|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 13 March 2008
doi: 10.1242/dev.019646
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
* Author for correspondence (e-mail: takeichi{at}cdb.riken.jp)
Accepted 18 February 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Shroom, Rho kinase, Myosin, Neural tube, Epithelial remodeling, Chicken, MDCK cells, Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). It
is thus crucial to understand the mechanisms governing the regulation of these
cell behaviors at the appropriate times and locations during development.
Apical constriction occurs in many morphogenetic processes, including
gastrulation and neurulation (Lecuit and
Lenne, 2007). In epithelial layers, the zonula adherens or
adhesion belt, which represents a form of the cadherin-based adherens
junction, encloses the cells near the apical end of their lateral cell-cell
contacts (Perez-Moreno et al.,
2003; Tepass et al.,
2001), organizing the apical junctional complex (AJC) together
with the tight junctions and desmosomes
(Farquhar and Palade, 1963;
Vogelmann and Nelson, 2005).
F-actin assembles at the cytoplasmic side of the adhesion belt, and its
contraction is thought to be a major mechanism for the apical constriction of
epithelial layers. When this occurs in embryonic cells, the cells assume a
wedge-like shape. This shape change is often used to force epithelial sheets
to invaginate. Apical constriction signals for Drosophila mesodermal
invagination have been extensively studied
(Lecuit and Lenne, 2007): upon
the binding of an apically secreted ligand, Fog, to its receptors,
G
12/13-Corcentina is activated
(Costa et al., 1994;
Parks and Wieschaus, 1991);
subsequently, RhoGEF2 becomes activated and anchored to the apical membranes
via its interaction with the transmembrane protein T48
(Barrett et al., 1997;
Kolsch et al., 2007;
Nikolaidou and Barrett, 2004;
Rogers et al., 2004). These
processes seem to induce the activation of Rho1 and Rho kinase at the apical
cell junction, which in turn leads to the invagination of the future
mesodermal layer.
In vertebrates, Shroom3, an actin-binding protein, is known to be a key
player for epithelial apical constriction. Shroom3 has been identified as a
gene product whose mutation disrupts neurulation
(Copp et al., 2003;
Hildebrand and Soriano, 1999).
This protein is localized at the AJC of the neural tube; and its depletion
causes neural tube closure defects in mice and Xenopus
(Haigo et al., 2003;
Hildebrand and Soriano, 1999).
When Shoom3 is overexpressed in epithelial cells, it induces their apical
constriction along with increased accumulation of myosin 2
(Haigo et al., 2003;
Hildebrand, 2005). Shroom3 was
also reported to recruit 
-tubulin to the apical areas in the
Xenopus neural tube (Lee et al.,
2007). Shroom3 belongs to the Shroom family, which includes
structurally related molecules, Shroom1, Shroom2 and Shroom4
(Hagens et al., 2006), each of
which shows unique functions (Dietz et al.,
2006; Fairbank et al.,
2006; Hagens et al.,
2006; Lee et al.,
2007; Yoder and Hildebrand,
2007). Shroom family members share similarity in several domains,
each having a PDZ domain, to Apx/Shrm-specific domains called ASD1 and ASD2,
and putative EVH1- and PDZ-binding sites
(Hildebrand and Soriano,
1999); and all the members have the ASD2 domain in common
(Hagens et al., 2006). The
ASD2 domain of Shroom3 has been shown to be essential for its apical
constriction activity (Dietz et al.,
2006); however, how this domain acts has yet to be understood.
Previous studies showed that the action of Shroom3 on epithelial
constriction required Rho kinases (ROCKs) and myosin 2
(Hildebrand, 2005), but how
these molecular systems work together remains unknown. In the present study,
we show that Shroom3 physically associated with the ROCKs via the ASD2 domain,
and recruited them to the apical junctions in epithelial cell lines, as well
as in the chicken neural tube. ROCKs are Ser/Thr kinases, which are activated
by binding to the GTP-bound form of RhoA
(Riento and Ridley, 2003).
Myosin regulatory light chain (MLC) is one of the targets phosphorylated by
ROCKs. Phosphorylation of MLC activates myosin 2, leading to the actomyosin
contraction (Matsumura, 2005).
ROCKs phosphorylate the MLC directly (Amano
et al., 1996) and also indirectly, for ROCKs also phosphorylate
and inactivate the MLC phosphatase (Kawano
et al., 1999). Although ROCKs are mainly cytoplasmic, some of them
localize at cell-cell junctions and regulate the assembly of tight
(Walsh et al., 2001) or
adherens (Sahai and Marshall,
2002) junctions. ROCKs have also been reported to be expressed in
the neural plate and involved in neurulation in the chicken embryo
(Wei et al., 2001). The
results obtained in this present study suggest that the main function of
Shroom3 is to recruit ROCKs to the apical junctions, so that Shroom3 can
induce their constriction. We also show a peculiar arrangement of
neuroepithelial cells and unique distribution of phosphorylated MLC in the
closing neural tube, which may explain the cellular basis of its bending
mechanisms.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Bacterial expression plasmids for glutathione S-transferase (GST)-tagged proteins were constructed by using the pGEX-4T vector (GE Healthcare). Tagged recombinant proteins were expressed in E. coli and purified by use of glutathione-Sepharose beads (GE Healthcare).
Antibodies
The cDNA fragments encoding amino acids 195-423 of mouse Shroom3, 562-836
of mouse Rock1 and 482-838 of chicken Rock1 were cloned into the pGEX-4T
vector. The antigens were expressed and purified as above. Antibodies against
mouse Shroom3 were generated by immunizing rabbits; and those to mouse Rock1
and chicken Rock1 were raised by immunizing rats. All antibodies were affinity
purified by use of antigen-coupled CH-Sepharose beads (GE Healthcare). The
specificity of each antibody was confirmed by western blotting (see Fig. S5 in
the supplementary material). The following antibodies were purchased: mouse
anti-Rock1 and anti-Rock2 (BD Biosciences); mouse anti-phospho-myosin light
chain (Ser19, Cell Signaling); mouse anti-ZO-1, rabbit anti-ZO-1 and mouse
anti-Lamin B1 (Zymed); mouse anti--tubulin clone DM1A, rabbit
anti-FLAG, mouse anti-FLAG clone M2 and rabbit anti-myosin IIA (Sigma); rabbit
anti-Par3 (Upstate); rat anti-hemagglutinin (HA) clone 3F10 and mouse anti-GFP
(Roche Diagnostics); mouse anti-HA clone 16B12 and mouse anti-GST clone 4C10
(Covance); rat anti-GFP (Nakarai); rabbit anti-GFP (MBL); Alexa Fluor 488-,
555-, 594- or 647-conjugated secondary antibody (Molecular Probes);
Cy3-conjugated anti-rat IgG (Chemicon); Cy2- or peroxidase-conjugated anti-rat
IgG (Jackson); and peroxidase-conjugated anti-mouse or anti-rabbit IgG (GE
Healthcare).
Pull-down assay
Mouse E9.5 embryos were homogenized in a lysis buffer consisting of 20 mM
Tris-HCl (pH 7.5) containing 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 5%
glycerol, 1 mM PMSF, a protease inhibitor cocktail (Roche Diagnostics) and 1
mM dithiothreitol, and centrifuged at 15,000 g for 10 minutes.
The supernatant was precleared with glutathione-sepharose 4B beads at 4°C
for 1 hour, and then incubated with the glutathione-sepharose 4B beads loaded
with GST-tagged ASD2 at 4°C for 2 hours. After washing the beads, the
bound proteins were subjected to SDS-PAGE followed by silver staining using a
Silver Stain MS kit (Wako).
Immunoprecipitation and western blotting
Cells were lysed at 4°C for 20 minutes in a lysis buffer consisting of
50 mM Tris-HCl (pH 7.5), containing 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA,
0.5 mM EGTA, 1.5 mM MgCl2, a protease inhibitor cocktail (Roche
Diagnostics) and 1 mM dithiothreitol. After centrifugation, the supernatants
were incubated with the appropriate antibodies at 4°C for 2 hours. Then
protein G-Sepharose beads (GE Healthcare) were added and incubation was
continued for an additional 30 minutes. After washing the beads, bound
proteins were subjected to SDS-PAGE and then western blotting. Signals on the
bots were detected by using the ECL or ECL Plus detection system (GE
Healthcare).
Cell culture and transfection
Madin-Darby canine kidney epithelial cells (MDCK, strain II) and COS7 cells
were grown in a 1:1 mixture of DMEM and Ham's F12 medium supplemented with 10%
fetal bovine serum (FBS). MDCK Tet-Off cells were purchased from BD
Biosciences and maintained in DMEM supplemented with 10% tetracycline-free
FBS, 1 µg/ml puromycin and 1 µg/ml doxycycline. MDCK or MDCK Tet-Off
Cells were transfected with expression plasmids by using Lipofectamine 2000
(Invitrogen). For isolating their stable transfectants, transfected cells were
selected and maintained in culture medium containing 200-400 µg/ml of
Hygromycin B. COS7 cells were transfected by using Effectene transfection
reagents (Qiagen).
Immunofluorescence microscopy for cultured cells
Cells grown on cover glasses were fixed with cold methanol at -20°C for
5 minutes, or with 4% (w/v) paraformaldehyde at room temperature for 20
minutes, and then made permeable with 0.5% Triton X-100 in Ca2+-
and Mg2+-free phosphate-buffered saline (PBS). The cells were
blocked with 10% (w/v) skim milk in PBS containing 0.1% Triton X-100 (PBS-T),
and incubated at room temperature for 2 hours with primary antibodies in PBS-T
containing 1% skim milk. After washes, the cells were incubated for 1 hour
with fluorescence-labeled secondary antibodies, and subsequently mounted with
FluoroSave (CalbioChem). The images were obtained by using a laser-scanning
confocal microscope LSM510 (Carl Zeiss) equipped with a Plan Apochromat
63x/1.4 lens (Carl Zeiss). The measurement of apical junctional length
and areas were performed by using an LSM4 image browser.
Manipulation of chicken embryos
Fertilized hens' eggs were purchased from Shiroyama Farm (Kanagawa, Japan)
and incubated at 38.5°C to the desired stages. For chemical treatments and
electroporation experiments, we used the modified New culture method, as
described (Nakazawa et al.,
2006). Electroporation was performed with an Intracel
electroporator (Intracel) at the following setting: 6 volts, 3 pulses for 50
ms at a 200 ms interval.
For immunohistochemical studies, whole chicken embryos were fixed for 1 hour with 4% PFA in PBS containing 1 mM EGTA at 4°C. After replacement of the fixative with 30% sucrose, the embryos were frozen in OCT compound (Sakura Finetek). Sections of 10 µm were prepared with a cryostat, air-dried for 3 hours and stored. Rehydrated sections were treated with 0.3% Triton X-100 and 0.2% bovine serum albumin (BSA) in TBS for 10 minutes at room temperature, and then blocked at room temperature for 2 hours in a solution of 3% BSA and 10% goat serum in TBS. The sections were subsequently incubated for 2 hours with primary antibodies in a solution of 0.1% Triton X-100, 0.2% BSA and 1% goat serum in TBS at room temperature. After washing, the sections were incubated at room temperature for 1 hour with fluorescence-labeled secondary antibodies, and mounted in FluoroSave. Microscopic images were obtained by using an Axioplan2 microscope (Carl Zeiss).
For whole-mount preparations, neural tubes were dissected, cut at the ventral midline and fixed with 4% PFA in PBS at room temperature for 15 minutes. Then the neural tubes were blocked at room temperature for 2 hours in a solution containing 3% BSA, 10% goat serum and 0.5% Triton X-100 in TBS, after which they were incubated with primary antibodies in Can Get Signal immunostain solution B (TOYOBO) at room temperature for 2 hours. After washes, the samples were incubated with fluorescence-labeled secondary antibodies at room temperature for 1 hour. After more washes, the neural tubes were mounted with their inner side up in glycerol gelatin (Sigma). Microscopic images were obtained by using the Axioplan2 microscope or LSM510 META multiphoton confocal system (Carl Zeiss) equipped with a Plan Apochromat 63x/1.4 lens (Carl Zeiss).
|
); in
natural epithelial cell sheets, we can also occasionally detect four-cell
vertices (see Fig. 1B as an
example), but rarely five- or six-cell vertices. Thus, we defined the cell
clusters with a vertex formed by more than five cells as `rosettes' organized
via non-random cell arrangement. |
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
ASD2;
Fig. 1A), introduced these
constructs into MDCK Tet-Off cells, the expression of which was controlled by
the Tet-Off promoter, and isolated their stable transfectants. Without
Shrm-Full expression in the presence of doxycycline, a suppressor of
expression, the apical cell-cell boundaries delineated by immunostaining for
ZO-1, a tight-junction protein, appeared slightly curved and/or serrated
(Fig. 1B). When Shrm-Full was
expressed by removal of doxycycline, the apical junctions of the transfectants
became tense and straightened (Fig.
1B). By contrast, expression of Shrm-ASD2 had no such
effects on the junctional morphology (Fig.
1B), confirming previous observations
(Dietz et al., 2006). To explore the function of the ASD2 domain (ASD2), we screened for molecules interacting with this domain by conducting pull-down assays. GST-tagged ASD2 (GST-ASD2) was incubated with a whole lysate of E9.5 mouse embryos, and the co-precipitated materials were analyzed by silver staining. The results showed that a protein of about 160 kDa interacted specifically with GST-ASD2 (see Fig. S1 in the supplementary material). Mass spectrometric analysis of this band showed that it contained the Rho kinases Rock1 and Rock2. To test the significance of this result, we incubated GST-ASD2 with a MDCK cell lysate, analyzed the co-precipitated materials by western blotting with anti-ROCK antibodies, and detected both Rock1 and Rock2 in this sample (Fig. 1C). Then, we examined whether the interactions between ASD2 and Rock1 or Rock2 also occurred in cells: FLAG-tagged ASD2 was co-expressed with either HA-tagged Rock1 or Rock2 in COS7 cells. Immunoprecipitates collected with anti-FLAG antibody from these cells contained both Rock1 and Rock2 (Fig. 1D). These results indicate that the ASD2 domain of Shroom3 interacted with Rock1 and Rock2.
Recruitment of ROCKs to the apical cell junctions by Shroom
To examine whether the distribution of ROCKs in cells could be regulated by
Shroom3, we immunostained MDCK cells inducibly expressing Shroom3 with
anti-Rock1 antibody. In the absence of expression of exogenous Shroom3, we
detected only diffuse Rock1 signals distributed in the cytoplasm
(Fig. 2A, upper), although its
faint signals were occasionally detected emanating from cell-cell junctions.
When the expression of Shroom3 was induced, Rock1 became condensed along the
apical junctions; and its signals were colocalized with those of Shroom3
(Fig. 2A, middle). However, the
expressed Shrm-ASD2 had no such effects on Rock1 distribution
(Fig. 2A, bottom). We also
introduced HA-tagged Rock2 into MDCK cells, and found that this construct was
localized at the apical junctions only when Shroom3 was expressed (data not
shown). Then, we tested whether Shrm-Full and Shrm-ASD2 were associated
with endogenous ROCKs, and found that Rock1 and, to a lesser extent, Rock2
were co-immunoprecipitated with Shrm-Full, but not with Shrm-ASD2, in
the lysates of the above MDCK transfectants
(Fig. 2B). All these results
demonstrate that Shroom3 recruited ROCKs to the apical junctions by their ASD2
domain.
|
The above results suggest that the C1 region of ROCKs competed with
endogenous ROCKs for their interactions with Shroom3. To further test this
possibility, we introduced EGFP-tagged RII-C1 or EGFP into MDCK Tet-Off
Shroom3 transfectants, and then immunoprecipitated Shroom3 from these cells.
The results showed that, when RII-C1 had been expressed, the
co-immunoprecipitation of Rock1 with Shroom3 was greatly suppressed
(Fig. 3C, left). In addition,
we immunostained these cells with anti-Rock1 antibody. In contrast to the
junctional accumulation of endogenous Rock1 in the Shroom3 transfectants
without RII-C1, those expressing RII-C1 lost the junctional Rock1 signal
(Fig. 3C, middle). The
activation of ROCKs is known to lead to the phosphorylation of myosin
regulatory light chains (MLCs), and this phosphorylation is crucial for the
activation of myosin 2 (Riento and Ridley,
2003). In Shroom3-expressing MDCK Tet-Off cells, MLC
phosphorylation was observed along the apical junctions, as described earlier
(Hildebrand, 2005). However,
when RII-C1 was expressed, MLC phosphorylation at these sites was
significantly reduced (Fig. 3C,
right). These results demonstrate that RII-C1 antagonized the interaction of
endogenous ROCKs with Shroom3, resulting in the suppression of the
tension-producing functions of Shroom3.
The Shroom3-Rock1 interaction is crucial for neural tube closure
To explore the role of the Shroom3-ROCK interaction in embryos, we first
observed their distributions in the developing chicken neural tube, choosing
the head regions at stages 9. As reported for other species
(Hildebrand, 2005), Shroom3
was concentrated along the apical junctional area of the neuroepithelial
cells. By using antibodies generated against the chicken Rock1, we found this
molecule to be also condensed along the apical surface of the neural tubes,
well colocalizing with Shroom3 (Fig.
4A). Phosphorylated myosin light chain (pMLC) was also colocalized
with the apical junctional signals of Shroom3 and Rock1 (see below).
To study the functional and spatial relationships of these molecules, we constructed RNAi vectors specific for chicken Shroom3, as well as control vectors. To evaluate the knockdown effect of these vectors, we transfected MDCK cells with the Shroom3-specific RNAi vector, RNAi vector for the scrambled sequence of Shroom3 RNAi (control RNAi) or empty vector, together with an EGFP-tagged chicken Shroom3 central region (ch-Shrm/M, which contains the Shroom3 RNAi sequence) or EGFP. By western blotting of the transfected cell lysates, we found that the Shroom RNAi vector suppressed the expression of EGFP-ch-Shrm/M, but not that of EGFP (Fig. 4B). Neither of the control RNAi nor empty vectors affected the expression of either EGFP-ch-Shrm/M or EGFP.
Then, we transfected the future neural plate with these vectors by
electroporation. Stage 3 chicken embryos were electroporated at the regions
anterior to the primitive streak with a mixture of the RNAi vectors and
EGFP-vector as a transfection marker, as illustrated in Fig. S3 in the
supplementary material. Electroporated embryos were cultured for about 1 day,
and fixed at stage 9. In embryos electroporated with the empty or control RNAi
vector, not only Shroom3 but also Rock1 and pMLC remained localized at the
apical surface of the neural tube (Fig.
4C, top and bottom rows). By contrast, in those electroporated
with the Shroom3 RNAi vector, in which Shroom3 immunoreactive signals
disappeared from the apical surface of the neural tube, the Rock1 signals were
also decreased at the apical region, although its diffuse cytoplasmic signals
remained (Fig. 4C, middle row).
In addition, the apical signals for MLC phosphorylation were also reduced. In
these Shroom3 RNAi-electroporated embryos, the neural plate failed to bend
normally, except at the floor plate, and did not close, suggesting that the
inner constriction of the tube was defective, as observed for other species
(Haigo et al., 2003;
Hildebrand and Soriano, 1999).
Importantly, the distribution of ZO-1 delineating the apical surface of the
neural tube was almost the same among these electroporated embryos, suggesting
that the apical surface structures were not particularly distorted by the
electroporation. These results suggest that Shroom3 recruited Rock1 to the
apical junctions of the neural tube, leading to the phosphorylation of MLC,
and that this process was important for neural tube closure.
|
|
|
Cell-level analysis of ROCK-dependent neural tube closure
The processes of neural plate invagination have not been fully analyzed
with regard to cell behavior in the 2-dimensional neuroepithelial plane. For
understating of the role of Shroom3 and ROCKs in neural tube closure at the
cellular level, we decided to observe the morphology of individual
neuroepithelial cells at their apical surfaces: we dissected the tubes from
embryos at stages 8 to 9 when neural tube closure is proceeding, opened them
by cutting along the dorsal midline, and then immunostained them for various
markers (Fig. 6A). As the
morphology of the cells comprising the neural tube varies to some extent with
the region of the tube, we selected the lateral wall of the midbrain for our
most detailed analysis, excluding the areas proximate to the dorsal and
ventral midlines.
ZO-1 immunostaining, which delineates the neuroepithelial cell-cell
junctions at their apical-most region, showed that cell morphology and
assembly pattern in the closing tubes was complex at first glance
(Fig. 6B). Close examinations
of these samples, however, suggested that some order existed within the cell
arrangement. For example, a cluster of cells often displayed a rosette-like
arrangement with condensed ZO-1 signals at their vertex points
(Fig. 6B,E, dotted circles),
which was reminiscent of that observed in epithelial layers undergoing cell
intercalation in Drosophila embryos
(Blankenship et al., 2006).
Some other cells were arranged in an arch-like or oval pattern
(Fig. 6B,E, dotted oval). Along
these ZO-1 signals, pMLC immunosignals displayed unexpected distributions
(Fig. 6B): the pMLC signals
were detected only at restricted cell-cell boundaries. These signals on an
individual cell were often contiguous to those on the neighboring cells,
resulting in a linear extension of the signals over four or five cells or even
more (Fig. 6B). Intriguingly,
these linear pMLC signals tended to be oriented along the dorsoventral axis of
the embryo, although not absolutely (Fig.
6C). Other pMLC signals were punctate and concentrated at the
converged or vertex points of the rosette-forming cells described above,
although the signals at a vertex were sometimes continuous to the above linear
pMLC signals. To quantify the ratio of cell clusters assuming such rosette
configuration, we counted the cells organizing a cluster in which six cells
and more were radially converged on a pMLC-condensed center (see Materials and
methods for the criteria for this scoring) and found that 30.8±5.1%
(the mean±s.d.; 127-250 cells were counted per sample, n=3) of
the neuroepithelial cells composing the midbrain lateral wall in stage 9
embryos displayed this configuration. We also counted the vertices, at which
six cell-cell borders or more meet; and estimated that 17.0±4.2% (the
mean±s.d.; 156-275 vertices were counted per sample, n=3) of
the vertices had this configuration. Stage 8 embryos gave similar values.
|
|
To test whether ROCK signals played any role in the above arrangements of
neural tube cells, we added Y27632, a ROCK inhibitor, to the culture medium of
embryos at 6 hours prior to the fixation at stage 9. As reported earlier
(Wei et al., 2001), this
inhibitor completely blocked neural tube closure. Double immunostaining for
ZO-1 and pMLC showed that pMLC signals, whether linear or punctate, were
drastically diminished; and, simultaneously, the apical surface of individual
neural tube cells was significantly expanded
(Fig. 6E). In these
Y27632-treated neural plates, cells with the rosette configuration became less
frequent; i.e. the vertices consisting of more than five cell-cell borders
were reduced to 4.9±3.0% (the mean ± s.d.; 152-202 vertices were
counted per sample, n=3) of the total vertices in the neural plate
region shown in Fig. 6E; this
value was originally 17% in the untreated samples (see above). These results
suggest that ROCK signals are crucial for the rosette-type arrangement of
neuroepithelial cells in the two-dimensional plane.
Blocking of the Shroom-ROCK interactions perturbs planar cell arrangement in the neural tube
To finally examine whether the above actions of ROCK on neuroepithelial
cells depended on the Shroom-associated pool of this molecule, we looked at
the effects of RII-C1 expression in the neural tube on cell arrangement, as
well as on pMLC localization. Embryos electroporated with the expression
vector for EGFP, EGFP-tagged RII-C1 or RII-C2, as described for the
experiments for Fig. 5, were
triple-immunostained for EGFP, pMLC and ZO-1. These vectors were expressed in
a mosaic fashion in the neuroepithelial layer. Analysis of the layers densely
transfected with these vectors showed that RII-C1 expression resulted in a
significant reduction in the pMLC-bearing cells
(Fig. 7), consistent with the
results obtained with tissue sections. Moreover, cells that had lost pMLC in
the RII-C1-expressing layer also lost the typical rosette configuration,
although residual pMLC signals, when detectable, still localized at the
convergent points of cells (Fig.
7, arrows). EGFP and RII-C2, however, showed no such effects.
These results support the idea that the Shroom-ROCK interaction is required
not only for the local phosphorylation of junctional MLC but also for the
specific two-dimensional arrangement of developing neural tube cells.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Our present results have provided evidence that Shroom3
physically associated with ROCKs via the ASD2 domain. We also identified a
Shroom3-binding region on Rock1 and Rock2. Peptides containing this region
competed with endogenous ROCKs in their interaction with Shroom3, leading to
the suppression of the junctional recruitment of ROCKs and a concomitant
relaxation of the apical junctions. These results suggest that Shroom3
functions as a scaffold to recruit ROCKs to the apical junctional complex
(AJC). How Shroom3 itself becomes localized at the AJC remains to be
elucidated, although Shroom2 was earlier shown to interact with ZO-1
(Etournay et al., 2007).
ROCKs induce the contraction of actin fibers by phosphorylating MLC as well
as by inhibiting myosin phosphatase
(Riento and Ridley, 2003). We
showed that the recruitment of ROCKs to the AJC well correlated with the
phosphorylation of MLC localized at the same sites in MDCK cells and that the
ROCK inhibitor Y27632 suppressed the MLC phosphorylation at the corresponding
sites in the neural tube. As the epithelial AJCs are lined with the
circumferential actin belts, it is likely that, by being recruited to the AJC,
the ROCKs directly control the contraction of these actin belts via the
classic MLC phosphorylation pathway. In the absence of Shroom3, the amount of
ROCKs at the AJC was only residual in MCDK cells and in the neural tube. These
observations suggest that ROCKs may not be able to regulate the apical
junction organization effectively without Shroom3 or other molecules having
similar functions.
Neural tube closure is not only a Shroom-dependent process but also a
ROCK-dependent one (Wei et al.,
2001; Ybot-Gonzalez et al.,
2007). We showed that the blocking of the Shroom3-ROCK interaction
with RII-C1 suppressed neural tube closure, concomitantly downregulating MLC
phosphorylation at the AJC. These findings strongly suggest that the
Shroom3-dependent recruitment of ROCKs to the neuroepithelial AJC is crucial
for the bending morphogenesis of neural tube. However, we suspect that simple
constriction of the apical junctions in the neuroepithelial layer would not be
sufficient for the control of its bending, as this process was polarized,
occurring along the dorsoventral axis. Thus, to close the neural tube
correctly, the ROCK-dependent contraction of neuroepithelial cell borders
would need to be spatially regulated.
To understand the cellular basis of polarized neural tube closure, we
analyzed the cell assembly pattern in the inner surface of the closing tube.
The pattern was complex, and the apical morphology of cells extensively varied
from cell to cell. Within the complexities, however, we could detect some
order; that is, the rosette-like or arch-type arrangement of neuroepithelial
cells. Rosette formation has been observed in Drosophila epithelial
cells undergoing intercalation during the germ band extension of embryos
(Blankenship et al., 2006).
During this intercalation, the boundaries between the anterior and posterior
cells become enriched in myosin 2; and other molecules such as Par3 complement
this pattern by localizing along the other sides of the junction on the same
cell (Bertet et al., 2004;
Zallen and Wieschaus, 2004).
Based on these observations, it is thought that the actomyosin-dependent
contraction of the cell-cell borders oriented along the dorsoventral axis,
coordinated with the concomitant expansion of the other cell boundaries
towards the anterior-posterior axis, leads the cells to intercalate and that
the rosette-like arrangement of cells represents an intermediate stage of this
process (Blankenship et al.,
2006). Our finding of rosettes suggests that vertebrate
neuroepithelial cells may use similar strategy for regulation of cell
rearrangement during the neural tube closure. This view is consistent with the
observations that neural tube morphogenesis involves convergent extension of
neuroepithelial cells (Ybot-Gonzalez et
al., 2007).
In the chicken neuroepithelial layer, however, myosin 2 and Par3 were rather ubiquitously localized along the cell-cell junctions. Instead, pMLC distribution was polarized: pMLC was concentrated only at restricted sites on the AJC surrounding a cell, and this pMLC signal was often contiguous to that on the neighboring cells, thus resulting in a linear extension of pMLC signals over a group of cells. Moreover, these pMLC signals tended to be oriented along the dorsoventral axis of the neural tube. Other pMLC signals were concentrated around the center of rosettes. The role of these unique distributions of pMLC remains to be further explored, but we can imagine a scheme such that the cell-cell junctions with the linear pMLC may contract at subsequent developmental stages; the rosette configuration of cells could have been a consequence of the contraction of these junctions. Such form of cell rearrangement, if it occurs, is reminiscent of the cell intercalation processes observed in Drosophila germ band extension, although the underlying molecular mechanisms appear to have diverged between these species.
However, we observed that even the dorsal ectoderm prior to invagination exhibited similar rosettes, indicating that rosette formation was not specific to the closing neural tube. Cells in the dorsal ectoderm may also be intercalating, e.g. for the anterior-posterior elongation of the embryo, so this may explain why they also have rosettes. Hereby, we could consider another view for the roles of pMLC localization. It is possible that rosette formation is generally involved in cell intercalation and that the linear pMLC has a separate role. It is tempting to speculate that the linear pMLC with the dorsoventral orientation could be used for the contraction of a group of cell junctions along this axis, leading to the polarized bending of the neuroepithelial sheet, and that this type of pMLC localization could have been elaborated specifically for closing a tube. The ROCK inhibitor and RII-C1 peptides abolished these pMLC signals, as well as inhibited neural tube closure and rosette formation, suggesting that the Shroom-dependent recruitment of ROCKs to the AJC and the ROCK-dependent contraction of AJC are necessary for all of these cell rearrangement processes.
Finally, how can the MLC phosphorylation be upregulated locally under the
ubiquitous distribution of myosin II along the AJCs? Although Sroom3 also
appeared to be localized ubiquitously along the AJCs, pMLC was not always
detected at the Shroom-positive junctions, suggesting that the association of
MLC with the Shroom-ROCK complex was not sufficient for inducing MLC
phosphorylation in the neural tube. Yet unidentified regulators may locally
activate ROCKs, possibly through the upregulation of some RhoGEFs. Planar
polarity genes expressed in the neural tube
(Ybot-Gonzalez et al., 2007)
may play a role in such regulation, because the local distribution of pMLC can
be considered sort of planar polarity phenomenon. It was also suggested that
the zebrafish Rho kinase 2 acts downstream of non-canonical Wnt11 signaling
(Marlow et al., 2002).
Identification of such local regulators for MLC phosphorylation is important
to test the hypotheses proposed in this work.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/8/1493/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano,
T., Matsuura, Y. and Kaibuchi, K. (1996). Phosphorylation and
activation of myosin by Rho-associated kinase (Rho-kinase). J.
Biol. Chem. 271,20246
-20249.
Barrett, K., Leptin, M. and Settleman, J.
(1997). The Rho GTPase and a putative RhoGEF mediate a signaling
pathway for the cell shape changes in Drosophila gastrulation.
Cell 91,905
-915.[CrossRef][Medline]
Bertet, C., Sulak, L. and Lecuit, T. (2004).
Myosin-dependent junction remodelling controls planar cell intercalation and
axis elongation. Nature
429,667
-671.[CrossRef][Medline]
Blankenship, J. T., Backovic, S. T., Sanny, J. S., Weitz, O. and
Zallen, J. A. (2006). Multicellular rosette formation links
planar cell polarity to tissue morphogenesis. Dev.
Cell 11,459
-470.[CrossRef][Medline]
Copp, A. J., Greene, N. D. and Murdoch, J. N.
(2003). The genetic basis of mammalian neurulation.
Nat. Rev. Genet. 4,784
-793.[CrossRef][Medline]
Costa, M., Wilson, E. T. and Wieschaus, E.
(1994). A putative cell signal encoded by the folded gastrulation
gene coordinates cell shape changes during Drosophila gastrulation.
Cell 76,1075
-1089.[CrossRef][Medline]
Dietz, M. L., Bernaciak, T. M., Vendetti, F., Kielec, J. M. and
Hildebrand, J. D. (2006). Differential actin-dependent
localization modulates the evolutionarily conserved activity of Shroom family
proteins. J. Biol. Chem.
281,20542
-20554.
Etournay, R., Zwaenepoel, I., Perfettini, I., Legrain, P.,
Petit, C. and El- Amraoui, A. (2007). Shroom2, a myosin-VIIa-
and actin-binding protein, directly interacts with ZO-1 at tight junctions.
J. Cell Sci. 120,2838
-2850.
Fairbank, P. D., Lee, C., Ellis, A., Hildebrand, J. D., Gross,
J. M. and Wallingford, J. B. (2006). Shroom2 (APXL) regulates
melanosome biogenesis and localization in the retinal pigment epithelium.
Development 133,4109
-4118.
Farquhar, M. G. and Palade, G. E. (1963).
Junctional complexes in various epithelia. J. Cell
Biol. 17,375
-412.
Hagens, O., Ballabio, A., Kalscheuer, V., Kraehenbuhl, J. P.,
Schiaffino, M. V., Smith, P., Staub, O., Hildebrand, J. and Wallingford, J.
B. (2006). A new standard nomenclature for proteins related
to Apx and Shroom. BMC Cell Biol.
7, 18.[CrossRef][Medline]
Haigo, S. L., Hildebrand, J. D., Harland, R. M. and Wallingford,
J. B. (2003). Shroom induces apical constriction and is
required for hingepoint formation during neural tube closure. Curr.
Biol. 13,2125
-2137.[CrossRef][Medline]
Hildebrand, J. D. (2005). Shroom regulates
epithelial cell shape via the apical positioning of an actomyosin network.
J. Cell Sci. 118,5191
-5203.
Hildebrand, J. D. and Soriano, P. (1999).
Shroom, a PDZ domain-containing actin-binding protein, is required for neural
tube morphogenesis in mice. Cell
99,485
-497.[CrossRef][Medline]
Honda, H. and Eguchi, G. (1980). How much does
the cell boundary contract in a monolayered cell sheet? J. Theor.
Biol. 84,575
-588.[CrossRef][Medline]
Kawano, Y., Fukata, Y., Oshiro, N., Amano, M., Nakamura, T.,
Ito, M., Matsumura, F., Inagaki, M. and Kaibuchi, K. (1999).
Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by
Rho-kinase in vivo. J. Cell Biol.
147,1023
-1038.
Kolsch, V., Seher, T., Fernandez-Ballester, G. J., Serrano, L.
and Leptin, M. (2007). Control of Drosophila gastrulation by
apical localization of adherens junctions and RhoGEF2.
Science 315,384
-386.
Lecuit, T. and Lenne, P. F. (2007). Cell
surface mechanics and the control of cell shape, tissue patterns and
morphogenesis. Nat. Rev. Mol. Cell Biol.
8, 633-644.[CrossRef][Medline]
Lee, C., Scherr, H. M. and Wallingford, J. B.
(2007). Shroom family proteins regulate gamma-tubulin
distribution and microtubule architecture during epithelial cell shape change.
Development 134,1431
-1441.
Marlow, F., Topczewski, J., Sepich, D. and Solnica-Krezel,
L. (2002). Zebrafish Rho kinase 2 acts downstream of Wnt11 to
mediate cell polarity and effective convergence and extension movements.
Curr. Biol. 12,876
-884.[CrossRef][Medline]
Matsumura, F. (2005). Regulation of myosin II
during cytokinesis in higher eukaryotes. Trends Cell
Biol. 15,371
-377.[CrossRef][Medline]
Nakazawa, F., Nagai, H., Shin, M. and Sheng, G.
(2006). Negative regulation of primitive hematopoiesis by the FGF
signaling pathway. Blood
108,3335
-3343.
Nikolaidou, K. K. and Barrett, K. (2004). A Rho
GTPase signaling pathway is used reiteratively in epithelial folding and
potentially selects the outcome of Rho activation. Curr.
Biol. 14,1822
-1826.[CrossRef][Medline]
Parks, S. and Wieschaus, E. (1991). The
Drosophila gastrulation gene concertina encodes a G alpha-like protein.
Cell 64,447
-458.[CrossRef][Medline]
Perez-Moreno, M., Jamora, C. and Fuchs, E.
(2003). Sticky business: orchestrating cellular signals at
adherens junctions. Cell
112,535
-548.[CrossRef][Medline]
Pilot, F. and Lecuit, T. (2005).
Compartmentalized morphogenesis in epithelia: from cell to tissue shape.
Dev. Dyn. 232,685
-694.[CrossRef][Medline]
Riento, K. and Ridley, A. J. (2003). Rocks:
multifunctional kinases in cell behaviour. Nat. Rev. Mol. Cell
Biol. 4,446
-456.[CrossRef][Medline]
Rogers, S. L., Wiedemann, U., Hacker, U., Turck, C. and Vale, R.
D. (2004). Drosophila RhoGEF2 associates with microtubule
plus ends in an EB1-dependent manner. Curr. Biol.
14,1827
-1833.[CrossRef][Medline]
Sahai, E. and Marshall, C. J. (2002). ROCK and
Dia have opposing effects on adherens junctions downstream of Rho.
Nat. Cell Biol. 4,408
-415.[CrossRef][Medline]
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R.
(2001). Epithelial cell polarity and cell junctions in
Drosophila. Annu. Rev. Genet.
35,747
-784.[CrossRef][Medline]
Vogelmann, R. and Nelson, W. J. (2005).
Fractionation of the epithelial apical junctional complex: reassessment of
protein distributions in different substructures. Mol. Biol.
Cell 16,701
-716.
Walsh, S. V., Hopkins, A. M., Chen, J., Narumiya, S., Parkos, C.
A. and Nusrat, A. (2001). Rho kinase regulates tight junction
function and is necessary for tight junction assembly in polarized intestinal
epithelia. Gastroenterology
121,566
-579.[CrossRef][Medline]
Wei, L., Roberts, W., Wang, L., Yamada, M., Zhang, S., Zhao, Z.,
Rivkees, S. A., Schwartz, R. J. and Imanaka-Yoshida, K.
(2001). Rho kinases play an obligatory role in vertebrate
embryonic organogenesis. Development
128,2953
-2962.
Ybot-Gonzalez, P., Savery, D., Gerrelli, D., Signore, M.,
Mitchell, C. E., Faux, C. H., Greene, N. D. and Copp, A. J.
(2007). Convergent extension, planar-cell-polarity signalling and
initiation of mouse neural tube closure. Development
134,789
-799.
Yoder, M. and Hildebrand, J. D. (2007). Shroom4
(Kiaa1202) is an actin-associated protein implicated in cytoskeletal
organization. Cell Motil. Cytoskel.
64, 49-63.[CrossRef][Medline]
Zallen, J. A. and Wieschaus, E. (2004).
Patterned gene expression directs bipolar planar polarity in Drosophila.
Dev. Cell 6,343
-355.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  J. Taylor, K.-H. Chung, C. Figueroa, J. Zurawski, H. M. Dickson, E. J. Brace, A. W. Avery, D. L. Turner, and A. B. Vojtek The Scaffold Protein POSH Regulates Axon Outgrowth Mol. Biol. Cell, December 1, 2008; 19(12): 5181 - 5192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Chung and D. J. Andrew The formation of epithelial tubes J. Cell Sci., November 1, 2008; 121(21): 3501 - 3504. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
