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First published online 31 October 2007
doi: 10.1242/dev.010397
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1 Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku
Kobe 650-0047, Japan.
2 Department of biology, Kobe University Graduate School of Science, Kobe,
Japan.
Author for correspondence (e-mail:
shayashi{at}cdb.riken.jp)
Accepted 7 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila melanogaster, EGFR, Cell intercalation, Invagination, Myosin, Trachea
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Leptin and
Grunewald, 1990; Oda and
Tsukita, 2001; Parks and
Wieschaus, 1991) and in the invagination of other organ placodes
(Llimargas and Casanova, 1999;
Myat and Andrew, 2000b;
Simoes et al., 2006).
Cell intercalation is thought to be the major force driving the global
transformation of epithelial shape, such as in embryonic body axis elongation
(Irvine and Wieschaus, 1994;
Keller, 2002) and gastrulation
(Ettensohn, 1985;
Hardin and Cheng, 1985). In
Drosophila, the orientation of intercalating individual cells or
multicellular rosettes of cells accompanies germ band extension under control
of the anterior-posterior (AP) patterning system
(Bertet et al., 2004;
Blankenship et al., 2006;
Zallen and Wieschaus, 2004).
Oriented cell intercalation is also observed in elongating tracheal branches
(Ribeiro et al., 2004). In
addition, cell intercalation and apical constriction depend on non-muscle
myosin, which is recruited to the cell-cell junctions and provides the
contractile force (Bertet et al.,
2004; Kiehart et al.,
2000).
The tracheal system of Drosophila is formed by the invagination
and branching of ectodermal epithelia
(Manning and Krasnow, 1993;
Samakovlis et al., 1996). The
tracheal fate is specified at stage 10, when the HLH-PAS protein Trachealess
(TRH) begins to be highly expressed in the tracheal placode
(Isaac and Andrew, 1996;
Wilk et al., 1996)
(Fig. 1A). Apical constriction
is observed in the dorsomedial region of the placode, where cells start to
invaginate until all the TRH-positive cells are internalized
(Fig. 1B). EGFR signaling is
activated within the tracheal placode under the control of TRH
(Gabay et al., 1997b). In
Egfr mutant embryos, the prospective tracheal cells fail to
concentrate F-actin at the constriction site
(Brodu and Casanova, 2006), and
the invagination is partially defective
(Llimargas and Casanova,
1999). It is not clear, however, to what extent apical
constriction and cell intercalation account for the highly coordinated process
of tracheal invagination or how EGFR signaling regulates these processes.
Progress in elucidating these events has been slow because of the lack of
precise knowledge about the cellular movements during invagination.
Here we used time-lapse imaging to trace the dynamic movements of cells and cell interfaces before and during tracheal invagination. We show that changes in the distribution of non-muscle myosin accompanies the rearrangement of cells in the tracheal placode, which encircle the invagination site by aligning in short rows arranged in arcs (a process called `boundary smoothing') and undergoing cell intercalation. These processes are controlled by EGFR, which is activated in an expanding circular pattern, thereby providing spatiotemporal information to cells in the tracheal placode. In the absence of EGFR, tracheal cells were internalized individually, suggesting that the key role of EGFR signaling is to coordinate the intrinsic ingression activity of the cells into the ordered process of invagination.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
trh-lacZ (Kassis et al.,
1992), MRLC-GFP (myosin-GFP)
(Royou et al., 2004),
btl-GFP-Moe (Kato et al.,
2004), rho-lacZ (a gift from Drs Kenji Matsuno and Yukio
Nakamura, Tokyo University of Science, Japan). Information on the following
stocks can be found in FlyBase
(http://flybase.net/):
rhodel1, Egfrf24, Dsor1Gp158,
pntdelta88, UAS-sspi, prd-Gal4, ovoD1 FRT101;
hs-FLP38, TM3, Kr-gal4 UAS-GFP.
Imaging
Imaging was performed as previously described
(Kato et al., 2004). Ten
1-µm-thick Z stacks were taken every 2 minutes over a period of up to 160
minutes. Embryos at this stage often rotated during imaging. Only images of a
few successful recordings that covered the entire invagination process were
used for cell tracing. The number of successfully imaged invagination events
and embryos (in parentheses) was: for GFP-Moesin-labeled embryos, control 9
(4), rhodel1 5 (2), Egfrf24 9 (4),
pntdelta88 7 (3), and for myosin-GFP-labeled control
embryos, 9 (3). Intercalating cells were marked and traced from still images,
and their centroid position was determined using the measurement function of
ImageJ (National Institutes of Health, USA). The angle and distance of cell
displacement was calculated. The invagination site was defined as the position
of the first cell to internalize. For the Egfrf24 embryos,
only cases in which the invagination took place only at one position were used
for analysis. The orientation of mitosis was determined in a similar manner,
except that the position of invagination was defined as the centroid of the
tracheal pit. Mutant embryos were identified by the absence of green balancer.
Non-randomness of the orientation of cell displacement and the cell division
axis were assessed by the Kolmogorov-Smirnov test.
Antibody staining
The primary antibodies were against the following proteins: DLG, E-cadherin
(Developmental Studies Hybridoma Bank), anti-double phosphorylated ERK
(dp-ERK; Sigma), KNI (Kosman et al.,
1998), GFP (rabbit, MBL; chick, Chemicon), myosin heavy chain
(MHC: a gift from Fumio Matsuzaki, RIKEN, Center for Developmental Biology,
Kobe, Japan) and ß-galactosidase (Cappel). The dp-ERK signal was
amplified with a TSA indirect kit (Perkin Elmer Life Sciences). Signal
intensity of MHC-stained cell boundaries were quantified by the use of ImageJ
and the statistical significance was tested by Student's t-test
(two-sample assuming equal variances, two-tail).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or myosin marker MRLC-GFP
(Royou et al., 2004)
(hereafter called myosin-GFP; Fig.
2, control, see Movies 1, 2 in the supplementary material) and
compared the results with the images of fixed preparations
(Fig. 1A,B)
(Brodu and Casanova, 2006;
Isaac and Andrew, 1996;
Llimargas and Casanova, 1999;
Manning and Krasnow, 1993;
Wilk et al., 1996). The use of
GFP markers allowed us to compare the dynamic distribution of F-actin and
myosin during invagination. About 50-70 minutes prior to the start of invagination, at cycle 15 of embryogenesis, the dorsal ectodermal cells entered mitotic quiescence (Fig. 2A and see Movie 1 in the supplementary material). Then, about 10 cells in the dorsal-medial part of the placode started to show apical constriction (Fig. 1Ba; Fig. 2B, yellow; defined as a reduction of apical cell surface below 5 µm2) and shifted basally about 3 µm (Fig. 1Bb, defined as time 0'). The cells with the constricted apical surfaces then became internalized, leaving a cleft called the tracheal pit (Fig. 1Bc, Fig. 2B, pink). Subsequently, cell internalization continued without apparent apical constriction (Fig. 1Bd), and mitotic activities resumed around the tracheal pit (Fig. 1Bc,d, Fig. 2B, asterisks). Time course analyses of apical constriction showed that those cells that internalized with sharply constricted apices (<1 µm2) were clustered at the center of the placode (Fig. 1C, red outline). We also mapped the timing of cell internalization within the tracheal placode and found that cells that internalize early were clustered at the center, surrounded by later internalizing cells in a stepwise manner, forming concentric circles (Fig. 1C). The cell size and temporal analyses of cell internalization demonstrated that the cells around a prospective tracheal pit internalized first with apical constriction (phase 1), followed by those surrounding them, with less extensive apical constriction (Fig. 1C). The timetable of the tracheal invagination events is shown in Fig. 1E.
Oriented cell intercalation and cell division in the tracheal placode
To examine how the concentric groups of cells formed, we analyzed the
pattern of cell intercalation in the 30-minute period prior to invagination,
by tracing the relative positions of neighboring cells
(Fig. 3A, blue and red arrows).
The direction of cell displacement was quantified by measuring the angle of
the line connecting the centroids of the two displaced cells
(Fig. 3B). We tested two
alternative hypotheses, (1) that cell intercalation follows a polar coordinate
system with the center situated at the point of invagination, or (2) that it
follows a Cartesian coordinate system defined by the AP and DV
(dorsal-ventral) embryonic axes. The displacement angle relative to the polar
coordinate (|
|) was 24.4±3.4° [average and
standard error (s.e.m.), n=21]. This value greatly deviates from the
average of 45° that would be expected if displacement occurred randomly
(Kolmogorov-Smirnov test, P<0.05). However, the average angle
relative to the AP axis (|'|) was
41.8±6.1° (n=18), which was judged to be random.
Therefore, the result favors the polar coordinate model.
After the initiation of invagination, the tracheal cells entered a final
wave of mitosis. The orientation of the cell-division axis of the placodal
cells was significantly biased toward the center of the tracheal pit
(||=14.6±2.78°, n=22,
P<0.01; Fig. 1D). A
radial orientation of cell division might help direct cells to flow into the
site of invagination. Taken together, these observations indicate that the
orientation of the cell displacement prior to invagination (phase 1), and the
cell division axis in phase 2 of invagination in the tracheal placode are both
aligned toward the site of invagination, suggesting that the planar cell
polarity in the tracheal placode is polarized toward the invagination
site.
Cell-boundary smoothing correlates with transient myosin accumulation
In the neighborhood of the tracheal placode cells undergoing intercalation
and we frequently noted groups of four to six cells forming arc-like rows that
collectively surrounded the future invagination site
(Fig. 3A, time 2'). By tracing
the contours of the cells in these rows, we found that these smooth arcs of
well-aligned cells arose from rows of cells whose contours formed zig-zagging
boundaries (Fig. 3A; green and
orange lines). We call this process `cell-boundary smoothing' and
characterized its cellular properties by monitoring the accumulation pattern
of myosin-GFP (Royou et al.,
2002) as a marker for contractility of the cell-cell junctions
(Bertet et al., 2004;
Kiehart et al., 2000) (see
Fig. 6 for quantitative
assessment of this process). Myosin-GFP was highly concentrated at the dorsal
border of the ectoderm that had begun to form the contractile supracellular
actomyosin purse string (Kiehart et al.,
2000) (Fig. 3C),
suggesting that myosin-GFP accumulation is a hallmark of contractile activity
at cell junctions. When dorsal ectodermal cells entered mitotic quiescence
before invagination, the myosin-GFP level in apical cell interface was
generally low except for a high accumulation between the straight rows of
cells abutting the segment boundary (see Movie 2 in the supplementary
material, time -40'). The prominent dot-like signals in each cell appeared to
be an artifact of the GFP fusion protein, because they were not detected with
anti-myosin heavy chain (MHC) antibody staining
(Fig. 6B) and were not
considered further. In the next 20 minutes, myosin-GFP accumulated at the
apical cell junctions of other ectodermal cells in non-uniform and rapidly
changing patterns. The signal was especially upregulated in shrinking cell
junctions undergoing cell intercalation
(Fig. 3E)
(Bertet et al., 2004). When the
arc-like rows of cells started to appear in the tracheal placode, the
myosin-GFP accumulation was often elevated in the smoothed boundaries of the
cellular arcs surrounding the invagination site
(Fig. 3C and see Movie 2 in the
supplementary material). We also noted a number of cell intercalation events
associated with smoothing boundaries (Fig.
3A).
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Spatial and temporal changes in ERK activation during invagination
As a candidate signal for coordinating the boundary smoothing and cell
intercalation, we studied the expression pattern of EGFR signaling. Active ERK
MAP kinase was visualized with an anti-double phosphorylated ERK (dp-ERK)
antibody (Gabay et al.,
1997a). dp-ERK was first detected in both the cytoplasm and nuclei
of the cells in the tracheal placode before they entered mitotic quiescence
and started to constrict apices (Fig.
4A), in a pattern roughly overlapping with the expression of the
EGFR activator rhomboid (rho;
Fig. 4F; detected by the
expression of rho-lacZ), within the cells in the tracheal placode
expressing trh-lacZ and KNI (Fig.
4F,G). The dp-ERK expression then expanded to cover the dorsal
half of the placode (Fig. 4B).
A y-z sectional view of the tracheal placode doubly labeled
for dp-ERK and nuclei (Fig.
4Bb) showed that the dp-ERK in the central domain was concentrated
in the apical cell cortex, and in the peripheral domain it accumulated in both
the cytoplasm and nuclei. Thus, cells with nuclear dp-ERK signals formed a
ring in a horizontal optical section at the basolateral level
(Fig. 4Bc). We found no sign of
apical constriction and cell boundary smoothing was partial at this stage
(Fig. 4Bd,e). During the next
stage, cell boundary smoothing proceeded and the central cells with an apical
accumulation of dp-ERK showed constricted apices and invaginated, and then
rapidly lost the dp-ERK signal during phase 2 of invagination, when mitotic
activity resumed (Fig. 4C and
see Fig. S1 in the supplementary material). dp-ERK was reduced in rho
mutants (data not shown), and lost completely in Egfr mutants
(Fig. 4D,E). We concluded that
the intracellular location of the dp-ERK changed from nuclear and cytoplasmic
to the apical cortex in about a 30-minute period prior to invagination, and
dp-ERK was subsequently eliminated after the onset of invagination. In the
tracheal placode, the pattern of nuclear dp-ERK expression was
spatiotemporally regulated by EGFR: it originated from a central spot and
expanded into a ring that encircled the prospective site of the apical
constriction and invagination.
|
;
Wappner et al., 1997). In
mutants of rho, a positive regulator of EGFR signaling, some tracheal
precursor cells fail to invaginate and remain at the epithelial surface
(Llimargas and Casanova, 1999)
(Fig. 5B). A similar phenotype
is observed in mutants of EGFR and the ETS domain transcription factor Pointed
(PNT), a key target of ERK signaling
(Brunner et al., 1994;
O'Neill et al., 1994)
(Fig. 5C,D), suggesting that
EGFR signaling and its nuclear transduction are essential for proper
invagination.
To investigate the role of EGFR signaling in the tracheal placode
invagination events, time-lapse imaging of rho, Egfr and pnt
mutants was performed, and their phenotypes were compared with that of control
embryos. As a staging reference, we used the cell division in the dorsal
epidermis that normally resumes about 60 minutes after the onset of
invagination (Fig. 2B-E). We
found that defects in the Egfr mutants were already apparent at the
onset of invagination. In the Egfr mutants apical constriction was
nearly absent (Fig. 2C,
compared with the yellow-colored cells in 2B), and the remaining apical
constriction event did not correlate with the position of invagination. These
defects in apical constriction are consistent with a previous report
implicating EGFR signaling in the initiation of local F-actin assembly
(Brodu and Casanova, 2006).
Furthermore, we found that the onset of invagination was delayed in the
Egfr mutants, and some cells resumed mitosis before the start of
invagination (Fig. 2C, 46
minutes, asterisk), suggesting that the temporal order of invagination and
mitosis was miscoordinated. The delayed invagination was confirmed by
examining fixed mutant embryos. In control embryos, tracheal precursor cells
started to constrict their apices and shift basally when the groove between
the maxilla and labium ingressed deeply, but no such morphological changes
were detected in the tracheal placodes of Egfr mutants at the
equivalent stage (data not shown). We also imaged rho and
pnt mutant embryos and found that invagination was delayed and apical
constriction was less extensive than in control embryos
(Fig. 2D,E and see Movies 4, 5
in the supplementary material). In addition, we monitored cell boundaries in
pnt mutants and found some degree of smoothing (see Fig. S2 in the
supplementary material). The phenotypes of rho and pnt
mutants are weaker version of the Egfr mutant phenotype. These data
suggest that EGFR signaling is required for the apical constriction and
specification of the timing of invagination.
|
). In the
expanded tracheal placode of the Egfr mutants, the position of the
tracheal pit was more variable and tended to shift ventrally, and occasionally
two tracheal pit-like indentations were observed
(Fig. 5G,I). To determine
whether ERK signaling was involved in this process, we studied the phenotype
of maternal-zygotic mutants of Dsor1, which completely lack ERK
kinase activity (Hou et al.,
1995; Tsuda et al.,
1993). These mutants had misplaced and duplicated tracheal pits
(Fig. 5J). Time course analyses
of the Egfr mutants revealed multiple incidences of one or two cells
being internalized prior to the full invagination of the larger group of cells
forming the visible tracheal pit (Fig.
2C and see Movie 3 in the supplementary material). Most of the
cells undergoing these individual internalization events were adjacent to
mitotic cells.
Although frequent mitosis appeared to interrupt the cell rearrangement
process, we were able to detect some arc-like cellular rows in the
Egfr mutant placode (Fig.
2C, time 60'; see Movie 3 in the supplementary material). However,
the pattern of these arc-like rows did not correlate with the position of the
invagination site (Fig. 2C). We
also detected a limited number of cell intercalation events, and measurement
of their orientation revealed a || that was judged to be
non-biased (43.9±7.2°, P>0.05). Taken together, these
results suggest that EGFR signaling is required for the proper patterning of
cell rearrangement and to restrict the site of tracheal invagination to a
single focus at the dorsal region in tracheal primordia.
|
To quantify the differential distribution of myosin, we classified cell boundaries of class T junctions into horizontal and vertical (Fig. 6D), and their intensity was compared. Intensity of horizontal class T boundaries was 1.52±0.11(mean±s.e.m., arbitrary unit, n=33), that was significantly increased compared to 1.00±0.11 in vertical boundaries (n=37, P<0.002, Student's t-test). Such enrichment of myosin to horizontal boundaries was observed in class T junctions both in arcs or not. Since two-thirds of class T junctions formed arcs, we conclude that myosin is preferentially enriched in cell junctions consisting of arcs.
To test whether EGFR was capable of inducing this cortical myosin
accumulation, we ectopically activated EGFR in otherwise flat epithelia by
expressing the secreted (activated) form of an EGFR ligand, Spitz (sSPI)
(Schweitzer et al., 1995). The
expression of sSPI by the paired enhancer
(Brand and Perrimon, 1993)
activated ERK kinase in broad bands that covered the even-numbered
parasegments. At the interface of cells expressing ectopic dp-ERK and their
neighbors, we often observed an elevated myosin accumulation
(Fig. 6E, arrowheads).
Comparison of myosin accumulation in this type of border (DV borders,
Fig. 6F) versus intersecting
borders (AP borders) demonstrated that dp-ERK activation specifically enriched
myosin accumulation in the interface with low dp-ERK-expressing cells
(Fig. 6F). The result suggests
that the juxtaposition of cells with high and low levels of EGFR activity can
trigger myosin accumulation at the cell interface. Furthermore, within the
region of high dp-ERK activity, we noted the formation of deep epidermal
depressions at the sites where segmental furrows would form
(Fig. 6E, asterisk): the
depressed region had abundant cells with constricted apices and became
continuous with the expanded tracheal pit. The odd-numbered parasegments
remained flat (arrow). These results suggest that a high level of EGFR
signaling promotes the apical constriction of epidermal cells and precocious
epidermal depression.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Apical constriction converts cells into a bottle-like
shape and is thought to be an important process in invagination
(Myat and Andrew, 2000a).
However, the observation that cells can be internalized without undergoing
apical constriction during normal invagination of the Drosophila
mesoderm (Oda and Tsukita,
2001) and trachea (this work), and as a result of mutations that
disturb apical constriction but allow the invagination of a majority of
placodal cells (Llimargas and Casanova,
1999; Oda and Tsukita,
2001; Sweeton et al.,
1991) suggest that the critical role of apical constriction is not
in invagination per se, but is likely to be modulatory. We have shown here
that invagination of the tracheal placode involves, prior to the start of
invagination, the rearrangement of cells to form multicellular arcs with
smooth boundaries. We have also shown here that polarized cell intercalation
and boundary smoothing take place simultaneously in multiple waves, and
together with apical constriction, coordinate the cells' intrinsic
internalization activities to allow precisely regulated invagination. Our
findings suggest a model in which EGFR signaling regulates this process
(Fig. 6G).
The role of localized myosin activity in invagination
We have shown that the accumulation of myosin-GFP was elevated in the
boundaries of cells that were arranged in arc-like rows. The linkage of a
cortical actomyosin network through such a chain of cells via a cell-cell
adhesion complex would result in the formation of supracellular actomyosin
cables similar to those observed at the leading edge of the dorsal epidermis
(Hutson et al., 2003;
Kiehart et al., 2000).
Although the cables formed were transient and weak compared with their
counterparts in the epidermal leading edge, their contractile force in convex
cell contours would help smooth the boundary and compress cells toward the
center, thereby accounting, at least in part, for the dense cell packing in
the prospective tracheal pit. In addition, polarized cell intercalation
contributes to cell packing by pushing cells toward the center and connecting
cell boundaries to form larger arcs. These packed, apically constricted cells
were first to internalize (Fig.
6G).
|
), it
was not clear whether EGFR influences cells surrounding the invagination site.
Here we showed that the nuclear transduction of EGFR signaling monitored by
dp-ERK formed a transient wave that swept from the center to the periphery of
the placode. Since PNT, the major nuclear target of EGFR-ERK signaling, was
required in this process, nuclear dp-ERK must represent the major EGFR
signaling output required for invagination. Our observation that nuclear
dp-ERK was already extinguished at the time of invagination suggests that the
effect of EGFR in invagination might be indirect. Since the spreading front of
the dp-ERK often correlated with high myosin accumulation, we speculate that
the circular propagation of EGFR signaling in the peripheral region helps to
specify the cell-cell interface where myosin accumulates.
A good candidate for the link between nuclear dp-ERK and myosin regulation
is the RHO GAP gene cv-c, which is transcriptionally upregulated by
EGFR (Brodu and Casanova,
2006). Since RHO GAP is linked to the regulation of myosin
contractility through the RHO-RHO kinase-myosin regulatory light-chain pathway
(Amano et al., 1997;
Winter et al., 2001), the
transcriptional activation of cv-c within a region of high EGFR
activity should downregulate myosin activity. Although the mechanism is not
known, a difference in myosin activity between neighboring cells might trigger
contractile activity. It follows that the circular spread of EGFR activity
would cause the sequential modulation of myosin contractility throughout the
tracheal placode. The requirement for pnt for proper invagination
indicates transcriptional regulation is essential. However, the relatively
weaker invagination phenotype in pnt mutants compared to
Egfr mutants suggests that another nuclear target, or a non-nuclear
pathway of EGFR signaling through apically accumulated dp-ERK regulates
invagination.
In Egfr mutants, the onset of invagination was delayed and took place at variable positions, and phase 1 of invagination was apparently skipped. These phenotypes can be explained by the loss of the putative centripetal force driven by the myosin-dependent contraction of the arc-like rows of cells. One striking observation in the Egfr mutants was the internalization of multiple individual cells prior to invagination. This observation indicates that EGFR is dispensable for cell internalization per se, but is essential for focusing the cell internalization activity, thus specifying the time and place of the initial invagination. This result also indicates that cells in the tracheal placode possess an EGFR-independent cell-ingression activity. Although the origin of this activity is not understood, it may be under the control of genes specifying the tracheal placode, such as trh and vvl. It is interesting to note that the cell internalization events observed in the Egfr mutants were often associated with mitosis, which might alter the local surface tension in the placode to permit neighboring cells to ingress. Thus, EGFR seems to restrict the invagination site to one place through two mechanisms: first by coordinating the circular cell rearrangement, and second by suppressing cell division to stabilize the surface tension of the placode.
The activation of EGFR signaling started from one spot and spread out in a
ring that encircled the prospective invagination site. Since the pattern of
rho transcription was not sharply focused in the tracheal placode, it
is likely that the secreted Spitz ligand is broadly distributed. The circular
spread of EGFR activation from this crude distribution of EGFR ligand might
require feedback regulation of EGFR signaling
(Shilo, 2005), as has been
shown in other cases (Brodu et al.,
2004; Wasserman and Freeman,
1998).
In this study, the precise mapping of dp-ERK and cell behaviors allowed us
to demonstrate the role of EGFR signaling in cell rearrangement.
Drosophila leg development bears several similarities to tracheal
invagination: it develops by evagination, a process fundamentally similar to
invagination, and requires graded EGFR activity
(Campbell, 2002;
Galindo et al., 2002) and TRH
(Tajiri et al., 2006). FGF
signaling is known to have a role in the invagination of the lens and otic
placode in the chick and mouse (Faber et
al., 2001; Ladher et al.,
2005). The concept of cell rearrangement at the signal propagation
front may be useful for understanding the cellular mechanisms underlying the
shape transformation of other organ placodes under the control of receptor
tyrosine kinases.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4273/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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1.5 fold in the
Egfr-/- (F) compared with Egfr+/- (E)
embryos. (G) In the Egfr mutants, the invagination position
(arrows) was variable and sometimes duplicated. (H-K) Position of
invagination sites (% position measured from the dorsal edge) and the number
of sites in controls, and in Egfr, Dsor1 and pnt mutants.
The incidence of double invagination observed in Egfr and
Dsor1 embryos was counted separately, without reference to their
position. Scale bars: 10 µm.