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First published online 1 October 2003
doi: 10.1242/dev.00867
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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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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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Key words: Drosophila embryos, Segmentation, Boundaries, hedgehog, engrailed, TEM
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Introduction |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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)
(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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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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),
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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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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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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). 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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;
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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; 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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) 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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;
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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Discussion |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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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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;
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.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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