1 Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany
2 University College London, Department of Anatomy and Developmental Biology,
Gower Street, London WC1E 6BT, UK
3 Programa de Morfologia, Instituto de Ciencias Biomedicas, Facultad de
Medicina, Universidad de Chile, PO Box 70079, Santiago de Chile, Chile
4 University of Iowa, Department of Biological Sciences, Iowa City, IA 52242,
USA
5 Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35 / III,
72076 Tübingen, Germany
6 University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2
3DY, UK
Author for correspondence (e-mail:
heisenberg{at}mpi-cbg.de)
Accepted 21 July 2003
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SUMMARY |
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Key words: Wnt signalling, Cell migration, Gastrulation, Zebrafish
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Introduction |
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The molecular basis underlying cell movements during vertebrate
gastrulation is only beginning to be unravelled. Several studies have shown
that Wnt genes are important for normal gastrulation movements, both in
Xenopus and in zebrafish (reviewed by
Keller, 2002;
Tada et al., 2002
;
Wallingford et al., 2002
). The
signalling pathway through which these Wnt ligands transmit their
morphogenetic activity shares several components with the Frizzled (Fz)
signalling cascade involved in the establishment of epithelial PCP in
Drosophila. Such shared components include the Wnt receptor Frizzled
(Fz), the intracellular signalling mediator Dishevelled (Dsh), the small
GTPases RhoA, Rac and Cdc42, the Rho effector Rho Kinase 2 (Rok2), the
transmembrane protein Strabismus/van Gogh (Stbm/Vang) and the ß'
subunit of Protein Phosphatase 2A (PP2A), Widerborst (Wdb)
(Darken et al., 2002
;
Djiane et al., 2000
;
Goto and Keller, 2002
;
Habas et al., 2001
;
Habas et al., 2003
;
Hannus et al., 2002
;
Heisenberg et al., 2000
;
Marlow et al., 2002
;
Park and Moon, 2002
;
Sumanas and Ekker, 2001
;
Sumanas et al., 2001
;
Wallingford et al., 2000
).
In zebrafish, several mutants that exhibit defects in cell movements during
gastrulation have been identified. In pipetail
(ppt)/wnt5, knypek (kny)/glypican4/6 and
trilobite (tri)/stbm mutants, CE movements are
predominantly affected in posterior regions of the gastrula, whereas in
slb/wnt11 (slb) embryos, CE movements in both anterior and
posterior parts of the gastrula are defective
(Hammerschmidt et al., 1996;
Heisenberg et al., 2000
;
Jessen et al., 2002
;
Kilian et al., 2003
;
Rauch et al., 1997
;
Solnica-Krezel et al., 1996
;
Topczewski et al., 2001
).
Epistasis experiments indicate that kny/glypican4/6 can function in
the slb/wnt11 signalling pathway, whereas tri/stbm appears
to act in a parallel pathway (Heisenberg
and Nüsslein-Volhard, 1997
;
Topczewski et al., 2001
).
However, the way in which these genes regulate gastrulation movements on a
cellular basis is not yet fully understood.
Comparison of the functions of the Wnt/PCP pathway during vertebrate
gastrulation and the Fz/PCP pathway in Drosophila reveals conserved
and divergent signalling mechanisms. In the Drosophila wing disc, the
Fz/PCP pathway determines polarity of cells along the proximal-distal axis,
which results in the directed outgrowth of a single wing hair at the distal
tip of those cells (reviewed by Adler,
2002). Proximal-distal cell polarization is preceded by an
asymmetric localization of various components of the PCP pathway, such as Fz,
Dsh, Fmi, Diego and Wdb, to the proximal and/or distal cortical domains of
these cells (reviewed by Strutt,
2002
). During vertebrate gastrulation, components of the Wnt/PCP
pathway, such as Dsh and Stbm/Vang, are localised to the cell membrane
(Park and Moon, 2002
;
Wallingford et al., 2000
).
However, no asymmetric distribution of these proteins has been observed.
Morphologically, ectodermal and mesendodermal cells undergoing CE movements
are elongated along their medio-lateral axis at late stages of gastrulation
(Concha and Adams, 1998
;
Elul and Keller, 2000
;
Keller et al., 1992
). Several
studies in Xenopus and zebrafish demonstrate that the medio-lateral
elongation of these cells is regulated by components of the Wnt/PCP pathway
such as Dsh, Kny/Glypican4/6, Tri/Stbm, Rok2 and Ppt/Wnt5
(Darken et al., 2002
;
Goto and Keller, 2002
;
Jessen et al., 2002
;
Kilian et al., 2003
;
Marlow et al., 2002
;
Topczewski et al., 2001
;
Wallingford et al., 2000
).
Thus, it is possible that the Wnt/PCP pathway, like its Drosophila
counterpart, is involved in the regulation of cell polarity during vertebrate
gastrulation. However, while in the Drosophila wing epithelium the
ultimate readout of planar cell polarisation is the unidirectional (monopolar)
orientation of one wing hair per cell, no equivalent Wnt/PCP-dependent
monopolar cell polarisation has been observed during vertebrate
gastrulation.
In this study, we analysed the role of slb/wnt11 in regulating cell movements and morphology during zebrafish gastrulation. From 3D reconstruction and motion analysis of individual cells, we present evidence that slb/wnt11 is required for the directionality and velocity of movements of hypoblast cells in the forming germ ring at the onset of gastrulation. We further demonstrate that slb hypoblast cells that have impaired migratory cell movements also exhibit defects in the orientation of cellular processes along their individual movement directions. This indicates that process orientation mediated by slb/wnt11 is crucial for facilitating and stabilising hypoblast cell movements at the onset of gastrulation. These observations provide the first direct evidence of a role of the slb/wnt11 signalling pathway in regulating process orientation and migratory cell movements at early stages of zebrafish gastrulation.
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Materials and methods |
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In situ hybridisation and gelatine sectioning
Whole-mount in situ hybridisation was performed as previously described
(Barth and Wilson, 1995). For
sections, in situ-stained embryos were equilibrated in gelatine/albumen
solution (0.49% gelatine, 30% egg albumen, 20% sucrose in PBS), transferred
into an embedding form coated with fresh polymerisation solution (albumen, 25%
glutaraldehyde, 10:1) and kept 15 minutes at room temperature to allow
polymerisation. 20-µm serial sections were taken using a Leica Vibratome
VT1000S.
mRNA misexpression
For ubiquitous overexpression, mRNA was injected into the yolk of
zygote/one-cell stage embryos as previously described
(Barth and Wilson, 1995). For
scatter labelling of wild-type and slb mutant embryos, a mixture of
30-100 pg cytosolic GFP mRNA and 60-150 pg GAP43-GFP mRNA was injected into
single blastomeres of 8-32-cell stage embryos. Transgenic gscGFP embryos were
scatter labelled by injecting 250 pL of 0.5% rhodamine-dextrane with
Mr =2.000x103 (Molecular Probes, Eugene)
in 0.2 M KCl into single blastomeres of embryos at the 8- to 32-cell
stage.
Confocal imaging
Shield-stage embryos were manually dechorionated and mounted in 1% agarose
in E3 fish medium. Life images were obtained at room temperature with a
60xwater-immersion objective on a BioRad Radiance 2000 Multiphoton
Confocal Microscope setup.
For dual channel confocal timelapses, 488 nm Ar Laser and 543 nm He/Ne laser lines were used simultaneously. The emitted light passed through a 560 nm dichroic mirror/long path filter. Image zstacks were obtained by scanning areas of 204.8 µmx204.8 µm (0.4 µm pixel1) with 166 lines per second and 1.5 µm steps over a total vertical distance of 66 µm. For each experiment, 12-20 image stacks were acquired in 4-minute time intervals.
For single channel timelapse recordings, z-stacks were acquired by scanning an area of 102.4 µmx102.4 µm (0.2 µm pixel1) with 750 lines second1 and 0.5 µm steps over a total vertical distance of 50 µm. Stacks were taken continuously with no time gap in between. A mode-locked infrared laser line between 890 and 910 nm with an average power of 500 mW was used, originating from a Mira 900 two-photon Ti::Sapphire laser. A 532 nm laser source with 5 W output power was used as a pump laser (Coherent, California).
Image analysis
The acquired confocal z-stacks were volume rendered in 3D over time using
the program Volocity (Improvision, UK). The cell movement analysis was also
carried out using this software by manually measuring the positions of the
geometric centre of the cells (the cell centroid) in three dimensions. Tracing
was done manually by outlining the cell borders, using a newly developed
version of the 3D-DIAS software (Soll et
al., 2000; Heid et al.,
2002
). The cell bodies were always traced separately from cellular
processes. Every cellular extension that emerged from the cell body at an
angle of <135° and a width of >2 µm was defined as a pseudopodial
extension. Epiblast and prechordal plate precursor cells were identified
according to their position and net movement direction. The cell traces were
rendered in three dimensions and measured using newly developed JAVA-based
3D-DIAS software (E.V. and D.R.S., unpublished), which allowed the
quantification of several morphometric parameters such as the surface area,
volume, centroid position and roundness
(rnd=6xVx
0.5xA1.5) of
single cells in three dimensions.
The subsequent analysis of the obtained morphological data was carried out using Microsoft Excel and ProFit (Quantum Soft, Basel). To account for the resolution limit in z-direction and because the manual traces could not always be perfect, processes that had <5% of the length of the corresponding cell body, were <2 µm long or had a volume <1.25x102% and a roundness >0.7 were not analysed.
Statistical analysis
Student's t-test (Microsoft Excel) with a two-tailed distribution
was used to analyse significance between two mean values. For analysis of
correlation between process orientation and movement persistence, a special
Microsoft Excel plug-in was used that tested a linear dependence between the
parameters (Müller et al.,
2002).
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Results |
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slb mesendodermal cells exhibit defects in the orientation
of their cellular processes along their individual movement directions
To investigate the cellular mechanisms that underlie the cell-movement
defects in slb mutants, we recorded the morphology of individual
cells labelled with a mixture of cytosolic and membrane-anchored GFP
(Okada et al., 1999) using
two-photon confocal microscopy. Parameters of cell morphology, such as the
number, shape and orientation of cellular processes, were measured in 3D over
time. Process orientation was calculated relative to the individual movement
direction of each cell by determining the points on the cell surface where
processes emerged. Temporal changes in the morphology of cellular processes
were quantified by calculating the total length and roundness, which measures
how efficiently a surface encloses a volume
(Heid et al., 2002
), of each
process at different timepoints (see Materials and methods).
First, we analysed the morphology of axial hypoblast cells at the leading edge of the presumptive prechordal plate. We chose those cells because they have more distinct processes than cells in more posterior parts of the prechordal plate (data not shown) and are, therefore, easier to analyse in respect to process formation and orientation. By analysing dynamic changes in the orientation of cellular processes relative to changes in the movement directions of single cells, we found that wild-type prechordal-plate precursor cells form pseudopod-like processes (average roundness <0.7) that are oriented preferentially towards their individual directions of movement (+x axis, Fig. 4A-C; see Movie 4 at http://dev.biologists.org/supplemental/; average percentage of processes per cell in +x versus x direction at all timepoints, 70% versus 30%; s.d., 38%; P=1.6x105). By contrast, these cells show no such process orientation in slb mutant embryos (Fig. 4D-F and data not shown), but preferentially orient their processes towards the underlying cells or substrate (z direction; Fig. 4D-F; see Movie 4 at http://dev.biologists.org/supplemental/; average percentage of processes per cell in +z versus z direction over all timepoints, 39% versus 61%; s.d., 39%; P=9.0x103). Comparison of the dynamic changes in the distribution of pseudopod lengths between wild-type and slb hypoblast cells along the x and z axes indicates that most elongated processes point into the +x direction in wild-type embryos but not in slb mutants (Fig. 4B',B'',E',E''). The average number, shape and length of processes revealed no obvious differences between wild type and slb hypoblast cells (data not shown).
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Taken together, these findings indicate that slb/wnt11 is needed in hypoblast cells to orient pseudopod-like processes towards their individual movement directions. In the absence of slb/wnt11 activity, both hypoblast and epiblast cells reorient their processes towards the underlying cells or substrate. The requirement for slb/wnt11 appears to be specific to process orientation, because the general shape of the cell body in epiblast and hypoblast cells is unchanged in slb mutants.
The slb mesendodermal cell morphology defect is linked to
less directed movements of these cells and can be rescued by ubiquitous
overexpression of slb/wnt11
To investigate if the change in the preferential orientation of cellular
processes in slb mutant cells was linked to the cell movement defect
in these cells, we determined the movement persistence of individual hypoblast
cells (Fig. 3 and see above) in
relation to the percentage of processes oriented towards their individual
movement directions in wild-type and slb mutant embryos. Wild-type
hypoblast cells that have processes preferentially oriented towards their
individual movement directions also show highly persistent movements, whereas
in slb mutant cells, a reduction in the percentage of processes
oriented towards their individual movement directions is accompanied by less
persistent movements (Fig. 6).
Testing the correlation between process orientation and movement persistence
for all cells analysed (wild type and slb) shows that these values
are statistically linked (P=0.02; r=0.36)
(Müller et al., 2002).
This indicates that slb/wnt11-mediated orientation of cellular
processes is needed in hypoblast cells for the directionality of cell
movements at the onset of gastrulation.
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Discussion |
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The cellular function of slb/wnt11 at the onset of
gastrulation
The main finding of this study is that slb/wnt11 is required for
both cellular process orientation and directed cell movements of hypoblast
cells at the onset of gastrulation. To obtain insight into the relationship of
process orientation and cell movement in single cells, we developed an assay
that allows us to compare dynamic changes in process orientation with changes
in the direction of cell movements. We showed that in wild-type hypoblast
cells, process orientation and movement direction are aligned, whereas in
slb mutant cells, no such alignment is observed. The misalignment of
process orientation and movement direction in slb mutants is linked
to less efficient movements of hypoblast cells towards the animal pole.
However, although these cells move slower with more frequent changes in the
movement direction (`less persistent movement'), the net direction of their
movement appears to be unaffected. This indicates that the role of
slb/wnt11-mediated orientation of cellular processes in the direction
of individual cell movements is to facilitate and stabilise these movements,
rather than to determine the overall direction of movement.
How do these findings relate to previous studies that implicate
slb/wnt11 and the Wnt/PCP pathway in the regulation of CE movements
at later stages of gastrulation (reviewed by
Tada et al., 2002;
Wallingford et al., 2002
)? We
analysed the function of slb/wnt11 in cells that were
morphogenetically distinct from cells undergoing CE movements. Hypoblast cells
in the region of the forming germ ring at the onset of gastrulation do not
show medio-lateral cell intercalation behaviour nor elongate along their
medio-lateral axes (M.L.C. and C.P.H., unpublished), as described for cells
undergoing CE movements (Concha and Adams,
1998
; Glickman et al.,
2003
). Instead, they move as loosely associated cells in a
straight path towards the animal pole, similar to the behaviour of cells with
directed migration on a substrate [this study and Warga and Kimmel
(Warga and Kimmel, 1990
)].
Consequently, we found that slb/wnt11 is required for the orientation
of cellular processes along the movement axis of individual cells, a feature
that is associated commonly with migrating cells in vitro and in vivo
(Lauffenburger and Horwitz,
1996
).
By contrast, the cellular functions of the Wnt/PCP pathway in controlling
cell movements during CE have not been fully addressed. Reduced medio-lateral
cell elongation in different mutant and morphant phenotypes of the Wnt/PCP
pathway has been associated with slower, less persistent cell movements at
late stages of gastrulation (Darken et al.,
2002; Goto and Keller,
2002
; Jessen et al.,
2002
; Marlow et al.,
2002
; Park and Moon,
2002
; Topczewski et al.,
2001
; Wallingford et al.,
2000
). However, the specific changes in cell elongation have not
been correlated with the dynamic changes in the individual movement directions
of these cells. Moreover, these cells have been analysed mainly in two
dimensions (x-y plane). This limits the view on those cells and, consequently,
the interpretation of these observations because cells undergoing CE movements
can also exhibit distinctive movements along the z-axis
(Glickman et al., 2003
) (F.U.
and C.P.H., unpublished). Future studies to compare the role of the Wnt/PCP
pathway in cellular dynamics during early versus late stages of gastrulation
are needed to reveal the common and divergent aspects of Wnt/PCP signalling
during the course of gastrulation.
Potential target processes of slb/wnt11 at the onset of
gastrulation
How does slb/wnt11 control the orientation of cellular processes
and directed cell movements in the germ ring at the onset of gastrulation? Our
observation that slb/wnt11 is expressed within the epiblast, although
it is required predominantly in the hypoblast, indicates that
slb/wnt11, produced in epiblast cells, directly or indirectly
influences hypoblast cell movement and morphology. It is possible that
slb/wnt11, secreted by epiblast cells, might exert direct control
over hypoblast cell morphogenesis by regulating rearrangements of the
hypoblast cytoskeleton that control the formation and orientation of processes
in these cells. slb/wnt11 might function either permissively, by
allowing these cells to extend and stabilise cellular processes in their
preferred orientation, or it might function instructively, by determining the
orientation of these processes. Our observation that ubiquitous overexpression
of slb/wnt11 rescued the cell morphology and movement phenotype of
slb mutants argues in favour of a more permissive function of
slb/wnt11 in this process. Findings from recent studies in zebrafish,
which show that Rok2, which directly regulates cytoskeletal elements such as
myosin in Drosophila, is a downstream component of the
slb/wnt11 signalling pathway support a function of slb/wnt11
in regulating cytoskeletal dynamics
(Marlow et al., 2002).
Alternatively, slb/wnt11 might also indirectly affect
morphogenesis of hypoblast tissue by regulating the differential adhesiveness
of hypoblast and epiblast cells, which would have a secondary effect on
process orientation and directed cell movement. The finding that hypoblast
(and epiblast) cells in slb mutants reorient their processes towards
the underlying cell(s) or the yolk-cell surface indicates that, in the absence
of slb/wnt11 function, either the adhesion of these cells to their
respective substrates is increased or adhesion between these cells is
decreased. Indeed, our own preliminary observations show that slb
hypoblast cells in culture display a reduced tendency to `cluster' (F.U. and
C.P.H., unpublished), which indicates that there are defects in cell-cell or
cell-substrate adhesion. Further support for a role of slb/wnt11 in
regulating cell adhesion comes from recent studies in Xenopus, which
show that the presumptive receptor for Wnt11, Fz7, is required for the
effective separation of mesoderm from ectoderm at the onset of gastrulation
(Winklbauer et al., 2001).
It is likely that slb/wnt11 uses both of these, and possibly other unknown, processes to control cellular morphology and movements during gastrulation. Future studies should identify the specific requirements of these target processes for the function of slb/wnt11 in different tissues during the course of gastrulation.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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