Dingwell, K. S., Holt, C. E. and Amaya, E.
(2001). Xenopus Sprouty2 inhibits FGF-mediated
gastrulation movements but does not affect mesoderm induction and patterning.
Genes Dev. 15,1152
-1166.
Papan, C. and Campos-Ortega, J. A. (1994). On the formation of the neural keel and neural tube in the zebrafish Danio (Brachydanio) rerio. Dev. Biol. 203,178 -186.[CrossRef]
Schulte-Merker, S., van Eeden, S. F., Halpern, M. E., Kimmel, C.
B. and Nüsslein-Volhard, C. (1994). no tail
(ntl) is the zebrafish homologue of the mouse T (Brachyury) gene.
Development 120,1009
-1015.
Shih, J. and Keller, R. (1992a). Cell motility
driving mediolateral intercalation in explants of Xenopus laevis.Development 116,901
-914.
Shih, J. and Keller, R. (1992b). Patterns of
cell motility in the organizer and dorsal mesoderm of Xenopus laevis.Development 116,915
-930.
Smith, J. C., Conlon, F. L., Saka, Y. and Tada, M. (2000). Xwnt11 and the regulation of gastrulation in Xenopus.Philos. Trans. R. Soc. Lond. B Biol. Sci. 355,923 -930.[CrossRef][Medline]
Solnica-Krezel, L. (1999). Pattern formation in zebrafishfruitful liaisons between embryology and genetics. Curr. Top. Dev. Biol. 41, 1-35.[Medline]
Solnica-Krezel, L., Stemple, D. L., Mountcastle-Shah, E.,
Rangini, Z., Neuhauss, S. C. F., Malicki, J., Schier, A. F., Stainier, D. Y.
R., Zwartkruis, F., Abdelilah, S. et al. (1996). Mutations
affecting cell fates and cellular rearrangements during gastrulation in
zebrafish. Development
123, 67-80.
Tada, M. and Concha, M. L. (2001). Vertebrate gastrulation: calcium waves orchestrate cell movements. Curr. Biol. 11,R470 -R472.[CrossRef][Medline]
Tada, M. and Smith, J. C. (2000). Xwnt11 is a
target of Xenopus Brachyury: regulation of gastrulation movements via
Dishevelled, but not through the canonical Wnt pathway.
Development 127,2227
-2238.
Tada, M. and Smith, J. C. (2001). T-targets: clues to understanding the functions of T-box proteins. Dev. Growth Differ. 43,1 -11.[CrossRef][Medline]
Talbot, W. S., Trevarrow, B., Halpern, M. E., Melby, A. E., Farr, G., Postlethwait, J. H., Jowett, T., Kimmel, C. B. and Kimelman, D. (1995). A homeobox gene essential for zebrafish notochord development. Nature 378,150 -157.[CrossRef][Medline]
Wallingford, J. B. and Harland, R. M. (2001).
Xenopus Dishevelled signaling regulates both neural and mesodermal
convergent extension: parallel forces elongating the body axis.
Development 128,2581
-2592.
Wallingford, J. B., Rowning, B. A., Vogeli, K. M., Rothbacher, U., Fraser, S. E. and Harland, R. M. (2000). Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405,81 -85.[CrossRef][Medline]
Wallingford, J. B., Ewald, A. J., Harland, R. M. and Fraser, S. E. (2001a). Calcium signaling during convergent extension in Xenopus. Curr Biol 11,652 -661.[CrossRef][Medline]
Wallingford, J. B., Vogeli, K. M. and Harland, R. M. (2001b). Regulation of convergent extension in Xenopus by Wnt5a and Frizzled-8 is independent of the canonical Wnt pathway. Int. J. Dev. Biol. 45,225 -227.[Medline]
Warga, R. M. and Kimmel, C. B. (1990). Cell movements during epiboly and gastrulation in zebrafish. Development 108,569 -580.[Abstract]
Westerfield, M. (1995). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio). Eugene: University of Oregon Press.
Wilson, E. T., Helde, K. A. and Grunwald, D. J. (1993). Something's fishy here rethinking cell movements and cell fate in the zebrafish embryo. Trends Genet. 9, 348-352.[CrossRef][Medline]
Yamamoto, A., Amacher, S. L., Kim, S. H., Geissert, D., Kimmel,
C. B. and de Robertis, E. M. (1998). Zebrafish paraxial
protocadherin is a downstream target of spadetail involved in
morphogenesis of gastrula mesoderm. Development
125,3389
-3397.
Zalik, S. E., Lewandowski, E., Kam, Z. and Geiger, B. (1999). Cell adhesion and the actin cytoskeleton of the enveloping layer in the zebrafish embryo during epiboly. Biochem. Cell Biol. 77,527 -542.[CrossRef][Medline]