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First published online 28 May 2008
doi: 10.1242/dev.019802
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Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, M5S 3G5, Canada.
* Author for correspondence (e-mail: tony.harris{at}utoronto.ca)
Accepted 2 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: Cell adhesion, Cell polarity, Cytoskeleton, Drosophila embryogenesis, Tissue morphogenesis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Like isolated cells, cells in tissues can change shape through cytoskeletal
rearrangements. For example, actin-myosin contractility drives apical
constriction and tissue invagination
(Leptin, 1999
), or can
constrict specific cell-cell contacts driving cell intercalation
(Lecuit and Lenne, 2007).
Actin-based protrusions drive cell migration during convergent extension
(Keller et al., 2003) and
wound repair (Van Aelst and Symons,
2002). Microtubule (MT) protrusion has been implicated in cell
elongation along the apicobasal axis during tissue invagination
(Lee et al., 2007), but MT
functions during other tissue rearrangements are unclear.
Cytoskeletal systems integrate with adherens junctions (AJs) to regulate
cell-cell interactions and coordinate tissue-wide morphogenesis. AJs form from
Cadherin receptors and adaptor proteins β-catenin (Armadillo, Arm) and
-catenin, which associate directly or indirectly with actin and MTs. In
Drosophila, actin-myosin-AJ links are evident during apical
constriction (Dawes-Hoang et al.,
2005) and cell intercalation
(Bertet et al., 2004).
Moreover, interplay between actin and MTs at AJs appears to control symmetric
AJ positioning around the apical domain during cell intercalation. These
interactions are regulated by the polarity proteins aPKC and PAR-6
(Blankenship et al., 2006;
Harris and Peifer, 2007).
Additionally, Bazooka (Baz/PAR-3) plays an earlier role in initial apical AJ
positioning (Harris and Peifer,
2004; Harris and Peifer,
2005). However, mechanisms that regulate AJ disassembly or
assembly as specific cell-cell contacts are lost or expanded remain poorly
understood.
To understand core epithelial morphogenesis mechanisms, it is important to
study how similar molecular machinery produces different types of epithelia.
Epithelial cells can be columnar, cuboidal or squamous. Squamous epithelial
cells are flat and elongated in the plane of the epithelial sheet. They are
often in stratified epithelia such as the skin, esophagus, trachea, cervix and
cornea, where their unique morphology probably provides protective barriers
resistant to physical stress. Squamous epithelia can also be monolayers. These
are often associated with developmental or tissue remodeling processes.
Examples include the trophectoderm of the mammalian embryo
(Yamanaka et al., 2006) and
the peripodial epithelium of Drosophila imaginal disks
(Gibson and Schubiger, 2001).
Cells also flatten and spread during epiboly
(Solnica-Krezel, 2006) and
wound healing (Van Aelst and Symons,
2002).
Here, we report cellular and molecular mechanisms that control
morphogenesis of the amnioserosa (AS), a squamous epithelial monolayer
connected to the main epidermis of the Drosophila embryo. The AS
plays a central role in guiding major embryo tissue rearrangements, including
germband retraction and dorsal closure
(Jacinto and Martin, 2001).
However, little is known about how the AS first forms. The AS forms during
gastrulation from columnar epithelial cells generated at cellularization (the
simultaneous formation of the 5000-6000 cells of the first embryonic
epithelium). TGF-β signaling specifies AS cells on the dorsal embryo
surface (O'Connor et al.,
2006), but the cellular mechanisms driving their morphogenesis are
unclear. We find that AS cells flatten and elongate autonomously. This
elongation occurs through a novel rotation of cellular components converting a
columnar morphology into a squamous morphology. Apical MT protrusion appears
to initiate the rotation, and actin constrains and helps orient these MTs. As
the cells elongate, they maintain contacts with their original neighbors, and
AJs at these contact sites are rapidly remodeled through the coordinated
activity of Myosin II and Baz.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), Moesin ABD-GFP
(Kiehart et al., 2000),
YFP-endoplasmic reticulum (ER) targeting sequence [Bloomington Drosophila
Stock Center (BDSC)], DE-Cad-GFP (Oda and
Tsukita, 2001), Spaghetti squash (Sqh)-GFP
(Royou et al., 2002), EB1-GFP
[a gift from H. Ohkura (Edinburgh University, UK)] and Baz-GFP (Flytrap).
Arm-CFP generated as Arm-GFP has been described previously
(McCartney et al., 2001).
armXP33 maternal-zygotic mutants were made as before
(Harris and Peifer, 2004).
bazXi106 mutants were a gift from A. Wodarz
(Göttingen University, Germany). bcdE1
nosL7 tsl146 mutants were a gift from
E. Wieschaus (Princeton, NJ, USA). dl1,
zen7 and zip1 mutants were from BDSC.
The wild type used was yellow white.
Embryo staining and treatment
For tubulin and phalloidin staining, embryos were fixed for 10 minutes in
1:1 10% formaldehyde/PBS:heptane and were de-vitellinized by hand peeling. For
other staining, embryos were fixed for 20 minutes in 1:1 3.7%
formaldehyde/PBS:heptane and were de-vitellinized with methanol. Blocking and
staining was carried out in PBS/1% goat serum/0.1% Triton X-100. Antibodies
used were: mouse Dlg [1:100; Developmental Studies Hybridoma Bank (DSHB)],
-tubulin (1:350; Sigma), acetylated tubulin (1:350; Sigma) and tubulin
(1:100; DSHB); rabbit Baz (1:350) and GFP (1:2000; Abcam); and rat DE-Cad
[1:100 (Oda et al., 1994)].
F-actin staining was carried out with phalloidin-Alexa488 (Invitrogen).
Colchicine and cytochalasin D treatments were carried out as previously
described (Harris and Peifer,
2005).
Image acquisition, quantification and manipulation
Fixed embryos were mounted in Aqua Polymount (Polysciences) and were imaged
using a 510 confocal microscope (Carl Zeiss) at room temperature. Lenses used
were 40x (Plan-NeoFluor; NA 1.3) and 63x (Plan-Apochromat; NA 1.4)
objectives. Images were analyzed using LSM 510 AIM software. Secondary Abs
were Alexa488, 546 and 647 (Invitrogen). Image deconvolution and projections
were carried out by Volocity software (Improvision). Fluorescence intensity
measurement was made using Image J (NIH). Unless otherwise noted, input levels
were adjusted by Adobe Photoshop for the main signal range to span full output
grayscale. Image resizing was carried out using bicubic interpolation (minimal
change at normal magnifications).
Time-lapse microscopy
Dechorionated embryos were mounted in halocarbon oil (series 700;
Halocarbon Products) on petriPERM dishes (Sigma). Analysis was carried out
using a Quorum spinning disk confocal microscope (Quorum Technologies) at room
temperature with 40x (Plan-NeoFluor; NA 1.3) and 63x
(Plan-Apochromat; NA 1.4) objectives. Images were captured in 300 nm
z-steps using a piezo top plate and Hamamatsu EM CCD camera linked to
Volocity software.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
First, the cephalic furrow separates head and body, and the ventral furrow
internalizes the mesoderm. Second, the posterior midgut invaginates from the
posterior pole, and the lateral germband undergoes convergent extension along
the anteroposterior (AP) axis (germband extension). Simultaneously, the AS
forms on the dorsal surface. The AS initially extends along the DV axis,
filling space left by the germband (Fig.
1A, the AS is bracketed). Next, the AS spreads posteriorly,
filling space between the ventral and dorsal halves of the extended germband
(Fig. 1B,C, the AS is
bracketed).
To overview AS cell shape changes, we examined AJ, MT and actin
localization in fixed tissue. After cellularization, AS cells, like other
blastoderm cells, are hexagonal and columnar with DE-Cadherin (DE-Cad; Cad in
figures) in spot junctions around the cell apex
(Fig. 1D)
(Tepass and Hartenstein,
1994). The cells have apical diameters of 5.1±1.0 µm
(100 cells from 10 embryos) and depths of 18.9±2.9 µm (60 cells from
12 embryos) (Fig. 1D,M, two
cells outlined, inset shows cell height). Lateral MT bundles run from the
level of AJs basally (Harris and Peifer,
2007) (Fig. 1D,E).
Actin partially colocalizes with AJs
(Pilot et al., 2006)
(Fig. 1F) but is also in apical
surface microvilli (Grevengoed et al.,
2003) and lateral (data not shown). After germband extension, AS
cells are fully elongated, with average lengths of 52.7±12.7 µm,
widths of 4.9±1.6 µm (32 cells from four embryos) and depths of
4.27±0.75 µm (21 positions from 11 embryos at the cell centre - the
cells taper at both ends) (Fig.
1J,M, two cells are outlined, inset shows cell height). AJs form
more continuous belt junctions during this transition
(Fig. 1G,J)
(Tepass and Hartenstein,
1994), and remain around the full cell circumference
(Fig. 1G,J). MT bundles
reorganize to run along the axis of cell elongation
(Fig. 1H,K, arrows). Actin
localizes around the full cell circumference
(Fig. 1I,L). Overall, AS cell
elongation requires AJ and actin reorganization for continual positioning
around the growing apical circumference, and MT reorganization from a series
of bundles along the apicobasal axis to a perpendicular series of bundles
along the long axis of the flattened cell.
Autonomy of the amnioserosa cell shape change
Because AS morphogenesis coincides closely with germband extension and
ventral furrow formation, we wondered which elements of AS morphogenesis are
cell autonomous (i.e. orchestrated by changes within AS cells themselves), and
which might involve surrounding tissues. Thus, we examined mutants affecting
each tissue. To assess whether germband extension and ventral furrow formation
are sufficient for elongating dorsal cells, we analyzed
zerknüllt (zen) zygotic mutants in which AS
differentiation is blocked but ventral furrow formation and germband extension
occur (Arora and Nusslein-Volhard,
1992). Stage 7-10 zen mutant embryos displayed no dorsal
cell elongation relative to zen mutant germband cells
(Fig. 1N, dorsal cells
bracketed; Fig. 1T,U show
higher magnification and cell length quantification). However, the ventral
midline of the extended germband was observed below the non-elongated dorsal
cells in 3D projections (Fig.
1O, arrow) - the zen mutant germband has previously been
shown to extend beneath the non-differentiated dorsal cells
(Arora and Nusslein-Volhard,
1992). Thus, ventral furrow formation and germband extension are
not sufficient for changing dorsal cell shape, indicating that amnioserosa
specification is either instructive or permissive for amnioserosa
morphogenesis.
To test whether amnioserosa specification is instructive, we examined
arm maternal/zygotic mutants. These form the first embryonic
epithelium with apicobasal polarity and proper embryonic axes, but at
gastrulation epithelial structure is disrupted and ventral furrow formation
and germband extension fail (Cox et al.,
1996; Harris and Peifer,
2004). In gastrulating arm mutants, we observed
elongating dorsal cells (Fig.
1P, brackets; ventral and ventrolateral markers identified the
dorsal surface). In stage 9-11 arm mutants, highly elongated dorsal
cells were present, but were restricted to a dorsal patch in which they formed
abnormal swirls of cells (Fig.
1Q, brackets; Fig.
1T,U show higher magnification and cell length quantification).
Thus, individual AS cells can elongate without ventral furrow formation and
germband extension.
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).
In gastrulating and stage 9-11 bcd nos tsl mutants, dorsal cells
elongated individually but the tissue remained in a dorsal patch similar to
arm mutants (Fig.
1R,S, brackets; Fig.
1T,U show higher magnification and cell length quantification). AS
patches were significantly larger in stage 9-11 bcd nos tsl mutants
versus stage 9-11 arm mutants (130.4±19 µm versus
60.2±21.7 µm lengths, n>10, P<0.001,
t-test), suggesting defects in forming/maintaining full patch size
without AJs. Intriguingly, stage 9-10 bcd nos tsl mutant AS cells
preferentially elongated along the DV axis
(Fig. 1S, quantified in 1Y),
although there was more tissue disorganization versus wild type
(Fig. 1B,C). Live bright-field
imaging of bcd nos tsl mutants revealed initial cell elongation along
the DV axis (Fig. 1V) and later
disorganization. We suspected that DV patterning might initially orient the
cells, and thus analyzed dl mutants that fail to form ventral tissue
and instead form a ring of AS around the full DV axis at the centre of the
embryo (Ray et al., 1991)
(Fig. 1W,X; bracketed). Here,
AS cells also elongated but reoriented along the AP axis
(Fig. 1W,X, quantified in
Fig. 1Y) towards ectopic
furrows (Fig. 1W,X, arrows).
Thus, AS cells can elongate with disruption of either germband extension or
the ventral furrow in bcd nos tsl mutants and dl mutants,
respectively, further confirming the autonomy of individual AS cell shape
change. However, mechanisms involving outside tissues may orient the
cells.
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To further evaluate the cell rotation, we examined the full MT network by 3D timelapse imaging of Tubulin-GFP. First, we analyzed 3D projection embryo surface views (see Movie 1 in the supplementary material). Before elongation, lateral MT bundles run along the apicobasal axis and are seen end-on (Fig. 2B, 0:00, arrows), with minimal links to the centrosomes (Fig. 2B, 0:00, arrowheads, above nuclei). Then, lateral bundles smoothly rotate into the axis of cell elongation, progressively revealing the long axis of the bundles in embryo surface views (Fig. 2B, 4:24-26:20, arrows). Notably, the cells could rotate in either direction along the axis of tissue extension (dorsally or ventrally) (Fig. 2C). These 3D projection analyses allowed detection of the overall rotation of the MT arrays, but the overlapping of structures with this imaging hindered analysis of individual elements of the arrays.
To clearly view individual elements of the arrays, we analyzed single sections from our 3D imaging. We viewed cells in cross-section and found individual lateral MT bundles progressively rotating from the apicobasal axis to the axis of tissue extension (Fig. 2D, two bundles marked individually with red and green asterisks). We also evaluated the rotation of the full MT network. Before elongation, embryo surface views revealed a ring of MT bundles in cross-section per cell (Fig. 2E, arrows, section at top of nuclei), whereas embryo cross-section views revealed long axes of the bundles (Fig. 2F, arrows). With cell elongation, embryo surface views revealed long axes of the bundles (Fig. 2G, arrows), whereas embryo cross-section views reveal a ring of MT bundles in cross-section per cell (Fig. 2H, arrows, section at end of nuclei). Before rotation, full MT arrays were 17.7±3.0 µm long, equaling cell depths. After 10 minutes rotation, the MT arrays had similar lengths (18.3±4.9 µm) but cell depths were significantly reduced (12.8±3.6 µm, P<0.001, t-test, 15 arrays from three embryos). Thus, the MT network appears to rotate as a unit as the cells flatten.
With rotation of the MT array, organelles initially polarized along the apicobasal axis should become planar polarized. Thus, we examined the positioning of the nucleus, centrosomes, ER and Golgi. At the end of cellularization nuclei are elongated along the apicobasal axis (Fig. 2F, seen by Tubulin-GFP nuclear exclusion), and two centrosomes localize above each nucleus (Fig. 2F,I, arrowheads, inset). With cell elongation, the nucleus long axis turns perpendicularly into the DV axis of the embryo (Fig. 2G), and the two centrosomes become positioned to the dorsal or ventral end of the cell (Fig. 2G, arrowheads; in fixed tissues in Fig. 2J,K, arrows). A bulk of ER is also above the nuclei following cellularization (Fig. 2L, inset), whereas the Golgi is non-polarized (not shown). During initial AS cell elongation, the ER is also positioned at ventral or dorsal ends of the cells (Fig. 2M, arrows), but then becomes non-polarized (Fig. 2N). These results indicate that the centrosomes, nuclei and ER also rotate as AS cells elongate. The apicobasal axis of the main MT network appears to rotate 90° to extend along the DV axis of the embryo: we call this `rotary cell elongation'.
Amnioserosa cell elongation is microtubule dependent
We hypothesized that MTs actively drive AS cell elongation. As the cells
elongated non-centrosomal MT bundles extended past the centrosomes in the
direction of cell elongation (compare Fig.
2F with 2G,
brackets; Fig. 2B, 26:20,
brackets). We evaluated MT organization in 3D image projections of the apical
region to assess how these protruding MTs develop. Immediately before cell
elongation, we observed apical MTs oriented perpendicularly to lateral MT
bundles but without specific AP or DV directionality
(Fig. 2O, 0:00, arrowheads). As
the cells elongated, these apical MTs then extended preferentially along the
DV axis (Fig. 2O, arrows). By
contrast, cortical actin is relatively evenly distributed during the process
(Fig. 2P).
To understand how the MTs protrude, we probed them for acetylated tubulin to detect older MTs, and imaged EB1-GFP to detect new MT growth. Lateral bundles present before elongation contained acetylated MTs (Fig. 2Q). During AS cell elongation, MT bundles running across the apical domain also contained acetylated MTs (Fig. 2R, arrows), but double staining for total tubulin also suggested the presence of non-acetylated MTs (Fig. 2R). Indeed, EB1-GFP imaging revealed puncta moving in both directions along the long axis of extending AS apical domains (kymogram in Fig. 2S; see Movie 2 in the supplementary material). Thus, apical MT bundles appear to protrude through rotational re-positioning of older lateral MTs and new bi-directional MT growth.
To test MT function during AS cell elongation, we treated embryos for 30 minutes with colchicine, fixed immediately and stained for DE-Cad as a marker for cell shape. During initial cell elongation (cells were still columnar in transverse furrows that form across the early AS, Fig. 3A), the colchicine treatment reduced elongation of the apical domain (Fig. 3C) versus control (Fig. 3B). Elongation was quantified by dividing the longest cell axis by the cell width at half the long axis (Fig. 3D, inset). At this stage, the control ratio was 1.92±0.53 (60 cells from six embryos) and colchicine treatment produced a significantly lower ratio of 1.46±0.34 (74 cells from nine embryos) (P<0.001, t-test) (Fig. 3D). Thus, MTs function in initiating AS cell elongation.
To test whether MTs maintain cell elongation, we examined later stages. In stage 8-9 control embryos, elongated cells were between two regions of less elongated cells, and in AS transverse furrows (Fig. 3E). In stage 8-9 colchicine-treated embryos, elongated cells lost their embryo surface position, but were in transverse furrows beneath the less elongated cells (Fig. 3F). In stage 9-10 control embryos, elongated cells were arranged in parallel over the embryo surface (Fig. 3G). In stage 9-10 colchicine-treated embryos, AS cells were elongated but had smaller surface-exposed regions (Fig. 3H). Thus, MTs appear to affect cell-cell interactions at later stages, but may not be needed for maintaining AS cell elongation.
Actin restrains microtubule protrusion during cell elongation
As actin often controls cell shape, we examined its role in early AS cell
shape change. We treated embryos for 30 minutes with cytochalasin D, fixed
immediately and stained for DE-Cad. At stage 7-8, initial cell elongation was
evident in carrier controls (Fig.
4A,C). Remarkably, cytochalasin D treatment produced excessive AS
cell protrusion (Fig. 4B,D,
arrows) - cells extended single abnormally long protrusions with improper
orientation (Fig. 4D,H; seen in
13/13 embryos, four experiments). DE-Cad often clustered at these protrusions
(Fig. 4D,H), contrasting its
normal symmetric distribution (Fig.
4C,G). Germband cells displayed less severe cell shape change with
the treatment (Fig. 4A,B),
whereas ventral furrow cells rounded up but did not display abnormal cell
protrusions (not shown). Phalloidin staining indicated residual filamentous
actin after the treatment (not shown). Thus, AS cells are uniquely sensitive
to partial actin disruption at this stage, producing abnormally long cell
protrusions.
As MTs function in normal AS cell elongation, we hypothesized that MTs might be responsible for the abnormal apical protrusions with actin disruption. Thus, we disrupted actin and MTs simultaneously. This suppressed cell protrusions otherwise produced by actin disruption, and restored AJ symmetry [double treatment in Fig. 4F is more similar to colchicine treatment alone (Fig. 4E) than to cytochalasin D treatment alone (Fig. 4D); clear suppression in 10/12 embryos, two experiments]. Thus, actin may normally counteract MTs during early AS cell elongation.
Weakening actin triggers MT-based flattening and elongation of normally columnar epithelial cells
Although cytochalasin D has a relatively specific effect on AS cells during
gastrulation, it induced dramatic reorganization of stage 9-11 germband cells.
DE-Cad-positive apical domains were abnormally extended and clustered, forming
large rosettes of cells (Fig.
4J, bracketed; seen in 31/31 embryos, five experiments; control in
Fig. 4I). The cells appeared to
re-orient towards tissue regions that normally have greater apical
constriction (Fig. 4I, arrow),
and lateral cell surfaces became exposed to the embryo surface [observed with
Dlg staining (not shown), and MT staining
(Fig. 4L)]. With actin
disruption, these cells appeared to rotate perpendicularly relative to their
original axis but did not distribute AJs around their subsequently
surface-exposed perimeter.
As this abnormal cell shape change resembled normal AS cell shape change, we suspected that MTs might be involved. Thus, we disrupted actin and MTs simultaneously. This suppressed the cytochalasin D-induced germband cell shape change (Fig. 4K; clear suppression in 25/26 embryos, three experiments). Moreover, with actin disruption alone, MTs protruded into the abnormally extended apical domains of the germband cells (Fig. 4L, an arrow marks an apical protrusion and a broken line indicates the surface-exposed lateral region of the cell). Live DE-Cad-GFP imaging after cytochalasin D treatment revealed minimal movement of the extended apical domains (data not shown), suggesting that they remain adherent to each other. Together, these results indicate that actin inhibits apical MT protrusion to maintain columnar versus elongated epithelial cell shape in the later germband. This suggests that normal, MT-driven AS cell elongation involves regulated reduction of apical actin activity.
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Next, we examined AJ changes at both AP and DV contacts. Remarkably, the length of AP contacts doubled in 5-10 minutes and quadrupled in 10-20 minutes [Fig. 5B; both DE-Cad-GFP and Moesin actin-binding-domain (ABD) GFP expressing embryos analyzed]. DE-Cad-GFP levels per membrane area at AP contacts were approximately half those in the same area at DV contacts at fourfold cell elongation (Fig. 5C), suggesting AJ redistribution along AP contacts. However, AJs were not static at DV contacts. In 3D projections, DV contacts displayed dynamic looping of DE-Cad-GFP, suggesting AJ remodeling at these sites as well (Fig. 5D, arrows; Arm-CFP loops, Fig. 6G, arrows). Thus, the translation of rotary cell elongation into tissue extension involves planar polarized AJ remodeling at both AP and DV contacts.
Myosin II and Bazooka show transient opposite planar polarization in the amnioserosa
Planar polarized junctional remodeling also occurs in the germband. Here,
Myosin II is enriched at AP cell contacts, and the polarity and AJ regulator
Baz is enriched at DV cell contacts
(Zallen and Wieschaus, 2004).
To determine whether AS cells share this organization, we localized Myosin II
and Baz. Before AS cell elongation, Myosin II localized relatively
symmetrically, with AJs and apical to AJs
(Fig. 6A, apical surface
bracketed), and Baz also localized relatively symmetrically
(Fig. 6D). As AS cells
elongated, Myosin II became enriched along AP cell contacts
(Fig. 6B, arrows; see Movie 4
in the supplementary material), versus DV contacts, and also localized to two
ends of AP contacts (Fig. 6B,
yellow arrowheads), suggesting loss from the contact length. Baz displayed the
reciprocal pattern, with enrichment along DV cell contacts
(Fig. 6E, arrows) versus AP
contacts [also seen live with Baz-GFP (see Movie 5 in the supplementary
material)]. Intriguingly, double live imaging revealed D/V Baz-GFP partly
colocalizing with Arm-CFP loops at this stage
(Fig. 6G, arrows). With full AS
cell elongation, both Myosin II and Baz were lost from the cell cortex, versus
germband cells (Fig. 6C,F).
Myosin II and Bazooka synergize to regulate amnioserosa AJ remodeling
To assess whether Myosin II and Baz affect AJ localization during AS cell
elongation, we examined DE-Cad localization in zygotic mutants for
zipper (zip), the Myosin II heavy chain gene and
baz. DE-Cad appeared normal in stage 8-10 AS cells in both homozygous
zygotic zip mutants and hemizygous zygotic baz mutants
(baz is X-linked) (data not shown). However, abnormal AS AJ
clustering occurred in hemizygous zygotic baz mutants derived from
females carrying a GFP-marked balancer chromosome. This probably resulted from
an unknown balancer chromosome mutation. Thus, we tested whether a mutant
zip allele also dominantly enhances the baz mutant phenotype
by generating double heterozygote females and crossing them with wild-type
males (Fig. 7A). In contrast to
zygotic baz mutants alone, which had no AJ phenotype (0/90 embryos),
17/86 of the progeny of the double heterozygote females displayed AJ defects
(approximately the expected Mendelian ratio). At stage 8, DE-Cad formed
abnormal small puncta along AP contacts
(Fig. 7B,C arrowheads) and
abnormal larger clusters at DV contacts
(Fig. 7B, arrows), contrasting
with wild type (Fig. 7F,
Fig. 1G). Similarly, stage 10
embryos displayed small DE-Cad puncta (Fig.
7D,E, arrowheads) and abnormal DE-Cad clusters
(Fig. 7D, arrows), in contrast
to wild type (Fig. 7F). Small
puncta were apparent at all elongated contacts observed (enhanced images:
Fig. 7C,E). Twenty-five out of
28 isolated large DE-Cad clusters were at non-elongated contacts (eight stage
8-10 embryos counted). In addition, the effects were relatively specific for
AS cells, as only 3/17 of the embryos with AS defects displayed AJ defects in
other tissues. Thus, Myosin II and Baz function together to coordinate AJ
positioning at AP and DV contacts during AS morphogenesis. However, Myosin II
and Baz probably act at different contact sites and other players are
undoubtedly involved. Implicating AP patterning, stage 7-8 bcd nos
tsl mutants displayed no or abnormal Baz planar polarity
(Fig. 7G, arrow) and stage 9-11
bcd nos tsl mutants displayed abnormal DE-Cad clustering at
non-elongated contacts colocalized with abnormally persistent Baz
accumulations (compare Fig. 7H,
arrows with Fig. 6F).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Warn and
Warn, 1986). With AS morphogenesis, the cells elongate and
flatten. AS cells could change shape by symmetrically re-positioning cellular
contents (full cytoskeleton and/or membrane reorganization) or by
perpendicularly rotating cellular components to reorient the long axis of the
cell into the plane of tissue extension. To distinguish these possibilities,
we analyzed 3D cell organization over time and discovered that the MT array
plus the nucleus, centrosomes and ER rotate, apparently as a unit, into the
plane of tissue extension - we term this `rotary cell elongation'
(Fig. 2). As discussed below,
more symmetric AJ and cortical reorganizations appear to accompany the
rotation. MT arrays also reorient to polarize cells during chemotaxis and
tissue migration (Siegrist and Doe,
2007), and to reposition cell contents as occurs during cortical
rotation in early Xenopus embryos
(Weaver and Kimelman, 2004).
Our results reveal rotation of the MT cytoskeleton linked to cell shape change
and AS morphogenesis. Similar mechanisms may underlie the development of other
squamous epithelial monolayers.
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As MTs extend apically the actin cytoskeleton appears to inhibit them, as
weakening actin leads to excessively long and randomly oriented MT-based
protrusions. Actin is normally found around the full apical circumference as
AS cells elongate (Figs 1 and
2). By contrast, Myosin II
becomes enriched at AP contacts and is gradually lost from the full cell
cortex (Fig. 5)
(Bertet et al., 2004). Thus,
different pools of actin may regulate MT protrusion. We speculate that the
gradual overall loss of cortical actin-myosin complexes permits, and may help
orient, regulated MT protrusion (Fig.
8A). Actin also antagonizes cortical MTs in other systems.
MT-based primary axons form where cortical actin is weakest
(Bradke and Dotti, 1999). Actin
inhibits cortical MT protrusion in neutrophils
(Eddy et al., 2002) and Myosin
IIA inhibits cortical MTs in mammalian cells
(Even-Ram et al., 2007). Actin
might physically block MT protrusion, but direct or indirect molecular
interactions may also be involved
(Etienne-Manneville, 2004;
Rodriguez et al., 2003).
In Drosophila embryos, MT-actin interactions also affect germband
cells. At stage 7-8, actin disruption enhances AJ planar polarity at DV
contacts (Harris and Peifer,
2007). MT disruption suppresses this, suggesting that actin
inhibits MT-based AJ positioning in these cells - however, germband cells show
minimal shape change with actin disruption at this stage
(Harris and Peifer, 2007).
Remarkably, the same actin disruption causes stage 9-10 germband cells to
rotate analogously to early AS cells. Their apical domains elongate and their
lateral regions rotate perpendicularly, becoming exposed to the embryo
surface. Implicating MTs in this change, lateral MT bundles run into the
extended apical domains and simultaneous MT disruption suppresses the cell
shape change. Thus, actin may inhibit apical MTs to regulate tissue structure
in many parts of the embryo. This MT inhibition may also involve coordination
with AJs, as disrupted germband cells in armm/z mutants
also display MTs protruding into extended apical domains
(Harris and Peifer, 2004).
How do MTs elongate the apical domain and how is this linked to the
rotation of the full MT cytoskeleton in the AS? We propose that rotary cell
elongation occurs in two phases. In phase one, our MT imaging and inhibitor
studies indicate that regulated MT protrusion elongates the apical domain.
This may involve a combination of physical force, membrane delivery and/or
relaxation of actin-myosin contractility
(Etienne-Manneville, 2004). The
AJ clustering we observed at abnormal apical MT protrusions formed with actin
inhibition in both early AS cells and the later germband suggests that MTs may
apply force to AJs. Consistent with this idea, MT inhibition affected both
initial AS cell elongation and later AS cell-cell interactions
(Fig. 3E-H). However, AS cell
elongation in armm/z mutants suggests that MTs may not
necessarily engage AJs directly. As Baz localizes apically in early
armm/z mutants (Harris
and Peifer, 2004), and is enriched at DV AS cell contacts to which
MTs rotate in wild type, it is a strong candidate for coordinating these
interactions. However, severe early defects in bazm/z
mutants (Harris and Peifer,
2004; Muller and Wieschaus,
1996) would confound analysis of AS development - this may require
conditional mutants. Phase two of rotary cell elongation requires full
perpendicular rotation of the MT array, apical and basal membrane growth, and
lateral membrane removal. Although it is unclear how full rotation occurs, MT
rotation and cortical remodeling may occur in concert. For example, membrane
remodeling may explain how AS cells remain elongated with MT disruption during
later development (Fig.
3E-H).
Planar polarity and AJ remodeling during rotary cell elongation
For rotary cell elongation to translate into tissue extension, cell
contacts and AJs must be remodeled. Remarkably, AS cells maintain their
neighbor relationships as they elongate, and two contact types develop; highly
elongated AP contacts and lesser elongated DV contacts. Each appears to
involve unique AJ remodeling. Intriguingly, Myosin II and Baz localize to AP
and DV contacts, respectively - the same reciprocal planar polarized
relationship displayed in the germband
(Bertet et al., 2004;
Zallen and Wieschaus, 2004).
In the AS, Myosin II and Baz synergize to control overall AJ positioning, a
regulatory interaction that, to our knowledge, has not been shown
elsewhere.
Myosin II and Baz may regulate specific AJ remodeling events occurring at
AP and DV contacts, respectively (see model in
Fig. 8B). AS cells increase
their apical circumference 
10-fold, initially doubling the length of
their AP cell contacts every 5-10 minutes. Remarkably, AJs localize around the
full circumference as this occurs. This contrasts elongating
Drosophila follicle cells, which lose AJ continuity
(Grammont, 2007), suggesting
specific mechanisms for maintaining AJ continuity during AS morphogenesis. AS
AJs do lose continuity with actin disruption, suggesting a role for actin.
More specifically, AJ fragmentation in baz zip double mutants
suggests a role for Myosin II. In the neighboring ventral furrow and germband,
actin-myosin contractility is coupled to AJs during apical constriction
(Dawes-Hoang et al., 2005) and
cell intercalation (Bertet et al.,
2004), respectively. The actin-myosin complexes enriched along AS
AP contacts may also be contractile, but here they may counterbalance MT
protrusion (Fig. 8B). Slowing
apical elongation may indirectly allow AJ remodeling. However, Myosin II may
also have direct affects on AJs, as evident in Drosophila
(Bertet et al., 2004;
Dawes-Hoang et al., 2005) and
elsewhere (Ivanov et al.,
2007; Miyake et al.,
2006; Shewan et al.,
2005; Yamada and Nelson,
2007).
Baz may regulate distinct AJ re-modeling at DV contacts. We hypothesize
that MT protrusion applies force to the DV contact at the cell `front', and
that cell elongation may also pull the `rear' contact
(Fig. 8B). Either force could
detach AJs and necessitate AJ remodeling. We observed dynamic looping of
DE-CadGFP and ArmCFP at D-V contacts, and BazGFP partly colocalized with these
loops. Although further experiments (e.g. photobleaching) are needed to
understand this and other AS AJ remodeling, Baz localization at DV contacts
and abnormal AJ aggregation at DV contacts in baz zip double mutants
suggests a role for Baz in AJ remodeling at these sites. Baz appears to
interact with MTs and Dynein to initially position AJs during
Drosophila cellularization
(Harris and Peifer, 2005), and
Baz might re-position AJs at DV AS contacts in a similar way
(Fig. 8B).
Orienting rotary cell elongation
In four different cases, we observed cells elongating towards potential
sources of pulling forces. First, wild-type AS cells elongate along the DV
axis towards the germband (potential source of DV pulling forces during
convergent extension) and the ventral furrow (potential source of DV pulling
forces during invagination). Second, AS cells elongate along the DV axis of
bcd nos tsl mutants (Fig.
1S,Y), in which germband extension fails
(Irvine and Wieschaus, 1994),
but ventral furrow formation occurs. Third, in dl mutants in which
the ventral furrow does not form and the AS forms a ring around the DV axis
(Ray et al., 1991), AS cells
reoriented along the AP axis towards ectopic contractile furrows
(Fig. 1X,Y). Fourth, in the
stage 9-11 wild-type germband, cells artificially induced to flatten and
elongate did so in coordinated groups oriented towards contractile regions of
the germband (Fig. 4I,J). Thus,
polarized pulling forces across a tissue may orient rotary cell elongation. In
wild-type embryos, these forces may come from germband extension and/or
ventral furrow formation. However, Zen must first trigger the AS cell shape
change (Fig. 8A), while AP
patterning may specifically regulate AJ remodeling
(Fig. 8B).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/13/2227/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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