Segment boundary formation in Drosophila embryos
Camilla W. Larsen,
Elizabeth Hirst,
Cyrille Alexandre and
Jean-Paul Vincent*
National Institute for Medical Research, The Ridgeway Mill Hill, London
NW7 1AA, UK
*
Author for correspondence (e-mail:
jvincen{at}nimr.mrc.ac.uk)
Accepted 14 August 2003
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SUMMARY
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In Drosophila embryos, segment boundaries form at the posterior
edge of each stripe of engrailed expression. We have used an HRP-CD2
transgene to follow by transmission electron microscopy the cell shape changes
that accompany boundary formation. The first change is a loosening of cell
contact at the apical side of cells on either side of the incipient boundary.
Then, the engrailed-expressing cells flanking the boundary undergo
apical constriction, move inwards and adopt a bottle morphology. Eventually,
grooves regress, first on the ventral side, then laterally. We noted that
groove formation and regression are contemporaneous with germ band retraction
and shortening, respectively, suggesting that these rearrangements could also
contribute to groove morphology. The cellular changes accompanying groove
formation require that Hedgehog signalling be activated, and, as a result, a
target of Ci expressed, at the posterior of each boundary (obvious targets
like stripe and rhomboid appear not to be involved). In
addition, Engrailed must be expressed at the anterior side of each boundary,
even if Hedgehog signalling is artificially maintained. Thus, there are
distinct genetic requirements on either side of the boundary. In addition,
Wingless signalling at the anterior of the domains of engrailed (and
hedgehog) expression represses groove formation and thus ensures that
segment boundaries form only at the posterior.
Key words: Drosophila embryos, Segmentation, Boundaries, hedgehog, engrailed, TEM
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Introduction
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The establishment of boundaries between groups of cells is a general
feature of developing animals. Preventing populations of cells to intermingle
allows patterning and growth to be controlled in well-defined compartments.
Moreover, boundaries are ideally suited to be a source of morphogen
(Basler and Struhl, 1994
)
(reviewed by Lawrence and Struhl,
1996
). One classic example of a compartment boundary is the border
that divides Drosophila imaginal disks into anterior and posterior
compartments (also known as the AP boundary). This boundary is established
early in embryogenesis at the anterior of each stripe of engrailed
expression and is maintained throughout the life of the fly
(Garcia-Bellido et al., 1973
;
Vincent and O'Farrell, 1992
).
The role of Engrailed in compartment boundary maintenance in the wing imaginal
disc was recognized nearly 30 years ago
(Morata and Lawrence, 1975
).
It is now established that this role is dual. On the one hand, Engrailed
imparts a specific `affinity' to posterior cells and thus encourages them to
sort out from cells in the A compartment
(Blair and Ralston, 1997
;
Dahmann and Basler, 2000
). On
the other hand, Engrailed activates the expression of Hedgehog, which signals
across the boundary and renders receiving cells immiscible with posterior,
engrailed-expressing cells (Blair
and Ralston, 1997
; Rodriguez
and Basler, 1997
). That Hedgehog signalling is indeed required in
anterior cells is demonstrated by the behavior of anterior cells that lack
either cubitus interruptus (ci) or smoothened
(smo), two essential components of the Hedgehog signal transduction
pathway. Such clones no longer respect the boundary even if engrailed
is expressed normally on the other side
(Blair and Ralston, 1997
;
Rodriguez and Basler, 1997
).
Because Ci is the transcription factor that mediates Hedgehog signaling, it
appears that the effect of Hedgehog on boundary maintenance is mediated by the
transcriptional activation of one or several genes in anterior cells lining
the boundary.
Cell sorting in imaginal discs could depend on a difference in affinity
between cells on either side of the boundary
(Lawrence, 1993
). Differential
adhesion models such as that proposed by Steinberg
(Steinberg, 1962
) state that
cells with similar affinity adhere preferentially with each other and sort out
from cells of different affinity. Differences in adhesion between two cell
populations could result from either a difference in concentration of one type
of adhesion molecule or the differential expression of distinct adhesion
molecules (Dahmann and Basler,
2000
). So far, no specific adhesion molecule has been identified
that is required for maintaining the boundary between the anterior and
posterior compartment. At the dorsoventral (DV) boundary of imaginal disks,
two putative cell adhesion molecules, the single pass transmembrane proteins
encoded by tartan and capricious, have been shown to
contribute to boundary maintenance (Milan
et al., 2001
). However, as yet, compartmental expression of
tartan and capricious does not fully account for boundary
maintenance as loss-of-function clones still respect the boundary. In the
vertebrate hindbrain, another class of membrane-associated proteins have been
implicated in boundary formation. There, lack of cell mixing across rhombomere
boundaries depends on the interaction between Eph receptors and their
GPI-anchored ligands, the ephrins, which are expressed in a complementary
fashion in alternate segments (reviewed by
Wilkinson, 2001
). Current data
suggest that these molecules control cell affinities by activating downstream
signalling, which leads to active repulsion between cells in neighbouring
rhombomeres.
The Drosophila embryo is another system where boundaries can be
studied both genetically and morphologically. During early development, the
embryonic epidermis becomes divided into a series of repeated patterning units
termed parasegments (Lawrence and Struhl,
1996
; Martinez-Arias and
Lawrence, 1985
). Parasegment boundaries are clonal boundaries that
form at the anterior edge of each stripe of engrailed expression as
soon as cellularization is complete
(Vincent and O'Farrell, 1992
).
They are maintained throughout the life of the fly and indeed give rise to
compartment boundaries in imaginal disks
(Garcia-Bellido et al., 1973
).
Around stage 11 of embryonic development, another boundary forms at the
posterior edge of each engrailed stripe. This boundary is easily
recognisable as deep grooves in the epithelium and marks the edge of each
segment. As a foundation to uncover the cell biological basis of segment
boundary formation, we have studied the morphological changes that accompany
this process and its genetic requirements.
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Materials and methods
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Fly stocks
The following mutant alleles were used: wgCX4
(Baker, 1987
),
hhAC (Lee et al.,
1992
), Df(2R)enE
(Tabata et al., 1992
),
ci94 (Methot and
Basler, 2001
), ciCell
(Slusarski et al., 1995
),
stripeDG4, rhomboid7M43
(Jurgens et al., 1984
),
zipper1 and hindsightE8. The
wgcx4 Df(2R)enE recombinant was a kind
gift from Peter Lawrence. The following Gal4 drivers and responders were used.
engrailed-Gal4 and UAS-lacZ (gift from Andrea Brand,
Cambridge, UK), tubulin-Gal4
(Pignoni and Zipursky, 1997
),
buttonhead-Gal4 (gift from Gines Morata, Madrid),
paired-Gal4 (gift from C. Desplan, NYU, USA), UAS-wingless
(Lawrence et al., 1995
),
UAS-armS10 (Pai et
al., 1997
), UAS-engrailed
(Guillen et al., 1995
) and
UAS-hedgehog (Fietz et al.,
1995
). UAS-CiVP16 was made by inserting DNA encoding the
activation domain of HSV VP16 in the BclI site of ci located
three codons upstream of the stop codon. This C-terminal fusion was then
transferred into pUAST. UAS-CD2-HRP was constructed as follows: DNA coding for
HRP along with the signal peptide from Wingless was amplified by PCR from
UAS-wingless-HRP (Dubois et al.,
2001
). This was ligated in frame to a PCR fragment encoding most
of CD2 (from Lys25 to the C terminus) and then transferred into pUAST.
Embryo staining and in situ hybridisation
Standard protocols were used for immunocytochemical staining. Antibodies
used were rabbit anti-ß-galactosidase (Sigma), mouse anti-Engrailed (4D9)
and mouse anti-wingless (4D4) (both from the Developmental Studies Hybridoma
Bank), and goat anti-HRP (Sigma). In situ hybridisation was performed as
described by Jowett (Jowett,
1997
), except that fixed embryos were kept at 100% methanol and no
proteinase K treatment took place. The probe was made from a hedgehog
cDNA obtained from M. van den Heuvel (Oxford, UK).
Scanning and transmission electron microscopy
Visualisation of HRP as well as post-fixation and embedding for TEM was
performed as described by Dubois et al.
(Dubois et al., 2001
) except
for the following modifications. The vitelline membrane was permeabilised
before fixation by incubating embryos in n-Octane for 3 minutes. Embryos were
then washed in 0.1 M sodium cacodylate buffer and fixed in 2% gltuteraldehyde
in 0.1 M sodium cacodylate buffer for 20 minutes. After fixation embryos were
washed in 0.1 M sodium cacodylate (pH 7.2) buffer and then devitelinised by
hand in PBS. For SEM, embryos were fixed and processed in the same way as for
TEM and then post-fixed in 1% osmium tetroxide in a 0.1 M sodium cacodylate
(pH 7.2) buffer. Dehydration was through a graded ethanol series. After
dehydration embryos were critical point dried from carbon dioxide and sputter
coated with 10 nm gold and viewed in a Jeol 35CF SEM.
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Results
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Morphogenesis of segmental grooves
Segmental boundary formation is initiated shortly after germ-band
retraction has begun. They are recognisable as periodic indentations in the
epidermis that separate cells expressing engrailed at the anterior
from those expressing rhomboid at the posterior
(Fig. 1A). To understand the
mechanisms involved in boundary formation, we examined the changes in cell
morphology before and during boundary formation by transmission electron
microscopy (TEM). To allow identification of cells in electron micrographs, we
devised a transgenic membrane marker based on horseradish peroxidase (HRP),
which catalyses the production of an electron-dense product from
diaminobenzidine (DAB). HRP was fused to the transmembrane protein CD2 so that
the marker would outline cells and thus reveal cell shapes
(Fig. 1B). This inert fusion
protein was expressed under the control of engrailed-Gal4, so that
the membrane of engrailed-expressing cells appears dark under the
electron microscope.

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Fig. 1. Morphological changes during segmental boundary formation. (A) Schematic
drawing of gene expression patterns in a horizontal section through one
segmental unit. The position of the segmental boundary is marked with a
vertical bar. (B) Schematic drawing of the fusion protein used to label cell
outlines under the EM (under UAS control). It comprises the signal peptide
from Wingless, human CD2 (without signal peptide) and HRP. (C-K) TEM images
showing the changes in cell morphology as segmental grooves form and regress.
Embryos were stained with DAB and sectioned horizontally through the ventral
aspect of the embryo. Although some staining appears at the surface of
non-expressing cells (maybe as a result of membrane shedding from expressing
cells), we were able to confidently identify expressing cells after a bit of
practice. An annotated version of this figure highlighting expressing cells is
provided at
http://dev.biologists.org/supplemental/.
(C) Shortly after germ-band retraction is initiated, a small dip (arrow)
appears between engrailed-expressing and non-expressing cells. (D)
Apical contact appears to loosen (arrow). (E) The most posterior
engrailed-positive cell constricts apically and moves inwards in
relation to surrounding cells. (F) This cell finds itself at the bottom of the
forming groove and neighbouring cells follow this inward motion. (G) More
cells have moved in and the groove is now two to three cell diameters deep.
(H) The groove at its deepest reaches at least three cell diameters in depth.
(I) At this stage the bottom engrailed-expressing cell is bottle
shaped and severely constricted apically (arrow). (J) The disappearance of
grooves is a very rapid event, which allows the cells to return to their
original position. (K) The embryo eventually becomes almost flat ventrally.
(L-N) Grooves are much deeper laterally (L) than ventrally (M), and posterior
grooves (between abdominal segments 2 and 3; N) are not as deep as anterior
ones (between abdominal segments 7 and 8; M). Scale bars: 500 nm.
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Cell shape changes during groove formation were studied in horizontal
sections through the ventral aspect of the embryo at the level of parasegment
9 (the boundary between abdominal segments 3 and 4). Groove formation begins
shortly after initiation of germ band retraction as a slight splaying between
HRP-positive and HRP-negative cells (arrow in
Fig. 1C). As this slit matures
into the boundary, we refer to the cells on either side as `groove founder
cells'. The groove founder cells further lose contact apically, and a groove
forms between them (Fig. 1D).
Subsequently, in any one section, the cell at the anterior of the incipient
boundary (the one expressing engrailed) appears to constrict its
apical surface. At the same time, it moves towards the interior of the embryo
(Fig. 1E), seemingly pulling
neighbouring cells along. As boundary formation proceeds, this cell becomes
positioned at the bottom of the groove and begins to adopt a bottle shape
(Fig. 1F). The cells
neighbouring the groove founder cells follow this inward movement, and also
display partial apical constriction. The groove continues to deepen
(Fig. 1G), until the bottle
cell, which is still HRP positive, ends up three to four cell diameters below
the surface of the embryo (Fig.
1H). This cell remains at the bottom of the groove with its apex
constricted (arrow Fig. 1I)
until late stage 13, coinciding with the onset of dorsal closure. After this
stage, in the ventral region, the groove regresses
(Fig. 1J) until stage 15, when
it has practically disappeared (Fig.
1K). At lateral positions, a similar sequence of events is seen,
but with two quantitative differences. Lateral grooves dig deeper into the
embryo and regress later than ventral ones (compare
Fig. 1L with 1M). In
conclusion, groove formation involves specific changes in cell contact between
the groove founder cells, apical constriction of the most posterior
engrailed-expressing cells, and inward migration of cells surrounding
the groove.
As indicated above, the most posterior engrailed-expressing cells
display a distinctive behaviour during groove formation. So far we have not
been able to track the fate of this cell as the grooves disappear. However, we
have obtained evidence that it ceases to express Engrailed around the time
when grooves are deepest. Embryos expressing HRP-CD2 under the control of
engrailed-Gal4 were stained for HRP (green) and Engrailed protein
(red) (Fig. 2). As the groove
grows deeper, Engrailed and HRP are co-expressed
(Fig. 2A,B) as expected.
However, at later stages, Engrailed protein is no longer detectable in the
bottle cell, whereas HRP membrane stain remains, presumably because HRP is
relatively stable (white arrow in Fig.
2C). Thus, during groove formation the most posterior
engrailed-expressing cell changes morphology dramatically and, upon
completion of this process, stops expressing the Engrailed protein.

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Fig. 2. Loss of engrailed expression in the `bottle cells'. (A-C) Lateral
view (focused on the ventral midline) of wild-type embryos stained with
anti-Engrailed (red) and anti-HRP (green). (A,B) At stages 12 and 13,
Engrailed and HRP immunoreactivity co-localises (although this is not clear at
all focal planes). (C) By contrast, at stage 14 the so-called bottle cell
downregulates Engrailed expression although it remains labelled with HRP
(white arrow). No attempt was made to identify the staining detected inside
the embryo, which could be in the mesoderm or the nervous system.
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We note here that groove formation coincides with germ band retraction as
if segments were being compressed, much like an accordion. The first segments
to undergo such apparent compression are the most anterior ones and this is
where grooves are deepest (compare Fig.
1M,N). Another noteworthy temporal correlation is between the
disappearance of grooves and dorsal closure, a process whereby the epidermis
spreads dorsally to enclose the whole embryo. Thus, it could be that the need
for additional surface area during dorsal closure promotes groove regression.
To investigate this further, we looked at zipper mutants, which are
defective in dorsal closure, albeit with a variable penetrance
(Cote et al., 1987
). In those
zipper mutants that completely fail to undergo dorsal closure,
grooves persist longer. For example, ventral grooves can be seen well into
stage 15 (staging based on anterior morphology and time of egg laying) (black
arrows in Fig. 3C,D), a stage
when the ventral surface of wild-type siblings is relatively smooth
(Fig. 3A and black arrow in
Fig. 3B). Moreover, at lateral
positions, grooves appear to be deeper in zipper mutants (white arrow
in Fig. 3D) than in wild type
(white arrow in Fig. 3B).

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Fig. 3. Persistence of segmental grooves is affected by dorsal closure. (A-D) Wild
type and zipper mutants, which are defective in dorsal closure, at
stage 15 and oriented such that the ventral midline is at the bottom. (A)
Wild-type embryo stained with anti-Engrailed (black). (B) Wild-type embryo as
seen by scanning electron microscopy (SEM). The ventral epidermis is almost
flat (black arrow), whereas shallow grooves are still present laterally (white
arrow). (C) Brightfield image of a zipper mutant embryo stained with
anti-Engrailed (black). Note that grooves persist ventrally (black arrow). (D)
Persistent ventral grooves can also be seen by SEM (black arrow). Moreover,
lateral grooves appear deeper (white arrow) than in the wild type.
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Segment boundary formation requires Hedgehog signalling
There is circumstantial evidence that both Engrailed and Hedgehog could be
involved in segment boundary formation. Boundaries fail to form in
engrailed and hedgehog mutant embryos. Moreover, as
described in the Introduction, both Engrailed and Hedgehog are implicated in
maintenance of the compartment boundary in wing imaginal disks
(Rodriguez and Basler, 1997
;
Blair and Ralston, 1997
).
Because Engrailed activates hedgehog expression and hedgehog
signalling activates wingless expression, which is itself needed for
continued engrailed expression, expression of hedgehog and
engrailed are interdependent during embryogenesis
(di Nardo et al., 1988
;
Martinez-Arias et al., 1988
;
Lee et al., 1992
), thus
complicating the genetic analysis. To investigate the specific contribution of
each gene on boundary formation, we devised genetic combinations that allowed
expression of one without the other. To maintain continued engrailed
expression in a hedgehog null mutant, an activated form of Armadillo
(Arm*, armS10) (Pai et al.,
1997
) was expressed under the control of engrailed-Gal4,
thus artificially maintaining wingless signalling in the
engrailed domain and rendering engrailed expression
independent of Wingless. No segmental groove form in such embryos
(Fig. 4C). The surface of the
epidermis appears smooth at the time when deep grooves can be seen in wild
type siblings (Fig. 4A). As
expected, engrailed expression is sustained in these embryos, however
segmental organisation is disrupted (Fig.
4D). Engrailed-positive cells are no longer confined to sharply
delineated stripes as in the wild type
(Fig. 4B), but are randomly
positioned in small clumps of cells throughout the epidermis. We conclude that
Hedgehog signalling is required for segment boundary formation and also for
maintenance of segmental organisation.

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Fig. 4. Boundary formation requires Hedgehog and Ci. (A-H) Stage 13+ embryos
stained with anti-Engrailed (black or brown). (A,B) Wild-type embryos. Deep
grooves are easily seen (black arrows) in the lateral view in A, while the
ventral view shows the normal stripes of Engrailed expression (two to three
cell diameters wide). (C,D) Embryos lacking hedgehog but continuing
to express engrailed (full genotype is shown). No groove form as seen from the
lateral view focused on the ventral midline (C) and stripes of
engrailed-expressing cells are broken up into clumps as seen in the
ventral view (D). (E,F) In ci94 embryos (with artificially
maintained Engrailed), grooves form (black arrows in E) and Engrailed stripes
appear normal (F). (G,H) In ciCell embryos (with
artificially maintained Engrailed), grooves do not form (G) and there is
moderate disruption of the Engrailed stripes (H). (I-L) Cartoons summarising
the results shown in panels above. (M,N) SEM of stage 13 embryos. Compare the
wild type in M with a hedgehog mutant (with artificially maintained
Engrailed) in N.
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Canonical signalling by Hedgehog is mediated by the transcription factor
encoded by ci (Aza-Blanc et al.,
1997
; Methot and Basler,
2001
). In the absence of Hedgehog, full-length Ci is
constitutively processed to a repressor form, Ci[75]. In the presence of
Hedgehog, Ci[75] is no longer produced and full-length Ci[155] can activate
target genes. To test whether the role of Hedgehog signalling in boundary
formation requires ci, as is the case in the wing disk, we looked at
groove formation in ci mutant embryos. As above, engrailed
expression was artificially maintained (with engrailed-Gal4
UAS-arm*). Two alleles of ci were used: ci94,
which lacks all Ci protein (i.e. both the repressor and the activator forms)
and ciCell, which encodes only Ci[75], the repressor form
(Methot and Basler, 2001
). The
result differs for the two alleles. In ci94, segmental
grooves and segmental organisation appear normal
(Fig. 4E,F) as in the wild type
(Fig. 4A,B). By contrast, in
ciCell, grooves are lacking
(Fig. 4G) and the domain of
engrailed expression (artificially maintained) is disorganised
(Fig. 4H) much as in a
hedgehog mutant. This suggests that a target of Ci is required for
boundary formation and that, in the absence of signalling, expression of this
target is repressed by Ci[75].
Wingless signalling inhibits segmental boundary formation
Hedgehog signals to cells located both at the posterior and the anterior of
the engrailed-expressing compartment. Yet, segment boundaries only
form at the posterior. What could be the reason for this asymmetry? One
obvious possibility is that Wingless, which is active at the anterior of each
engrailed stripe, could prevent boundary formation there. Indeed,
such a regulatory mechanism ensures that rhomboid is only expressed
at the posterior of each stripe of hedgehog expression
rhomboid expression is activated by Hedgehog signalling and repressed
by Wingless signalling (Alexandre et al.,
1999
). To assess the role of Wingless signalling on segmental
grooves, we looked at wingless mutants in which engrailed
(and hedgehog) expression was artificially sustained with the
engrailed-Gal4 UAS Arm* system. In the ventral region,
engrailed expression is maintained in defined stripes
(Fig. 5A) and grooves form on
both sides (Fig. 5B) suggesting
that, indeed, Wingless signalling normally prevents Hedgehog from activating
groove formation at the anterior. More laterally, the segmental organisation
is disrupted and engrailed-expressing cells are often found in small
groups (Fig. 5C) surrounded by
grooves (arrow in Fig. 5D).
Disruption of the integrity of engrailed stripes at lateral positions
could be due to a failure to maintain parasegment boundaries in the absence of
Wingless and to a differential requirement for Wingless along the DV axis.
Importantly for the purpose of this paper, grooves forms around all
engrailed-expressing cells whether they are in stripes or loosely
arranged in groups. To confirm that these grooves are indeed due to the action
of Hedgehog; the same experiment was repeated in the absence of both
wingless and hedgehog (wingless
engrailed-Gal4 UAS-Arm* hedgehog). In these embryos,
grooves are abolished altogether (Fig.
5F,G). Furthermore the stripes of engrailed expression
are disrupted ventrally (Fig.
5E) as well as laterally.

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Fig. 5. Wingless signalling inhibits segmental boundary formation. All embryos are
at stage 13+ and stained by immunocytochemistry with anti-Engrailed (black).
(A-D) Removal of Wingless (while maintaining engrailed expression)
leads to duplication of segment boundaries. An `en face' view of the ventral
area (A) shows that engrailed stripes are sharply delineated on both
sides. In a side view of the ventral region (B), one can see grooves on both
sides of engrailed stripe (e.g. black arrows). In the lateral region,
an `en face' view (C) shows that Engrailed stripes are broken up into clumps.
(D) Grooves are generated around the islands of engrailed-positive
cells as seen in a side view. (E-G) In a double mutant
(wingless hedgehog), no groove
forms. (E) Engrailed stripes are disrupted throughout (en face view of the
ventral region as in A). (F) Ventral grooves are no longer generated, as seen
in a side view as in B. (G) Likewise no groove can be recognised laterally in
a side view similar to that in D. (H,I) Schematic drawings summarising the
results shown in A-G.
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Role of Engrailed in groove formation
So far our results demonstrate the requirement of hedgehog in
segment boundary formation but they do not exclude the possibility that
engrailed might also be required. By analogy with the experiments
above, where engrailed expression was artificially maintained in a
hedgehog mutant, we added back hedgehog expression in an
engrailed mutant to specifically test the requirement of Engrailed.
To drive hedgehog expression, we used paired-Gal4, a driver
whose posterior limit of expression correlates roughly with the position of
wild-type segment boundaries (wingless
engrailed paired-Gal4 UAS-hedgehog). As shown in
Fig. 6B, exogenous expression
of Hedgehog does not rescue segmental grooves in the absence of
engrailed function, and such embryos exhibit a flat surface. As
positive control, we asked whether grooves are rescued by adding exogenous
engrailed (thereby also inducing hedgehog expression) using
the same driver in the otherwise same genetic background
(wingless engrailed paired-Gal4
UAS-engrailed) and indeed they are
(Fig. 6A). Thus co-expression
of hedgehog and engrailed is required for grooves to
form.

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Fig. 6. Hedgehog-independent requirement of Engrailed in groove formation.
Engrailed is required in addition to Hedgehog for boundary formation. No
grooves form in an engrailed mutant (or in an engrailed
wingless double mutant). (A) Groove formation is rescued, at least in the
lateral epidermis (see legend of Fig.
7), by expressing engrailed with paired-gal4,
shown here in the wingless engrailed double mutant: grooves form on
both sides (arrows) of the expression domain because of the absence of
wingless. (B) By contrast, no such rescue is seen when Hedgehog is
expressed in the same genetic background. (C,D) Diagrams summarising the
results in A and B. (E) Stage 13 embryo expressing CiVP16 under the control of
engrailed-Gal4 stained with anti-Engrailed (brown) and a ci
RNA probe (purple). This embryo is expected to have active Hedgehog signalling
on both sides of the presumptive boundaries. Boundary formation is not
prevented. This is represented diagrammatically in F.
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Engrailed could contribute to segment boundary formation by regulating the
expression of one or several effector genes. A minimalist view is that the
only relevant target of Engrailed in this respect is ci. Engrailed is
known to repress ci expression
(Eaton and Kornberg, 1990
) and
this ensures that no Hedgehog signalling takes place where engrailed
is expressed. Conceivably, the juxtaposition of cells undergoing Hedgehog
signalling (HH ON) with cells that are unable to activate the pathway (HH OFF)
could be sufficient to cause segment boundary formation. However, artificial
activation of Hedgehog signalling in the Engrailed domain, using
engrailed-gal4 and UAS-CiVP16 (which encodes a powerful
activated form of Ci; C. A., unpublished) does not prevent boundary formation
(Fig. 6E). Thus, activation of
Hedgehog signalling on both side of the boundary is compatible with boundary
formation.
Continuous requirement of Engrailed and Hedgehog in groove
maintenance
We noticed that, on the ventral surface of the embryos described above
(wingless engrailed paired-Gal4
UAS-engrailed; Fig. 6A),
groove formation is initiated normally and maintained until stage 12
(Fig. 7A). Such grooves then
disappear prematurely, before stage 13
(Fig. 7B). At lateral
positions, in the same embryos, boundaries are maintained until at least stage
14 (Fig. 6A). The reason for
this spatial difference could be due to the expression of
paired-Gal4, which starts to decay around late stage 12 ventrally
(Fig. 7C) while laterally, it
is maintained until at least stage 14. Thus, the presence of grooves in this
genetic background (wingless
engrailed paired-Gal4 UAS-engrailed), correlates
temporally and spatially with the expression of engrailed and
hedgehog. This suggests that these two genes could be continuously
required throughout the lifetime of the groove. To test this possibility, we
performed an experiment analogous to that above, but with
buttonhead-Gal4, which is expressed in the ventral epidermis beyond
stage 14 (engrailed buttonhead-Gal4 UAS-engrailed).
Ventral grooves are concomitantly detectable until stage 14 in such embryos
(Fig. 7D). This confirms the
suggestion that continuous expression of engrailed and
hedgehog is required for groove maintenance.

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Fig. 7. Continuous requirement of engrailed and hedgehog in
groove formation. When driven by paired-gal4, expression of
engrailed and hedgehog rescues segmental grooves only
transiently (in an embryo lacking engrailed and also
wingless). (A) Side view of such an embryo at stage 12, focused on
the ventral midline. Shallow grooves can be seen (arrows). (B) At stage 13,
these grooves are no longer visible. (C) Schematic representation of the
domains of paired (Prd) and buttonhead (Btd) expression.
Note that, in the ventral region, expression of buttonhead persists
longer (up to stage 15) than that of paired (to stage 12+). (D)
buttonhead-driven engrailed expression rescues groove
formation (arrows) in an engrailed mutant even at stage 13 and
beyond. This is not true of paired-gal4-driven engrailed
(not shown).
|
|
Neither Stripe nor the EGFR pathway appears to be required for
boundary formation
Our results suggest that segment boundary formation requires the activation
of specific genes in cells on both sides of the boundary. One important
challenge for the future is to identify such target genes. No obvious relevant
targets of Engrailed have been reported so far. However, there are candidate
targets of Hedgehog signalling that could be involved in boundary formation.
In particular, expression of both rhomboid and stripe are
activated by Hedgehog signalling and repressed by Wingless signalling
(Alexandre et al., 1999
;
Piepenburg et al., 2000
), as
expected from a `boundary-forming gene'. However stripe null mutants
exhibit normal grooves (Fig.
8B) when compared with wild-type embryos
(Fig. 8A), although the spacing
of engrailed stripes is a little irregular. Likewise,
rhomboid mutants also make segmental grooves
(Fig. 8C). As Rhomboid is
limiting for the activation of Spitz, which itself activates the EGFR
(Guichard et al., 1999
;
Lee et al., 2001
), we also
looked at spitz mutants. They too form normal grooves
(Fig. 8D).

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Fig. 8. stripe, rhomboid and spitz are not required for groove
formation. Lateral views of stage 14 embryos. (A) Wild type. (B)
stripeDG4 mutant embryos (here stained with
anti-Engrailed) have grooves although they can be irregularly spaced. (C)
Normal grooves form in a rhomboid7M43 mutant (also stained
with anti-Engrailed). (D) spitz1 mutant. Again, grooves
form although the epidermis can be disorganised
|
|
This provides additional evidence against the possible requirement of EGFR
signalling, although further analysis of EGFR mutants is needed before a
definite conclusion can be reached.
 |
Discussion
|
---|
In this paper we have characterised the boundary that delineates individual
segments during Drosophila embryogenesis. We described the
morphological changes that accompany groove formation and identified two key
genetic requirements for this process. These are the presence of Engrailed at
the anterior of the boundary and the activation of Hedgehog signaling at the
posterior. In the absence of either, grooves do not form and, in addition, the
segmental organization of the germ band is disrupted.
Why boundaries and grooves?
The primary function of boundaries must be to ensure that distinct
populations of cells can be patterned separately during development. This is
evident from the classic clonal analysis of Drosophila appendages.
Because segment boundaries form after most embryonic mitoses have occurred,
clonal analysis is of limited use to demonstrate the separation of cells
between different segments in the embryo. Nevertheless, in the absence of
visible boundary grooves i.e. in the absence of Hedgehog,
engrailed-expressing cells are no longer confined to well-demarcated
stripes suggesting that segment boundaries are needed to maintain the
segmental organization of the epidermis. Therefore, segment boundaries, like
compartment boundaries in imaginal discs keep distinct cell populations
separate. However, unlike the compartment boundary in disks, segment
boundaries are associated with a groove, which could be functionally
significant. For example, it is conceivable that grooves contribute to muscle
attachment by bringing the appropriate epidermal cells (epidermal muscle
attachment) EMA cells (Becker et al.,
1997
; Frommer et al.,
1996
) in close proximity to the mesoderm, thus helping muscle
recognise its epidermal target.
Groove morphogenesis
Our morphological analysis reveals that groove formation involves apical
constriction within the most posterior engrailed-expressing cells and
the eventual acquisition of a bottle cell morphology
(Fig. 9). Such changes in cell
shape are encountered during many morphogenetic events. For example,
invagination of the Drosophila mesoderm is characterised by apical
constriction (Kam et al.,
1991
; Leptin and Roth,
1994
; Oda and Tsukita,
2001
). Likewise, a large reduction of the apical surface of eye
imaginal disks cells is seen in the morphogenetic furrow
(Wolff and Ready, 1991
). In
sea urchins, bottle cells have been shown to be required for invagination of
the ectoderm (Kimberly and Hardin,
1998
). In vertebrates, classic examples include the formation of
the neural tube in chick (Schoenwolf and
Franks, 1984
), and of the blastopore lip in amphibians
(Hardin and Keller, 1988
).
Thus, local changes in cell shape may be an important component of the
mechanics of groove formation, although in the case of segmental grooves,
specific ablation would be required to demonstrate the importance of the
bottle cells.

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|
Fig. 9. Morphological changes accompanying segmental groove formation. Schematic
representation of the changes in cell morphology and genetic interactions
before and during boundary formation. Here, drawings are oriented such that
the apical side of the epithelium is upwards, according to convention. (A)
Groove formation is initiated by Hedgehog signalling in cells adjoining the
most posterior engrailed-expressing cells. Signalling by Hedgehog
prevents repression by Ci[75], leading to the expression of gene(s)
x. (B) The groove founder cells loose contact on their apical side
and an unknown signal (Y) feeds back on the engrailed-expressing
cell. (C) The most posterior engrailed-expressing cell constricts its
apical surface and moves inwards. (D) It comes to lie at the bottom of the
forming groove while continuing to constrict its apical surface. (E) As the
groove reaches its deepest point, the most posterior
engrailed-expressing cell acquires a bottle shape. At the same time,
it turns off engrailed expression.
|
|
Segmental grooves, when they are deepest, include three or four cells on
either side of the bottle cells. It is therefore conceivable that additional
forces contribute to groove formation. One possibility is that muscles could
pull epithelial cells towards the interior of the embryo. However, grooves
still form in stripe mutants, which lack muscle attachment sites
(Becker et al., 1997
;
Frommer et al., 1996
). We can
therefore exclude a role of muscles in groove formation. Although local
changes occur at incipient segment boundaries, a large-scale epithelial
rearrangement called germ band shortening takes place and could contribute to
groove formation. For example, compression of the germ band by the amnioserosa
could conceivably lead to buckling of the epithelium at weak points. Indeed,
it has been proposed that convergence of cells toward the vegetal pole in sea
urchin embryos creates compression that causes the vegetal plate to buckle
(Ettensohn, 1985
). To assess
the role of germband shortening in groove formation, we looked at
hindsight mutants, which are deficient in germband retraction
(Yip et al., 1997
). We found
that such embryos do form grooves (data not shown). However, as some degree of
germ band shortening still occurs in these mutants, it could be that modest
compression of the germband is sufficient to cause groove formation.
Alternatively, as suggested by Shock and Perrimon
(Schock and Perrimon, 2002
),
groove formation could facilitate, but not be absolutely required for,
germ-band retraction. A definitive assessment of the role of germ band
shortening awaits the isolation of mutations that completely prevents it.
Although germ band shortening leads to a reduction of the exposed surface
area of the epidermis, dorsal closure has the opposite effect and this is
accompanied by groove regression. In this case, evidence for a causal relation
is better because, as we found, groove regression does not occur in mutants
such as zipper, which are defective in dorsal closure. This suggests
that the surface area needed for dorsal closure could be supplied by cells
that are buried in segmental grooves at stages 12-13. More importantly, it
shows that manipulating the total surface area of the germband does impact on
grooves, indicating that general morphological changes, in addition to local
cell shape changes, could be important in groove formation or maintenance.
In conclusion, we found that cells undergo specific morphological changes
at incipient boundaries, especially those cells that line the anterior side of
the boundary (the most posterior engrailed-expressing cells). At the
same time, it may be that global rearrangements within the epithelium also
contribute to groove formation.
Genetic requirements for groove formation
A parallel with the compartment boundary in wing imaginal disks
As described in the Introduction, Engrailed has both a cell autonomous and
a non-cell autonomous function in the establishment of the compartment
boundary in wing imaginal discs. Although the compartment boundary does not
trace its embryonic origin to segment boundaries (see Introduction), there is
a striking parallel between the two. As we have shown, for segmental grooves
to form, Hedgehog signaling is required in cells at the posterior of the
boundary, even if engrailed expression is artificially maintained at
the anterior side. Conversely, Hedgehog signaling is not sufficient as
exogenous expression of hedgehog in the absence of engrailed
does not lead to groove formation.
Two-way signaling across the boundary
As described above, it is the cells that line the anterior side of segment
boundaries (the most posterior engrailed-expressing cells) that
undergo the most distinctive behaviour during groove formation. This behaviour
requires Hedgehog signalling, and yet engrailed-expressing cells are
not responsive to this signal. Therefore, their morphological changes must be
in response to a signal originating from neighbouring non-engrailed
expressing cells. This could be achieved through standard paracrine signaling
or by contact-dependent signal mediated by cell surface proteins. Whatever the
mechanism, Hedgehog-responsive cells influence the behaviour of adjoining
engrailed-expressing cells across the boundary, and crosstalk between
the two cells takes place. This is reminiscent of the situation at rhombomere
boundaries where cross communication between neighbouring rhombomere cells are
required for their formation.
The role of ci
Because, as we have shown, boundaries form in the complete absence of Ci
(in ci94), we conclude that the activator form of Ci is
not required for segment boundary formation. However, no boundary forms in
ciCell mutant embryos indicating that the presence of
Ci[75] (the repressor) prevents boundary formation. We suggest therefore that
boundary formation requires the expression of a gene (x) that is
repressed by Ci[75] but does not require Ci[155] to be activated. Presumably,
an activator of x is constitutively present but, in the absence of
Hedgehog, it is prevented from activating x expression by Ci[75].
Hedgehog signaling would remove Ci[75] and thus allow activation to occur. Two
characterized target genes of Hedgehog (wingless and
rhomboid) follow the same mode of regulation. For example, expression
of wingless in the embryonic epidermis decays in
ciCell but is still present in the complete absence of Ci,
in ci94 embryos
(Methot and Basler, 2001
).
Repression of x expression by Wingless signalling
Although Hedgehog signaling is activated both at the anterior and the
posterior of its source, segment boundaries only form at the posterior. One
reason for this asymmetry is that Wingless signaling represses boundary
formation at the anterior. Indeed, in the absence of Wingless, boundaries are
duplicated, as long as expression of Engrailed and Hedgehog is artificially
maintained. We conclude that expression of x is repressed by Wingless
signalling. Two obvious candidates for x are rhomboid and stripe.
Both genes are activated by Hedgehog signaling and repressed by Wingless
signaling (Sanson et al.,
1999
; Alexandre et al.,
1999
; Piepenburg et al.,
2000
) and, indeed, both are expressed in cells that line the
segment boundary. To determine if either gene could mediate the role of
Hedgehog in boundary formation we looked at the respective mutants. No effect
on grooves could be seen. We conclude that neither rhomboid nor stripe is
required for boundary formation although we cannot exclude the possibility
that these genes could contribute in a redundant fashion. Overall our genetic
analysis suggests that additional targets of Hedgehog must be involved in
boundary formation. It will be interesting to find out whether any of these
targets will turn out to be implicated in compartment boundary maintenance as
well.
The cell-autonomous role of engrailed
Although we have emphasised the role of a Hedgehog target gene in boundary
formation, it is clear from our analysis that engrailed also has a
cell-autonomous role. We have provided evidence that, even though Engrailed
represses ci expression, its role in boundary formation is likely to
involve the transcriptional regulation of another target gene (see
Fig. 6E). One possibility is
that Engrailed could be a repressor of x and that boundaries would
form at the interface between x-expressing and non-expressing cells.
However, we think that instead, or in addition, Engrailed has a
Hedgehog-independent effect on cell affinity and that this could contribute to
boundary formation. Of note is the observation that
engrailed-expressing cells remain together in small groups even when
boundaries are lost for lack of hedgehog. This suggests that
engrailed-expressing cells have increased affinity for one another.
Thus, Engrailed could specify P specific cell adhesion independently of
Hedgehog. Clearly, future progress will require the identification of
Engrailed target genes that control such preferential affinity and/or
contribute to boundary formation.
 |
ACKNOWLEDGMENTS
|
---|
We thank Talila Volk for fly stocks and antibodies, Gines Morata for fly
stocks, Laurence Dubois for reagents and Veronique Brodu for sharing embryos.
Antibodies were also obtained from the Developmental Studies Hybridoma Bank.
Constructive comments on the manuscript were provided by Andrea Pasini. This
work was entirely supported by the UK's Medical Research Council.
 |
Footnotes
|
---|
Supplemental data available online
 |
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