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First published online 3 January 2007
doi: 10.1242/dev.02748
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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 20 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fog, G-alpha 12, G-alpha 13, Abl, Cta, Neural tube, S2 cell, Adherens junction, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drosophila embryogenesis is an attractive model in which to study
morphogenesis. Many morphogenetic events have been characterized, which are
likely to share mechanisms with vertebrate morphogenetic events. Through
loss-of-function studies, mutants in which particular processes are affected
have been identified. Mesoderm invagination in the ventral furrow (VF) is one
such event (reviewed in Pilot and Lecuit,
2005
). During gastrulation, a subset of ventral mesodermal
precursor cells apically constrict in a highly coordinated fashion. This
creates an invagination - the VF - which internalizes these cells as a tube;
they then undergo an epithelial-mesenchymal transition. Internalization of VF
cells also brings together two rows of mesectodermal cells of the CNS
midline.
Genetic screens have identified two VF regulators that give insight into
how the process works: the ligand Folded gastrulation (Fog)
(Costa et al., 1994) and the
G-
-protein Concertina (Cta) (Parks
and Wieschaus, 1991). In either mutant, apical cell constriction
occurs, but is uncoordinated, disrupting tube formation
(Parks and Wieschaus, 1991;
Sweeton et al., 1991). The
mesodermal transcription factors Twist and Snail specify fog
expression in the VF (Costa et al.,
1994), and Fog acts upstream of Cta
(Morize et al., 1998). The
G-protein-coupled receptor for Fog remains unidentified.
Another VF regulator, the Rho1 activator RhoGEF2, provides a link between
Fog-Cta signaling and the cytoskeleton
(Hacker and Perrimon, 1998;
Barrett et al., 1997). RhoGEF2,
with a G-proteininteracting RGS domain, acts downstream of Cta in
Drosophila embryos and cultured S2 cells
(Rogers et al., 2004;
Barrett et al., 1997). RhoGEF2
activates Rho1 to direct non-muscle myosin II (myosin) accumulation. Cta, Rho1
and RhoGEF2 are sufficient for myosin accumulation and cell constriction in S2
cells (Rogers et al., 2004),
and loss of fog, cta or RhoGEF2 disrupts apical myosin
localization in the VF (Dawes-Hoang et al.,
2005; Nikolaidou and Barrett,
2004).
These data provide an attractive model, but loss-of-function analysis suggests that unidentified players exist. fog- and cta-null mutants have uncoordinated cell constriction, but internalize mesoderm, albeit abnormally. By contrast, in RhoGEF2-null mutants, constriction fails entirely and mesoderm remains on the surface. This suggests that RhoGEF2 regulates apical constriction by both Cta-dependent and -independent mechanisms, but the Cta-independent mechanism remains mysterious.
Here we examine the role of Abelson (Abl) kinase in morphogenesis. Abl is
unique among non-receptor tyrosine kinases, as it contains C-terminal
actin-binding domains. Mammalian Abl regulates actin dynamics in cultured
cells (reviewed in Woodring et al.,
2003). Loss of both Abl and Abelson-related-gene (Arg, also known
as Abl2 - Mouse Genome Informatics) during mouse development disrupts neural
tube closure (Koleske et al.,
1998). Neural tube closure mirrors VF formation, with apical cell
constriction creating a tubular invagination that internalizes neurectoderm.
Interestingly, Rho signaling also regulates this process
(Brouns et al., 2000).
Drosophila Abl regulates both axon guidance and epithelial
morphogenesis. In the CNS, Abl negatively regulates the actin modulator
Enabled (Ena) (Gertler et al.,
1995) by cooperating with other axon guidance regulators,
including the Rho-family GEF Trio (reviewed in
Lanier and Gertler, 2000).
Loss of Abl disrupts actin organization in several epithelia, including in
ovarian follicle cells (Baum and Perrimon,
2001) and in the embryonic epidermis during dorsal closure
(Grevengoed et al., 2001). We
examined the mechanism of action of Abl during the simpler stages of
blastoderm development; there, Abl negatively regulates apical Ena
localization in forming cells, thus regulating the location and type of actin
polymerization. In abl mutants, actin accumulates in apical
microvilli and is depleted from invaginating membrane furrows
(Grevengoed et al., 2003).
Here, we describe a novel role for Abl in morphogenesis - that Abl regulates apical constriction of the VF. Our data further suggest a significant revision of the apical cell constriction model, helping explain phenotypic differences between fog-cta and RhoGEF2.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fly stocks
Mutations were as described at
http://flybase.bio.indiana.edu.
FRTRhoGEF204291 was from U. Hacker (Lund University, Lund,
Sweden) and ctaRC10 from E. Wieschaus (Princeton
University, Princeton, NJ, USA). cta embryos were from
ctaRC10/Df(2L)C' females. Germline clones used were
as in Grevengoed et al. (Grevengoed et
al., 2001). cta;abl embryos were from
hs-FLP/+;ctaRC10/Df(2L)C;FRT79D-Fabl4,
moesin::GFP/FRT79D-FovoD mothers. All experiments were at
25°C. Live-imaging used moesin::GFP
(Edwards et al., 1997).
S2 cells
Cell culture was as in Rogers et al.
(Rogers et al., 2002); and
RNAi as in Clemens et al. (Clemens et al.,
2000). Double-stranded RNA-templates for in vitro transcription
were generated by PCR introducing T7 promoters upstream of: abl(-)
5'-ACTGCATCTCCAGTTCCAGC-3',
5'-ACTGCATCTCCAGTTCCAGC-3'; control [pBluescriptSK(-)]
5'-TAAATTGTAAGCGTTAATATTTTG-3',
5'-GAATTCGATATCAAGCTTATCGAT-3'. For ransient transfections the
Effectene kit (Qiagen) was used. Expression was driven by co-transfecting
metallothionein-Gal4. DNAs used were UAS-ctaR277HMyc,
UAS-rhoV14 (S. Rogers, UNC-CH, Chapel Hill, NC, USA), and
UAS-abl::GFP. The metallothionein promoter was induced with 500 µM
CuSO4 for 24 hours.
Immunofluorescence and immunoblotting
For fixation, the following were used: myosin, RhoGEF2, heat-methanol
(Muller and Wieschaus, 1996);
phalloidin/Ena 5 minutes, 37% formaldehyde; S2 cells, as in Rogers et al.
(Rogers et al., 2004); all
others, 20 minutes, 1:1 heptane:3.7% formaldehyde. Embryos were methanol- or
hand-devitellinized (for phalloidin), blocked/stained in PBS/1% goat
serum/0.1%TritonX-100. For antibodies/probes see
Table 1. Embryo
cross-sectioning was as in Dawes-Hoang et al.
(Dawes-Hoang et al., 2005).
Sample mounting was in Aqua-Polymount (Polysciences). For fixed sample
imaging, Zeiss LSM510 or Pascal confocal microscopes and LSM software was
used. When comparing the effects of Abl etc. on actin or myosin in S2 cells,
all transfected cells within regions of the slide were analyzed. The LSM
range-indicator feature was used on two control cells, and set such that the
levels of actin or myosin in the control cells were just below saturation.
Transfected cells were scored as increased if the range indicator said that
they were above saturation. For live imaging, Perkin-Elmer UltraVIEW
spinning-disc confocal, ORCA-ER digital camera, Metamorph software was used.
All images were acquired at 40x. Adobe Photoshop7.0 was used to adjust
brightness and contrast. When protein levels were compared, compared images
were equally adjusted. Immunoblotting was carried out as in Grevengoed et al.
(Grevengoed et al., 2001).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Using this mechanistic insight, we revisited the roles of Abl in
morphogenesis. Prior to morphogenesis, embryos that are maternally mutant for
abl4, a protein-null allele (see Fig. S1A in the
supplementary material) (Bennett and
Hoffmann, 1992), have cellularization defects. However, phenotypic
severity varies with temperature: at 18°C embryos have many multinucleate
cells, but at 25°C many embryos have few/no multinucleate cells
(Grevengoed et al., 2003).
We took advantage of this to follow abl mutants with weak
cellularization phenotypes into morphogenesis. abl mutants progress
through morphogenesis relatively normally until germband retraction and dorsal
closure, when obvious defects arise
(Grevengoed et al., 2001).
However, during this analysis, we uncovered a novel, fully penetrant phenotype
earlier in development, during mesoderm invagination.
Gastrulation involves two main types of cell-shape change: apical
constriction internalizes VF and posterior-midgut cells
(Sweeton et al., 1991), and
cell-cell intercalation extends the germband in the anterior-posterior axis
(Irvine and Wieschaus, 1994).
abl mutants showed no obvious germband extension defects
(Fig. 1A vs 1B). To confirm
that intercalation does not require Abl, we imaged live embryos expressing
Moesin::GFP and therefore highlighting F-Actin
(Edwards et al., 1997). The
cell-cell contact rearrangements driving germband extension
(Bertet et al., 2004) occurred
normally in abl mutants (Fig. 1C
vs 1D).
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).
VF formation is triggered by regional expression of the Twist transcription
factor, which specifies mesodermal fates and triggers morphogenesis. The first
stage in VF formation is the apical constriction of individual cells scattered
throughout the Twist-expressing domain
(Sweeton et al., 1991). In
wild-type fly embryos, central mesodermal precursors transition quickly from
uncoordinated to coordinated constriction as the VF forms. We analyzed
wild-type VF formation in detail in both fixed and living embryos. Central
cells (Fig. 1E, arrow) apically
constricted to a wedge shape (Fig.
1K, arrow), while more lateral VF cells
(Fig. 1G, arrow) did not
constrict but adopted an asymmetrical morphology, positioning their apices
towards the invagination (Leptin and
Grunewald, 1990) (Fig.
1K, arrowhead). Most remarkable were the outermost VF cells, which
extended their apices far over neighboring cells towards the center of the VF
(Fig. 1I, arrow). The
difference in morphology between central constricting cells and more lateral
cells probably reflects the lack of fog expression in lateral
mesoderm (Costa et al.,
1994).
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We first tested whether Abl is required for mesoderm specification, by examining expression of the mesodermal marker Twist. We observed no change in Twist expression in abl mutants (Fig. 1M vs 1N), and flanking mesectodermal cells continued to express Single-minded (a mesodermal marker; Fig. 1O' vs 1P'), suggesting that cell fates were not altered.
We next examined VF formation live, examining how individual cell behaviors
drive the overall process. This also allowed us to address a possible caveat
not ruled out in our fixed embryo analysis: that non-constricting cells in
abl mutants could be multinucleate cells from earlier blastoderm
defects. To ensure that multinucleate cells do not cause the
abl-mutant defects, we only imaged embryos with few/no multinucleate
cells (Fig. 1R, t=0
min) (Grevengoed et al.,
2003). Wild-type mesodermal cells constricted uniformly and
internalized synchronously. The difference between central and lateral cells
of the furrow was evident; central cells uniformly constricted during
internalization (Fig. 1Q,
arrow, t=10 minutes; also see Movie 1 in the supplementary material);
whereas lateral cells did not constrict, but elongated their apical ends
towards the furrow during internalization
(Fig. 1Q, arrow, t=15
minutes). By contrast, cell constriction in abl mutants was
uncoordinated, and some cells never constricted. Defects arose when cell-shape
changes initiated in the central, constricting cells (compare insets in
Fig. 1Q vs 1R, t=5 and
10 minutes, respectively; also see Movie 2 in the supplementary material).
When groups of neighboring cells constricted
(Fig. 1R, t=10
minutes, arrows), they often appeared to pull non-constricting neighbors
towards them (Fig. 1R,
t=10 minutes, arrowhead), carrying them inside the embryo. Some
unconstricted cells persisted on the surface after most cells internalized
(Fig. 1R, t=15, 20 and
25 minutes, arrow). These cells may contribute to midline cell-shape
irregularities in abl mutants
(Fig. 1O vs 1P). However, most
abl mutants eventually internalized all mesoderm, allowing the two
rows of Single-minded-expressing midline cells to join
(Fig. 1O' vs 1P').
Despite defects in coordinated constriction, furrow formation in abl
mutants occurred roughly as quickly as in wild type
(Fig. 1Q vs 1R; also see Movies
1 vs 2 in the supplementary material).
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).
Although these actin patches were common in abl mutants, they did not
correlate with sites of failed constriction. We describe the contribution of
actin localization to the VF phenotype in abl mutants in detail
below. Overall, these results suggest that, during gastrulation, Abl regulates
apical constriction in the VF but is dispensable for germband extension.
Abl localizes and is activated apically in embryonic epithelia
To better understand the mechanism of action of Abl during apical
constriction, we investigated Abl localization. We took two approaches to
examine Abl localization and to test the hypothesis that Abl is active
apically. First, we added a C-terminal GFP-tag to Abl, joined by a linker
peptide (Fig. 2A), as had been
demonstrated to work with mammalian Arg::YFP
(Wang et al., 2001). We cloned
Abl::GFP downstream of 2 kb of the abl 5' flanking sequence
previously used in a rescuing transgene
(Henkemeyer et al., 1987).
Abl::GFP rescued abl mutants to viability and fertility (see Fig. S1B
in the supplementary material), suggesting that it replicates endogenous Abl
expression and localization. Second, we used a Phospho-specific antibody that
recognizes a tyrosine in the activation loop that is phosphorylated during Abl
activation. This tyrosine is conserved in Drosophila Abl, and the
antibody recognizes active Drosophila Abl (T. Stevens, D.F. and M.P.,
unpublished data). Both reagents revealed a pool of Abl at the apical cortex
of epithelial cells. We began analysis during the blastoderm stages, where we
hypothesized Abl to act apically. Indeed, both Abl::GFP and phospho-Abl
localized apically (Fig.
2C,D,F-H, arrows) and cortically
(Fig. 2B,E). At
mid-cellularization, some Abl also localized to basal furrow canals
(Fig. 2G, arrowhead). Apical
Abl overlapped adherens junctions (AJs), colocalizing with DE-Cadherin
(Fig. 2H-H''), but also
extended more apically, where other actin-associated proteins reside
(Harris and Peifer, 2005).
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Apical myosin fails to uniformly contract in abl mutants
In embryos and S2 cells, RhoGEF2 promotes myosin organization into an
apical contractile ring in constricting cells
(Nikolaidou and Barrett, 2004;
Rogers et al., 2004). To
explore how Abl regulates apical constriction, we compared myosin and RhoGEF2
localization in wild-type and abl-mutant VFs. Myosin and RhoGEF2
largely colocalized in wild-type embryos. During cellularization, myosin and
RhoGEF2 localized basally in furrow canals
(Fig. 3A,I, arrows), as
previously described (Dawes-Hoang et al.,
2005; Grosshans et al.,
2005; Padash Barmchi et al.,
2005). Following cellularization, most cells retained myosin in
basal yolk canals (remnants of cellularization contractile rings). However,
mesodermal precursors strikingly relocalize myosin apically at, or immediately
prior to, constriction (Nikolaidou and
Barrett, 2004) (Fig.
3C,E,G, arrows). We also examined active myosin, using an antibody
against the conserved myosin-lightchain Ser-19 phosphorylation site.
Constricting cells exhibited elevated apical phosphorylated myosin
(myosin-P) (Fig. 3Q,
arrow). Unlike myosin, RhoGEF2 disappeared from basal furrow canals at the
onset of gastrulation (Fig. 3J;
this transition occurred slightly earlier in the mesoderm,
Fig. 3I, arrowhead), exhibiting
diffuse apical localization in all cells. Next, similar to myosin, RhoGEF2
accumulates at apical AJs of mesodermal precursors
(Grosshans et al., 2005)
(Fig. 3K, arrow). Apical
RhoGEF2 was seen in cells before constriction
(Fig. 3K), suggesting that, as
with myosin (Nikolaidou and Barrett,
2004), apical RhoGEF2 precedes constriction. It was much easier to
identify embryos with apical RhoGEF2 than apical myosin prior to constriction,
suggesting that RhoGEF2 may precede myosin at AJs. RhoGEF2 localization during
mesoderm internalization parallels myosin and Abl, concentrating at sites of
apical constriction (Fig. 3M,
arrow, O). Many features of myosin and RhoGEF2 localization are normal in
abl mutants, including basal myosin in furrow canals
(Fig. 3B, arrow), and apical
myosin and RhoGEF2 relocalization in mesodermal precursors
(Fig. 3D,L, arrows). In
cross-sections, constricting cells of abl mutants exhibited normal
myosin and RhoGEF2 localization (Fig.
3F,N, arrows), and normal myosin-P levels
(Fig. 3Q vs 3R, arrows). By
contrast, however, in unconstricted cells of abl mutants, although
the constriction machinery localized apically, it did not assemble into an
effective contractile ring. In grazing sections, myosin and RhoGEF2
concentrated apically in constricting cells
(Fig. 3H,P, arrows) but were
diffuse and discontinuous in unconstricted cells
(Fig. 3H,P, arrowheads); we saw
similar results in cross-section (Fig.
3F,N, arrowheads). This abl phenotype contrasts with
embryos lacking AJs, in which actomyosin rings constrict without constricting
cells (Dawes-Hoang et al.,
2005). Taken together, our results suggest that, in abl
mutants, constriction machinery localizes apically but fails to constrict in
some cells.
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;
Sweeton et al., 1991). To
understand how Abl function relates to other VF regulators, we examined the
cta-mutant phenotype in detail, revealing striking similarities
between abl and cta, and new details about cta VF
defects. cta mutants closely resemble abl mutants in VF cell
morphology and phenotypic severity. In cta mutants, constricted cells
appear next to unconstricted cells (Parks
and Wieschaus, 1991) (Fig.
4A,C, arrows vs arrowheads), and mesodermal cells internalized at
varying angles (Fig. 4B) and
exhibited aberrant morphology after internalization
(Fig. 4D, arrow).
We next examined the localization of the apical constriction machinery in
cta mutants. Nikolaidou and Barrett
(Nikolaidou and Barrett, 2004)
reported lower levels of apical myosin in constricting cells in cta
mutants than in wild type. However, when we compared wild-type and mutant
embryos stained and imaged together, we saw no decrease in apical myosin in
constricting cells (Fig. 4G vs
4H, arrows). We did, however, find sections with little or no
apical myosin, which represent sections through few or no constricting cells.
Myosin failed to assemble into continuous contractile structures in
unconstricted cells of cta mutants
(Fig. 4E), as in abl
mutants. RhoGEF2 localization was non-uniform in the VF, concentrating where
cells successfully constricted (Fig.
4F, arrows in 4I vs 4J), as in abl mutants, consistent
with the idea that defects in RhoGEF2 localization in cta mutants
disrupt myosin activation. Thus, both abl and cta mutants
have unconstricted cells where RhoGEF2 and myosin fail to assemble a
contractile network.
In S2 cells Abl regulates actin while Cta regulates myosin
Our data suggest that both Abl and Cta promote actomyosin-based contraction
during VF formation. To better understand the mechanisms by which they
function, we used a single-cell assay for actin and myosin organization during
cell constriction in Drosophila S2 cells. Overexpression of
RhoGEF2-pathway components, including activated Cta or Rho, promotes myosin
reorganization in S2 cells (Rogers et al.,
2004). Constitutively active RhoV14 also promotes actin
organization (S. Rogers, personal communication). We thus examined both myosin
and actin localization in S2 cells in response to Abl (using UAS-Abl::GFP,
Fig. 5A), Cta and Rho
overexpression. We also used RNAi loss-of-function to test whether Abl can
function in the RhoGEF2 pathway.
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)
(Fig. 5D'', arrow, 30/50
cells). By contrast, these cells did not exhibit consistent changes in actin
organization, either in the cell periphery, as seen for Abl, or in a ring-like
structure, as described below for Rho (Fig.
5D', absent in 43/50 cells). Thus, in S2 cells Abl and Cta
promote actin and myosin organization, respectively.
Overexpression of RhoV14 altered both actin
(Fig. 5E', 37/50 cells)
and myosin-P localization (Fig.
5E'', 49/50 cells). In contrast to UAS-Abl cells, actin in
RhoV14 cells localized to a ring above the cell center that
colocalizes with myosin (arrows in Fig.
5E'-E'''). Myosin localization in RhoV14
cells differed qualitatively from most CtaR277H cells, forming a
tighter ring (Fig. 5E'' vs
5D''), as was previously observed
(Rogers et al., 2004). Thus,
Rho overexpression combines elements of both Abl and Cta overexpression,
concentrating both actin and myosin-P into a ring-like contractile
structure.
We next asked whether Abl is required for the gain-of-function phenotype of
Rho by performing abl RNAi in RhoV14 S2 cells. Loss of Abl
had no obvious effects on F-actin in wild-type S2 cells (data not shown)
(Rogers et al., 2003). Despite
efficient Abl knockdown (Fig.
5I), the RhoV14 phenotype was unaffected by
abl RNAi (arrows in Fig. 5G'
vs 5H'). This suggests that Abl does not act downstream of
Rho signaling in S2 cells.
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One key Abl target in other contexts is the actin regulator Ena. We next examined whether Ena activity is regulated during VF formation and whether this is crucial for apical constriction. Prior to apical constriction, Ena localized diffusely (Fig. 6A', arrowhead), but, during VF formation, it exhibited a striking difference in localization between mesoderm and non-mesoderm, concentrating at apical AJs in non-mesodermal cells (Fig. 6D, arrow). However, Ena remained noticeably absent in VF cells as apical constriction initiates (Fig. 6D, arrowhead), suggesting that it is downregulated in these cells. Thus, actin and Ena show opposite changes in localization in VFs.
Abl can regulate both Ena and actin, and we thus tested the hypothesis that Abl regulates these changes in actin and Ena localization. In cellularizing abl mutants, actin and Ena accumulated ectopically along the apicolateral membrane (Fig. 6E,E', arrowheads; Fig. 1R, t=0 minutes, arrow). During VF formation, actin reorganization in the mesoderm failed in abl mutants. In wild type, actin was tightly focused to the apex of constricting cells (Fig. 6C, yellow arrowhead), while it localized to a broader band in non-mesodermal cells (white arrowhead). By contrast, in abl mutants, actin was found all along the apicolateral membrane of mesodermal cells (Fig. 6F,G, yellow arrowheads). However, cells that successfully constricted accumulated elevated apical actin (Fig. 6F, red arrowhead).
The normal downregulation of cortical Ena observed in wild-type mesoderm did not occur in abl mutants. Instead, Ena accumulated at high levels at the apicolateral cortex of mesodermal cells (Fig. 6H, arrowhead; ectopic Ena also accumulated apicolaterally in non-mesodermal cells). Thus, Abl is essential for the downregulation of Ena in VF cells, and this appears essential for the correct organization of apical actin in constricting cells.
To test the hypothesis that Ena-deregulation underlies actin and apical constriction defects in abl mutants, we genetically reduced Ena levels in abl mutants and asked if this rescued the VF phenotype of abl mutants. In embryos from maternally ena210/+; abl females, the VF phenotype was largely suppressed (normal VF in 14/16 examined; Fig. 6S vs 6T). Together, these results suggest that Abl-dependent Ena-downregulation in VF cells is essential for ordered apical actin assembly and coordinated cell constriction.
RhoGEF2, but not Cta, regulates ventral furrow actin localization
These results suggest that, in addition to myosin regulation, organization
of the actin network is a key mechanistic input into ordered apical
constriction. We next asked whether other VF regulators also direct actin
localization, examining actin and Ena localization in cta and
RhoGEF2 mutants. As actin localization defects arise prior to
gastrulation in abl mutants
(Grevengoed et al., 2003), we
examined both cellularization and gastrulation. cta mutants showed no
obvious actin localization defects during either cellularization
(Fig. 6I) or gastrulation
(Fig. 6J,K), paralleling our
results in S2 cells, where Cta activation did not effect actin localization
(Fig. 5). Furthermore, Ena
downregulation occured normally in cta mutants
(Fig. 6I',L, arrowhead vs
arrow). Thus, although abl and cta mutants have similar VF
phenotypes, they appear to regulate this process via distinct mechanisms.
In contrast to cta mutants, RhoGEF2 mutants display actin
mislocalization very similar to that of abl mutants. During
cellularization, actin accumulates ectopically in apicolateral regions
(Padash Barmchi et al., 2005)
(Fig. 6M, arrowhead), as in
abl mutants. This actin localization defect persisted in all cells
during VF formation (Fig.
6N,O), similar to abl. Excess actin accumulated along
apicolateral membranes (Fig.
6N,O, yellow arrowhead), resulting in a less-focused actin network
(Fig. 6O). However, Ena did not
accumulate apically during either cellularization
(Fig. 6M') or VF
formation (Fig. 6P), thus
differing from abl (in later RhoGEF2 mutants, actin and Ena
did co-localize outside the mesoderm to a distinct apical domain;
Fig. 6Q-R'', arrows).
Thus, both Abl and RhoGEF2 are essential for proper apical actin organization
prior to and during VF formation. Whereas Abl acts via Ena, RhoGEF2 appears to
act via a distinct mechanism.
Abl and Cta work in parallel to promote ventral furrow formation
Our results suggest that the assembly of an apically polarized actin
network is a key mechanistic input into apical constriction. Further, the
normal actin localization in cta mutants suggests that actin
organization is independent of Fog-Cta signaling. This led us to ask whether
the role of RhoGEF2 in actin organization could account for the difference in
VF phenotypes between cta and RhoGEF2 mutants. To examine
this mechanistic model, we disrupted both actin organization and Fog-Cta
signaling, the two cues lacking in RhoGEF2 mutants, by generating
cta;abl double mutants. We compared their phenotype to
RhoGEF2 mutants. In RhoGEF2 mutants, the two lateral rows of
midline cells fail to join (Fig. 7A vs
7D, arrows), due to failure of mesoderm internalization
(Fig. 7D, arrowhead). This was
not seen in either cta or abl single mutants
(Fig. 7B,C). However,
cta;abl mutants phenocopy RhoGEF2 mutants. In double
mutants, mesoderm remains on the surface
(Fig. 7E,F, arrowheads) and
midline cells do not join, a phenotype that was never observed in either
single mutant (Fig. 7E,F
arrows). Thus, both apical actin organization and Fog-Cta signaling appear to
be key mechanistic inputs into mesoderm internalization.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A mechanistic model of apical cell constriction
Previous work established myosin as a key output of RhoGEF2 signaling
during mesoderm internalization
(Dawes-Hoang et al., 2005;
Nikolaidou and Barrett, 2004;
Rogers et al., 2004). However,
ambiguities remained regarding the circuitry of this pathway, as the
RhoGEF2 phenotype is much more severe than that of cta or
fog mutants, suggesting that a simple linear pathway is unlikely. Our
data suggest that RhoGEF2 plays dual roles in actin and myosin regulation, and
thus its inactivation has more severe effects.
From our data, we developed a mechanistic model for the regulation of
apical constriction during VF formation
(Fig. 8). The regulation of
actin localization by Abl and RhoGEF2 promotes organization of the apical
actin network in constricting cells. We suggest that Abl regulates actin by
actively downregulating cortical Ena in mesoderm, thus leading to polarized
actin accumulation, similar to the role that it was shown to play in follicle
cells (Baum and Perrimon,
2001). RhoGEF2 plays a distinct, Cta-independent role in the
effective assembly of organized apical actin. While RhoGEF2 and Abl are
modulating actin assembly, the mesodermal transcription machinery activates
Fog-Cta signaling (Costa et al.,
1994), apically stabilizing RhoGEF2. This allows the efficient
activation of apical myosin. Coupling of these two cues - an organized apical
actin ring at AJs and stable apical myosin activation - cooperate to ensure
highly coordinated actomyosin constriction throughout the sheet of mesodermal
cells in a short timeframe.
This model helps explain the mutant phenotypes observed in this and
previous studies. In abl mutants, Fog-Cta allow RhoGEF2 stabilization
and myosin contraction, but the lack of organized mesodermal actin in these
mutants (Fig. 6F), which
results from inappropriate Ena regulation
(Fig. 6H,T), prevents the
uniform assembly of actin-based contractile rings. cta mutants lack a
stabilizing signal for RhoGEF2, preventing uniform apical myosin activation
(Dawes-Hoang et al., 2005;
Nikolaidou and Barrett, 2004)
and uniform constriction. However, some cells can constrict without Fog-Cta
(Sweeton et al., 1991),
accumulating apical myosin levels comparable to those in wild type
(Fig. 4H). In RhoGEF2
mutants, the combined failure to stabilize/activate myosin
(Dawes-Hoang et al., 2005;
Nikolaidou and Barrett, 2004)
and a lack of organized apical actin (Fig.
6N) severely compromises apical constriction. The similarity
between RhoGEF2 and cta;abl mutants supports this model, as
both processes should be compromised.
Actin regulation during mesoderm internalization
Our model suggests that organized apical actin is an essential prerequisite
for cell constriction. Although both Abl and RhoGEF2 regulate actin
localization, our data argue that each acts independently. First, actin
defects arise during cellularization, when Abl and RhoGEF2 have
non-overlapping localizations (Fig.
2H vs Fig. 3I).
Second, whereas Abl clearly acts through Ena, loss of RhoGEF2 disrupts actin
without altering Ena localization (Fig.
6M'). Finally, Abl is not a Rho effector in S2 cells
(Fig. 5H).
Several unanswered questions remain. With respect to abl, a major
question is why do some cells apically constrict while others fail? This
phenotype resembles the cellularization defects of abl mutants
(Grevengoed et al., 2003), in
which only some cells fail to reorganize actin into furrows. However, all
cells exhibit excess apical Ena and thus form abnormally long, apical
microvilli. Perhaps, in some cells, furrow actin assembly drops below a
crucial threshold and furrows fail. In the VF, the absence of Abl may have
similar effects. VF defects could result from both competition for cellular
actin and recruitment of other regulators (e.g. the formin Diaphanous) to
ectopic locations, preventing their action in VF formation. This may reduce
actin assembly into contractile rings. When constriction initiates, stochastic
variations in ring strength may lead some rings to fail, leading to
unconstricted cells. Future work is needed to identify the full set of actin
regulators involved, and to assess how they work. Interestingly, recent work
implicates Abl in epithelial-mesenchymal transitions
(Yang et al., 2006). Whereas
Abl disrupts VF formation, Twist is normally localized in abl mutants
(Fig. 1M,N), suggesting that
this major regulator of such transitions is not an Abl target in flies.
Our data also reveal the importance of mesodermal Ena downregulation. This may result from increased mesodermal Abl activity, suggested by elevated levels of mesodermal Abl relative to non-mesoderm; however, this remains to be tested. We also need to identify the mechanism by which Abl regulates Ena. In some places, such as the syncytial blastoderm, Abl localizes to sites where Ena is normally absent and, in the absence of Abl, ectopic Ena is found at these sites. This suggests that Abl actively antagonizes Ena localization. At other times and regions, however, such as the leading-edge during dorsal closure, Abl co-localizes with Ena, and thus may hold it in an inactive state. In VFs, Abl localizes to the apical-lateral cortex, and Ena localizes to this site in its absence. Further studies of Abl action will be needed to clarify the mechanisms by which it downregulates Ena.
Interestingly, manipulating mammalian Ena/VASP can affect cell
contractility (Galler et al.,
2006; Hoffman et al.,
2006). Thus, Ena-downregulation may permit proper VF cell
contractility. Testing this hypothesis will be important.
Our results also raise questions regarding RhoGEF2. Our model suggests that
RhoGEF2 acts via two mechanisms, only one of which is Cta-dependent. Perhaps
another upstream cue acts on RhoGEF2 to promote actin organization. Because
RhoGEF2 mutants have actin-organization defects in all cells, this
regulator may act in all cells prior to gastrulation. However, our data do not
rule out a second mesoderm-specific RhoGEF2 regulator acting in parallel to
Cta (see Note added in proof). Although Rho-Kinase is a potential Rho effector
with respect to myosin (Dawes-Hoang et al.,
2005; Rogers et al.,
2004), another effector may regulate actin organization.
Attractive candidates are the Formins, which reorganize actin in many
processes (Faix and Grosse,
2006).
Abl is required for specific cell-shape changes
Our data strengthen the idea that different cytoskeletal regulators direct
distinct morphogenetic processes. Both Abl (our data) and Fog
(Sweeton et al., 1991;
Bertet et al., 2004) regulate
mesodermal apical constriction but are dispensable for germband cell-cell
intercalation. Thus, although both processes require dynamic myosin
reorganization (Nikolaidou and Barrett,
2004; Zallen and Wieschaus,
2004), distinct regulators act in each.
The picture becomes more complex when considering other roles of Fog, Cta
and RhoGEF2. All are required for internalization of the posterior midgut and
salivary glands (Nikolaidou and Barrett,
2004; Barrett et al.,
1997; Sweeton et al.,
1991), but these cells internalize in abl mutants (data
not shown). Thus, different types of apical constriction may be regulated
differently. It will be interesting to explore the roles of Fog, Cta and
RhoGEF2 during dorsal closure, which requires Abl.
Conserved roles for Abl and Rho during apical constriction
Our work supports mechanistic connections between VF formation and neural
tube closure. Both involve actin-based apical constriction to internalize a
sheet of cells into a tube. Mice lacking Abl and Arg kinases have neural tube
defects, and actin organization in neuroepithelial cells appears altered
(Koleske et al., 1998);
interestingly, these cells have ectopic actin that is less polarized than
normal, similar to what we observed in abl-mutant VFs. Furthermore,
double-mutant analysis suggests that mammalian Ena plays a role in neural tube
closure in conjunction with Profilin
(Lanier et al., 1999). Thus,
Abl-Ena signaling may represent a conserved mechanism of actin regulation
during apical constriction. Our mechanistic insights can now be pursued in
mammals.
Rho also regulates neural tube closure. Mice lacking p190RhoGAP have neural
tube defects (Brouns et al.,
2000). Interestingly, p190RhoGAP is an Arg substrate in the brain
(Hernandez et al., 2004),
suggesting possible direct links between Abl and Rho in apical constriction.
The role of Drosophila p190RhoGAP in the VF has yet to be examined,
but RhoGAP68F is implicated in VF formation
(Sanny et al., 2006). Future
work in both flies and mice will provide further mechanistic insights into
conserved mechanisms of apical cell constriction.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/3/567/DC1
Note added in proof
We highlighted the potential for a second mesoderm-specific RhoGEF2
regulator that acts in parallel to Cta in this paper. Since submission of this
manuscript, Kölsch et al.
(Kölsch et al., 2007)
have identified such a regulator - a transmembrane protein that can bind
RhoGEF2.
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