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First published online 11 June 2008
doi: 10.1242/dev.014704
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1 Department of Biology, University of Virginia, Charlottesville, VA 22903,
USA.
2 Department of Cell Biology, University of Virginia, Charlottesville, VA 22903,
USA.
* Author for correspondence (e-mail: frogman{at}virginia.edu)
Accepted 13 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Morphogenesis, Xenopus, Myosin, Mesoderm, Notochord
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Keller et al., 2003;
Myers et al., 2002;
Zallen, 2007). Any example of
CE in these diverse systems could be due to passive stretching of the tissue
as a consequence of external forces, or could be active and autonomously
driven by internal force-generating processes
(Keller et al., 2000). CE of
the Drosophila germband, an epithelial sheet of cells, occurs by
intercalation of cells to form a narrower, longer array in a process involving
apical junctional remodeling, either between pairs of neighboring cells or
between multiple cells in rosettes, and is dependent on the polarized
localization of myosin II in these junctions
(Bertet et al., 2004;
Blankenship et al., 2006;
Zallen and Wieschaus, 2004).
CE of the dorsal axial and paraxial mesoderm of vertebrates, a mesenchymal
tissue that therefore lacks apical junctions, also occurs by the polarized
intercalation of cells to form a longer, narrower array, and this process
depends on a characteristic polarized protrusive activity
(Glickman et al., 2003;
Keller et al., 2000;
Sepich et al., 2000) for which
the vertebrate non-canonical Wnt/planar cell polarity (PCP) pathway is
essential (Goto and Keller,
2002; Heisenberg et al.,
2000; Tada and Smith,
2000). In both epithelial
(Iwaki et al., 2001;
Lengyel and Iwaki, 2002;
Zallen and Wieschaus, 2004)
and mesenchymal systems (Ninomiya et al.,
2004), patterning cues in the future axis of extension are
necessary for intercalation of cells in the axis of convergence. It is not
known in any system how the forces that drive the active intercalation of
cells, and thus CE, are actually generated and transmitted from the molecular
to the cellular and tissue level. Here we examine the function of conventional
myosins in this process as it occurs in the dorsal mesoderm of Xenopus
laevis, a system in which the cellular- and tissue-level biomechanics are
at least partially understood.
In Xenopus, active cell intercalation during CE is accompanied by
a characteristic suite of polarized cell behavior [mediolateral intercalation
behavior (MIB)] (Shih and Keller,
1992a), which is thought to exert traction on adjacent cells and
pull them between one another. As the cells actively wedge between one
another, they force the elongation of the tissue
(Keller et al., 1992). At the
tissue level, the axis of mediolateral intercalation, and thus the axis of
tissue convergence, describes an arc across the dorsal lip of the blastopore.
These convergence forces are generated by this polarized, oriented cell
behavior, and are associated with the development of circumblastoporal cell
intercalation leading to the shortening of these arcs of hoop stress around
the blastopore, squeezing the blastopore shut, as well as driving an
orthogonal anterior-posterior axial extension
(Keller et al., 2000;
Shih and Keller, 1992a;
Shih and Keller, 1992b). These
arcs of tension must run through cells and, at least at the notochord-somite
boundary, through the extracellular matrix (ECM), but the molecular mechanisms
and subcellular functional elements that generate or support this tension have
not been identified. Disruption of the mechanical continuity of these arcs of
convergence blocks the development of these circumblastoporal forces and thus
blastopore closure (Keller,
1981; Schectman,
1942; Skoglund and Keller,
2007). Here we report that myosin IIB is essential for the
organization of a previously undescribed cortical actin cytoskeletal network
that underlies the development of these circumblastoporal convergence
forces.
Cytoskeletal (non-muscle) myosin II complexes are molecular motors that can
both bind to and cross-link actin filaments into higher-order structures, as
well as translate chemical energy into force production inside the cell by
coupling ATP hydrolysis to movement along an actin filament
(Geeves and Holmes, 2005;
Rayment and Holden, 1994).
Vertebrates have three myosin IIs (A, B and C), characterized by distinct
heavy chain isoforms (MHC-A, -B and -C)
(Berg et al., 2001;
Golomb et al., 2004). Myosins
are hexamers composed of pairs of myosin heavy chains along with two
regulatory and two essential light chains to form a bivalent actin-binding
unit, which further aggregates into higher-order bipolar filaments to
facilitate actin cross-linking and thereby regulate cytoskeletal architecture
in the cell (Landsverk and Epstein,
2005). Myosin IIA and IIB exhibit distinct biochemical rates for a
conserved set of activities, including on/off rates for actin binding, rates
of actin-dependent ATP hydrolysis and duty cycle characteristics governing the
balance between cytoskeletal assembly activity and force production
(Geeves and Holmes, 1999;
Holmes and Geeves, 2000;
Kelley et al., 1996). They can
be differentially located even when expressed in the same cell
(Kelley et al., 1996;
Kolega, 1998;
Maupin et al., 1994;
Rochlin et al., 1995),
suggesting that each isoform has specific roles.
We have investigated the role of myosin IIB in convergence and extension.
In Xenopus, myosin IIB is the best candidate to be involved in CE
because MHC-B mRNA is expressed in dorsal mesoderm and is upregulated by
activin in animal cap experiments
(Bhatia-Dey et al., 1993;
Bhatia-Dey et al., 1998;
Kelley et al., 1996), and thus
is expressed at the right time and place for involvement in CE of presumptive
mesoderm. We find that myosin IIB is required for normal blastopore closure by
operating in the process of CE at the dorsal lip. We show that myosin IIB
organizes a cortical actin network in intercalating cells, that this
cytoskeletal structure is polarized with respect to the embryonic axis and
that misregulation of this network is likely to underlie the range of
phenotypes observed in MHC-B-depleted cells, including defects in regulation
of polarized cell motility and reduced cell-cell and cell-matrix adhesion.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
staged (Nieuwkoop and Faber,
1967) by conventional means. In situ hybridizations were performed
as described (Skoglund et al.,
2006), using XhoI and SP6 RNA polymerase to make the
brachyury probe from the plasmid pXBRA
(Amaya et al., 1993). Explants
to visualize notochordal cell behavior were cut as described in
Fig. 2C.
Whole-mount immunochemistry and western blotting
MHC-A and MHC-B were detected in whole-mount staining experiments and
western blotting with isoform-specific anti-peptide antibodies (Covance,
Berkeley, CA). Whole-mount immunochemistry followed
(Skoglund et al., 2006), using
a rhodamine-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch,
West Grove, PA) for confocal imaging after dehydration and clearing as
described (Skoglund et al.,
2006). Western blots were performed as described
(Skoglund and Keller, 2007),
using stage-19 embryo extracts for MHC-B and stage-13 embryos for MHC-A;
extracts were run on 5% polyacrylamide gels and detection was with anti-rabbit
HRP using Supersignal reagents (Pierce, Rockford, IL). Densitometry was
performed as described (Skoglund et al.,
2006).
Adhesion assays for C-cadherin and fibronectin and evaluation of surface expression of C-cadherin and integrin 
5
The C-cadherin (C-Cad) adhesion assay was performed as described
(Chen and Gumbiner, 2006;
Niessen and Gumbiner, 2002),
using 4 µg/ml purified recombinant C-Cad extracellular domain for coating
plastic as a substrate for cells; assays were performed in triplicate with
more than 400 cells per condition per experiment. The fibronectin (FN)
adhesion assay was similar, but used 20 µg/ml FN as substrate (Sigma, St
Louis, MO). Rescue of myosin IIB depletion-mediated adhesion to FN was with a
human myosin IIB-GFP fusion construct
(Vicente-Manzanares et al.,
2007), injected at 100 pg per embryo at the 4-cell stage. This
experiment was modified in that dorsal explants were cut at early gastrulation
and cultured until stage 18, requiring addition of 10 µl of 0.5 M EDTA to
facilitate dissociation to single cells but allowing visualization of
fluorescent cells in the adhesion assay. The biotinylation and trypsin
experiments to determine the amount of cell-surface C-Cad were as described
(Chen and Gumbiner, 2006), and
the same procedure was used to determine the amount of cell-surface integrin
5 using a polyclonal antibody directed against this protein.
Morpholinos and injections
A morpholino oligonucleotide (MO) directed against the translation start
site of MHC-B (5'-CTTCCTGCCCTGGTCTCTGTGACAT-3') was produced (Gene
Tools, Philomath, OR); the control MO varied at five nucleotides
(5'-CTTGCTCCCCTGCTCTCTCTGAGAT-3').
MO injections were into both cells at the 2-cell stage or into single
blastomeres at the 32-cell stage targeted to the dorsal marginal zone by
pigment cues (Lee and Gumbiner,
1995), using 0.03-5 nl of 1 mM MO to achieve 1-10 µM final
concentration in the injected cell. Dorsal cells were co-injected with 0.5 nl
of 30 ng/µl RNA in water in blastomeres at the 32-cell stage. C-terminal
moesin-GFP was from the RNA expression plasmid CS107-GFP-Moe constructed in Dr
John Wallingford's laboratory (Institute for Cellular and Molecular Biology,
University of Texas, Austin, TX, USA) from the original
(Litman et al., 2000). Capped
RNA was made using AscI/SP6 polymerase and the mMessage mMachine Kit
(Ambion, Austin, TX). In some experiment, 50 ng ruby-labeled dextran
(Invitrogen, Molecular Probes, Carlsbad, CA) was co-injected at the 32-cell
stage. Myosins were pharmacologically targeted by injecting 50 nl of 2 mM
(-)-blebbistatin (Calbiochem, San Diego, CA) in dimethylsulfoxide (DMSO;
Sigma), or DMSO alone, into the blastocoel of mid-gastrula (stage 10) embryos
and observing for defects in blastopore closure. Evaluating whether ectopic
C-Cad expression could rescue partially MHC-IIB morphant embryos was performed
in triplicate by injecting 1.5 ng C-Cad RNA
(Chen and Gumbiner, 2006) with
or without 5 µM MHC-IIB MO, and assaying embryos for blastopore closure
defects.
Imaging
Vegetal-view movies of whole embryos were recorded on an inverted Nikon
IX70 microscope running MetaMorph software and using a 4x objective.
Still images were captured using either a Hamamatsu color chilled CCD or a MTI
CCD camera mounted on a Zeiss stereoscope, and images processed though NIH
Image (NIH), ImageJ (NIH) or Photoshop (Adobe) software. Confocal imaging used
a Nikon IX70 with 60x and 100x 1.4 NA oil-immersion lenses, a
BioRad Radiance2100 system and software at the Keck Center for Cellular
Imaging at the University of Virginia. Framing intervals of 15 seconds to 3
minutes were used and image processing consisted of frame averaging and
brightness and contrast adjustment as described previously
(Shih and Keller, 1992a).
Quantitation of the motion of actin foci was by plotting foci position from
magnified confocal movies onto acetate sheets and then measuring
anterior-posterior and mediolateral displacement (A and M, respectively) for
each 1-minute interval. For notochord, ten cells with two to six foci per cell
(33 foci total) were followed for 5 minutes; for pre-involution axial mesoderm
[pre-notochord (PN)] and animal cap (AC), five cells with at least 20 foci
were followed. The average A and M displacement in arbitrary units was
generated for each cell, except for AC cells, for which it is not possible to
determine anterior-posterior position, so the direction of maximum excursion
of the first focus examined became M, and the orthogonal direction became A,
and the average of these is presented with s.e.m.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Skoglund et al., 2006). In
more-posterior notochordal regions, where cell intercalation and CE were still
in an earlier phase, MHC-B was distributed throughout the cortical region of
the cells as well, but it was also distributed in a polarized fashion within
the cortex, with high levels at the anterior and posterior edges of the cells
where they attach to the NSB (Fig.
1D, arrowheads), and at triple cell junctions where the large
lamellipodia that lead the process of active cell intercalation are formed
(Shih and Keller, 1992a)
(Fig. 1D, asterisks). This
pattern is consistent with a role for myosin IIB as a mechanical element in
the mediolateral arcs of tension that are postulated to accompany dorsal
mesodermal CE (Keller et al.,
2000). Imaging the ventral surface of a neural deep-over-mesoderm
explant for MHC-B revealed staining over the basal surfaces of notochordal and
somitic cells (Fig. 1E), and
indicated that the myosin IIB enrichment at these tissue boundaries is not due
entirely to a generalized increased concentration of myosin IIB in the cortex
of cells facing tissue boundaries, but also includes a component of myosin IIB
organized in a linear or fibrillar pattern at these tissue boundaries.
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; Keller et al.,
2003), and thus this phenotype is consistent with a specific
failure of CE in the MHC-B MO-injected embryos. Other morphogenic processes
occurred normally in morphant embryos, including cell division through the
cleavage stages and the apical constriction that is associated with bottle
cell formation (as revealed by the characteristic pigment accumulation;
Fig. 2C,G, insets)
(Hardin and Keller, 1988;
Keller, 1981), indicating that
they do not depend on zygotic MHC-B protein. Blastopore closure was also
perturbed by acute application of the myosin II inhibitor blebbistatin to the
embryo at early gastrulation stages, with 41% of embryos failing to close
their blastopores and 28% exhibiting delayed blastopore closure
(n=32), whereas embryos treated with DMSO alone did not exhibit
significant developmental defects (5% delayed blastopore closure,
n=40).
MHC-B MO-mediated failure of gastrulation is dose dependent
We titrated MHC-B MO injection to create a series of embryos with declining
MHC-B protein levels. We found that the severity of failure of blastopore
closure is dependent on the dose of MO injected, and thus on MHC-B levels
(Fig. 2B). Unmanipulated
embryos close their blastopores within 5 hours of dorsal lip formation, the
normal site for closure being closer to the ventral lip than to the dorsal lip
(Fig. 3A, asterisks). By
contrast, embryos injected with 2.5 µM MHC-B MO delayed blastopore closure
by 2.5 hours, and exhibited a more dorsally localized closure point
(Fig. 3B, asterisks).
Increasing the MHC-B MO dose to 5 µM further delayed blastopore closure,
which occurred in an abnormal, symmetric fashion. In some cases it failed, and
a small, symmetrical blastopore remained open
(Fig. 3C). At 10 µM MO, we
observed the highly penetrant, large open blastopore phenotype characterized
by a complete block of axial morphogenesis as described above
(Fig. 3D). When the extent of
notochordal extension was assayed by whole-mount in situ hybridization for the
expression of brachyury (Bra), which identifies preinvolution
mesoderm and postinvolution notochord, morphant embryos exhibited
dose-dependent deficiencies in axial development, with the length of the
developing notochord being inversely related to the amount of MHC-B MO
injected, and were thus related to the levels of MHC-B protein in the embryo.
The pattern of Bra expression in a normal early-neurula (stage 13)
embryo reveals the normal extent of notochordal development
(Fig. 3E), whereas a 5 µM
MO-injected sibling embryo exhibited both delayed blastopore closure and a
shorter notochord (Fig. 3F).
This dose-dependent shortening of the notochord was slightly variable at 5
µM MO and these embryos often exhibited a notch or asymmetry at the dorsal
lip of the blastopore (Fig.
3G); although these embryos did develop relatively normally, by
the tailbud stages they exhibited a distinct dorsal bend or flexure (data not
shown). By contrast, embryos injected at 10 µM MO had little or no apparent
notochord, a large open blastopore and expressed Bra in a pattern
reminiscent of that seen in normal embryos at early gastrula stages
(Fig. 3H). Because the abnormal
pattern of Bra expression in 10 µM MO morphants is similar to the
normal fate map of dorsal mesoderm at the beginning of gastrulation
(Keller, 1976), and because
the 5 µM MO morphants exhibit Bra expression in an intermediate
pattern, this suggests an MHC-B MO dose-dependent failure of normal cell
movements to generate the dorsal axis at gastrulation, rather than a failure
of tissue specification.
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). This
`boundary capture' stabilizes and defines notochordal separation from the
somitic mesoderm (Keller and Danilchik,
1988; Shih and Keller,
1992b). When embryos mosaic for 10 µM MHC-B MO were examined,
most morphant cells were excluded from the otherwise normal embryo by late
gastrula stages, apparently owing to their relatively low adhesion (see below;
data not shown). Reducing the concentration of MO injected to 5 µM leads to
embryos mosaic for partially MHC-B-depleted cells that remain in the notochord
until neurula stages, and these cells can be imaged during morphogenesis in
dorsal, axial explants in which the endodermal epithelium covering the
notochord is removed (Fig. 4A).
In these partially MHC-B-depleted cells, protrusive activity was upregulated
at the ends of cells that bound the NSB. These protrusions were abnormally
large, superficially resembling blebs exhibited by dying cells. However,
unlike blebs, which are hemispherical, these protrusions adopted whatever
shape the space around the cells allowed and so they were variably loboform,
lamelliform and circuitously channeled between other cells
(Fig. 4B, see Movie 3 in the
supplementary material). Partially morphant cells were eventually shed from
the notochord. Although they often fragmented similar to myosin-null
Dictyostelium cells in mechanically restricted environments
(Laevsky and Knecht, 2003),
the partially morphant cells continued to exhibit cell motile behaviors in
culture for more than 5 hours after shedding, indicating that they are not
necrotic (data not shown). Just before being shed, the internal ends of these
morphant cells were the last part of the cell to adopt this behavior and, on
doing so, the cells left the notochord to take up residence in whatever space
was available to them, which was usually the groove formed at the NSB
(Fig. 4C, see Movie 4 in the
supplementary material). This phenotype represents both an increase in
protrusive activity and an abnormal distribution of protrusive activity, and
indicates a polarization defect in these cells
(Fig. 4D). These behaviors
suggest a loss of cortical mechanical integrity, loss of the normal
boundary-mediated constraint on protrusive activity, loss of cell adhesion to
the boundary ECM, and loss of cell-cell adhesion in morphant cells.
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MHC-B-depleted cells show dose-dependent loss of cortical actin cytoskeletal integrity
We wished to determine whether myosin IIB regulates the cortical actin
network in notochordal cells. We found that in notochordal cells morphant for
MHC-B MO at a dose of 5 µM, the cortical actin network is perturbed,
indicating that the proper regulation of this network requires myosin IIB in
these cells (Fig. 5D, see Movie
6 in the supplementary material). During this behavior, actin cables were
absent or few in number, and those that were present were thickened and
sinuous. Moreover, the cells appeared able to make large, rapidly extending
protrusions but could not retract them or effectively pull the cell body
toward the protrusions. The transition between normal and perturbed cortical
actin organization in morphant cells correlated with the loss of the normal,
polarized protrusive activity of the notochord cells and the eventual
exclusion of MHC-B-depleted cells from the notochord
(Fig. 5E, see Movie 7 in the
supplementary material). This change in protrusive activity was accompanied,
or closely followed, by disruption of the characteristic hub-and-cable pattern
of cortical actin microfilament bundles. Instead of foci connected by straight
(and thus apparently taut or tensioned) actin cables, there were fewer,
thicker cables that were often sinuous, unconnected to foci and appeared to be
under little or no tension. These observations suggest that the normal
foci-cable array of cortical actin cytoskeleton depends on myosin IIB.
Both the disruption of the cortical actin cytoskeleton and the cellular exclusion phenotype are dependent on MHC-B levels, as morphant notochordal cells with a low-level MHC-B depletion (1 µM MO) both remained in the notochord and had a relatively intact cortical actin cytoskeleton (Fig. 5F,G, see Movie 8 in the supplementary material). However, these morphant cells had more actin-rich filopodial protrusions than normal cells, indicating that the polarized protrusive activity characterizing these intercalating cells is sensitive to MHC-B levels.
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;
Shewan et al., 2005;
Xu et al., 1996). Because
regulation of C-cadherin (C-Cad) is necessary for mesodermal CE in
Xenopus embryos (Brieher and
Gumbiner, 1994; Lee and
Gumbiner, 1995; Zhong et al.,
1999), we assayed C-Cad-mediated adhesion in MHC-B-depleted cells.
We found a dramatic dose-dependent reduction in adhesion
(Fig. 6A). However, morphant
cells maintained their cell-surface C-Cad protein levels when assayed by
trypsin digestion (Fig. 6B) or
surface biotinylation (data not shown). Because expressing exogenous C-Cad
protein did not rescue the blastopore closure phenotype of partially morphant
embryos, but rather made this phenotype worse, this again suggests that C-Cad
protein levels are not limiting in morphant embryos
(Fig. 6E). Morphant cells also
exhibited a reduction in adhesion to fibronectin (FN)
(Fig. 6C), indicating that
integrin-receptor-mediated adhesion is dependent on myosin IIB in these cells.
Similar to what was observed for C-Cad, morphant cells maintain their
cell-surface integrin 5 protein levels when assayed by trypsin
digestion (Fig. 6D) or surface
biotinylation (data not shown). Expression of a human myosin IIB heavy
chain-GFP fusion rescued FN adhesion in morphant cells
(Fig. 6F), indicating that the
MO specifically targets the myosin IIB heavy chain. Thus, myosin IIB is
necessary for both cell-cell and cell-matrix adhesion, either directly or
indirectly through its role in organizing the cortical actin network, as the
assembly state of actin affects cadherin function
(Jaffe et al., 1990). |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Kelley et al.,
1996). Second, we find MHC-B protein distributed in a polarized
fashion in notochordal cells undergoing CE. Third, we show that reducing MHC-B
protein in the embryo leads to the failure of gastrulation. We find that
myosin IIB organizes a polarized cortical actin network in notochordal cells,
and that this network is required for CE. Because this network is required for
both polarized expression of cell motility and regulation of cell adhesion,
these defects are likely to be responsible for the failure of morphogenesis.
This work identifies the dynamic, myosin IIB-dependent cortical actin network
as the cell component integrating adhesion and polarized motility during
CE.
In Drosophila, myosin II is also involved in the epithelial cell
intercalation that occurs during germ band CE
(Bertet et al., 2004;
Zallen and Wieschaus, 2004).
Cell intercalation in germ band extension involves remodeling of the
circumapical adherens junctions during neighbor changes among pairs of cells
(Bertet et al., 2004) or in
multicellular rosettes of cells
(Blankenship et al., 2006),
whereas the cell intercalation in the Xenopus dorsal mesoderm
involves deep, non-epithelial mesenchymal cells, which are attached to each
other at foci and lack an apical junctional complex. In Xenopus, we
find myosin IIB distributed cortically in involuting mesodermal cells, with
higher medial and lateral concentrations, whereas in the Drosophila
germ band myosin II is localized asymmetrically adjacent to anterior and
posterior apical contacts (Bertet et al.,
2004; Zallen and Wieschaus,
2004), indicating that although these two examples of CE both use
myosin II, they are mechanistically distinct processes.
Embryos morphant for MHC-B fail to close their blastopores in a fashion
that indicates failure of CE in the involuting dorsal axial and paraxial
mesoderm (Keller et al., 2000;
Keller et al., 2003).
Normally, CE of the axial and paraxial mesoderm constricts the involuting
marginal zone, which closes the blastopore, and simultaneously elongates the
body axis by extension. This occurs because the polarized protrusive activity
and traction of the deep mesodermal cells, the resulting cell intercalation,
and the consequent convergence, all occur along the presumptive mediolateral
axis of the dorsal tissues, which in the involuting marginal zone of the
embryo is described as an arc-like pattern around the blastopore
(Keller et al., 1992;
Keller, 1984;
Shih and Keller, 1992b).
Shortening these arcs by cell intercalation results in constriction of the
blastopore and the resulting extension in the perpendicular direction results
in axial elongation, all in one stroke
(Keller et al., 2003;
Keller and Shook, 2004).
Morphant embryos show a dose-dependent gradation of success in constricting
the blastopore, with stronger depletions failing entirely and weaker
depletions showing limited closures and specific defects in dorsal, axial
extension. This behavior is strong evidence for the myosin IIB dependence of
CE. By contrast, other morphogenic processes occur normally, including cell
division during cleavage and bottle cell formation, suggesting that the
cellular mechanisms underlying CE in dorsal mesodermal tissue are those most
dependent on myosin IIB, consistent with a morpho-mechanical role for this
myosin isoform in CE.
Cellular MHC-B morphant phenotypes show that MHC-B is essential for a specialized cortical actin cytoskeleton necessary for the development of convergence forces
Protrusive activity of the type displayed in a bipolar, oriented fashion by
the deep mesodermal cells is invariably associated with traction forces in
cell culture on deformable substrates
(Beningo and Wang, 2002;
Harris et al., 1980;
Lo et al., 2004;
Lo et al., 2000). This
suggests that these cells are exerting traction on adjacent cells or on the
matrix and are under tension in the mediolateral axis, which is also the axis
of convergence. Notochordal cell elongation and intercalation in the presence
- and only in the presence - of their characteristic bipolar protrusive
activity further supports this contention. What generates the tensile forces
at the cell level that drives cell intercalation and convergence and what
integrates these forces over long distances along the arcs of convergence are
long-standing questions. The anisotropic dynamic behavior of the cortical
actin cytoskeleton described here and its perturbation in myosin IIB morphant
cells suggest that this cytoskeletal architecture and associated cortical
tension play a large role in this process. First, the predominant movement of
the actin nodes, and thus the predominant change in length of the actin cables
connecting them, is in the mediolateral axis. This is also the axis of cell
intercalation, cell traction generation and tensile force generation,
suggesting that this cytoskeleton functions in these processes. These tensile
forces across the involuting marginal zone rise to 1.5 µN during
gastrulation (D. Shook, personal communication). Second, the sequence of
defects appearing in cells partially depleted for MHC-B include loss of
integrity of this cortical actin network, abnormal protrusive activity, loss
of the elongate polarized morphology, apparent loss of cortical integrity as
judged by the inability of the cell to maintain its shape, accommodating
instead to surrounding spaces, and, finally, the loss of adhesion. Third,
myosin IIB is localized to the cell cortex, which is expected to be under
tension along cell surfaces between protrusions
(Kolega, 1986), and is thus
important in developing mediolateral traction or resisting the cell elongation
it might produce. Myosin IIB is also concentrated in the cell-cell,
cell-matrix junctions of cell surfaces facing the mesodermal NSB boundary, a
region of stable anchorage and suppression of protrusive activity that is
required for the integrity of the boundary and for directed extension
(Shih and Keller, 1992b;
Skoglund et al., 2006;
Skoglund and Keller, 2007).
Myosin IIB levels are also elevated at triple cell junctions found in the
interior of the notochord, an increase that probably reflects the presence of
the polarized, medial and lateral protrusions thought to generate the traction
for cell intercalation. Finally, the vigorous but aberrant protrusive
activity, the inability to constrain their shape in a mechanically resistant
environment, and the tendency for fragmentation are all characteristics that
myosin IIB morphant Xenopus cells have in common with myosin II-null
mutants in Dictyostelium, in which this molecule functions
principally as an actin cross-linker and is important in the maintenance of
cortical integrity necessary for migration along mechanically restricted
environments (Laevsky and Knecht,
2001; Laevsky and Knecht,
2003; Xu et al.,
2001). These data combine to suggest that myosin IIB has a
mechanical role in the generation or transmission of the mediolateral tensile
forces thought to underlie CE, and thereby blastopore closure and axial
elongation (Keller et al.,
2000).
Myosin IIB is essential for cell-cell and cell-matrix adhesion
Severely depleted morphant cells eventually appear to lose adhesion to
adjacent cells and ECM, a loss of adhesive ability that was confirmed by
decreased adhesion to defined cadherin and FN substrates. This suggests a role
for myosin IIB in adhesion, a notion supported by the fact that myosin IIB is
important for clustering E-cadherin at adhesion sites and for forming
adhesions in cultured epithelial cells
(Shewan et al., 2005). In
these mesenchymal Xenopus cells, the important cadherin in CE appears
to be C-cad (Brieher and Gumbiner,
1994; Zhong et al.,
1999), and we that show myosin IIB regulates Xenopus
cell-cell adhesion by a mechanism distinct from regulating the surface
expression of C-cad. The fact that both cadherin-mediated adhesion to cadherin
and integrin-mediated adhesion to ECM are affected in morphant cells suggests
a general and perhaps indirect effect of myosin on adhesion. This consequence
of myosin IIB depletion might be mediated by loss of the actin cytoskeletal
organization, as it parallels the loss of adhesion that occurs upon disruption
of microfilaments with cytochalasin (Jaffe
et al., 1990), and suggests that a normal function of the dynamic
cortical actin network is to dynamically regulate cell-cell and cell-matrix
adhesion on notochordal cells in a manner appropriate to support CE.
Both integrin (O'Toole et al.,
1994) and cadherin (Marsden
and DeSimone, 2003) adhesive functions can be modulated by
cytoplasmic signals, and modulation of one of these adhesion systems can
affect the other (Finnemann et al.,
1995; Winklbauer,
1998). Our results allow for the possibility that this
interdependency could be partially mediated by local modifications of the
cortical actin network, because we show that both adhesion systems depend on
this network. This view is supported by the observation that Xenopus
C-Cad intracellular tail interactions with p120 catenin regulate the local
pattern and density of the cortical actin network in blastomeres
(Tao et al., 2007).
Summary of myosin IIB function in CE
A summary of how we think myosin IIB functions in the context of what is
known about CE in Xenopus is shown in
Fig. 7. The mediolateral
intercalation of cells occurs as the initially unpolarized, isodiametric deep
mesodermal cells become polarized to form large filo-lamelliform protrusions
at their medial and lateral ends, which display repeated cycles of extension
(Fig. 7A, black arrows) and
shortening (Fig. 7A, gray
arrows) (Keller et al., 1989;
Shih and Keller, 1992a). As
this type of protrusive activity is invariably associated with exertion of
traction on the substrate (Beningo et al.,
2002; Harris et al.,
1980; Lo et al.,
2004; Lo et al.,
2000), which in this case is either adjacent cells or ECM, this
protrusive activity is very likely to generate the traction that first
elongates the cells and then pulls them between one another, thus directly
shortening the mediolateral axis of the tissue and, indirectly, by wedging the
cells between one another, extending the anterior-posterior axis
(Fig. 7C, large arrows). The
myosin IIB-dependent dynamic cortical actin network consisting of actin cables
(Fig. 7B, green) meeting at
foci or nodes (Fig. 7B, black)
serves as a tensile element in the cell cortex that limits the elongation of
the cell, driven by the polarized traction, and transmits the tension from one
adhesion site to another inside the cell. Tension is transmitted from cell to
cell through adhesion sites, generating arcs of tension across the tissue
fabric. The dynamic oscillation of the length of the actin cables and movement
of the nodes is along the mediolateral axis, which is also the axis of
development of the convergence forces and suggests an active contractile role
for the cortical actin network in CE. The cortical distribution of myosin IIB
and the apparent lack of cortical tension in the morphant cells are consistent
with involvement of this molecule in cross-linking the cortical actin
cytoskeleton. In addition to maintaining cortical integrity, myosin IIB might
also participate in active contraction by reeling actin filaments into the
foci and thus generating the force for intercalation, but this remains to be
determined by imaging these molecules and assaying their activity state in
living cells. The concentration of myosin IIB in the anterior/posterior
corners of the cell surfaces contacting the ECM structure at the NSBs
(Fig. 7B, red) might reflect a
mechanical reinforcement at this critical boundary. The apparent concentration
at the interior, triple cell junctions
(Fig. 7B, red) might reflect
the same thing, although in this case the increased staining may not reflect
an increase in myosin IIB per unit area of cortex, but instead the close
apposition of four cortical regions or the presence of the molecule in the
lamellipodia at these ends of the cells. In either case, it reflects a high
concentration of myosin IIB per cell volume, which again may reflect a
mechanical reinforcement. Myosin IIB is involved in polarized cell behavior
(Lo et al., 2004), but whether
its involvement is direct or is mediated by its role in organizing the actin
cytoskeleton remains to be determined.
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ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Department of Anatomy and Developmental Biology,
University College London, Gower Street, London, WC1E 6BT, UK 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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