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First published online 19 July 2006
doi: 10.1242/dev.02492
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Department of Biology, McGill University, 1205 Doctor Penfield Avenue, Montréal, QC H3A 1B1, Canada.
* Author for correspondence (e-mail: laura.nilson{at}mcgill.ca)
Accepted 14 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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Key words: Echinoid, Epithelial morphogenesis, Actomyosin cable, Follicular epithelium, Dorsal closure, Epithelial tube
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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;
Schöck and Perrimon,
2002b). These changes typically arise from modulation of the actin
cytoskeleton. Distinct cell behaviors are associated with specific cell types,
but how the determination of cell fate engages the subcellular mechanisms that
drive morphogenesis is not well understood. We have used the
Drosophila ovary and embryo as model systems to study how
interactions between cell types lead to local changes in the cytoskeleton that
mediate epithelial morphogenesis.
In the ovary, the follicular epithelium surrounds individual cysts of
germline cells, each of which gives rise to a single egg
(Spradling, 1993). Late in
oogenesis, the follicle cells secrete the eggshell, which exhibits pronounced
asymmetries produced by specialized follicle cell domains
(Berg, 2005;
Dobens and Raftery, 2000). The
most prominent features are the two appendages that project from the dorsal
anterior region of the eggshell (Fig.
1A). The follicle cell primordia that produce these appendages are
specified in mid-oogenesis and flank the dorsal anterior midline of the
epithelium. Subsequently, the two cell types that comprise the primordium
undergo a series of coordinated cell shape changes that remodel the flat
primordia into epithelial tubes that then extend anteriorly. Secretion of
chorion into the lumen of each tube produces the appendages
(Dorman et al., 2004;
Ward and Berg, 2005). The
signals that specify the fate and position of the appendage primordia are well
understood (Berg, 2005;
Nilson and Schüpbach,
1999; Roth, 2003),
but the changes in cell shape and organization that occur during the
morphogenesis of these primordia have only recently been described in detail
(Dorman et al., 2004;
Ward and Berg, 2005).
Mutations that disrupt appendage morphology downstream of primordia
specification have been identified, and thus may affect factors that
contribute specifically to tube formation
(Berg, 2005), but the molecular
mechanisms that control these morphogenetic movements remain unknown.
By contrast, the cytoskeletal forces driving epithelial movements during
Drosophila embryonic dorsal closure have been well characterized.
Dorsal closure occurs when two lateral epidermal sheets move dorsally over the
extra-embryonic amnioserosa and converge at the dorsal midline, sealing the
dorsal side of the embryo. These movements are driven by multiple forces,
including tissue-specific changes in the shape of individual cells as well as
the tension generated by a supracellular contractile actin cable that arises
at the interface of the lateral epidermis and amnioserosa
(Kiehart et al., 2000;
Jacinto et al., 2002;
Martin and Parkhurst, 2004).
Differential activity of the Jun N-terminal kinase pathway between the
epidermis and amnioserosa is involved in generating these forces
(Reed et al., 2001), but how
this difference in activity produces a local effect on the cytoskeleton at the
interface between these tissues is not understood.
We demonstrate that Echinoid (Ed), a cell-adhesion molecule and adherens
junction component (Bai et al.,
2001; Islam et al.,
2003; Spencer and Cagan,
2003; Wei et al.,
2005) is required for the epithelial sheet movements that occur
during appendage primordia morphogenesis and dorsal closure. Specifically, we
find that the juxtaposition of cells expressing and lacking Ed induces the
assembly of a contractile actomyosin cable at their interface. We initially
identified a mutant allele of ed, based on the smooth borders
exhibited by homozygous mutant follicle cell clones, and found that the apical
clone border displays morphological and molecular characteristics of a
contractile actomyosin cable. Strikingly, we demonstrate that Ed is absent
from some cell types during development, generating endogenous interfaces
between cells with and without Ed; these Ed expression borders display
contractile features identical to those of ed mutant clones. In
wild-type ovaries, Ed is absent from one of the two cell types of the
appendage primordium, generating an Ed expression border within the
primordium. In the embryo, Ed becomes undetectable in the amnioserosa prior to
dorsal closure, resulting in an Ed expression border that coincides with the
well-characterized contractile actomyosin cable between these tissues. In both
cases, elimination of Ed results in the absence of the contractile structure
and in defective morphogenesis. Taken together, these data suggest that
differential Ed expression between cell types induces the formation of a
contractile actomyosin cable at their interface. These observations may
identify a general morphogenetic mechanism that converts a difference in
protein expression into a local effect on the cytoskeleton.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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), edlF20 P{neoFRT}40A (gift of J.-C.
Hsu), rho-lacZ R1.1 line (Ip et
al., 1992), w; al dp P{NM}31E P{neoFRT}40A, and y w
P{hsFLP}122; P{NM}31E P{neoFRT}40A, sqh-GFP-moesin (SGMCA) (gift of D.
Kiehart).
Mapping and identification of the F72 mutation
The F72 mutation was mapped through meiotic recombination between the
parental strains w; al dp P{NM}31E P{neoFRT}40A and w;
edF72 P{neoFRT}40A using single nucleotide polymorphisms as
molecular markers, as described previously
(Berger et al., 2001;
Hoskins et al., 2001;
Martin et al., 2001). After
mapping the F72 phenotype to an interval containing ed, all nine
ed exons, their splice sites and 
1 kb of flanking genomic DNA
were amplified by PCR, in fragments of 800 bp, and sequenced. The only
difference between the F72 chromosome and the parental chromosome was an A to
T substitution at position 1043 of the transcript, generating a premature stop
at codon 205 of the ed open reading frame. The
edF72 allele is therefore predicted to encode a protein
truncated in the second of seven immunoglobulin-like domains predicted by the
ExPASy ScanProsite proteomics algorithm.
Generation of Ed antiserum
The cDNA RE66591 (Drosophila Genome Resources Center) was used as
template to amplify by PCR the fragment encoding the C-terminal domain of Ed,
which was then cloned in frame into the pGEX2T-His6 vector (gift of
S. Gunderson). After expression in E. coli BL21 cells, the
recombinant protein was purified by selection for the His6 tag and
used to immunize rats.
Mitotic recombination
Mitotic follicle cell clones were induced in females of the genotype y
w P{hsFLP}122; P{NM}31E P{neoFRT}40A/ed P{neoFRT}40A by
incubating pupae at 37°C for 1 hour on three consecutive days. Prior to
dissection, well-fed mosaic females were incubated at 37°C for 80 minutes
to induce expression of N-myc (NM) clone marker
(Xu and Rubin, 1993). Germline
clones homozygous for either edF72 or edlF20
were generated and imaged as described previously
(Chou and Perrimon, 1996;
Schöck and Perrimon,
2002a).
Immunohistochemistry
Fixation and staining of ovaries and embryos was performed as described
previously (Van Buskirk and
Schüpbach, 2002;
Wieschaus and Nusslein-Volhard,
1986). Antibodies used were anti-Ed (1:1000 for ovaries, 1:10,000
for embryos), anti-c-Myc supernatant 9E10 (1:100, Developmental Studies
Hybridoma Bank (DSHB)), anti-phospho-myosin light chain 1 Ser19 (1:250, Cell
Signaling Technology), anti-DE-cadherin DCAD2 supernatant (1:100, DSHB),
anti-Arm N2 7A1 supernatant (1:100 for ovaries; 1:500 for embryos, DSHB),
anti-ß-galactosidase 40a1 supernatant (1:50, DSHB), anti-Broad Core
supernatant (1:50, DSHB), anti-Enabled 5G2 (1:500, DSHB) and
anti-phosphotyrosine (1:200, Upstate Cell Signaling Solutions). All secondary
antibodies (Molecular Probes) were pre-blocked against ovaries and used at a
final concentration of 1:500 for 2 hours at room temperature or 1:1000
overnight at 4°C. For F-actin labeling, tissues were incubated for 2 hours
at room temperature with 0.5 U/ml of Alexa Fluor 546 phalloidin (dried of
methanol; Molecular Probes).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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; Nilson and
Schüpbach, 1998;
Wieschaus et al., 1981), the
dec-1-marked imprints were contained exclusively within the smooth
borders, indicating that these borders occur at the interface of imprints
produced by mutant and non-mutant cells
(Fig. 1C). Consistent with the mosaic eggshell phenotype, clones of homozygous mutant follicle cells exhibit smooth borders with adjacent heterozygous or homozygous wild-type cells. Interfaces between mutant cells within the clone, however, appear normal (Fig. 1E,E'). Interestingly, the smooth clone border is detectable only at the apical side of the epithelium, while the basal aspect of the clone displays no obvious phenotype (compare Fig. 1E with 1E'). This mosaic phenotype also exhibits a surprising temporal profile. The smooth clone border phenotype is completely penetrant in early stage egg chambers (data not shown) but, during stage 10 of mid-oogenesis, the border of F72 mutant clones becomes indistinguishable from adjacent intercellular interfaces (Fig. 1F,F'). The disappearance of the phenotype is transient, however, and by stage 11 the marked smoothness of the clone border is again readily detectable and completely penetrant (Fig. 1E,E'), and persists for the remainder of oogenesis.
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;
Escudero et al., 2003).
Follicle cell clones homozygous for edlF20 exhibit a
smooth border phenotype indistinguishable in all respects from F72 mutant
clones (Fig. 1D; data not
shown). Together these data confirm that the phenotype is the result of the
mutation in ed.
ed encodes a 1332 amino acid transmembrane protein with seven
immunoglobulin (Ig) domains, a fibronectin type III domain and a cytoplasmic
tail with a PDZ-binding domain (see Materials and methods)
(Bai et al., 2001;
Wei et al., 2005). The
edF72 allele is predicted to encode an Ed protein that is
truncated in the second Ig domain, thus lacking most of the extracellular
domain, as well as the transmembrane and intracellular domains, and therefore
unlikely to retain Ed function. The Ed extracellular domain resembles that of
Ig-type cell-adhesion molecules, and recently Ed has been reported to be a
component of adherens junctions (Wei et
al., 2005). The molecular nature of Ed is therefore consistent
with the ed mosaic phenotype, and suggests that the absence of this
molecule from the cell surface affects the interaction between wild-type and
mutant follicle cells.
ed clone borders induce assembly of a contractile actomyosin cable
Our initial observations of ed mutant follicle cell clones
revealed that the apical clone circumference is markedly reduced relative to
the basal circumference (compare Fig. 1E
with 1E' and Fig. 2A with
2A'; Fig.
2B,B'). In addition, filamentous actin (F-actin) appears
enriched at the apical interface between wild-type and ed mutant
cells (Fig. 2A,A'), but
not at the basal interface (see Fig. S1 in the supplementary material).
Together with the reduced apical circumference, this observation suggested the
presence of a contractile actin cable at the clone border. Consistent with
this hypothesis, the active, phosphorylated form of the light chain of
non-muscle myosin II (p-MLC) is also enriched at the apical clone border
(Fig. 2C,C'), suggesting
that non-muscle myosin II is activated at the interface between wild-type and
ed mutant cells (Sellers,
1991; Trybus,
1991). Closer inspection of the clone border resolves two `rings'
of p-MLC immunoreactivity (Fig.
2D,D'), raising the possibility that one contractile
structure assembles within the ed mutant cells at the clone border
and another in the adjacent wild-type cells. Identical mosaic phenotypes were
observed with both edF72 and
edlF20.
The reduced circumference and enrichment of F-actin and p-MLC at the apical
ed clone border are characteristic of the supracellular contractile
actomyosin structures that mediate the epithelial movements observed in
processes such as wound healing and embryonic epithelial closure
(Bement, 2002;
Martin and Parkhurst, 2004).
We propose that the juxtaposition of wild-type and ed mutant cells is
sufficient to trigger the assembly of such a structure at their interface,
resulting in a contractile force at the border that generates the apically
constricted smooth circumference of ed mutant follicle cell clones.
This phenotype is similar to that reported for ed mutant clones in
the wing imaginal disc epithelium (Wei et
al., 2005). However, although ed mutant cells in the wing
disc required a genetic growth advantage to recover sufficient clones for
analysis, ed mutant follicle cells display no detectable defects in
growth or viability. This difference may reflect tissue specificity in the
requirement for ed, or a difference in the ed mutant
chromosome studied in the wing.
Adherens junctions are destabilized at the border of ed mutant clones
Because the smooth border of ed mutant follicle cell clones
suggested possible differential adhesion with the neighboring wild-type cells
(Lawrence, 1997;
Dahmann and Basler, 1999;
Tepass et al., 2002), we
determined whether ed mutant clones exhibit altered levels of the
cell-adhesion molecule DE-cadherin (DE-cad). At stage 10, when the smooth
border phenotype is not detectable, the level and distribution of DE-cad
appeared normal at the border of 38/38 ed mutant clones examined
(data not shown). After stage 11, DE-cad immunoreactivity was strongly reduced
or absent at the clone border in 6/20 clones observed
(Fig. 2E,E'). This effect
was less dramatic in 11/20 clones (Fig.
2F,F'), where DE-cad at individual interfaces along a single
clone border appeared either undetectable
(Fig. 2F', arrow), wild
type or discontinuous (Fig.
2F', arrowhead). In 3/20 clones, there was no detectable
effect. Levels of Armadillo (Arm), the Drosophila homolog of
ß-catenin and an intracellular component of adherens junctions, were
similarly affected (data not shown). In both cases, the degree of disruption
did not correlate with clone size or position, and was variable even between
egg chambers of the same stage. These data indicate that the juxtaposition of
wild-type and ed mutant cells can affect the distribution of DE-cad
and Arm. However, given the variability of this effect, it remains unclear
whether alteration of adherens junction components is the cause of the
ed smooth border phenotype.
Ed exhibits a dynamic expression pattern in the follicular epithelium
To visualize the distribution of Ed in the follicular epithelium, we
generated an antiserum against the Ed intracellular domain. We detected no
immunoreactivity in ed mutant follicle cells, confirming the
specificity of the antiserum (Fig.
3A,A'). In individual follicle cells, Ed levels appear
highest apically with lower levels detectable in lateral membranes
(Fig. 3B, inset), resembling
the distribution of DE-cad and Arm and consistent with recent evidence
implicating Ed as an adherens junction component
(Wei et al., 2005). As
reported previously, Ed immunoreactivity is either discontinuous
(Fig. 3A, arrowhead) or absent
(Fig. 3A, arrow) from the
surrounding wild-type cells at the interface with ed mutant cells,
consistent with previous observations that Ed molecules on adjacent cells can
interact homophilically (Islam et al.,
2003; Spencer and Cagan,
2003).
Ed exhibits a spatially and temporally dynamic expression pattern in wild-type ovaries. In early stages of oogenesis, similar levels of Ed are detectable in all follicle cells (Fig. 3B, left), but begin to decline by stage 8 (Fig. 3B, middle). By early stage 10B, little or no Ed is detectable above background levels (Fig. 3B, right). Interestingly, the absence of detectable Ed at this stage coincides with the transient disappearance of the ed mosaic phenotype, supporting the hypothesis that the smooth border of ed mutant clones is triggered by the juxtaposition of cells with and without Ed; presumably ed mutant clones do not exhibit a smooth border at this stage (see Fig. 1F,F') because Ed is also absent from the surrounding wild-type cells.
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The absence of Ed from specific follicle cell populations generates endogenous interfaces between cells with and without Ed, which we refer to as Ed expression borders, that resemble the ectopic interfaces generated by ed mutant clones. Indeed, we found that these endogenous Ed expression borders are smooth and exhibit the same apical enrichment of F-actin and p-MLC associated with ed mutant clones (Fig. 3C',D'; data not shown). At later stages, however, the enrichment of actin at this endogenous border appears less pronounced, owing to increased F-actin levels in the individual cells within this domain (Fig. 3D'). These data further support the hypothesis that juxtaposition of cells with and without Ed results in formation of a contractile actin cable at their interface. Moreover, the spatial and temporal regulation of the appearance of this Ed expression border suggested that it may have a developmental function.
Ed is absent from the roof cells of the appendage primordia
The dorsal-anterior Ed expression pattern suggested a correlation between
Ed expression and the specification or behavior of the dorsal appendage
primordia, which are located in the same region of the epithelium. Each
primordium consists of two cell types, which are specified coordinately during
midoogenesis (Dorman et al.,
2004; Ward and Berg,
2005). The majority of the cells in the primordium will form the
roof of the appendage-producing tube, while the cells in a single `L'-shaped
row at the anterior and medial edges of the primordium will form the tube
floor. The roof cells are distinguished by high nuclear levels of the Broad
protein, a zinc-finger transcription factor
(Bayer et al., 1996;
Deng and Bownes, 1997;
DiBello et al., 1991;
Tzolovsky et al., 1999), while
the floor cells express a lacZ reporter driven by a region of the
rhomboid promoter (rho-lacZ)
(Dorman et al., 2004;
Ip et al., 1992;
Ward and Berg, 2005). The
rho-lacZ marker is detectable throughout the floor cell cytoplasm and
thus also highlights the changes in floor cell shape that occur during
appendage morphogenesis (Fig.
4A,B).
We found that the follicle cell domains that lack Ed expression (see
Fig. 3D) coincide precisely
with the two roof cell populations, which express high levels of Broad
(Fig. 4C-C''). In
addition, visualization of the rho-lacZ floor cell marker revealed
that Ed is present apically in the adjacent floor cells
(Fig. 4A,B,D-D''). The
border of the domain lacking Ed therefore aligns precisely with the roof/floor
interface, which has been observed previously to be smooth
(Ward and Berg, 2005). Our
data show that an endogenous Ed expression border corresponds to a contractile
interface between the two cell types that populate the appendage
primordium.
The dorsoventral pattern of follicle cell fates, including both the
appendage primordia and the dorsal midline, is established through dorsally
localized activation of the Drosophila epidermal growth factor
receptor (Egfr) pathway (Berg,
2005; Nilson and
Schüpbach, 1999; Roth,
2003), and Ed has been reported to downregulate Egfr signaling in
the eye imaginal disc (Bai et al.,
2001; Rawlins et al.,
2003b; Spencer and Cagan,
2003). However, large ed mutant follicle cell clones
encompassing the dorsal midline and one or both appendage primordia
(n=6) have no detectable effect on the pattern of Broad expression
(data not shown). This observation indicates that Ed functions downstream of
cell fate determination and does not regulate Egfr signaling in this
patterning process.
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To determine whether the endogenous Ed expression border is required for the remodeling of the appendage primordia into epithelial tubes, we analyzed ed mosaic egg chambers at later stages, when the tubes are being formed. In mosaic epithelia with one mutant (Fig. 5B, top) and one wild-type primordium (Fig. 5B, bottom), the nascent ed mutant tube was shorter (Fig. 5B') and exhibited a wider opening (Fig. 5B'', arrows) than the wild-type tube, suggesting a defect in tube morphogenesis in the absence of an Ed expression border (n=3).
We further characterized this tube defect using the rho-lacZ
reporter to visualize the floor cell movements that occur during tube
formation. In wild-type primordia, at the onset of tube morphogenesis the
floor cells elongate and the apices of the anterior and medial floor cell
domains begin to approach each other, moving underneath the roof cells, which
constrict apically at this stage (Fig.
5C,C'; see Fig.
4A,B). The floor cell apices meet first at the intersection (or
hinge) between the anterior and medial domains and progressively converge,
closing the tube floor (Fig.
5C',C'',D-F). As floor closure nears completion, the
wedge-shaped floor cell domain (Fig.
5D) adopts a more rounded appearance
(Fig. 5E,F)
(Dorman et al., 2004), and
moves anteriorly. By the time the floor cell domain begins to overlap the
nurse cell cluster, the tube floor is closed. The completed tube then
continues to extends anteriorly.
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This tube formation defect results in abnormal eggshell appendages. Appendage morphology can be visualized in egg chambers at stage 14; this is the final stage of oogenesis, when egg chambers have completed dorsal appendage formation. We examined stage 14 egg chambers from ed mosaic females, focusing on those that retained an intact follicular epithelium and lacked detectable Ed. Of 14 ed mutant appendages recovered, 12 were severely reduced in length or had failed to extend from the main body of the eggshell (Fig. 5J,J'). These mutant appendage phenotypes confirm that the defects in floor closure observed in the absence of ed result in improperly formed epithelial tubes.
Differential Ed expression promotes embryonic dorsal closure
The morphogenetic movements of the floor cells are reminiscent of those
observed during embryonic dorsal closure, where the lateral epidermal sheets
of the embryo move dorsally and ultimately fuse at the embryonic dorsal
midline, covering the extra-embryonic amnioserosa
(Jacinto et al., 2002;
Martin and Parkhurst, 2004).
Interestingly, the leading edge cells of the lateral epidermis assemble a
supracellular contractile actomyosin cable that provides one of the forces
driving epithelial closure (Young et al.,
1993; Hutson et al.,
2003; Kiehart et al.,
2000). Given the contractile nature of endogenous and ectopic Ed
expression borders in the follicular epithelium, we asked whether the
actomyosin cable between the amnioserosa and lateral epidermis is also
associated with differential Ed expression. We found that during dorsal
closure Ed is present in the lateral epidermis but undetectable in the
amnioserosa (Fig. 6A,A'),
generating an endogenous Ed expression border at the interface of these two
cell types.
To determine whether this differential Ed expression is necessary for the generation of the actomyosin cable and for dorsal closure, we generated embryos lacking both maternal and zygotic contributions of ed (edMZ). Fixed edMZ embryos exhibit apparent irregularities in the progression of the leading edge during dorsal closure stages (14/14; Fig. 6B), as well as gaps and segment misalignments at the dorsal midline in later stage embryos (14/14; Fig. 6C).
We also used time-lapse confocal microscopy to image live wild-type and
edMZ embryos expressing a transgene encoding the actin
binding fragment of Drosophila moesin fused to GFP (GFP-moesin), a
well-characterized marker that labels F-actin
(Edwards et al., 1997). In
wild-type embryos during dorsal closure, this marker highlights the actin
cable at the interface of the lateral epidermis and amnioserosa
(Fig. 6D,D', arrows) and
allows visualization of the progressive dorsal movement of the epidermis in
live embryos (Fig.
6D-D''). In contrast to wild-type embryos,
edMZ embryos during dorsal closure stages fail to exhibit
pronounced accumulation of GFP-moesin at the leading edge of the lateral
epidermis (Fig. 6E,E',
arrows), suggesting that the actomyosin cable fails to assemble. Moreover, in
these mutant embryos, the dorsal epidermis appears to buckle towards the
amnioserosa, suggesting that a lack of tension prevents the formation of a
taut interface with the amnioserosa
(Grevengoed et al., 2001). As
development proceeds, these embryos exhibit defective dorsal closure. The
dorsal movement of the lateral epidermis is delayed compared with wild-type
embryos, and discontinuities and puckering at the dorsal midline and
misalignment of opposing segments are ultimately observed. These data support
the hypothesis that the Ed expression border is required for assembly of the
supracellular actomyosin cable, and that the absence of this structure leads
to defective morphogenesis.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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The apical constriction associated with the loss of Ed appears to be restricted to the Ed expression boundary itself; individual ed mutant follicle cells that do not contact the clone border do not display pronounced apical constriction. Although the apical circumference of follicle cells in the interior of ed mutant clones occasionally appears reduced (see Fig. 2A',C'), this effect is not observed in larger clones (see Fig. 2E',F'). The reduction of apical circumference observed in individual ed mutant cells may therefore be a secondary consequence of the contractile force generated at the clone border, rather than a direct effect of the absence of Ed.
Although a smooth border has been reported previously for ed
mutant clones in the wing imaginal disc
(Wei et al., 2005), our data
are the first to reveal a developmentally regulated absence of Ed in specific
cell types associated with epithelial sheet movements. We show that Ed is
absent from the presumptive roof cells of the appendage primordia prior to
tube morphogenesis, and from the embryonic amnioserosa prior to dorsal
closure. In both cases, the resulting endogenous Ed expression borders are
smooth and display features of a contractile actomyosin cable, and loss of Ed
results in defects in epithelial closure. Because generation of ectopic Ed
expression borders is sufficient to generate a smooth contractile
intercellular interface, we interpret these defects as a result of the
elimination of the endogenous Ed expression borders between these tissues. We
propose that the juxtaposition of cells with and without Ed at these
endogenous interfaces induces local contractility of the actin cytoskeleton
that in turn drives the convergence of opposing epithelial domains during
morphogenesis.
Ed does not appear to play a role, however, in the generation of the
actin-rich smooth interface observed at the boundary between dorsal and
ventral compartments of the wing imaginal disc
(Major and Irvine, 2005).
Differential expression of Ed between dorsal and ventral compartments is not
detected, and ed mutant clones in either compartment exhibit smooth
borders (Rawlins et al.,
2003a; Wei et al.,
2005). Therefore, despite a general morphological similarity,
differential Ed expression does not appear to play a role at this epithelial
boundary.
Multiple forces contribute to morphogenesis
Although our data demonstrate that differential Ed expression generates a
contractile interface that is required for proper appendage tube formation and
dorsal closure, other forces also contribute to these processes. The
involvement of multiple forces is best understood for dorsal closure where, in
addition to the contractile actin cable at the epidermis/amnioserosa
interface, apical constriction of the individual amnioserosa cells also drives
the movement of the leading edge, particularly in the initial stages of the
process. In later stages, interactions between filopodia of opposing leading
edge cells also contribute to the completion of closure
(Jacinto et al., 2002;
Kiehart et al., 2000).
Consistent with the involvement of multiple forces, the lateral epidermal
edges do ultimately approach the dorsal midline in edMZ
embryos, suggesting that the elimination of the Ed expression border
specifically disrupts the actin cable, while the other forces remain
functional.
The cell movements and shape changes associated with the morphogenesis of
the appendage primordia appear very similar to those observed in dorsal
closure. In addition to the convergence of opposing floor cell domains to form
the tube floor, the individual roof cells constrict apically
(Dorman et al., 2004), similar
to the amnioserosa cells. This roof cell behavior is probably a consequence of
roof cell fate determination rather than the absence of Ed, as ed
mutant cells outside of this domain do not exhibit this same pronounced
reduction in apical circumference. Presumably the epithelial groove generated
by the coordinated apical constriction of the roof cells, together with the
elongation of the floor cells, can generate the rudimentary tubes that give
rise to the severely shortened and malformed appendages observed in the
absence of floor closure in ed mutant primordia.
Given the proposed role of Ed as a homophilic adhesion molecule
(Islam et al., 2003;
Rawlins et al., 2003a;
Spencer and Cagan, 2003),
selective affinity may also contribute to morphogenesis. For example, as the
anterior and medial floor cells elongate towards the midline of the
primordium, preferential affinity for the opposing floor cells, which also
express Ed, over the roof cells, which lack Ed, may favor floor cell
association. In dorsal closure, Ed-mediated interactions between opposing
leading edge cells could play a similar role. It is also possible that
differential Ed expression may have a dual function, contributing to
morphogenesis through generation of both a contractile interface and
differential affinity between cell types.
ed mutant follicle cells do not undergo premature cell death
In irradiated cultured epithelia, a smooth contractile interface has been
observed between apoptotic epithelial cells and their neighbors, suggesting
that active extrusion of dying cells preserves the integrity of the epithelium
(Rosenblatt et al., 2001).
This effect resembles the ed mosaic phenotype, but the presence on
the eggshell surface of imprints produced by ed mutant cells
indicates that these cells do not die before the secretion of eggshell at the
end of oogenesis. Moreover, ed mutant clones are not detectably
smaller than their associated twin spots and we detect no evidence of DNA
fragmentation or the active form of the proapoptotic enzyme caspase 3 in
ed mutant follicle cells (data not shown), confirming that the
contractile border of ed mutant clones is not induced by premature
cell death.
Ed expression borders affect adherens junction components
We have observed reduced levels and altered distribution of DE-cad and Arm
at the border between cells with and without Ed. By contrast, the distribution
and level of DE-cad and Arm at the interfaces between ed mutant
follicle cells within a clone appear normal. This observation demonstrates
that, although recent evidence suggests that Ed is a component of adherens
junctions (Wei et al., 2005),
Ed is not generally required for adherens junction stability.
A border effect on adherens junction components has also been reported in
ed mutant clones in the wing disc epithelium, where it has been
proposed to play a causative role in the generation of a smooth clone border
by mediating cell sorting (Wei et al.,
2005). However, at the border of ed mutant follicle cell
clones, this effect is frequently mild and occasionally undetectable, whereas
the contractile phenotype is completely penetrant. This difference could
suggest that a functionally relevant alteration in adherens junction
distribution is only occasionally reflected by diminished immunoreactivity.
Alternatively, this effect on adherens junction components could be instead a
consequence of contraction of the actin cable assembled at the Ed expression
border. Indeed, an actomyosin-based contractile force has been proposed to be
capable of disrupting adherens junctions
(Bertet et al., 2004;
Sahai and Marshall, 2002).
However, we have not observed a disruption of adherens junction components at
endogenous Ed expression borders (data not shown), raising the possibility
that this effect is not involved in Ed expression border function.
Local effect of Ed expression borders on the actin cytoskeleton
How an Ed expression border induces the local assembly of a contractile
actin cable remains unclear. A potential connection between Ed and the actin
cytoskeleton is suggested by the reported interaction between Ed and Canoe
(Cno), which is homologous to mammalian Afadin and contains a actin filament
binding domain, suggesting that Ed may function as a Nectin, the Afadin
binding partner (Takai and Nakanishi,
2003; Wei et al.,
2005). However, in ed mosaic wing imaginal discs, Cno
distribution is altered throughout ed mutant clones, not just at the
border (Wei et al., 2005).
This observation does not exclude a role for Cno in Ed function but, because
this effect on Cno is not restricted to the clone border, it alone cannot
explain the localized effect on the actin cytoskeleton. Interestingly, an
interaction with Ed does not appear to be strictly required for proper
membrane localization of Cno, as Ed is lost from the amnioserosa during dorsal
closure (see Fig. 8E') while Cno remains detectable
(Boettner et al., 2003;
Takahashi et al., 1998).
An obvious distinguishing feature of Ed expression borders is the absence of Ed from the apposing face of the Ed-expressing population, presumably owing to the absence of trans homophilic interactions. The mechanism that removes or redistributes Ed from this interface, rather than the absence of Ed itself, might therefore mediate the border-specific effect on the actin cytoskeleton. If the machinery that removes Ed, e.g. through endocytosis, is not completely specific, such a model could also account for altered levels of DE-cad and Arm at these interfaces. Alternatively, the absence of homophilic interactions across Ed expression borders could favor the interaction of Ed with other factors, which could in turn mediate border specific effects.
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/16/3255/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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