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First published online 6 February 2008
doi: 10.1242/dev.016337
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1 Department of Biology, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599-3280, USA.
2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599-3280, USA.
* Author for correspondence (e-mail: peifer{at}unc.edu)
Accepted 10 January 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: Myosin, Formins, RhoGEF2, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Cell-cell adhesion and the cytoskeleton must be tightly coordinated during
morphogenesis. Cell shape changes require remodeling of adherens junctions
(AJs) and the actomyosin cytoskeleton. AJs mediate cell-cell adhesion via
transmembrane cadherins, with β-catenin (Drosophila Armadillo;
Arm) and 
-catenin bound to their cytoplasmic tails
(Halbleib and Nelson, 2006
).
-Catenin also interacts with actin. AJs and actin are intimately
interrelated, as disrupting one disrupts the other (e.g.
Cox et al., 1996;
Quinlan and Hyatt, 1999), but
mechanisms regulating this coordination are not well understood.
During morphogenesis, many epithelial cells polarize in the plane of the
epithelium, orthogonal to the apicobasal axis. This planar cell polarity
involves asymmetric distribution of AJ and cytoskeletal proteins around the
apical circumference (Zallen,
2007). For example, during Drosophila germband extension,
F-actin enrichment at anterior/posterior (A/P) cell borders is the first break
in symmetry. Nonmuscle Myosin 2 (hereafter Myosin) then becomes enriched at
A/P cell borders, while Bazooka/PAR-3 and AJ proteins accumulate at reciprocal
dorsal/ventral (D/V) cell borders (Bertet
et al., 2004; Blankenship et
al., 2006; Zallen and
Wieschaus, 2004). Planar polarization of AJs, actin and Myosin
also occur during dorsal closure, when epidermal cells elongate along the D/V
axis and leading-edge cells construct an actomyosin cable along their dorsal
borders (Kaltschmidt et al.,
2002; Kiehart et al.,
2000).
Actin and Myosin regulators, like Rho family GTPases, direct cell movement,
shape changes and planar cell polarity. Rho regulates the cytoskeleton and
AJs, and Drosophila Rho1 mutants have dorsal closure defects
(Magie et al., 1999). Two
major Rho effectors are Rho kinases (ROCKs), which are Myosin regulators, and
Diaphanous-related formins (DRFs), which are actin regulators. Embryos
zygotically lacking Drosophila ROCK are normal, owing to maternal
contribution, while removing maternal ROCK blocks oogenesis
(Verdier et al., 2006b;
Winter et al., 2001). Myosin
heavy chain [MHC; encoded by zipper (zip)] is required for
proper dorsal closure (Franke et al.,
2005). However, zip zygotic mutants retain maternal MHC
and thus do not reveal the full spectrum of functions of Myosin.
DRFs, a second class of Rho effectors, nucleate actin filaments and promote
filament elongation (Kovar,
2006). DRFs normally are autoinhibited via intramolecular
interactions between the N-terminal GTPase-binding domain (GBD) and C-terminal
DAD domain. Rho binds the GBD, activating DRFs. DRFs are essential regulators
of cytokinesis in yeast, nematodes and flies
(Castrillon and Wasserman,
1994; Severson et al.,
2002; Swan et al.,
1998). Although DRFs exact mechanism of action is not clear, they
are crucial in assembling/stabilizing the contractile actomyosin ring. In
Drosophila Diaphanous (Dia) is the sole DRF. Dia is essential in
conventional cytokinesis and in more specialized events of early
embryogenesis, when Dia localizes to tips of syncytial and cellularization
furrows, coordinating actin assembly as cells form
(Afshar et al., 2000;
Grosshans et al., 2005).
Formins also have roles outside of cytokinesis. DRFs can affect
transcription by activating MAL/SRF through G-actin depletion, and can
stabilize polarized microtubules (Faix and
Grosse, 2006). In cultured mammalian cells, both Dia1 and Formin 1
promote AJ stability; AJ stabilization by Formin 1 requires its actin
polymerization function but is independent of microtubule binding
(Kobielak et al., 2004;
Sahai and Marshall, 2002;
Carramusa et al., 2007).
DRF roles in morphogenesis remain largely unknown. Two challenges impede
progress. First, mammals have three DRFs that are at least partially
redundant. Mouse Dia1 mutant mice have hematopoiesis defects, but are
otherwise normal (Eisenmann et al.,
2007; Peng et al.,
2007). Human DIA1 mutations result in deafness
(Lynch et al., 1997), but
these mutations may be gain of function. Uncovering the full function of
mammalian DRFs will require multiple knockouts. The second, more difficult
challenge is their essential role in cytokinesis. Although flies and nematodes
have only a single DRF, its inactivation disrupts cytokinesis from the onset
of development (Afshar et al.,
2000; Swan et al.,
1998), preventing analysis of morphogenesis.
To address this challenge, we used loss and gain-of-function genetic tools available in Drosophila to study the function of Dia during morphogenesis, and to address its mechanisms of action. These studies suggest that Dia plays an important and unexpected role in coordinating actomyosin contractility and adhesion at AJs, and provide evidence that it acts upstream of Myosin as well as actin.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), modified by T.
Murphy for Gateway cloning with an N-terminal EGFP tag
(www.ciwemb.edu/labs/murphy/Gateway%20vectors.html).
The entire Dia ORF was cloned into this vector, with a short linker as follow;
M-EGFP-HRYTSLYKKAGSAAAPFT-dia (see Fig. S6 in the
supplementary material). Flies were generally grown at 25°C. Temperature
shifts of dia5M/Z mutants were done as follows. For fixed
embryos, embryos were collected for 8 hours at 18°C, aged at 18°C for
4 hours and then shifted to 25°C and aged until the desired stage. For
ventral furrow imaging embryos were collected for 8 hours at 18°C and
shifted to room temperature for filming. Females with dia mutant
germlines were generated by heat-shocking 48-72 hour
hsflp12; FRT40A dia/FRT40A
ovoD1 larvae for 3 hours at 37°C.
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).
Signal was detected by ECL (Amersham). Immunoblots were quantitated using
Image J; we quantitated at least two exposures in each case, with similar
results. RNA was prepared and analyzed as described by D'Avino et al.
(D'Avino et al., 1995). For
anti-myosin, embryos were heat-methanol fixed
(Müller and Wieschaus,
1996); otherwise embryos were prepared and imaged as described by
Fox and Peifer (Fox and Peifer,
2007). Antibodies, actin and RNA probes are in
Table 1. Adobe Photoshop 7.0
was used to adjust input levels to span entire output grayscale, and to adjust
brightness and contrast. When protein levels were compared, images were
equally adjusted. For fluorescence quantitation, images were acquired from
live embryos using a spinning-disc confocal (Perkin-Elmer CSU10), a 12-bit
camera, and Metamorph. Time-lapse imaging was as described previously
(Grevengoed et al., 2001).
Images were acquired every 15 seconds. Fluorescence-intensity measurements
were as described previously (Harris and
Peifer, 2007), using single planes; all fluorescent measurements
were conducted on images with a grayscale range of 200-700 (no saturated
pixels), using ImageJ.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and live
imaging of EGFP::Dia. UAS-EGFP::Dia driven by e22c-Gal4 can partially rescue
dia mutants to adult viability (data not shown), and is expressed at
levels roughly similar to those of endogenous Dia
(Fig. 1L), suggesting it
replicates endogenous Dia localization. During cellularization, Dia
colocalizes with actin and Myosin at tips of invaginating furrows
(Afshar et al., 2000;
Grosshans et al., 2005)
(Fig. 1A) but its subsequent
localization was unknown. We found that as cellularization ends Dia
re-localizes to the apicolateral domain
(Fig. 1B,C), overlapping AJs
(Fig. 1B), and remains apical
during gastrulation (Fig.
1D,E). During germband extension, when Myosin [visualized using
Sqh::GFP (Myosin regulatory light chain)] and AJs take on opposing
planar-polarized localizations, Dia is cortical but not notably
planar-polarized (Fig. 1F).
Both Dia (Fig. 1H,I) and
EGFP::Dia (Fig. 1M,N) remain
cortically enriched in epidermis and amnioserosa. Consistent with its role in
cytokinesis, Dia co-localizes with Myosin in invaginating cytokinetic furrows
(Fig. 1G, arrows), but does not
remain in midbodies (Fig. 1G,
arrowheads). The basal-to-apical re-localization of Dia suggests that it has
new functions during morphogenesis, and its overlap with AJs makes it a
candidate to regulate actin there.
Dynamic localization of Dia during dorsal closure suggests roles in cell shape change
As a model for morphogenesis, we used dorsal closure. Prior to this stage,
the amnioserosa, a squamous epithelium, covers the dorsal surface. During
closure amnioserosal cells apically constrict in a radially symmetric fashion,
while polarized shape changes cause epidermal cells to nearly double their D/V
length, enclosing the embryo. At their leading-edge, epidermal cells construct
a contractile actomyosin cable joined cell-cell by AJs. Amnioserosal cell
constriction, epidermal cell elongation and actomyosin cable constriction
combine to drive dorsal closure (Kiehart
et al., 2000). Although most epidermal cells elongate, one row of
cells per segment, segmental groove cells, do not elongate like their
neighbors, and thus form grooves around the embryo
(Larsen et al., 2003).
Dia has a dynamic localization during dorsal closure. In elongating
epidermal cells, Dia and EGFP::Dia are cortical
(Fig. 1I,J,N arrowheads) and
strongly enriched in leading-edge AJs (Fig.
1J,N arrows), where the actomyosin cable is anchored. In segmental
groove cells, EGFP::Dia is planar polarized, preferentially accumulating at
D/V borders (Fig. 1O1 - because
they form a groove these cells are basal to their neighbors, seen in
Fig. 1O2). Quantitation of
EGFP::Dia at D/V versus A/P borders revealed that this polarization is
statistically significant (Fig.
1P). Dia is not planar polarized in other epidermal cells
(Fig. 1O2,P). Amnioserosal
cells apically constrict during dorsal closure - most do so in concert, but a
subset ('drop-out' cells) constricts more rapidly than its neighbors
(Kiehart et al., 2000). Dia is
cortical in all amnioserosal cells (Fig.
1I,K,N), but particularly accumulates in drop-out cells, mirroring
Myosin (Fig. 1K arrows). Apical
accumulation of Dia in constricting amnioserosal cells and planar polarization
in groove cells suggests that it may regulate their cell shape changes during
dorsal closure.
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).
We first expressed DiaCA in amnioserosal cells. Their
constriction requires an actomyosin contractile ring (diagrammed in
Fig. 2I)
(Franke et al., 2005). Dia
accumulation in wild-type constricting amnioserosal cells
(Fig. 1K) suggests that Dia may
regulate constriction. We tested this by examining how Dia activation affects
amnioserosal cell behavior, using GAL4 drivers to induce
HA-tagged-DiaCA expression in the entire amnioserosa [c381-GAL4
(Table 1); e.g.
Fig. 2B,F], in large groups of
amnioserosal cells (seen in a small subset of c381-GAL4-driven embryos; e.g.
Fig. 2H,J), or in individual
cells in a mosaic pattern [engrailed(en)-GAL4
(Table 1); e.g.
Fig. 2C,D]. DiaCA is
cortically enriched (Fig.
2B,C), and thus positioned to modulate apical constriction.
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We next explored mechanisms by which DiaCA induces apical
constriction. Apical constriction is mediated by an actomyosin ring
(Fig. 2I)
(Franke et al., 2005). Dia
stimulates actin polymerization (Wallar
and Alberts, 2003). As expected, cortical F-actin is increased in
individual DiaCA-expressing amnioserosal cells
(Fig. 2D'', arrowheads),
and when DiaCA is expressed in all amnioserosal cells (compare
Fig. 2F with
2G). However, DiaCA
also had an unexpected effect on Myosin localization and levels. In wild-type,
Myosin accumulates cortically at low levels in most amnioserosal cells
(Fig. 2K) and at high levels in
drop-out cells undergoing rapid apical constriction
(Fig. 1K;
Fig. 2K, arrows).
DiaCA-expressing amnioserosal cells had increased cortical Myosin;
this was most obvious in embryos expressing DiaCA in many but not
all amnioserosal cells, providing an internal control
(Fig. 2J,J'), but was
also seen in individual DiaCA-expressing cells
(Fig. 2C, arrowheads). To
assess whether this involves more Myosin protein, we compared MHC levels by
immunoblotting. DiaCA expression using the ubiquitous epithelial
driver e22c-GAL4 (Table 1)
elevated total Myosin 
2.5-fold (Fig.
2L); as only half the embryos receive both driver and
UAS-DiaCA, this underestimates the magnitude of the effect. Thus,
DiaCA coordinately elevates both cortical actin and Myosin.
During apical constriction, actomyosin contractile rings are linked to AJs
(Fig. 2I)
(Dawes-Hoang et al., 2005).
AJs and actin have a reciprocal relationship; AJs organize and anchor
contractile rings and actin stabilizes AJs. We thus assessed whether
DiaCA affects AJs. Mosaic DiaCA expression in the
amnioserosa elevated cortical Arm levels
(Fig. 2H) and stabilized
-catenin, as assessed by immunoblotting
(Fig. 2L). This suggests that
DiaCA stabilizes AJs and underlying actomyosin rings.
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In epidermal cells DiaCA induces groove cell-like behavior without changing cell fate
We next examined planar-polarized elongating epidermal cells and segmental
groove cells, contrasting the effects of DiaCA to those in
radially-symmetric amnioserosal cells. As amnioserosal cells constrict, most
epidermal cells elongate in the D/V axis, while segmental groove cells
elongate less.
DiaCA expression in segmental stripes induced abnormally deep and persistent segmental grooves [compare Fig. 3A with 3B,C; expression was either in broad stripes in every other segment (paired(prd)-GAL4) or narrow stripes in every segment (en-GAL4)]. Wild-type segmental grooves disappear during dorsal closure (Fig. 3A) but in DiaCA-expressing embryos, grooves persist much longer and become very deep (Fig. 3B,C). Epidermal cells expressing DiaCA are wider in the A/P axis and shorter in the D/V axis than are non-expressing neighbors (Fig. 3E,F), thus forming a shorter belt of cells, explaining the deeper grooves. Interestingly, wild-type segmental groove cells have a similar shape (Fig. 3D), suggesting that DiaCA-expressing epidermal cells mimic groove cell behavior.
As DiaCA-expressing cells behave like groove cells, we tested whether DiaCA alters epidermal cell fate. Odd-skipped is a groove cell marker (S. Vincent and J. Axelrod, personal communication). Odd-skipped expression is not altered by DiaCA expression; it remains restricted to normal groove cells (a single cell row per segment flanking the DiaCA-expressing cells), and is not misexpressed in cells adopting groove-cell behavior (Fig. 3M). Thus, DiaCA affects cell shape without changing cell fate.
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). AJs
are slightly D/V planar-polarized in all epidermal cells, but this is
accentuated in groove cells (Fig.
3I; quantitated in
3O). Interestingly, wild-type
Dia is D/V planar-polarized in groove cells
(Fig. 1O,P;
Fig. 3I) but not strikingly
polarized in other epidermal cells, consistent with differential Dia function
in these two cell types. DiaCA accumulates cortically
(Fig. 3F''') and at late
stages is also D/V planar polarized (Fig.
3J). Epidermal DiaCA expression dramatically alters the cytoskeleton and AJs. DiaCA triggers elevated cortical F-actin, as expected (Fig. 3E,F). Strikingly, all DiaCA-expressing epidermal cells accumulate cortical Myosin at higher levels than their neighbors, and it is especially enriched at D/V cell borders (Fig. 3K, arrows; quantitation is shown in Fig. 3N). DiaCA also triggers accentuated AJ D/V planar-polarization (Fig. 3K,L; quantitation is shown in Fig. 3O). Thus, DiaCA cells take on both groove cell behavior and planar-polarity properties, with elevated actin and myosin accumulating at stabilized AJs at D/V cell borders.
Thus, DiaCA triggers actin and Myosin accumulation, and stabilizes AJs in both amnioserosa and epidermis. In radially symmetric amnioserosal cells, this triggers premature apical constriction. By contrast, in epidermal cells, actin and Myosin are selectively stabilized at D/V cell boundaries where AJ proteins are already enriched. Perhaps segmental groove cells normally activate Dia, thus regulating their cell shape.
The phenotype of DiaCA cannot be mimicked by increasing Myosin levels
The most surprising effect of DiaCA was its effect on Myosin
levels. We thus assessed mechanisms by which this occurs, and whether these
are necessary or sufficient to produce the DiaCA phenotype. We
tested two hypotheses: (1) Dia increases myosin transcription,
possibly through the transcription factor Serum Response Factor (SRF), a Dia
target (Tominaga et al.,
2000); or (2) Dia activates or stabilizes phosphorylated, active
Myosin at AJs.
We hypothesized that SRF might regulate Myosin levels. To test this, we
used hyperactive SRF (fused to the strong VP16 activation domain
(Guillemin et al., 1996). When
SRF-VP16 is expressed in segmental stripes (compare
Fig. 4A with
4C) or the amnioserosa (compare
Fig. 4E with
4G), it markedly elevates
Myosin, mimicking DiaCA (Fig.
4F). Interestingly, although both SRF-VP16 and DiaCA
increase Myosin protein, this does not occur at the transcriptional level (see
Fig. S1G,H in the supplementary material).
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We also tested whether myosin transcription is necessary for the
effects of DiaCA, by expressing DiaCA in MHC
zygotically-null mutants (zip; selected by absence of a GFP-tagged
Balancer chromosome). zip mutants are normal until late morphogenesis
(Franke et al., 2005), owing
to maternally contributed Myosin, but cannot upregulate myosin
transcription in response to DiaCA as they lack a functional
myosin gene. DiaCA expression in zip-null embryos
still generated cell shape changes in both epidermis (compare
Fig. 4J with
4I; deep grooves) and
amnioserosa (Fig. 4K; uniformly
rounded cells). Thus, the effects of DiaCA do not require elevated
myosin transcription.
DiaCA is partially mimicked by Myosin activation
We next explored whether Myosin activation mimicked the DiaCA
phenotype. We first tried constitutively active Rho Kinase
(Verdier et al., 2006a), but
this did not have obvious phenotypic effects (data not shown), perhaps because
expression levels were too low. We next induced Myosin activation using
constitutively active Myosin light chain kinase [MLCKCA
(Kim et al., 2002) derived
from chicken MLCK, allowing its specific detection]. MLCKCA
expression in the entire amnioserosa led to uniform amnisoserosal cell
rounding (Fig. 4H), similar to,
though weaker than, that caused by DiaCA
(Fig. 4F), and expression in
individual amnioserosal cells triggered premature apical constriction
(Fig. 5A,B, arrows).
Furthermore, MLCKCA expression in epidermal stripes triggered deep
segmental grooves (Fig. 4D),
mimicking DiaCA (Fig.
4B). Thus, MLCKCA and DiaCA have similar
morphological consequences, stimulating apical constriction, although the
effects of DiaCA are somewhat stronger.
We next examined whether MLCKCA and DiaCA act by similar mechanisms. Some effects were similar, whereas others were distinct. In the amnioserosa, both elevated Myosin (compare Fig. 4F,H with 4E; Fig. 5A) and actin (Fig. 5B), but levels and subcellular localization differed. In epidermal cells, MLCKCA and DiaCA had even more distinct effects. MLCKCA elevated cortical actin, but less so than DiaCA (compare Fig. 5F with Fig. 3E). MLCKCA also elevated cortical myosin, but this was not planar polarized (compare Fig. 5C,D with Fig. 3K), and the cell shape changes (Fig. 5E) were not as extreme as those induced by DiaCA (Fig. 3K) - MLCKCA-expressing cells may simply apically constrict rather than adopting groove-cell-like shapes. Thus, MLCKCA mimics the morphological phenotypes of DiaCA, but does not replicate its cell biological effects. This is consistent with a model in which Dia triggers Myosin activation at specific sites like AJs, so that global Myosin activation does not precisely mimic its effects.
Severely reducing Dia function disrupts morphogenesis
These data are consistent with the hypothesis that regulated Dia activity
helps coordinate adhesion with actomyosin assembly/activity at AJs. To test
this mechanistic hypothesis, we examined how reducing Dia function affects
morphogenesis. To circumvent difficulties caused by the key role of Dia in
cellularization, we sought to reduce but not eliminate Dia function. This was
facilitated by our discovery that dia5 is not a null
allele, as was thought (Afshar et al.,
2000), but produces severely reduced amounts of wild-type Dia, and
its phenotype is temperature sensitive (see Fig. S2 in the supplementary
material). This allowed us to use temperature shifts to reduce Dia function
after cellularization and explore its roles in morphogenesis. We crossed
females with dia5 homozygous germlines to males
heterozygous for the null allele dia2; half the progeny
are zygotically `rescued' (dia5M) and half are
dia5M/Z mutant (distinguishable by late extended germband
using GFP-marked Balancers). As dia5M/Z mutants retain
residual wild-type Dia, we can only draw conclusions from processes that are
defective - residual Dia may suffice for other events.
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Dia is required for AJ maintenance
These defects in morphogenesis in embryos with reduced Dia function are
consistent with several possible mechanisms of action. Our DiaCA
gain-of-function experiments suggested that Dia helps coordinate actomyosin
activity with adhesion at AJs. These data, along with previous work
implicating formins as AJ regulators (see Introduction) led us to test the
hypothesis that Dia regulates AJ assembly/maintenance.
We compared Arm and DE-cadherin (DE-cad) localization in wild-type (histone-GFP-marked), dia5M/Z and zygotically-rescued dia5M mutants (identified with a twist-GFP Balancer), all stained and imaged together to allow direct comparison of protein levels. By the end of gastrulation, wild-type AJs are evenly distributed around the apical cortex (Fig. 7A,B). AJs were normal in zygotically rescued dia5M mutants (Fig. 7C,D,I1,I2). In dia5M/Z, AJs formed (Fig. 7E,F) and cortical F-actin and Myosin were not grossly disrupted (see Fig. S3 in the supplementary material). Thus, this degree of reduction in Dia function does not prevent initial AJ assembly.
However, Dia is essential for AJ maintenance. In
dia5M/Z mutants, Arm (compare
Fig. 7E,F with
7A,B) and DE-cad (compare
Fig. 7J2,K with
7I2) cortical localization were
weaker and more discontinuous (apical AJ positioning was unaltered; compare
Fig. 7I2 with
7J2, insets). AJ maintenance
was abnormal in embryos with and without ventral furrow disruption (compare
Fig. 7G,H with
7E,F), so this is not a
secondary consequence of disrupted morphogenesis. To confirm the effect on
AJs, we examined DE-cad levels by immunoblotting. DE-cad was noticeably
reduced in dia5M/Z (hand-selected using a GFP-marked
Balancer; Fig. 7L; Arm was
slightly reduced and -catenin unaffected). AJ destabilization is
accompanied by Arm and DE-Cad accumulation in cytoplasmic puncta
(Fig. 7M,N, arrows). We next
stained dia5M/Z mutants for Rab5, which marks early
endosomes. Rab5 vesicles are normally largely basal to AJs
(Fig. 7O). In
dia5M/Z mutants, Rab5 vesicles accumulate at the apical
cortex, partially colocalizing with AJs
(Fig. 7P; most internal DE-cad
puncta are not Rab5 positive - they may be later endocytic intermediates).
Reduction in Dia and consequent reduction in apical actomyosin may expose AJs
to endocytic machinery - further work is needed to test this hypothesis.
Interestingly, AJ destabilization was accompanied by cortical blebbing on the
basolateral cortex just below AJs [Fig.
7R,S, arrows; Discs Large (Dlg) (a basolateral marker)]. Thus,
strong reduction of Dia function destabilizes AJs and increases
protrusiveness. These data are consistent with the model from our
gain-of-function experiments: that Dia promotes mutual stabilization of AJs
and junctional actomyosin.
dia and Rho1 exhibit very strong genetic interactions
These data suggest that Dia stabilizes AJs. Drosophila Rho1 plays
a similar role (Bloor and Kiehart,
2002; Magie et al.,
2002). As DRFs are regulated by Rho binding
(Watanabe et al., 1997), this
suggested that Rho may work, in part, through Dia.
To test this hypothesis, we examined whether Rho1 and dia
genetically interact. We found a very strong genetic interaction. Progeny of
heterozygous dia2 +/+ Rho1 females (subsequently
referred to as dia/Rho1 mutants for simplicity) grow up to be
infertile adults, as they lack pole cells, precursors of germ cells, they thus
resemble dia5M mutants (see Fig. S4H,I in the
supplementary material) (Afshar et al.,
2000). dia/Rho1 mutants also have partially penetrant
cellularization defects (see Fig. S4B,E in the supplementary material),
similar to but much less severe than those of dia5M
mutants at 25°C (see Fig. S4C,F in the supplementary material)
(Afshar et al., 2000). Thus,
reducing both Dia and Rho1 levels by only 50% leads to defects in development,
demonstrating that dia and Rho1 exhibit non-allelic
non-complementation. This is quite rare and suggests an intimate
relationship.
This allowed us to explore how Dia and Rho work together during
morphogenesis, by examining what events are compromised when Dia and Rho1
function are reduced. Embryonic cuticles provide an easy means to assess
morphogenesis. Zygotic dia2-null mutants are embryonic
viable (Castrillon and Wasserman,
1994), dying as late larvae. However, if we reduced maternal Rho1
by 50% in dia zygotic mutants (dia2 +/+
Rho1 females crossed to dia2/+males), nearly all
of dia zygotic mutants died as embryos (23% of total progeny), with
defects in head involution, ventral cuticle or the completion of dorsal
closure (Tables 2,
3). Zygotic Rho1
mutants are embryonic lethal with head defects and dorsal cuticle bowing
(Magie et al., 1999). Maternal
heterozygosity for dia substantially enhanced the phenotype of
Rho1 (Tables 2,
3), disrupting the ventral
cuticle. This may indicate problems during mesoderm invagination or epithelial
integrity, consistent with defects in dia5M mutants. Even
milder reductions in Rho1 and Dia affect viability; 13% of progeny of
dia2 +/+ Rho1 females die as embryos, largely
with defects in head involution (both single mutant heterozygotes are fully
viable; less than 7% lethality). These data suggest that Dia and Rho1 work
together in morphogenesis, with head involution, dorsal closure and ventral
epidermal integrity particularly sensitive to their reduced function.
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), with D/V shortening and A/P elongation of amnioserosal
cells driving tail retraction. This is facilitated by stable adhesion between
the two tissues across a well-defined tissue boundary
(Fig. 8A,C,I, arrows), in which
the amnioserosa adheres to and extends over the caudal germband, and extends
broad lamellipodia under it (Schock and
Perrimon, 2003), while anterior germband cells abut and adhere to
lateral amnioserosal cells. dia/Rho1 mutants exhibit striking differences from wild type. During germband retraction, amnioserosal cells all along the germband of dia/Rho1 embryos form long persistent cell extensions extending over neighboring epidermal cells (Fig. 8B-E). dia5M/Z mutants have similar protrusions (Fig. 8F), demonstrating that reduced Dia activity is key. Protrusions are long, thin and stable to harsh fixation (Fig. 8B,E), have Myosin enriched at their tips (Fig. 8E), and contain F-actin (Fig. 8D). Analysis of germband retraction live, using Moesin::GFP (Fig. 8H,I see Movies 3-5 in the supplementary material; Moesin::GFP binds F-actin) allowed us to examine cell behavior and visualize actin dynamics. This further highlighted altered cell behaviors in dia/Rho1 mutants, revealing protrusions from amnioserosal cells over the epidermis all along the germband. They were especially prominent caudally (Fig. 8H, yellow arrows), but were also observed laterally and anteriorly (Fig. 8H, red arrows). Amnioserosal cells also extended abnormal processes over one another (Fig. 8H, blue arrows), and we saw occasional long protrusions from epidermal cells (Fig. 8L). Alterations in protrusive behavior continued into dorsal closure. Amnioserosal cells normally send out fine filopodia during closure (Fig. 9A, arrows). In dia/Rho1 mutants these were longer and sometimes emerged from broad lamella (Fig. 9B, arrows). This was even more exaggerated in dia5M/Z mutants (Fig. 9C, arrows). dia/Rho1 amnioserosal cells also occasionally produced broad processes over one another (Fig. 9E) or over the epidermis (Fig. 9D). In dia/Rho1, dorsal closure was otherwise normal, with one exception: drop-out cells appeared much earlier in dorsal closure than in wild type (compare Fig. 9F with 9G). However, in dia5M/Z mutants, dorsal closure was more defective, with cell misalignment as the epidermal sheets met at the dorsal midline (Fig. 9H). Thus, amnioserosal cells are most sensitive to Dia and Rho1 reduction; it disrupts their stable tissue interface with the epidermis, triggering abnormal protrusiveness.
|
;
Hacker and Perrimon, 1998). We
also explored whether dia genetically interacts with
RhoGEF2. The progeny of dia +/+ RhoGEF2 females had
defective pole cells (see Fig. S4J in the supplementary material), albeit not
as defective as those of dia/Rho1, and also had abnormal protrusions
of amnioserosal cells over the adjacent epidermis
(Fig. 8G), similar to those of
dia/Rho1 embryos. This non-allelic non-complementation is consistent
with RhoGEF2 working together with Dia and Rho in morphogenesis.
Dia and Myosin regulation
To examine mechanisms by which Dia affects protrusiveness, we first
examined localization of β-integrin, which is key for germband retraction
(Schock and Perrimon, 2003).
However, we saw no obvious differences in β-integrin levels or
localization in dia/Rho1 mutants in amnioserosa or epidermis (compare
Fig. S5A with S5B in the supplementary material; data not shown).
Our gain- and loss-of-function analyses suggested that Dia coordinates adhesion at AJs with actomyosin contractility. We thus tested the hypothesis that abnormal amnioserosal protrusions occur because reduced Dia activity reduces Myosin activity at AJs, destabilizing them and increasing protrusiveness. We activated Myosin by expressing MLCKCA in both the germband and amnioserosa of dia/Rho1 mutants using e22c-GAL4, detecting misexpression with anti-MLCKCA. Interestingly, inducing Myosin phosphorylation rescued the abnormal protrusiveness (compare Fig. 8J with 8K). This is consistent with reduced Dia activity leading to reduced Myosin activity at AJs, destabilizing the normal tissue boundary and triggering protrusive behavior across it.
|
DISCUSSION |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Dia helps coordinate adhesion and actomyosin contractility during morphogenesis
Previous analysis of DRF function in morphogenesis was hampered by
mammalian DRF redundancy and the key role of DRFs in cytokinesis. We used the
genetic tools available in Drosophila to partially circumvent these
difficulties. We knew that Dia regulates cellularization
(Afshar et al., 2000;
Grosshans et al., 2005). Our
analysis revealed new roles during morphogenesis. In embryos with severely
reduced Dia function, gastrulation is disrupted. Apical constriction of
central furrow cells is delayed or blocked in a subset of cells, suggesting a
possible role for Dia in regulated apical constriction. Previous work
suggested that an unknown Rho effector regulates actin during this process
(Fox and Peifer, 2007);
perhaps this is Dia. Adjacent cells, which are stretched during invagination,
become massively multinucleate in dia5M mutants. This may
reflect the fact that the cells of the blastoderm undergo an incomplete form
of cytokinesis, remaining connected to the underlying yolk by actin-lined yolk
canals. These canals normally close at the onset of gastrulation in ventral
furrow cells; this may prevent cell membrane rupture initiated at the yolk
canal under stress. Dia may regulate yolk canal closure; it localizes there at
the end of cellularization (Afshar et al.,
2000; Grosshans et al.,
2005) and similar defects are seen in mutants lacking the septin
Peanut, a yolk canal component (Adam et
al., 2000). Alternately, Dia may stabilize cortical actomyosin or
its connections to AJs, with its absence weakening cortical integrity under
stress.
Dia also plays a key role during germband retraction. The most striking
effect of reduced Dia/Rho1 function is altered amnioserosal cell
protrusiveness. They normally make stable AJs and form a tight tissue boundary
with the epidermis; this is destabilized in dia/Rho1 and
dia5M/Z mutants. We also observed altered protrusive
activity in other contexts: dia5M/Z ectoderm exhibited
cortical blebbing, and dia/Rho1 and dia5M/Z
amnioserosal cells had altered protrusiveness during dorsal closure. Reduced
Dia function destabilized AJs, and the striking increase in cell
protrusiveness in dia/Rho1 mutants was substantially rescued by
increasing Myosin phosphorylation. These data support the hypothesis that Dia
normally helps restrict actomyosin contractility to AJs, and in its absence,
AJs are destabilized and myosin activity outside AJs stimulates protrusiveness
(Fig. 10A). Interestingly, in
cultured cells, fully assembled AJs inhibit Rac1 and lamellipodial activity
while increasing Rho and contractility (e.g.
Yamada and Nelson, 2007), and
myosin is required for mouse Dia1-induced AJ strengthening
(Carramusa et al., 2007).
|
;
Carramusa et al., 2007). There
are intimate connections between AJs and cortical actomyosin - AJs help
organize the apicolateral actin ring while cortical actin stabilizes AJs.
Interestingly, in cultured cells Myosin-mediated contraction of actin
filaments is essential for cell-cell contact expansion
(Shewan et al., 2005;
Yamada and Nelson, 2007), and
mouse Dia1 AJ stabilization requires myosin activity
(Carramusa et al., 2007),
providing further evidence that contractility, AJ stability and Dia activity
are mechanistically linked. The actomyosin network may also stabilize AJs by
preventing endocytosis. Perhaps Dia, like forming 1
(Kobielak et al., 2004),
directly binds AJs. Linear actin filaments promoted by Dia at AJs might
recruit -catenin, which then might inhibit Arp2/3-induced actin
branching (Drees et al.,
2005), facilitating formation of a belt of unbranched actin. Thus,
Dia is poised to act at the interface between AJs, cortical actin and
contractility.
The Yin-Yang relationship between adhesion and protrusiveness is a very
interesting one, underlying epithelial-mesenchymal transitions. Stable AJs
probably actively inhibit protrusiveness, and Dia may assist this, acting in a
reinforcing loop that concentrates actomyosin activity at AJs. There are
interesting parallels with the role of Dia in cytokinesis. Dia stabilizes
Myosin at the midzone. In its absence Myosin is delocalized around the cortex,
leading to abnormal protrusiveness (Dean
et al., 2005). Future work will reveal how Dia fits into the
regulatory network coordinating adhesion and cytoskeletal regulation.
A mechanistic model of roles of Dia in actin and Myosin regulation
From our data, we developed a mechanistic model for the regulation of
actin, Myosin and AJs by Dia during cell shape change
(Fig. 10A), in which Dia
activation stabilizes actin and active Myosin at AJs. In radially symmetric
amnioserosal cells, Dia promotes organization/activation of an apical
actomyosin network linked to AJs, inducing precocious apical cell
constriction. Activation of endogenous Dia may regulate normal amnioserosal
constriction: this now needs to be tested. Dia activation in cells where AJs
are planar polarized, such as epidermal cells, promotes actomyosin
organization preferentially at cell borders where AJs are enriched. This leads
to cell widening or helps cells resist elongation, generating groove-cell-like
morphology. Once again, Dia may be normally activated specifically in groove
cells to modulate their shape.
How does Dia activation activate Myosin? It does not occur primarily
through SRF, which triggers Myosin accumulation but not cell shape changes.
Myosin activation via MLCK partially mimicked DiaCA, suggesting
that Myosin activation is an important part of the process. However, MLCK did
not precisely mimic DiaCA, suggesting that Dia acts preferentially
at specific sites such as AJs rather than globally activating Myosin. The dual
effects on actin and Myosin suggests the speculative possibility that feedback
mechanisms exist to coordinate the Rho-regulated actin and Myosin pathways
(Fig. 10B). Recent work
revealed that active Dia1 can activate RhoA by binding the Rho-GEF LARG
(Kitzing et al., 2007), and we
saw strong genetic interactions between dia and the LARG relative
RhoGEF2, making this idea more plausible. Future work is needed to
test this hypothesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1005/DC1
|
ACKNOWLEDGMENTS |
|---|
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RESULTS
DISCUSSION
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