1 Department of Molecular Biology and Biochemistry, Simon Fraser University,
8888 University Drive, Burnaby, BC, V5A 1S6, Canada
2 Drosophila Neurobiology, Institute of Molecular and Cell Biology, 30 Medical
Drive, Singapore 117609, Republic of Singapore
3 Glaxo-IMCB Laboratories, Institute of Molecular and Cell Biology, 30 Medical
Drive, Singapore 117609, Republic of Singapore
4 Department of Neurochemistry, Institute of Neurology, 1 Wakefield St., London
WC1 1PJ, UK
Present address: MRC Centre for Developmental Neurobiology, King's College
London SE1 1UL, UK
* Author for correspondence (e-mail: nharden{at}sfu.ca )
Accepted 21 February 2002
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Summary |
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Key words: Drosophila, Rac, Small GTPase, Crumbs, Amnioserosa, Dorsal Closure, Cytoskeleton, Morphogenesis
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Introduction |
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Numerous genes have been identified that are required for correct dorsal
closure, and these can be grouped into several categories including small
GTPases (p21s), members of a Jun-amino-terminal kinase (JNK) cascade, members
of a Decapentaplegic (Dpp) pathway, members of the Wingless pathway and
various proteins associated with the cytoskeleton and/or cell junctions
(Noselli and Agnes, 1999;
McEwen et al., 2000
).
Disruption of the function of these genes leads to defects in dorsal closure
that are frequently accompanied by failures of cell shape change in the
epidermis and abnormalities in the distribution of F-actin and myosin at the
leading edge. These results strongly support the idea that the leading edge
cytoskeleton is driving dorsal closure through effecting cell shape change in
the epidermis. Studies in mammalian cells and model organisms have
demonstrated the involvement of the Rho subfamily of Ras-related small GTPases
in regulation of the actin cytoskeleton and cell shape, and we and others have
characterized the involvement of Ras1 and three Rho subfamily members, Drac1,
Dcdc42 and RhoA (Rho1), in dorsal closure
(Harden et al., 1995
;
Harden et al., 1996
;
Harden et al., 1999
;
Magie et al., 1999
;
Ricos et al., 1999
;
Riesgo-Escovar et al., 1996
;
Strutt et al., 1997
).
Expression of dominant-negative versions of these p21s (and evaluation of
loss-of-function Rho1 alleles) during embryogenesis demonstrates that only
dominant-negative Drac1 is capable of preventing the accumulation of the
leading edge actomyosin contractile apparatus. The other small GTPases have
roles in the epidermis during dorsal closure, but impairment of their function
does not completely disrupt the leading edge cytoskeleton.
We had previously seen a reduction in peripheral F-actin and spectrin
staining in amnioserosa cells following heat shock induction of a
dominant-negative Drac1 transgene, Drac1N17, and we wondered
if Drac1 might be regulating the cytoskeleton and morphology of the
amnioserosa during dorsal closure (Harden
et al., 1995). In this study, we address small GTPase function in
the amnioserosa further by using the GAL4-UAS system
(Brand and Perrimon, 1993
) to
drive expression of constitutively active and dominant-negative transgenes
during dorsal closure with various GAL4 drivers. We show that, of the p21s
tested, only Drac1 appears to be required for morphogenesis of the
amnioserosa. A constitutively active version of Drac1 causes excessive
contraction of the amnioserosa, whereas a dominant-negative version of Drac1
retards amnioserosa morphogenesis. We present evidence that Crumbs (Crb), a
determinant of apical-basal polarity
(Wodarz et al., 1995
), is a
component of Drac1-mediated amnioserosa morphogenesis.
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Materials and Methods |
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Heat shock expression of Drac1V12
Embryos were collected and aged at 25°C until 8 to 12 hours after egg
laying (AEL), placed in vials and heat-shocked for 10 minutes in a water bath
set at 37°C. Following heat-shock, embryos were either aged for 7 hours at
21°C and fixed for immunohistochemistry or aged at 21°C for at least
48 hours and subjected to cuticle preparation.
Immunohistochemistry
Fixed embryos were staged independently of the degree of dorsal closure
using the extent of head involution and/or central nervous system development
instead (Campos-Ortega and Hartenstein,
1985). Unless otherwise stated, all staining procedures were
carried out at room temperature. After dechorionation in 50% household bleach
in 0.01% Triton X-100, embryos were washed in 0.01% Triton and fixed for 25
minutes in 4% paraformaldehyde in PBS (0.1 M NaCl, 10 mM phosphate buffer, pH
7.4)/heptane. Vitelline membranes were then removed by washing with methanol
or 80% ethanol if embryos were to be phalloidin stained. Embryos were washed
for 1 hour in three changes of PBT (PBS with 0.1% Triton X-100) and blocked
for 1 hour in PBT containing 1% bovine serum albumin (BSA). Primary antibody
incubations in PBT containing 1% BSA were done overnight at 4°C, and
embryos were then washed for 1 hour in PBT. Fluorescent detection of primary
antibodies was done using either secondary antibodies directly labeled with
Texas Red or FITC, or biotinylated secondary antibodies and streptavidin
labeled with Texas Red or FITC (all materials from Vector Laboratories). All
secondary antibodies were diluted 1:200 in 1% BSA in PBT. Secondary antibody
incubation was done for 2 hours, and embryos were then washed for 1 hour in
three changes of PBT and incubated with a 1:1000 dilution of labeled
streptavidin in PBS for 1 hour. If F-actin staining was required, FITC-labeled
or TRITC-labeled phalloidin (Sigma) was added to a final concentration of 1
µg/ml 30 minutes into the streptavidin incubation. Embryos were washed for
20 minutes in several changes of PBS, mounted in Vectashield and examined
using either a Bio-Rad MRC 600 or Zeiss LSM confocal laser scanning
microscope. Confocal images were assembled from a series of Z-sections.
Detection of primary antibodies for Nomarski microscopy was done using the
glucose oxidase-DAB-nickel method (Hsu et
al., 1988
).
Cuticle preparations
Cuticles were prepared as described by Ashburner
(Ashburner, 1989), except that
the fixation step was omitted. At least 100 embryos were examined from each
cross.
Plasmid rescue
Isolation of genomic sequences flanking the crbS010409
P-element insertion was done as described
(O'Kane, 1998).
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Results |
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Drac1V12 expression in the amnioserosa induced elevated levels of
F-actin, myosin and phosphotyrosine in amnioserosa cells and contraction of
this tissue
Embryos in which Drac1V12 had been induced with the
GAL4332.3 driver were stained for phosphotyrosine, myosin
or F-actin and the amnioserosa compared to wild-type embryos of a similar age.
As a result of Drac1V12 expression, the amnioserosa early in dorsal closure
was transformed from a flat sheet of cells occupying the entire dorsal hole
into a contracted tissue of greatly constricted cells occupying less than half
the dorsal hole at its posterior end. In these embryos it appeared that all
amnioserosa cells had constricted, rather than only the end cells as occurs in
wild-type embryos (compare Fig.
2A with Fig. 1C). A
similar, excessive contraction of the amnioserosa was induced through heat
shock expression of Drac1V12 with the Hs-GAL4M-4 driver
(Fig. 2C). As the amnioserosa
pulled away from the leading edge in Drac1V12-expressing embryos, it retained
attachments to the epidermis, which were pulled taut
(Fig. 2C,
Fig. 3D). Optical sectioning of
the amnioserosa in these embryos by confocal microscopy indicated that the
tissue is no longer a monolayer of cells but rather a `ball' of cells (data
not shown), and we suspect that the amnioserosa cells that disappear from the
anterior end of the dorsal hole are contained within this. The contraction of
the amnioserosa was followed by `bunched' closure of the epidermis around this
tissue, as seen in embryos late in dorsal closure
(Fig. 2B,D). The bunching of
the epidermis causes embryos to become bowed, with the head and tail pulled in
towards each other.
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|
The contraction of the amnioserosa following induction of Drac1V12 was
caused by a striking change in cell shape. Cell shape change in the wild-type
amnioserosa is accompanied by the accumulation of F-actin, myosin and
phosphotyrosine. It is not unexpected, therefore, that the levels of these
cytoskeletal components in the amnioserosa of Drac1V12-expressing embryos were
often clearly higher than wild-type, even taking into account the fact that
staining will look stronger in a compacted tissue
(Fig. 2B, Fig. 3). As with our earlier
studies on phosphotyrosine staining at the leading edge
(Harden et al., 1996), we
found through double-labeling studies of the amnioserosa in both
Drac1V12-expressing and wild-type embryos that levels of phosphotyrosine
staining correlated extremely well with the levels of F-actin and myosin (for
example, compare Fig. 1C with
1D).
Expression of Drac1N17 in the amnioserosa impedes both amnioserosa
morphogenesis and migration of the epidermis during dorsal closure
Having found that expression of Drac1V12 with the
GAL4332.3 driver caused excessive contraction of the
amnioserosa, we checked to see if amnioserosa-specific expression of a
dominant-negative Drac1 transgene, UAS-Drac1N17, would have
any effect on this tissue. GAL4332.3 was used to drive
Drac1N17 expression in embryos that were then subjected to cuticle
preparation. 61% of such embryos failed to secrete cuticle; of the embryos
that did secrete cuticle, 8% showed holes in the dorsal surface whereas the
rest appeared to be wild-type (data not shown). We then expressed Drac1N17
with the GAL4332.3 driver in the presence of a
UAS-lacZ reporter gene. These embryos were fixed and double-stained
for phosphotyrosine and ß-galactosidase in parallel with control embryos
in which the UAS-lacZ reporter gene alone had been expressed with the
GAL4332.3 driver. Examination of stained
GAL4332.3;UAS-Darc1N17 embryos revealed impairment of both
amnioserosa morphogenesis and migration of the epidermis. The amnioserosa of
control embryos examined late in dorsal closure was in the process of
narrowing from the original elliptical shape
(Fig. 4A,C). The amnioserosa of
similarly aged GAL4332.3;UAS-Drac1N17 embryos was still
elliptical and was frequently ruptured by the hindgut
(Fig. 4B,E). A lack of
morphological change in the amnioserosa in
GAL4332.3;UAS-Drac1N17 embryos appeared to impede the
movement of the lateral epidermis, such that the dorsal hole was larger than
in wild-type embryos of similar age (compare
Fig. 4E with 4C and 4F with
4D). Occasionally, within a single embryo, there were patches of
amnioserosa cells that had changed shape appropriately and patches of cells
that had not (Fig. 5A). The
amnioserosa cells towards the top of the dorsal hole in
Fig. 5 have successfully
elongated in the A-P direction and have a small apical surface area, whereas
amnioserosa cells towards the bottom of the dorsal hole have retained a large
surface area. Additionally, the epidermis has progressed further towards the
dorsal midline on the side of the embryo, where the amnioserosa cells have
changed shape effectively, than on the side where amnioserosa cell shape
change is retarded. This finding supports the idea that lack of morphogenesis
in the amnioserosa impedes movement of the epidermis. A consistent finding
from the examination of many embryos was that there was an excellent
correlation between the extent of amnioserosa morphogenesis and the degree of
closure of the epidermis. The closure of the epidermis was clearly impaired in
GAL4332.3;UAS-Drac1N17 individuals, but the leading edge
cytoskeleton was not disrupted, and many embryos completed dorsal closure,
although they frequently had mild defects in the dorsal surface
(Fig. 5B).
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Dominant-negative or constitutively active forms of the small GTPases
Dcdc42, Ras1 or RhoA have little effect on amnioserosa morphogenesis
Many of the processes regulated by the Rac proteins, including dorsal
closure, involve other small GTPases, and therefore we checked to see if other
p21s had a role in amnioserosa morphogenesis. We expressed activated and
dominant-negative versions of Dcdc42, Ras1 and human RhoA in the amnioserosa
using the GAL4332.3 driver and examined
transgene-expressing embryos by cuticle preparation or staining for
phosphotyrosine. None of these transgenes caused a significant frequency of
dorsal cuticle defects, nor did any of them have substantial effects on
amnioserosa morphology (data not shown). Expression of a dominant-negative
Drosophila RhoA transgene with the GAL4332.3
driver had no discernible effect on amnioserosa morphology (data not shown).
These results suggest that Dcdc42, Ras1 and RhoA play little or no role in
regulating amnioserosa morphogenesis.
Overexpression of Crumbs in the amnioserosa causes premature apical
cell constriction
Are there any candidate participants in Drac1-mediated amnioserosa
morphogenesis? To our knowledge only one gene has been previously described
for which there is evidence for a role in this process. Crumbs (Crb) is a
determinant of cell polarity expressed in epithelia, including the amnioserosa
(Tepass et al., 1990).
Overexpression of Crb in the amnioserosa using the
GAL4332.3 driver leads to contraction of the tissue into a
dumbbell-shaped structure (Wodarz et al.,
1995
). We repeated this experiment using the transgene
UAS-crbwt and examined the resulting embryos with cuticle
preparations or staining for phosphotyrosine and myosin. 34% of
GAL4332.3/UAS-crbwt embryos failed to form
cuticle, whereas 44% had dorsal holes (Fig.
6B) similar to those induced by Drac 1N17 expression in the
epidermis (Harden et al.,
1995
). In stage 13
GAL4332.3/UAS-crbwt embryos at the onset of
dorsal closure, the end cells of the amnioserosa were apically constricted
relative to the middle cells and showed elevated levels of phosphotyrosine and
myosin staining (Fig. 7A, C).
In wild-type embryos of this age, there is no such distinction between the end
cells and the middle cells of the amnioserosa
(Fig. 1A). The ends cells of
the amnioserosa of stage 13 GAL4332.3/UAS-crbwt
embryos looked similar to the end cells of late stage 14 wild-type embryos
(compare Fig. 7C with
Fig. 1E), suggesting that
overexpressed Crb induced premature morphogenesis of this tissue. In older
GAL4332.3/UAS-crbwt embryos, a dumbbell-shaped
amnioserosa was seen, as previously described
(Fig. 7B). Distinct from
Drac1V12-expressing embryos, the epidermis did not close up around the
contracted amnioserosa in Crb-expressing embryos, and the gut and dorsal
vessel were exposed in the large dorsal hole that remained. This failure of
epidermal migration may have been caused by disruption of the leading edge
cytoskeleton and a lack of epidermal cell elongation, as revealed by
myosin/phosphotyrosine staining (Fig.
8A,B).
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Characterization of a P-element-induced hypomorphic allele of
crb reveals a requirement for Crb in dorsal closure and germband
retraction
The fact that overexpression of Crb led to premature cell constriction in
the amnioserosa prompted us to consider a role for Crb in amnioserosa
morphogenesis and dorsal closure. It is not possible to evaluate dorsal
closure in embryos homozygous for complete loss-of-function alleles of
crb, as these embryos have disorganized epithelia and almost
completely lack cuticle (Tepass and Knust,
1990). In a search of third chromosome P-element-induced lethals
(Deak et al., 1997
) for new
participants in dorsal closure, we identified five insertions,
l(3)S010409, l(3)S025807, l(3)S025817, l(3)S025819, and
l(3)S050920, that all showed defects in dorsal closure and germband
retraction and belonged to a single complementation group. Some of these
insertions have been mapped by chromosome in situ hybridization to the
vicinity of crb at 95F and are uncovered by the deficiency
Df(3R)crbS87-5 (Tepass and Knust,
1990
). We confirmed that these five insertions are all alleles of
crb through the finding that they all failed to complement the
strong, amorphic allele crb2
(crb11A22) (Tepass and
Knust, 1990
), and we have named these new crb alleles
crbS010409 etc. We chose one of these alleles,
crbS010409, for further analysis. Chromosome in situ
hybridization on this line with a P-element probe shows an insertion at
95F3-10. To check that the lethality of this line is caused by P-element
insertion, we mobilized the P-element by crossing in the transposase source
P(
2-3) and established excision lines. Some of these excisions are
homozygous viable, indicating that the lethality in the
crbS010409 line is caused by the P-element insertion. We
performed plasmid rescue on the P-element in the
crbS010409 line and obtained a genomic sequence flanking
the insertion. The P-element is inserted in an exon of the crb locus,
182 bp upstream of the predicted initiator methionine codon
(Tepass et al., 1990
). Cuticle
preparations on the crbS010409 line revealed a high
frequency of embryos with dorsal holes and germband retraction failures. 27%
of embryos showed one or both of these defects, which was close to the
expected frequency of 25% crbS010409 homozygotes. Most
defective embryos showed both of these phenotypes together
(Fig. 6D), although embryos
could be found with either phenotype alone. Comparison of these results with
cuticle data on other crb alleles indicated that
crbS010409 is a hypomorphic allele, weaker than
crbS87-2 (Tepass and
Knust, 1990
). We stained homozygous crbS010409
embryos with anti-phosphotyrosine antibodies or with phalloidin to detect
F-actin and examined them by confocal microscopy.
crbS010409 embryos had an intact leading edge cytoskeleton
that was comparable to wild-type but during dorsal closure consistently had a
dorsal hole larger than wild-type embryos of similar age
(Fig. 4G,H). The morphogenesis
of the amnioserosa of crbS010409 embryos did not proceed
correctly, and the sheet of amnioserosa cells was frequently ruptured by the
hindgut. The epidermis was well organized in crbS010409
embryos and, except for the germband retraction defect, was wild-type in
appearance. This is in contrast to embryos that are homozygous for amorphic
alleles of crb, which show extensive disorganization and cell death
in the epidermis (Tepass and Knust,
1990
; Tepass et al.,
1990
). As with the cuticle preparations, we found stained
crbS010409 embryos with defects in dorsal closure but
without abnormalities in germband retraction (data not shown). This result
indicated that the dorsal closure failures in crbS010409
embryos were not caused by germband retraction defects. Overall, the dorsal
closure phenotypes of crbS010409 embryos and
GAL4332.3; UAS-Drac1N17 embryos were similar in
that both show impaired morphogenesis of the amnioserosa in the presence of an
intact leading edge cytoskeleton. In comparison with
GAL4332.3;UAS-Drac1N17 embryos, however,
crbS010409 embryos tended to be less successful with their
final degree of closure, and many late embryos were seen with a persistent
dorsal hole and extrusion of the gut. This finding was consistent with the
high frequency of dorsal holes seen in crbS010409 cuticle
preparations.
Co-expression of Drac1N17 in the amnioserosa does not prevent
induction of premature apical cell constriction by overexpressed Crb
The finding that both Drac1 and Crb participate in amnioserosa
morphogenesis led us to look for interdependence of these proteins in
regulating this process. To test for a requirement for Drac1 in induction of
premature cell constriction by Crb, we created a line homozygous for both the
UAS-crbwt and UAS-Drac1N17 transgenes and mated
these flies to the GAL4332.3 line. All the progeny from
this cross will overexpress Crb in the amnioserosa and have impaired Drac1
signaling. Progeny from the cross were examined as embryos with
anti-phosphotyrosine staining. As shown in
Fig. 7D, impairment of Drac1
signaling did not prevent Crb from inducing premature apical cell
constriction.
Contraction of the amnioserosa by Drac1V12 is weakened to a phenotype
of premature apical cell constriction in a crbS010409
mutant background
To check for a requirement for Crb in Drac1V12-induced amnioserosa
contraction, we expressed Drac1V12 by heat shock using the
Hs-GAL4M-4 driver in embryos homozygous for the
crbS010409 allele and examined
anti-phosphotyrosine-stained embryos by confocal microscopy. When Drac1V12 was
expressed in the crbS010409 mutant background, it no
longer generated contraction of the entire amnioserosa. Rather, embryos showed
a premature apical constriction of cells at the anterior end of the tissue
prior to the onset of dorsal closure in a manner very similar to that seen
with Crb overexpression (Fig.
7E). The status of cells at the posterior end of the amnioserosa
could not be assessed because of impaired germband retraction.
High frequencies of dorsal closure defects occur when Drac1
and crb transgenes are expressed in the amnioserosa with the
GAL4c381 driver
A problem with expression of the Drac1 and crb transgenes
with the GAL4332.3 driver is that many embryos fail to
form cuticle and their final degree of dorsal closure cannot be assessed. We
believe that this failure to form cuticle is caused by expression of the
driver in the epidermis following dorsal closure
(Fig. 1H). This expression will
lead to alterations in Drac1 or Crb function in the epidermis during cuticle
secretion. Crb overexpression has previously been shown to have severe effects
on the cuticle (Wodarz et al.,
1995). During the course of this work we became aware of another
amnioserosa driver, GAL4c381
(Manseau et al., 1997
). We
checked the expression of the GAL4c381 driver using a
UAS-LacZ reporter gene and saw no ß-galactosidase staining in
the epidermis during or after dorsal closure
(Fig. 9A-C). The
UAS-Drac1V12, UAS-Drac1N17 and UAS-crb transgenes were each
crossed with the GAL4c381 driver and the progeny evaluated
by cuticle preparation or as embryos stained with anti-phosphotyrosine. With
the GAL4c381 driver, none of the transgenes caused
failures of cuticle formation, and each transgene induced a very consistent
phenotype, which occured in greater than 90% of embryos. Drac1V12-expressing
embryos failed to undergo germband retraction but did complete dorsal closure,
although their cuticles showed `puckers' extending out from the dorsal surface
(Fig. 9D). Drac1N17-expressing
embryos did not complete dorsal closure and had a large dorsal hole in their
cuticle extending form the middle of the dorsal surface to the rear of the
embryo (Fig. 9E).
Crb-overexpressing embryos also failed to close and had a cuticle phenotype
very similar to that seen with Drac1N17. Examination of embryos stained for
phosphotyrosine revealed that each transgene had similar effects on
amnioserosa morphology to when expressed with the
GAL4332.3 driver (Fig.
9G) (data not shown), but two major differences were noted in the
final morphology of embryos. Consistent with cuticle preparations,
UAS-Drac1V12;GAL4c381 embryos showed a high frequency of
impaired germband retractions (Fig.
9G), and the epidermis failed to migrate over the amnioserosa in
UAS-Drac1N17;GAL4c381 embryos (data not shown).
|
As described earlier, the behavior of the leading edge and dorsal epidermis following expression of Drac1V12 with the GAL4332.3 driver was distinct from that seen following overexpression of Crb in the same manner. These distinct epidermal phenotypes were also seen when these transgenes were expressed with the GAL4c381 driver. UAS-Drac1V12;GAL4c381 embryos showed bunched closure of the epidermis around the amnioserosa (Fig. 9G). This bunched closure made it difficult to evaluate the leading edge cytoskeleton, but those portions of the leading edge that could be assessed had an accumulation of phosphotyrosine comparable to wild-type embryos, and there was pronounced elongation of the epidermal cells along the dorsoventral axis (Fig. 9H). UAS-crbwt;GAL4c381 embryos showed disruption of leading edge phosphotyrosine, and there was little elongation of the epidermal cells (Fig. 9I).
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Discussion |
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Excessive Drac1 activity induces a dramatic contraction of the amnioserosa
such that it shrinks to occupy less than half the dorsal hole, and this is
accompanied by elevated levels of myosin, F-actin, and phosphotyrosine in this
tissue. Our interpretation is that Drac1V12 is driving premature and excessive
amnioserosa cell constriction through its effects on the cytoskeleton. We
propose that Drac1 participates in amnioserosa morphogenesis by driving the
assembly of an apical actomyosin contractile apparatus that constricts the
amnioserosa cells, first at the ends of the tissue and possibly later in the
middle. Contraction of an apical actomyosin belt has been implicated in
diverse types of epithelial morphogenesis
(Fristrom, 1988;
von Kalm et al., 1995
)
including Drosophila gastrulation
(Leptin, 1999
), which shows
some similarity to amnioserosa morphogenesis in that both processes involve
apical construction of a monolayer of cells that then invaginates.
During the course of this work, Kiehart et al.
(Kiehart et al., 2000)
reported the results of a study that used cell ablation to address the
contributions of the epidermis and amnioserosa to dorsal closure. They
demonstrated that the amnioserosa is under tension, as ablation of cells in
the amnioserosa causes the tissue to recoil away from the wound site, and the
leading edge is pushed back away from the dorsal midline. They conclude, as we
do, that there is active cell shape change in the amnioserosa that contributes
to dorsal closure, rather than the tissue being simply compressed by the
movement of the leading edge. Their finding that the recoiling of the
amnioserosa after wounding pushes back the leading edge is consistent with our
result that impairing amnioserosa morphogenesis through Drac1N17 expression
hinders leading edge migration.
Crumbs, a determinant of epithelial apical-basal polarity,
participates with Drac1 in establishing apical cell constriction in the
amnioserosa
Overexpression of Crb in the amnioserosa leads to contraction of the tissue
and failure of dorsal closure (Wodarz et
al., 1995). We examined this phenotype in more detail and found
that excessive Crb activity induces a premature constriction of cells at the
ends of the amnioserosa. We have identified five P-element-induced
crb alleles that are hypomorphic mutations, causing defects in dorsal
closure and germband retraction. We have characterized one of these
crb mutations, crbS010409, in detail. Embryos
homozygous for crbS010409 show a dorsal closure defect
similar to that seen with expression of Drac1N17 in the amnioserosa:
amnioserosa morphogenesis is impaired, but the leading edge cytoskeleton is
intact. In contrast to amorphic crb alleles, the epidermis is not
disorganized in crbS010409 mutants and it secretes
cuticle. Amnioserosa morphogenesis and germband retraction may be particularly
sensitive to the level of Crb activity. Our interpretation is that Crb
activity in the amnioserosa is required for amnioserosa morphogenesis,
although we cannot exclude the possibility that loss of Crb activity elsewhere
in the embryo is affecting this process. Crb is a transmembrane domain protein
with extracellular EGF-like and laminin A G-domain-like repeats that plays a
key role in determining apical-basal polarity in epithelial cells
(Tepass et al., 1990
;
Wodarz et al., 1995
).
Drac1 may act through Crb in regulating the cytoskeleton, as the
Drac1V12-induced phenotype of excessive contraction of the amnioserosa is
weakened in a crbS010409 mutant background. This weaker
Drac1V12 phenotype of premature constriction of the end cells of the
amnioserosa is very similar to that caused by Crb overexpression. There may be
sufficient Crb in the crbS010409 mutant embryos for
Drac1V12 to be able to prematurely constrict cells at the ends of the
amnioserosa but not to excessively contract the tissue. Crb overexpression
does not appear to require Drac1 to cause premature constriction of
amnioserosa cells, as it can achieve this in the presence of Drac1N17. The
excessive contraction of the amnioserosa caused by Drac1V12 expression in
embryos with wild-type Crb activity, and the dumbbell-shaped amnioserosa
induced by Crb overexpression, could both result from excessive constriction
of amnioserosa cells to produce a tissue that only occupies a fraction of the
dorsal hole. Such excessive constriction may be driven by ectopic accumulation
of a normally apically localized actomyosin contractile apparatus. A role for
Crb in defining the location of the actomyosin contractile apparatus is
consistent with the idea that Crb defines the range of the apical membrane
cytoskeleton (Grawe et al.,
1996; Wodarz et al.,
1993
; Wodarz et al.,
1995
). The actin-crosslinking protein
ßHeavy(ßH)-spectrin normally has an
apicolateral distribution, but upon overexpression of Crb is also found at the
basolateral membrane, indicating a redistribution of the membrane cytoskeleton
(Thomas and Kiehart, 1994
;
Wodarz et al., 1995
).
ßH-spectrin is required for apical constriction of follicle
cells during Drosophila oogenesis and may participate in organization
of an actomyosin contractile apparatus
(Zarnescu and Thomas, 1999
).
It is conceivable that the ectopic localization of
(ßH)-spectrin domain following Crb overexpression could be
accompanied by an ectopic accumulation of F-actin and myosin. Future goals in
studying Drac1-Crb function in amnioserosa morphogenesis will include
addressing the nature of the interaction between the two proteins and defining
which portion(s) of the Crb protein are required. The short cytoplasmic domain
of Crb appears sufficient to execute all Crb functions studied to date
(Klebes and Knust, 2000
). No
definitive role has been found for the large extracellular domain, although
there is evidence that the Drosophila and human Crb proteins have
non-cell-autonomous functions (Rashbass
and Skaer, 2000
).
Although Drac1 and Crb both generate premature contraction of the
amnioserosa when their activity is experimentally upregulated in this tissue,
their phenotypic effects are not identical. Drac1V12 expression drives
constriction of all amnioserosa cells early in closure, whereas, at the same
stage, Crb overexpression only promotes constriction of the end cells. A
plausible explanation for this is that constriction of the middle cells
requires Drac1 to activate Crb-independent processes and that Crb function is
necessary but not sufficient for middle cell constriction. Crb overexpression
in the amnioserosa causes disruption of the leading edge cytoskeleton and a
failure of cell shape change in the epidermis, suggesting that a signal from
the amnioserosa required for dorsal closure is disrupted. That communication
between the amnioserosa and the epidermis is a component of dorsal closure is
demonstrated by two recent reports. Downregulation of JNK signaling in the
amnioserosa is required for phosphotyrosine accumulation at the leading edge
and dorsalward migration of the epidermis, and leading edge cells are
specified wherever an interface of amnioserosa and dorsal epidermis occurs
(Reed et al., 2001;
Stronach and Perrimon, 2001
).
Drac1V12 expression in the amnioserosa does not disrupt the leading edge
cytoskeleton or prevent closure of the epidermis, and this result suggests
that Drac1V12 cannot activate a function of Crb that influences communication
between the amnioserosa and the epidermis.
Amnioserosa morphogenesis as a system for studying small GTPase
regulation of epithelial morphology
Evidence is emerging that Drac1 is a key regulator of epithelial cell
morphology in Drosophila. Drac1 transgene expression can affect head
involution, germband retraction, border cell migration in the oocyte and, in
the wing disc, adherens junction actin and planar polarity
(Eaton et al., 1995;
Eaton et al., 1996
;
Harden et al., 1995
;
Murphy and Montell, 1996
). The
present study and earlier work (Harden et
al., 1995
; Harden et al.,
1999
) indicate that Drac1 is essential for the morphogenesis of
both the epidermis and the amnioserosa during dorsal closure. There is an
enormous body of evidence showing that different small GTPases act in concert
with each other to regulate diverse cellular events. However, none of the
other small GTPases we have tested appear to have a major role in amnioserosa
morphogenesis, as activated and dominant-negative versions of Dcdc42, RhoA and
Ras1 do not substantially affect amnioserosa morphology. Thus, unlike the
morphogenesis of the epidermis during dorsal closure, which is regulated by
Drac1 and all these other small GTPases, only Drac1 may be required for
amnioserosa morphogenesis. The finding that, of the p21s tested, only Drac1
has a substantial effect on amnioserosa morphology suggests that there may be
a Drac1-specific control of amnioserosa morphogenesis and that this process
may be a good system for identifying and characterizing Rac-specific
effectors. Although Drac1, Dcdc42, RhoA and Ras1 all participate in dorsal
closure (Harden et al., 1999
),
there is no evidence for the Cdc42
Rac
RhoA hierarchy of p21
activity demonstrated in some cultured mammalian cells
(Allen et al., 1997
;
Nobes and Hall, 1995
). Our
results on p21 regulation of cell shape in the amnioserosa are a further
indication that Rho subfamily hierarchies may often not be utilized in
developmental cell shape change.
Our findings on Drac1-Crb regulation of amnioserosa morphology, taken
together with other recent results indicating an active role for the
amnioserosa in dorsal closure (Kiehart et
al., 2000; Reed et al.,
2001
; Stronach and Perrimon,
2001
), have important implications for the use of dorsal closure
as a system for studying signal transduction and epithelial morphogenesis. It
is now clear that one must consider the potential effects on the amnioserosa
when interpreting the phenotype of any mutant with a dorsal closure defect.
Genes acting solely in the amnioserosa may contribute to epidermal migration
and, in this respect, we have identified a number of P[lacZ] insertion lines
with dorsal closure defects that show high levels of ß-galactosidase
staining in the amnioserosa (N.H. and M.R., unpublished). The characterization
of such genes should yield further insight into the control of amnioserosa
morphogenesis and the role of the amnioserosa in dorsal closure.
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