Cell movements controlled by the Notch signalling cascade during foregut development in Drosophila
Bernhard Fuss*,
Frank Josten*,
Maritta Feix and
Michael Hoch
Universität Bonn, Institut für Molekulare Physiologie und
Entwicklungsbiologie, Abteilung für Molekulare Entwicklungsbiologie,
Poppelsdorfer Schloss, D-53115 Bonn, Germany
Author for correspondence (e-mail:
m.hoch{at}uni-bonn.de)
Accepted 16 December 2003
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SUMMARY
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Notch signalling is an evolutionarily conserved cell interaction mechanism,
the role of which in controlling cell fate choices has been studied
extensively. Recent studies in both vertebrates and invertebrates revealed
additional functions of Notch in proliferation and apoptotic events. We
provide evidence for an essential role of the Notch signalling pathway during
morphogenetic cell movements required for the formation of the
foregut-associated proventriculus organ in the Drosophila embryo. We
demonstrate that the activation of the Notch receptor occurs in two rows of
boundary cells in the proventriculus primordium. The boundary cells delimit a
population of foregut epithelial cells that invaginate into the endodermal
midgut layer during proventriculus morphogenesis. Notch receptor activation
requires the expression of its ligand Delta in the invaginating cells and
apical Notch receptor localisation in the boundary cells. We further show that
the movement of the proventricular cells is dependent on the short
stop gene that encodes the Drosophila plectin homolog of
vertebrates and is a cytoskeletal linker protein of the spectraplakin
superfamily. short stop is transcriptionally activated in response to
the Notch signalling pathway in boundary cells and we demonstrate that the
localisation of the Notch receptor and Notch signalling activity depend on
short stop activity. Our results provide a novel link between the
Notch signalling pathway and cytoskeletal reorganisation controlling cell
movement during the development of foregut-associated organs.
Key words: Drosophila, Cell movement, Notch, Short stop
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Introduction
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The Notch signalling pathway has been shown to mediate cell fate decisions
through local interactions during animal development (for a review, see
Artavanis-Tsakonas et al.,
1999
). Studies in the Drosophila wing have demonstrated
that the range of Notch signalling is determined by the spatial and temporal
expression pattern of its ligands, Delta and the transmembrane protein Serrate
(Ser), and by the activity of the glycosyltransferase Fringe (Fng) (for a
review, see Blair, 2000
). Fng
modulates ligand affinity of Notch and plays a major role in the
Notch-dependent positioning of sharp compartment boundaries. It has been shown
to modify the glycosylation state of the receptor in the Golgi complex,
thereby lowering its sensitivity to Ser and enhancing its sensitivity to Delta
(Ju et al., 2000
;
Brückner et al., 2000
;
Moloney et al., 2000
). Ligand
binding to the Notch receptor results in a proteolytic intracellular
processing of Notch and gives rise to the Notch intracellular domain fragment
(NICD). NICD is released from the membrane and
translocates to the nucleus where it interacts as a transcriptional
co-activator with Supressor of Hairless [Su(H)], a ubiquitously expressed
DNA-binding protein. DNA-bound complexes containing of both Su(H) and
NICD are thought to activate the transcription of Notch target
genes in cooperation with other transcriptional activators (for a review, see
Bray and Furriols, 2001
). The
genes of the Enhancer of split [E(spl)] locus which encode
nuclear basic helix-loop-helix proteins, are primary target genes of Notch
signalling that repress neural cell fate (for a review, see
Greenwald, 1998
).
Phenotypic analyses in both vertebrates and invertebrates revealed that
apart from the well-documented involvement of Notch in cell fate decisions,
both proliferation and apoptotic events can also be affected by Notch
signalling. Notch activation appears to inhibit apoptosis in murine
erythroleukemia cells (Shelly et al.,
1999
) and the involvement of Notch activation in proliferation has
been demonstrated for wing and leg development in the fly
(Go et al., 1998
;
de Celis et al., 1998
).
Furthermore, recent studies on neural crest cells in the mouse have suggested
additional roles for the Notch signalling pathway during cell migration. Loss
of Delta-1 in mice causes severe disruption of neural crest migration
and neural crest cells become randomly dispersed through the somites instead
of following a restricted movement through the rostral portion of each
sclerotome (De Bellard et al.,
2002
). However, the mechanism by which Notch controls cell
migration is still rather elusive.
We provide evidence for an essential role of the Notch signalling pathway
for the morphogenetic cell movements during the formation of the
foregut-associated proventriculus organ in the Drosophila embryo.
Notch signalling activity is required in two rows of boundary cells in the
proventriculus primordium. These cells delimit a population of foregut
epithelial cells that undergo a coordinated series of cell shape changes and
cell movement events leading to the invagination of the ectodermal foregut
epithelium into the endodermal midgut layer. We further demonstrate that the
short stop gene, which encodes a cytoskeletal crosslinker protein of
the spectraplakin superfamily (Gregory and
Brown, 1998
; Strumpf and Volk,
1998
; Röper et al.,
2002
), is essential for cell movement in the proventriculus
primordium. short stop is transcriptionally activated in boundary
cells in response to Notch and we provide evidence that its activity is
required for Notch receptor localisation and Notch signalling. These results
connect the Notch signalling pathway with the modulation of cytoskeletal
organisation in key morphoregulatory cells that drive the formation of a
foregut-derived organ in Drosophila.
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Materials and methods
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Drosophila stocks and analysis of mutants
The Oregon R strain was used as wild type. For mutant analysis we used
N55e11/FM7 (Kidd et al., 1983);
cdc423/TM3 (R. Fehon); Dl9P/TM3 (M.
Muskavitch); Su(H)AR9/CyO (A. Preiss);
fng13/TM3 (T. Klein); kak65-2/CyO
(Prout et al., 1997
); and
shotk15606/CyO (Gao et
al., 1999
). Mutant alleles were kept on FM7ActGFP,
CyOwglacZ and TM3ActGFP balancers. Notch signalling activity
was detected by the Gbe-Su(H)m8-lacZ reporter construct
(Furriols and Bray, 2001
).
Ectopic expression studies were performed using the Gal4 driver lines 14-3
fkh-Gal4 (Fuss and Hoch,
1998
), dri-Gal4 (R. Saint) and hsGal4 (Bloomington Stock
Centre). The dri-Gal4 driver mediates expression in the posterior
boundary cells. 14-3 fkh-Gal4 drives expression from stage 10 onwards
in the oesophagus and in the endodermal part of the proventriculus primordium.
For heat-shock-induced expression, 0-3 hour egg collections were allowed to
age for 13 hours at 25°C. The heat shock was performed three times for 20
minutes in a 37°C waterbath, interrupted by two 15 minute breaks at room
temperature. Prior to fixation, embryos were allowed to age for 3 hours after
the heat shock protocol. As UAS effector strains, we used UAS-Dl (M.
Muskavitch), UAS-NICD (G. Struhl), UAS-NECD (T. Klein)
and UAS-Cdc42N17 (R. Schuh).
Immunostainings and in situ hybridisation
Embryos were staged and stained as described previously
(Fuss and Hoch, 1998
). As
antibodies we used: anti-NotchICD (1:10, mAbC17.9C6),
anti-NotchECD (1:10, mAb F461.3B), anti-Delta (1:5, mAbC594.9B);
antiFas3 (1:5, mAb7G10, Hybridoma Bank, Iowa), anti-Dve (1:1000)
(Nakagoshi et al., 1998
),
anti-Kakapo (1:300, T. Volk), anti-Forkhead (1:100, P. Carrera), anti-MHC
(1:50; D. Kiehart) and anti-ß-Gal (1: 100, Promega). Fluorescent
labelling was performed with Alexa543 and Alexa488
coupled secondary antibodies (Molecular Probes) or Cy2, Cy3 and Cy5 coupled
secondary antibodies (Dianova). Fluorescent images were recorded using a Leica
TSP2 confocal microscope (Leica, Wetzlar, Germany) and images of
multi-labelled samples were acquired sequentially on separate channels.
Digoxygenin-labeled RNA antisense probes were generated by in vitro
transcription of a shot/kakapo cDNA (kindly provided by N.
Brown), Su(H) cDNA (F. Schweisguth) and a Ser cDNA (cDNA
clone RE42104). For Su(H) and fng RNA expression, see also
http://www.fruitfly.org/cgi-bin/ex/insitu.pl
 |
Results
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The proventriculus is a multiply folded, cardia-shaped organ that functions
as a valve to regulate food passage from the foregut into the midgut of
Drosophila larvae (Strasburger,
1932
). It is derived from the stomodeum, which gives rise to the
foregut tube and to parts of the anterior midgut in the early embryo
(Campos-Ortega and Hartenstein,
1997
). Cell shape changes are initiated at stage 12 when cell
proliferation has been completed within the proventriculus primordium
(Pankratz and Hoch, 1995
;
Campos-Ortega and Hartenstein,
1997
). Anti-Forkhead (Fkh)/anti-Defective proventriculus (Dve)
double immunostainings which specifically visualise ectodermal and endodermal
cells, respectively (Fuss and Hoch,
1998
), reveal that the first step of proventriculus morphogenesis
involves the formation of a ball-like evagination at the ectoderm/endoderm
boundary of the posterior foregut tube
(Fig. 1A). The formation of
this evagination is initiated by a local constriction of apical membranes at
the ectoderm/endoderm boundary leading to an accumulation of
membrane-associated markers such as Arm towards the luminal (apical) side
(Fig. 1E). It is of note that
the ectodermal part of the ball-like evagination localises in a mesoderm-free
region, whereas the surrounding cells of the developing foregut and the midgut
are covered by visceral mesoderm (Fig.
1I,J) (Pankratz and Hoch,
1995
). At stage 14, a constriction forms at the boundary of the
ectodermal and the endodermal cells (Fig.
1B,F,J). This results in the formation of the `keyhole' structure
that we have described previously (Fig.
1B) (Pankratz and Hoch,
1995
; Fuss and Hoch,
1998
; Bauer et al.,
2002
). From stage 14 onwards, cells from the anterior portion of
the ectodermal keyhole part (in the mesoderm-free area) begin to move inwards
into the endodermal part of the keyhole and a heart-like structure is formed
(Fig. 1C,G,K). The ectodermal
keyhole cells continue to move inward until late stage 17
(Fig. 1D,H,L) and give rise to
the recurrent layer of the proventriculus; it links the outer endodermal layer
(derived from the endodermal keyhole cells) and the inner layer of the
proventriculus which is a continuation of the oesophagus
(King, 1988
). The cells at the
tip of the invaginating ectodermal keyhole cells which derive from the most
anterior region of the keyhole, are not covered by visceral mesoderm
(Fig. 1L). It has been observed
before that these cells assume a stretched appearance with long cytoplasmic
extensions (Pankratz and Hoch,
1995
). The different steps of proventriculus development are shown
schematically in Fig. 1M-Q.

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Fig. 1. Cell movement during proventriculus development. Developing proventriculi
of stage 12 (A,E,I), stage 14 (B,F,J), stage 15 (C,G,K) and stage 17 (D,H,L)
wild-type embryos. (A-D) Anti-Fkh (red)/anti-Dve (green) marking ectodermal
and endodermal cells, respectively. A constriction separates the ectodermal
and endodermal part of the keyhole (arrow in B, see also F). (E-H)
anti-Armadillo staining. Note the concentration of Arm towards the apical side
of the cells (arrow in E,F). (I-L) Anti-Arm(red)/anti-MHC (blue)
immunostaining. The mesoderm-free region that lacks MHC expression is marked
by arrows in I. (M-Q) Schematic representation of the different stages of
proventriculus development and the cell movements resulting in the
invagination of the ectodermal keyhole cells. Ectodermal cells in orange,
endodermal cells in green and mesodermal cells in blue.
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Notch signalling is required for cell movements in the proventriculus primordium
The Notch receptor ligands Delta (Fig.
2A-D) and Ser (not shown; Ser mutants do not show a
proventricular phenotype) (B.F. and M.H., unpublished) are specifically
expressed in the invaginating ectodermal keyhole cells from early stages of
proventriculus development until the end of organogenesis. From stage 15
onwards, Delta becomes downregulated in the most anterior and the most
posterior cells of the ectodermal keyhole domain (the latter are positioned
directly at the ectoderm/endoderm boundary, see arrowheads in
Fig. 2C,D). By contrast, Notch
receptor expression is strongly elevated in these two cell rows from stage 13
onwards compared with the surrounding epithelial cells, as revealed by
anti-Notch/anti-Dve double immunostainings
(Fig. 2E-L). The Notch receptor
continues to be upregulated in these two cell rows, which we designate as the
anterior and posterior boundary cells, respectively, until late stage 17. The
Notch receptor expression domain in the anterior boundary cells forms the tip
of the ectodermal keyhole cells that invaginate into the endodermal part of
the keyhole (Fig. 2G,H,K,L,
designated `ac'), whereas the Notch receptor expression domain in the
posterior cells becomes localised at the rim of the developing proventriculus
(Fig. 2G,H,K,L, designated
`pc'). Su(H), which encodes the only known transducing transcription
factor of the Notch signalling pathway, is rather ubiquitously expressed in
the developing proventriculus; the glycosyltransferase Fng is expressed in
domains anteriorly and posteriorly to Delta (data not shown; see also
http://www.fruitfly.org/cgi-bin/ex/insitu.pl).
The Notch-dependent genes of the E (spl) complex are not expressed
during proventriculus development (Welch
et al., 1999
).

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Fig. 2. Expression of members of the Notch signalling pathway during proventriculus
development. (A-D) Dl expression in the ectodermal cells of the
keyhole monitored by Anti-Dl (red)/anti-Dve (green) immunostaining at stage 12
(A), stage 15 (B), stage 16 (C) and late stage 17 (D). Delta localises to the
ectodermal keyhole domain which moves into the endodermal cell layer. Arrows
(C,D) mark the downregulation of Dl expression in anterior (ac) and posterior
(pc) boundary cells. (E-L) Dynamic Notch receptor expression visualised in a
single channel visualisation (E-H) and in an anti-Notch (red)/anti-Dve (green)
immunostaining (I-L). (E,I) Stage 13; (F,J) stage 14; (G,K) stage 15; (H,L)
late stage 17 wild-type embryos. The Notch receptor is upregulated in ac and
pc. (M-P) Relative localisation Delta (green) and Notch signalling activity
[Gbe-Su(H)m8-lacZ; red] during proventriculus development.
The epithelial gut tube is surrounded by broken lines; ac and pc are
highlighted by arrows. Notch signalling activity is restricted to the ac and
pc, which are adjacent to the Delta expression domain.
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To determine the range of Notch signalling in the ectodermal keyhole cells,
we used a lacZ-reporter construct carrying multiple Su(H)-binding
sites from the E(spl) m8 gene combined with binding sites for the
transcription factor Grainyhead (Grh)
(Furriols and Bray, 2001
;
Bray and Furriols, 2001
). In
cells, in which Notch signalling is active and Grh is expressed, Su(H)
cooperates with Grh to yield high levels of reporter gene expression, whereas
reporter gene expression is repressed in cells in which Notch is inactive
(Furriols and Bray, 2001
).
Reporter gene expression in corresponding transgenic embryos reflects the
range of Notch signalling. We used this construct previously to demonstrate
that Notch signalling is restricted to the boundary cells that separate the
dorsal from the ventral half of the hindgut
(Fuss and Hoch, 2002
). As
shown in Fig. 2M-P, the
Notch-dependent reporter construct is activated from stage 12 onwards until
late stages of proventricular development in two domains that are localised
directly adjacent to the Delta expression domain. Both Notch activity domains
encompass the cells of the anterior and posterior boundary cells in which the
Notch receptor is upregulated (compare the ß-Gal pattern in
Fig. 2P with Notch receptor
expression in L).
Notch signalling is required for cell movements in the developing proventiculus
In Delta, Notch, Fng and Su(H) mutants, the early steps
of proventricular development including the formation of the ball-like
evagination at the ectoderm/endoderm boundary occur normally
(Fig. 3A,D,G,J). However, the
anterior boundary cells of the ectodermal keyhole region do not initiate cell
movements to invaginte into the endodermal cell layer
(Fig. 3B,E,H,K). Rather, the
ectodermal keyhole cells arrest anteriorly and do not move inwards until late
stages of embryonic development (Fig.
3C,F,I,L). Furthermore we find a significant collapse of the
endodermal proventriculus rim (Fig.
3C,F, compare with wild type in
Fig. 2D) suggesting defective
function of posterior boundary cells in which the Notch receptor is expressed
(Fig. 2K,L). Note that the
number of ectodermal cells is not changed in both Delta and
Notch mutants, indicating that no cell death has occurred. A very
similar phenotype is also obtained in embryos in which Delta is ectopically
expressed at a high level in the cells of the posterior foregut and the
anterior midgut using the 14-3fkhGal4 driver and the UAS-Dl effector
line (Fig. 3M). This indicates
that restricted expression of Delta in the ectodermal cells of the keyhole and
its downregulation in the boundary cells may be necessary for the Notch
signalling-dependent inward movement. Notch-dependent reporter gene expression
of the Gbe-Su(H)m8 reporter is abolished in the anterior and
posterior boundary cells when Delta is ectopically expressed in these embryos
(B.F. and M.H., unpublished). When we ubiquitously express the Notch
extracellular domain (NECD), which acts as a dominant-negative form
of the Notch receptor, the ectodermal keyhole cells fail to complete the
inward movement. By contrast, ectopic activation of Notch signalling by
overexpressing the Notch intracellular domain (NICD) in the
proventricular cells causes ectopic cell movements. However, we do not observe
changes in endodermal or ectodermal cell fate in these embryos
(Fig. 3O). In summary, these
results suggest that the Notch signalling pathway controls cell movement of
the proventricular epithelial cells via its localised activity in the anterior
and posterior boundary cells.

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Fig. 3. Notch signalling is required for cell movements during proventriculus
morphogenesis. Anti-Fkh(red)/anti-Dve (green) immunostaining of Dl
(A-C), Notch (D-F), fng (G-I) and Su(H) (J-L)
loss-of-function mutants at stage 12 (A,D,G,J), stage 15 (B,E,H,K) and stage
17 (C,F,I,L) of proventriculus development. Specification of the early
proventriculus primordium is not affected in any of the mutants (compare with
wild type, Fig. 1A), whereas
cell movements leading to the keyhole structure at stage 15 (compare with wild
type, Fig. 1C) and to the
cardia structure at stage 17 (compare with wild type,
Fig. 1D) do not take place,
leading to block of invagination in mutants of the Notch signalling cassette.
(M) Ectopic 14-3fkh-Gal4 mediated expression of the Notch ligand Dl
causes a Notch-like phenotype, i.e. loss of invagination of
ectodermal cells, as shown by anti-Dl (red)/anti-Dve (green) double staining.
(N) Ectopic hsGal4 mediated expression of the Notch extracellular domain
(NECD) also abrogates infolding of ectodermal cells at late stages
of proventriculus development, visualised by anti-Dl(red)/anti-Dve(green)
double staining. (O) Anti-Fkh (red)/anti-Dve (green) double staining showing
that ectopic activation of the Notch signalling pathway causes ectopic cell
movements (arrow). However, we do not observe changes in endodermal or
ectodermal cell fate in these embryos.
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The expression of the Drosophila spectraplakin short stop is controlled by the Notch signalling pathway in the posterior boundary cells
In a search for further genes controlling cell movement in the
proventriculus, we identified the short stop (shot) gene as
a key regulator of proventriculus morphogenesis. shot is allelic to
kakapo (Gregory and Brown,
1998
; Strumpf and Volk,
1998
) and encodes a member of the recently named spectraplakin
superfamily of cytoskeletal linker proteins
(Röper et al., 2002
).
Shot is composed of a C-terminal microtubule-binding domain, a N-terminal
actin binding domain and a plakin-repeat domain that may interact with
transmembrane cell adhesion receptors
(Leung et al., 1999
;
Röper et al., 2002
). A
role of shot has been shown for cytoskeletal organisation in tracheal
cells (Lee and Kolodziej,
2002a
; Lee and Kolodziej,
2002b
), in neuronal support cells
(Kuang et al., 2000
), in
muscle attachment cells (Prokop et al.,
1998
; Stumpf and Volk, 1998) and for the adhesion between and
within germlayers in the embryo (Gregory
and Brown, 1998
).
Shot accumulates cortically on the apical side of all the ectodermal cells
of the ectodermal keyhole domain that will subsequently move inwards, as shown
by anti-Shot/anti-Dve antibody staining
(Fig. 4A,B,E,F). In the
adjacent endodermal cells, Shot is localised apically and also basally in a
spot-like pattern at the interface to the overlying visceral mesoderm,
reflecting most likely its requirement for the attachment of endodermal and
mesodermal germ layers (Fig.
4A, arrowheads) (Gregory and
Brown, 1998
). From the keyhole stage onwards, we find a high level
of Shot accumulation in the posterior boundary cells of the keyhole
(Fig. 4C,D,G,H), whereas its
expression in the anterior boundary cells
(Fig. 4D,H) becomes localised
to the tip of the invaginating cells. The accumulation of Shot in the
posterior boundary cells is specific and not due to the fold of the
epithelium, as other apical markers, such as Armadillo, are not upregulated in
the posterior boundary cells (Fig.
4I). In amorphic shot mutants, initial formation of the
ectodermal keyhole region is normal (Fig.
4J), but the inward movement of these cells into the endodermal
keyhole domain fails to occur (Fig.
4K). Furthermore, the rim of the proventriculus is significantly
reduced in size (Fig. 4L). The
different steps in the manifestation of this mutant phenotype are very similar
to Delta, Notch, fng and Su(H) mutants (compare
Fig. 4J-L with
Fig. 3A-L). Phalloidin
stainings reveal an enrichment of actin cytoskeletal structures towards the
apical sides of all the proventricular cells
(Fig. 4M,N). Whereas in the
anterior boundary cells, low levels of actin can be detected in the tip of the
invaginating cells (Fig. 4N,
ac), we find high levels of phalloidin staining on the apical side of the
posterior boundary cells (Fig.
4M, pc, arrowheads). This indicates the presence of abundant actin
cytoskeletal structures in an apical position in the posterior boundary cells,
in which we also find Shot to be localised
(Fig. 4M, compare with 4G). In
amorphic mutants of the small GTPase Cdc42, which controls F-actin
polymerisation in many developmental contexts
(Nobes and Hall, 1999
;
Hall, 1998
), the ectodermal
keyhole cells fail to invaginate into the proventricular endoderm, resulting
in a mutant phenotype that is reminiscent of shot and Notch
mutants (Fig. 4O). Notably,
ectopic expression of a dominant-negative form of Cdc42 in the posterior
boundary cells using the dri-Gal4 driver causes a similar
invagination defect of the ectodermal keyhole cells
(Fig. 4P; Materials and
methods).

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Fig. 4. Shot expression during proventriculus development. (A-H) Anti-Shot
(red)/anti-Dve (green) antibody stainings of wild-type embryos of stage 12
(A,E), 14 (B,F) and 17 (C,G, tangential section; D,H, sagittal section). (A-D)
Single channel visualisation of Shot expression. Shot localises to the apical
side of the ectodermal (ec) keyhole domain (lower arrow in A) and to the
apical and basal sides of the neighbouring endodermal cells that are covered
by visceral mesoderm (upper arrow in A). During invagination, Shot protein is
upregulated on the apical side of the posterior boundary cells (pc in C,D).
Shot expression is reduced in the ac (D,H). (I) Anti-Shot (red)/anti-Arm
(green)/anti-MHC (blue) immunostaining at stage 17 visualising uniform
expression of Arm throughout the proventriculus epithelium and locally
restricted elevation of Shot in the pc. (J-L) Anti-Fkh(red)/anti-Dve (green)
immunostaining of shot mutants at stage 12 (J), stage 15 (K) and
stage 17 (L) revealing the failure of ectodermal cells to invaginate and a
collapse of the proventricular endoderm. (M-O) Phalloidin stainings
visualising the actin cytoskeleton of stage 17 wild-type embryos (M,N) and a
cdc42 mutant embryo (O). In wild type, actin filaments accumulate on
the apical side of pc (M) whereas lower levels of actin filaments are seen in
the ac that move inward (N, arrow). (O) In cdc42 mutants, cell
movements leading to the keyhole structure are not initiated, the endodermal
proventriculus epithelium is collapsed and ectodermal cells fail to
invaginate. (P) Anti-Shot (red)/anti-Arm (green)/anti-MHC(blue) triple
staining of a late stage 17 embryo in which a dominant-negative form of the
GTPase Cdc42 was expressed in the posterior boundary cells. Note the failure
of invagination.
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As Shot accumulates at high levels on the apical sides of the posterior
boundary cells, we tested whether this accumulation is dependent on Notch
signalling. In amorphic Notch mutants, we still find basal levels of
Shot expression both apically and basally in all the proventricular cells, as
it is in wild-type embryos of the same stage. However, in Notch
mutants, no upregulation of Shot occurs in the posterior boundary cells
(Fig. 5B, compare with wild type in
A; see arrowheads), arguing that either shot
transcription or the accumulation/stability of the Shot protein may be
dependent on Notch signalling. As shown above, Notch signalling is confined to
the anterior and posterior boundary cells. To further test whether
shot transcription is dependent on Notch signalling, we ectopically
activated the Notch signalling pathway by expressing NICD using the
14-3fkh-Gal4 driver and the UAS-NICD effector lines (see
Materials and methods). Ectopic activation of the Notch signalling pathway
results in an ectopic activation of shot transcription, as determined
by in situ hybridisation experiments using an antisense shot RNA
probe (Fig. 5C,D). In summary,
the lack and gain-of-function experiments provide strong evidence that the
Notch signalling pathway directly or indirectly activates shot
expression.

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Fig. 5. shot is a Notch target gene and required for Notch signalling.
(A,B) Anti-Shot (red)/anti-Dve (green) immunostaining of stage 17 wild-type
(A) and Notch (B) mutant embryos. Upregulation of Shot expression in
the posterior boundary cells (pc) does not occur in Notch mutants
(arrow). (C) Shot mRNA expression in a wild-type embryo of stage 17
and in an embryo in which the Notch pathway has been ectopically activated
(D). Note the ectopic activation of shot transcription as compared
with wild type. (E,F) Anti-NotchECD immunostaining of the
proventriculus primordium of stage 14 wild-type (E) and shot mutant
embryos (F). Arrows in E and F indicate the keyhole. Notch receptor expression
is strongly reduced in the ectodermal keyhole (ky) domain in shot
mutants. (G,H) Anti-ß-Gal(red)/anti-Fas3 (green) double staining of late
stage 14 Gbe-Su(H)m8-lacZ embryos in a wild-type (G) and a
shot mutant embryo (H); note the loss of reporter gene expression in
pc. (I,J) Anti-ßGal(red)/anti-Dve (green) double staining of stage 17
Gbe-Su(H)m8-lacZ embryos in wild-type (I) and shot
mutant background (J). The activity of the Notch signalling pathway is
strongly reduced in the posterior boundary cells (pc).
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The cytoskeletal linker protein Shot is required for the localisation of the Notch receptor in the boundary cells
In the posterior boundary cells, high levels of Shot are localised at the
apical cortex of the cells (Fig.
4G,H). It is known from vertebrate studies that the plakin domain
of BPAG1e binds directly to the transmembrane protein BPAG2
(Hopkinson and Jones, 2000
).
To test whether the localisation of the Notch receptor is dependent on Shot,
we analyzed Notch expression in shot mutants. From the early stages
of proventriculus development onwards, Notch receptor expression is
specifically reduced in the ectodermal cells of the keyhole region which
undergo extensive cell movements, whereas there are basal levels of Notch
expression in adjacent cells of the developing oesophagus or the
proventricular endoderm (Fig.
5E,F; note that the ectodermal keyhole cells (ky) are still
present in shot mutants). This effect is seen both with an antibody
against the Notch intracellular domain (data not shown) and with an antibody
against the Notch extracellular domain
(Fig. 5E,F). To test whether
Shot-dependent localisation of the Notch receptor is required for Notch
signalling activity, we monitored the expression of the Gbe-Su(H)m8
reporter construct in amorphic shot mutants. As shown in
Fig. 5G,H, Notch dependent
reporter gene expression is already reduced prior to the onset of the
invagination movement in the posterior boundary cells of late stage 14
shot mutants (Fig.
5H); the reduction of Notch signalling in the posterior boundary
cells is observed until late stages (Fig,
5I,J). These results suggest that Shot is crucial for proper Notch
signalling in the posterior boundary cells.
 |
Discussion
|
---|
Previous work has shown that Notch signalling is an evolutionarily
conserved mechanism to control cell fates through local cell interactions
(Artavanis-Tsakonas et al.,
1999
). Our results suggest a crucial role of Notch for controlling
morphogenetic cell movements within the proventriculus primordium.
Furthermore, the activation of shot transcription in response to
Notch signalling provides a novel link between the Notch signalling pathway
and the modulation of cytoskeletal architecture during morphogenesis.
Notch signalling and the control of cell movement during proventriculus development
Immunohistochemical analysis demonstrates that the ligands of the Notch
receptor, Delta and Serrate are expressed in the ectodermal keyhole cells that
invaginate into the endodermal cell layer during proventriculus development.
Their expression becomes downregulated in the anterior and posterior boundary
cells in which the Notch receptor is elevated
(Fig. 2C,D) and in which the
Notch signalling pathway is activated, as demonstrated by the Notch-dependent
Gbe-Su(H)m8-lacZ reporter construct
(Fig. 2O,P). Whereas there is
no proventricular phenotype in Ser mutants, the invagination movement
of the ectodermal keyhole cells is defective in mutants of other components of
the Notch signalling pathway, such as Notch, Delta, fng or
Su(H) (Fig. 3). This
strongly suggests that the boundary cells play a crucial role for cell
movement during proventriculus development. A schematic model of the
activities of the regulators during proventiculus morphogenesis is shown in
Fig. 6. We do not know whether
the cell movements are driven by the anterior boundary cells, dragging the
oesophageal cells behind or whether the major force for the inward movement is
contributed by the ectodermal foregut cells changing their shapes from a
cuboidal to a more stretched appearance. The latter is known to occur during
mid and late stages of embryogenesis when the foregut and the hindgut elongate
dramatically increasing their size by two- to threefold
(Skaer, 1993
;
Lengyel and Liu, 1998
). It has
been shown for dorsal closure that multiple forces contribute to cell sheet
morphogenesis (Kiehart et al.,
2000
; Hutson et al.,
2003
). A similar scenario may apply for proventriculus
morphogenesis. Genetic mosaic studies have revealed that the activity of the
Notch receptor occurs in cells that are adjacent to the ligand-expressing
cells (Heitzler and Simpson,
1991
). Therefore, the downregulation of Delta in the boundary
cells may be a prerequisite for Notch signalling and cell movement, which
would be consistent with our observation that a Notch-like
proventriculus phenotype is induced when Delta expression is maintained in the
anterior and posterior boundary cells (Fig.
3M).

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|
Fig. 6. Model of Notch signalling controlling cell movement in the proventriculus.
(A) Schematic illustration of the proventriculus primordium in late stage 13
(left) and stage 15 (right), highlighting the expression domains of
proventriculus regulators and the cell movement events. Mesoderm in blue;
ectoderm in orange; endoderm in green; ac, anterior boundary cells; pc,
posterior boundary cells. Arrows highlight the inward movement of the ac. Note
that the number of cells in the mesoderm-free region is about 10. For a better
overview of the localisation of gene activities in the proventriculus
primordium, only maximum gene activities are highlighted in the cells. The
expression domains of regulators of proventricular development are shown in B.
For further information, see text.
|
|
Recent studies on neural crest cells in the mouse also have suggested a
role for the Notch signalling pathway during cell migration
(De Bellard et al., 2002
). The
neural crest cells in vertebrates give rise to a wide range of cell types,
including nerve cells, pigment cells, as well as skeletal and connective
tissue (Bronner-Fraser, 1986
).
These cells constitue a migratory cell population that leaves the dorsal
neural tube to migrate along specific tracks to their final destinations in
the periphery of the body. In Delta1 knockout mice, the local
expression of Ephrin receptors and ligands which are guiding molecules
(Flanagan and Vanderhaeghen,
1998
; Holder and Klein,
1999
) is reduced in the caudal region of the sclerotome, as well
as in neural crest-derived peripheral ganglia
(De Bellard et al., 2002
). A
connection of Notch signalling with the modulation of cytoskeletal
architecture has not been shown in these mutants. From our loss-of-function
experiments, we cannot totally exclude an alternative view that Notch
signalling may determine the fate of the boundary cells rather than directly
controlling cell movement. However, when we ectopically activate the Notch
pathway by misexpressing NICD in the proventricular endoderm, this
does not result in a change of cell fates of the endodermal cells towards
ectodermal boundary cell fates (Fig.
3O). Furthermore, the link between Notch signalling and the
activation of shot which is a known cytoskeletal regulator, provides
good evidence for a more direct role of Notch in controlling cell movements
rather than determining cell fates.
The spectraplakin Shot may be involved in Notch receptor localisation and is required for Notch signalling
Our results further demonstrate that the shot gene which encodes a
member of the spectraplakin superfamily of cytoskeletal linker proteins, is
directly or indirectly transcriptionally regulated by the Notch signalling
pathway (Fig. 5A-D). Members of
the spectraplakin superfamily such as Shot in flies or dystonin/BPAG1 or MACF1
in mammals share features of both the spectrin and plakin superfamilies and
produce a large variety of giant proteins of up to almost 9000 amino acids in
length (Röper et al.,
2002
). These proteins contain motifs interacting with all three
elements of the cytoskeleton, the actin, the microtubules and the intermediate
filaments, and they contribute to the linkage between membrane receptors and
the cytoskeletal elements. shot is strongly expressed during
embryogenesis at the muscle attachment sites, which are the most prominent
sites of position-dependent integrin adhesion
(Gregory and Brown, 1998
). An
essential role for Shot has been shown for muscle-dependent tendon cell
differentiation (Strumpf and Volk,
1998
; Prokop et al.,
1998
). In the shot mutant tendon cells, Vein, a
neuregulin-like factor that activates the EGF-Receptor signalling pathway,
fails to be localised properly at the muscle-tendon junctional site; Vein is
dispersed and its level is reduced
(Strumpf and Volk, 1998
). In
these cells, Shot is concentrated at the apical and basal sides. Similarly,
our results place shot both upstream and downstream of Notch
signalling during proventricular development. In the posterior boundary cells,
shot transcription is activated in response to Notch signalling; Shot
protein, in turn, is required in the posterior boundary cells for Notch
receptor localisation and/or stability as receptor expression and Notch
signalling activity in the posterior boundary cells are affected in
shot mutants (Fig.
5G-J). This indicates a feedback loop, as we have suggested
previously for Crumbs-dependent localisation of the Notch receptor in the
boundary cells of the hindgut (Fuss and
Hoch, 2002
). It is not clear how shot expression is
regulated in the ac cells, in which it may require additional inputs from
other yet unknown signalling pathways. Further molecular and biochemical
experiments will have to demonstrate whether there exists a direct interaction
between the Notch receptor and the cytoskeletal Shot protein.
Apical localisation of the actin cytoskeleton in the posterior boundary cells
In the tracheal system, Shot is required for the formation of the
RhoA-dependent F-actin cytoskeleton in the fusion cells and to form the
lumenal connections between tracheal branches
(Lee et al., 2000
;
Lee and Kolodziej, 2002a
). It
has been suggested that Shot may function downstream of RhoA to form
E-cadherin-associated cytoskeletal structures that are necessary for apical
determinant localisation. Our analysis of the actin cytoskeleton using
phalloidin staining reveal a strong apical localisation of F-actin filaments
in the posterior boundary cells in which Shot also accumulates apically to a
high level. By contrast, the density of the actin cytoskeleton is reduced in
the anterior boundary cells that move inwards and in which the contribution of
Shot for Notch signalling activity seems minor
(Fig. 4M,N). A stabilised
cytoskeletal architecture in the posterior boundary cells may be required to
provide stiffness/tension that may enable the inward movement of the anterior
boundary cells. Our lack- and gain-of-function results suggest that the small
GTPase Cdc42 that is one of several known cytoskeletal regulators
(Hall, 1998
), may play a major
role to control cytoskeletal architecture during the inward movement of the
proventricular cells (Fig.
4O,P). These results are consistent with the idea that Notch
signalling controls cytoskeletal organisation via the cytoskeletal linker
protein Shot and they suggest a role for Cdc42 in this process, the specific
involvement of which, however, has to be studied in more detail.
 |
ACKNOWLEDGMENTS
|
---|
We thank S. Bray, S. Hou, T. Volk, D. Harrison, N. Perrimon, N. Brown, J.
Gastelli-Gair, T. Klein, E. Knust, S. Cohen, R. Saint, M. Muskavitch, G.
Struhl, R. Schuh, U. Schäfer and H. Jäckle sharing flies and
antibodies; C. Müller for excellent technical assistance; and M. Josef
Pankratz for comments on the manuscript. F.J. is supported by the BIF. The
work was supported by a DFG grant to M.H. (FOR425) and the SFB 572 (TPA8).
 |
Footnotes
|
---|
* These authors contributed equally to this work 
 |
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