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Mechanisms of Development 87 (1999) 77±91
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Serotonin synchronises convergent extension of ectoderm with
morphogenetic gastrulation movements in Drosophila
Jean-FrancËois Colas a,1, Jean-Marie Launay b, Jean-Luc Vonesch a, Pierre Hickel a,
Luc Maroteaux a,*
b
a
IGBMC-CNRS-INSERM, Universite de Strasbourg, BP 163, 67404 Illkirch Cedex, France
CR C. Bernard `Pathologie expeÂrimentale et communications cellulaires', IFR HoÃpital LariboisieÁre, Service de Biochimie,
2 rue Ambroise PareÂ, 75475 Paris Cedex 10, France
Received 9 March 1999; received in revised form 31 May 1999; accepted 16 June 1999
Abstract
During Drosophila gastrulation, convergent extension of the ectoderm is required for germband extension. Adhesive heterogeneity within
ectodermal cells has been proposed to trigger the intercalation of cells responsible for this movement. Segmentation genes would impose this
heterogeneity by establishing a pair-rule pattern of cell adhesion properties. We previously reported that the serotonin receptor (5-ht2Dro) is
expressed in the presumptive ectoderm with a pair-rule pattern. Here, we show that the peaks of 5-ht2Dro expression and serotonin synthesis
coincide precisely with the onset of convergent extension of the ectoderm. Gastrulae genetically depleted of serotonin or the 5-ht2Dro receptor
do not extend their germband properly, and the ectodermal movements becomes asynchronous with the morphogenetic movements in the
endoderm and mesoderm. Associated with the beginning of this desynchronisation, is an altered subcellular localisation of adherens junctions
within the ectoderm. Combined, these data highlight the role of the ectoderm in Drosophila gastrulation and support the notion that serotonin
signalling through the 5-HT2Dro receptor triggers changes in cell adhesiveness that are necessary for cell intercalation. q 1999 Elsevier
Science Ireland Ltd. All rights reserved.
Keywords: Adherens junction; Cell intercalation; Ectoderm extension; Gastrulation; G protein; Pair rule; Serotonin
1. Introduction
The major morphogenetic movements during Drosophila
embryogenesis are observed at gastrulation immediately
after cellularisation of the blastoderm embryo. The ventral
mesoderm and the posterior endoderm primordium start
invaginating by cell shape changes to form the ventral
furrow and the proctodeal invagination, respectively
(Costa et al., 1993; Irvine and Wieschaus, 1994). Anteriorly,
the cephalic furrow forms whereas the dorsal side of the
embryo contracts as the result of pulling under the proctodeal invagination (Rickoll and Counce, 1981). The consequent dorsal and anterior shift of the posterior endoderm
invagination marks the beginning of the so-called germband
extension. During this process and within 1 h, the length of
the ectoderm along the antero-posterior axis doubles, while
* Corresponding author. Fax: 133-3-8865-3201.
E-mail address: [email protected] (L. Maroteaux)
1
Present address: Department of Neurobiology and Anatomy, University
of Utah School of Medicine, 50 N. Medical Drive, Salt Lake City, UT
84132, USA.
simultaneously its width along the dorso-ventral axis
narrows by half. By converging from the dorsal towards
the ventral side of the embryo and extending posteriorly,
ectodermal cells push the endoderm primordium in the same
direction as the pulling which generates the dorsal contraction. During the initial and most rapid phase of germband
extension, the convergent extension of ectoderm and dorsal
contraction operate as two independent forces acting in
concert (Irvine and Wieschaus, 1994). The dorsal pulling
of the endoderm primordium slightly precedes the onset of
ectodermal convergent extension and still occurs in acellular embryos indicating that ectodermal cells are not required
for the initiation of germ band extension (Costa et al., 1993).
Nevertheless, the pushing force given by the ectoderm
convergent extension is required for correct germband
extension. This force is generated exclusively by intercalation of ectodermal cells from their dorsal to their ventral
neighbours (Irvine and Wieschaus, 1994). In segmentation
gene mutant embryos, cell intercalation is reduced and
consequently the convergent extension of ectoderm and
the germ band extension are also reduced. In the model
proposed by Irvine and Wieschaus (1994), cell intercalation
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J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
in the ectoderm is dependent upon the establishment of
striped expression patterns for pair-rule genes which
would generate adhesive differences between alternate parasegments of cells. To date, mutational analysis has failed to
identify gene products that directly generate an adhesive
heterogeneity necessary for ectoderm convergent extension
although they can be predicted to have a pair-rule expression pattern.
We previously reported that the G-protein coupled receptor for serotonin (5-hydroxytryptamine, 5-HT), 5-ht2Dro, is
an orthologue of the mammalian 5-ht2 receptor subfamily
(Colas et al., 1995). 5-ht2Dro gene expression starts after
cellularisation at the blastoderm stage when major gastrulation movements begin. Simultaneously, a peak of [ 125I]DOI
(a speci®c ligand for 5-HT2 receptor subtype) binding sites
appears which are 5-ht2Dro-speci®c since they are pharmacologically indistinguishable from those of 5-ht2Dro transfected cells (Colas et al., 1995). As previously reported,
concomitant with this expression, there is also a transient
peak of 5-HT synthesis (Colas et al., 1995). The serotonin
receptor 5-ht2Dro together with tenascin Ten-m (Baumgartner et al., 1994; Meinhardt, 1995) are the only examples of
membrane proteins expressed similar to zygotic pair-rule
genes during early Drosophila gastrulation when ectodermal cells begin to move. Interestingly, in contrast to other
pair-rule genes whose expression surrounds the embryo
entirely, the seven stripes of 5-ht2Dro mRNA are restricted
to the presumptive ectoderm.
Serotonin, in adult vertebrates, is involved in vasoconstriction and neurotransmission. This compound has also
been detected during zygotic cleavage divisions, gastrulation and neurulation in embryos of sea urchins, frogs and
chickens. The presence of 5-HT and 5-HT receptors early in
embryogenesis and the ability of 5-HT-speci®c pharmacological agents to interfere with embryonic development
have led to the suggestion, that early embryos use 5-HT
prior to the onset of neurogenesis to regulate cell proliferation and/or morphogenetic movements (Buznikov et al.,
1996). However, the molecular effectors of these `prenervous' functions of 5-HT remain unresolved.
On the basis of its expression pattern, we speculated that
the 5-ht2Dro receptor might participate in germband extension during Drosophila gastrulation. Using a `reverse
genetic' approach, we identi®ed mutants defective in the
reception of the 5-HT signal and we show here that these
mutants are also defective in convergent extension of the
ectoderm.
2. Results
2.1. Peaks of serotonin and 5-ht2 binding sites correspond
precisely to stage-7 embryos
Embryos selected according to morphological criteria
de®ned by Campos-Ortega and Hartenstein (1985) were
Table 1
5-HT content and 5-ht2 binding sites of staged Drosophila embryos a
Morphology
Timing
5-HT
5-ht2 binding sites
Stage 1±4
Stage 5
Stage 6
Stage 7
Stage 8
Stage 9
,2h 30 min
2 h 30 min±3 h 10 min
3 h 10 min±3 h 20 min
3 h 20 min±3 h 35 min
3 h 35 min±4 h 00 min
4 h 00 min±4 h 30 min
,5
17.5 ^ 1.5
30.0 ^ 2.0
51.5 ^ 3.5*
39.5 ^ 2.5
31.0 ^ 4.0
,1
9.15 ^ 0.55
12.80 ^ 0.70
18.00 ^ 0.40*
10.90 ^ 0.40
9.00 ^ 0.60
a
In extracts from 100 embryos morphologically selected under oil at
248C (Campos-Ortega and Hartenstein, 1985; Costa et al., 1993), the
amount of 5-HT and of 5-ht2-speci®c binding sites (in fmol/mg protein)
was determined by radioenzymology and by [ 125I]DOI binding (Colas et al.,
1995). The values are representative of at least three experiments performed
in triplicate and expressed as means ^ SD.
*
P , 0:05 according to a Kruskal±Wallis test.
used to re®ne the exact period of the peaks of serotonin
and of 5-HT2 binding sites, previously detected at around
3 h of embryonic development (Colas et al., 1995). The
presence of receptor protein was investigated by analysing
the binding of a labelled 5-HT2 speci®c ligand, [ 125I]DOI, to
Drosophila embryo extracts. Speci®c binding can be
detected with a pharmacology indistinguishable from that
determined in 5-ht2Dro transfected COS-1 cells membranes
(Colas et al., 1995) and with a maximum of 18 fmol of
receptor/mg of total protein from stage-7 embryos (Table
1). The peak of 5-HT, corresponding to a global concentration of 51.5 fmol/mg of protein is also observed precisely at
stage 7 (Table 1). None of these serotonergic molecules can
be detected in stage 1 to 4 embryos suggesting that there is
no maternally-deposited 5-ht2Dro protein or 5-HT. Therefore,
measurements in morphologically staged wild-type
embryos reveal that both peaks of 5-HT and of 5-ht2Drospeci®c binding sites coincide precisely with the beginning
of germband extension at stage 7.
2.2. Deletion of the 5-ht2Dro locus
After our initial description of the 5-ht2Dro mRNA expression at the early gastrula stage (Colas et al., 1995), we
wanted to assess the role of the 5-ht2Dro receptor in germband extension. We initially mapped the 5-ht2Dro gene to the
82C-E genomic locus. Since the 5-ht2Dro pair-rule expression
is not affected by nearby transposon insertions, we used one
of them, l(3)j4D1, to generate two overlapping deletions
Df(3R)HTR6 and Df(3R)HTRI. Both are homozygous
lethal. Df(3R)HTRI eliminates the 5-ht2Dro receptor gene,
including 15 kb upstream and 25 kb of downstream
sequences which also include the lethal transposon insertions, j3A4 and j7E8 (Fig. 1). The j3A4, j7E8 and
Df(3R)HTR6 homozygous embryos are not embryonic
lethal but die only during late larval stages. Df(3R)HTR6
leaves the receptor intact, whereas homozygous embryos for
Df(3R)HTRI which deletes the receptor gene, present early
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
Fig. 1. Physical organisation of the 5-ht2Dro locus. Lethal transposons in the
82C-E genomic locus were localised by plasmid rescue and positioned on
the Asp 718 restriction map. Both large de®ciencies Df(3R)110 and
Df(3R)HTRE eliminate the 5-ht2Dro gene. In addition to sequences deleted
by Df(3R)HTR6, Df(3R)HTRI deletes the 5-ht2Dro transcribed region, 15 kb
of upstream and 25 kb downstream sequences. Overlapping genomic clones
were mapped and partially sequenced.
79
of a super®cial layer of the embryos in the video images,
laying the embryo ¯at, and accumulating these images over
time. This allows the quanti®cation of the speed and extent
of individual cell movement at the surface of an embryo and
the evaluation of the synchronisation of these movements.
Taking the initiation of ventral extension movements as a
reference time point, the reduced speed of the extension
movements appears clearly in 5-ht2Dro null embryos at
stage 7 (Fig. 3). After a short phase of contraction that
corresponds to mesodermal cell shape changes and invagination (210 min) and to the posterior midgut dorsal shift
(25 min), the ventral extension movements appear reduced
both in the initial forward movements and in all the subsequent backward movements. Only the endoderm invagination movement occurs at approximately normal speed (10
mm/min vs. 15 mm/min). The ectodermal cells move at a
speed close to that observed after 20 min of germband
extension in control embryos (about 4±5 mm/min). The
pole cells invaginate about 3 min late and are not centred.
These observations suggest that for mutant embryos, the
larval and embryonic lethality, and must, therefore, represent a null allele of the 5-ht2Dro gene.
2.3. Embryos lacking the 5-ht2Dro locus show abnormal
germband extension movements
In Df(3R)HTRI developing embryos, time-lapse video
recordings reveal an apparently correct cellularisation and
stage 6 progression: The cephalic furrow forms and both the
mesoderm and the proctodeum start to invaginate. The
germband initially extends dorsally in response to the pull
from the endoderm primordium on the dorsal side of the
embryo. However, in homozygous Df(3R)HTRI embryos
(genotyped after recording), the extension movements
become rapidly delayed (Fig. 2; 4 min). The rapid phase
of germband extension, in 5-ht2Dro null embryos, occurs
but at a clearly reduced speed. In the control embryos,
cells at the anterior part of the germband, initially move
both ventrally and anteriorly, pushing against the head
region which by resisting, induces a bending of the cephalic
furrow in the ventral region before disappearing. In contrast,
in the de®cient embryos the cephalic furrow remains
roughly straight and visible for most of the rapid phase of
germband extension (Fig. 2) suggesting that the pushing
force is impaired or lacking. This absence of a pushing
force is also evident later when extension is almost completely abolished in homozygous embryos (Fig. 2; 20 min).
Using image analysis of the digital video recording, we
assessed the speed of cell movements, near the ventral and
dorsal midline of the embryo. This was made possible by
using newly developed software that allows the `peeling off'
Fig. 2. Morphological analysis of germband extension by time-lapse video
recording of wild-type (WT) and of homozygous Df(3R)HTRI embryos
(Df(3R)HTRI). Matched images observed by time-lapse video are shown
at various time expressed in minutes and seconds, the zero time point is
de®ned as the initiation of extension movements and is shown in Fig. 3.
Embryos have been followed until the end of embryogenesis and then PCRgenotyped. This set of pictures reveals germband extension delay in homozygous Df(3R)HTRI. These embryos are typical of at least three independent recordings.
80
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
Fig. 3. Morphological analysis of germband extension of Df(3R)HTRI embryos. Computer generated image of a ¯at projection of the unrolled surface of the
video recorded embryos allows a direct visualisation and quanti®cation of gastrulation movements and their relative synchronisation. The cephalic furrow (cf)
formation at stage 6 is marked on the left for the dorsal and on the right for the ventral. PMG, posterior midgut and AMG, anterior midgut mark the initial limits
of the germband. The pole cells (PC) are surrounded by dorsal and ventral posterior endoderm which invaginate and form the proctodeal invagination. The
period covered by the peak of 5-HT (stage 7) starts with the zero time point which is positioned at the beginning of extension movements, and is marked on the
right as well as the time point corresponding to the images selected for Fig. 2. Tracing of the cell movements every 50 mm at zero time point are superimposed
as well as the limit of the proctodeal invagination and of the pole cells on the left. In the Df(3R)HTRI homozygous mutant (lower panel), the cell movements
are slowed down from the time extension starts and appears the origin of desynchronisation. Scales: vertical for the total time, 60 min for control (WT) and for
Df(3R)HTRI homozygous mutant (5-HT2Dro2/2), horizontal for the AP position on the embryo, total length 1000 mm.
dorsal contraction is not relayed properly by intercalation,
the movements appearing slower, delayed and ending
prematurely (Figs. 2 and 3). The resulting effect is desynchronisation between germband extension and mesodermal
and endodermal invaginations and a premature termination
of movements. Neither the homozygous embryos for the
partially overlapping de®ciency Df(3R)HTR6 nor for the
transposon insertions j3A4 and j7E8 displayed any of
these characteristic embryonic abnormalities
2.4. Stage-7 embryos lacking the 5-ht2Dro locus shows
abnormal morphology
Signs of desynchronisation can be visualised by scanning
electron microscopy (SEM). In stage-7 embryos, the pole
cells, rather than being centred in the forming proctodeal
invagination, are located posteriorly (Fig. 4F,H±J), suggesting an impairment of support cell movements dependent of
the ectoderm. Extreme desynchronisation leading to a
complete morphogenetic block is illustrated in older homozygous embryos. Here, elongated amnioserosa cells (typical
of stage 8±9) ®ll an abnormally large dorsal gap between the
front of the germband and the cephalic furrow, whereas pole
cells are still visible outside and posterior to the internalised
proctodeal invagination (Fig. 4H±J). Although in
Df(3R)HTRI homozygous de®cient embryos the mesoderm
invagination is apparently normal, defects in the ventral
midline closure are frequently observed (Fig. 4G vs. B).
2.5. Transgenic embryos lacking 5-ht2Dro binding sites
mimic Df(3R)HTRI abnormal germband extension
To assess the speci®c involvement of 5-ht2Dro in the
Df(3R)HTRI de®ciency, we abolished the expression of
the receptor using an antisense 5-ht2Dro cDNA expressed
from a heat-shock promoter (Y32 transgenic strain, see
Section 4). After establishing the conditions of minimal
heat-shock (Fig. 5A), we veri®ed that in Y32 embryos, the
5-ht2Dro-speci®c DOI-binding sites are lost at stage 7. The
result shown on Table 2 indicates that an 8 min heat-shock
induction is suf®cient to eliminate any 5-ht2-speci®c binding
sites (Table 2) and to trigger speci®c lethality associated
with embryonic defects phenocopying those of embryos
lacking the 5-ht2Dro locus. As in Df(3R)HTRI de®ciency,
we also observed slower germband extension movements,
abnormal dorsal pole cells positioning and incomplete
ventral closure (Fig. 5B±D).
In summary, the similar defects in extension movements
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
81
Fig. 4. Morphological analysis of germband extension by scanning electron microscopy. The selected images show the developmental sequence of germband
extension of a control embryo from (A) early stage 7 to (E) stage 9. All embryos have been genotyped using b -galactosidase expressing balancer chromosomes
and presented with anterior to the left and dorsal to the top except (B,G) ventral view. (A,C) Dorso-lateral course of proctodeal invagination (white arrowhead)
and of pole cells (small black arrowhead). Superposition of the two arrowheads illustrates the coincidence of these two events. (B) Stage-7 embryo showing
complete mesoderm closure. As the ectoderm elongates, at (D) stage 8, the proctodeal invagination carrying the pole cells is internalised. (E) Stars indicate
elongated cells of amnioserosa and dividing cells over the head region both typical for stage 9. (F±J) Homozygous Df(3R)HTRI embryos showing defects in
germband extension. (F) At early stage 7, the location of pole cells (small black arrowhead) is already out of phase from the proctodeal invagination (white
arrowhead). (G) Stage 7 with incomplete mesoderm closure. (H±J) In stage-9 embryo (stars, see also in E), proctodeal invagination (white arrowhead) is
internalised but misplaced pole cells (black arrowhead) stay on the surface of the embryo at a position typical for stage 7. Scale bars: …A±H† ˆ 100 mm,
…I† ˆ 20 mm, …J† ˆ 2 mm.
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J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
Fig. 5. Heat-shock conditions and phenotype induced by antisense 5-ht2Dro expression. (A) An 8 min heat shock (see Section 4) with the youngest embryo at 2 h
40 min generates the best contrast in lethality between transgenic embryos (Y32) expressing an `anti-sense cDNA' and w1118 control embryos. (B±D)
Scanning electron micrograph of Y32 embryos, heat-shocked and ®xed after 50 min latency (258C) with early morphological abnormalities similar to those of
homozygous Df(3R)HTRI (see Fig. 2). (B,C) Stage-7 embryo showing proctodeal invagination with abnormal position of pole cells, (C) High magni®cation of
(B), and (D) embryos with incomplete ventral midline closure. Scale bars: …B; D† ˆ 100 mm, …C† ˆ 20 mm.
strongly suggest that both Df(3R)HTRI and the Y32 transgenic strain have lost the same function, namely signalling
through 5-ht2Dro.
2.6. Lack of 5-HT synthesis in stage-7 embryos mimic
Df(3R)HTRI abnormal germband extension
Since the peaks of 5-HT and 5-ht2-speci®c binding sites
precisely coincided with stage 7 when the rapid phase of
germband extension begins, we searched for documented
mutations that could speci®cally affect this peak of 5-HT
synthesis in the Drosophila gastrula. We focused on alleles
of the Punch locus which encodes the GTP-CH enzyme
(Reynolds and O'Donnell, 1987). This enzyme synthesises
the pteridin cofactor required for aromatic amino acids
hydroxylases enzymatic activity including tryptophan
hydroxylase, that catalyses the limiting reaction in 5-HT
synthesis. One class of allele of the Pu locus, the embryospeci®c or class V, affects maternal and/or early zygotic
GTP-CH activity, suggesting that the resulting lethality is
due to a de®cit in early pteridin function. Using a capillary
electrophoresis technique to evaluate 5-HT content in single
class V rWE67 embryos, three populations (in morphologically selected stage-7 embryos) are distinguished: 25% with
no detectable 5-HT (less than 5 attomoles), 25% with wildtype dose and 50% with dose of 5-HT (Colas et al., 1999).
In embryos homozygous for the punctual mutation
rWE67 in the Punch locus, the pushing force generated by
ectoderm convergent extension seems also impaired or lacking. A desynchronisation of germband extension from
mesoderm and endoderm invaginations is revealed. Similar
SEM images to Df(3R)HTRI embryos were obtained. These
included embryos where pole cells, rather than being
centred in the forming proctodeal invagination, are located
posteriorly, together with defects in the ventral midline
closure and arrested extension (Fig. 6).
2.7. 5-ht2Dro ectopic expression affects ectodermal
movements
To further assess the ability of the 5-ht2Dro receptor to
control ectodermal cell movements, we tested the global
gain of function effect in transgenic embryos expressing a
sense 5-ht2Dro cDNA ectopically under heat-shock promoter
control. Strikingly, even after mild induction (heat shock of
8 min), the ubiquitous expression of the receptor strongly
disturbs the ectoderm layer elongation and cuticular organisation, both associated with a high level of lethality (not
shown). Therefore, this effect prevented us from performing
any reliable phenotypic rescue experiments.
We thus investigated the effect of a more restricted ectopic expression. In the Kr-UT1 strain which uses a KruÈppel
driver to express the Gal4 coupled to an UAS-5-HT2Dro
cDNA, a local overexpression of the 5-ht2Dro mRNA takes
place in the domain of the segmentation gap gene KruÈppel
(Fig. 7A±D). It starts almost synchronously with the 5-ht2Dro
receptor endogenous expression as a large domain (parasegTable 2
Number of 5-ht2 speci®c binding sites (fmol/mg protein) in embryos
expressing 5-ht2Dro antisense cDNA a
Latency after heat-shock (min)
Control (w1118)
Transgenic antisense (Y32)
20
4
,0.5
40
9
0.6
60
15.6
1.2
a
200 staged embryos were heat shocked (378C) for 8 min between 2 h 40
min and 3 h 00 min followed by a latency (258C) of 20, 40 and 60 min.
Number of [ 125I]DOI speci®c binding sites is expressed in fmol/mg protein
and is the average of at least two independent experiments with binding
assay performed in triplicate.
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
ment 5±8) in the region of the weakest endogenous receptor
expression (parasegment 8) and including the mesodermal
area. Drivers derived from the pair-rule genes generate
expression patterns that appear later (due to the delay in
synthesising the Gal4 protein) and therefore are not useful
for these studies (not shown). In spite of the low level of
lethality displayed by Kr-UT1, SEM (Fig. 7E±H), timelapse video observations reveal signi®cant perturbations at
the beginning of gastrulation (Fig. 8). The ®rst defects
appear as an abnormal anterior initiation of the extension
83
movements 7 min before the posterior initiation of movements. The midline ectodermal cells ®rst move forward and
only when the movements initiate at the posterior pole of the
germband does the backward movement start. The pole cells
are positioned ahead of endoderm invagination since dorsal
movement seems to initiate 1±2 min before the anterior
movements start (Fig. 9). The initial delay appears to be
compensated by a late increase in the speed of cell movement. Globally, the fact that the movement is not strikingly
abnormal in timing or extent may explain the low level of
Fig. 6. Morphological analysis of germband extension of Pu rWE67 embryos. (A±D) SEM of control embryos showing the developmental sequence of germband
extension from (A) early stage 7 to (D) stage 9. All embryos are genotyped and presented with anterior to the left and dorsal to the top except (B,F) ventral view.
(A,C) Dorso-lateral course of proctodeal invagination (white arrowhead) and of pole cells (small black arrowhead) and magni®ed in C. (B) Ventral view of
stage-7 embryo. (D) elongated cells of amnioserosa and dividing cells over the head region both typical for stage 9. (E±H) Homozygous Pu rWE67 embryos
showing defects in germband extension similar to those of embryos defective in 5-ht2Dro. (E) At early stage 7, the course of pole cells (small black arrowhead)
can also be observed out of phase from the proctodeal invagination (white arrowhead, magni®ed in G). (F) Stage 7 with incomplete mesoderm closure. (H)
Stage-9 embryo presenting only partial germband extension. Scale bars: …A; B; D±F; H† ˆ 100 mm, …C; G† ˆ 20 mm.
84
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
Fig. 7. Morphological analysis of germband extension in targeted Kr-Gal4-UAS-5-ht2Dro cDNA gain of function embryos. (A) The transgene expression site is
shown (black arrows) in stage 6 of Kr-UT1 embryos, as revealed by in situ hybridisation and include mesodermal expression. (B±D) Signal of 5-ht2Dro becomes
rapidly reinforced in parasegments 5±8 at (B) stage 7, (C) stage 8 and (D) stage 9. (E±H) Impairment of germband extension revealed by in SEM. (E) Kr-UT1
embryo at early stage 7 with delayed ventral closure of the mesoderm posterior to the KruÈppel domain (delimited by white arrows). (F) At stage 7, pole cells can
be located ahead of the proctodeal invagination, revealing a delay in support cell movement, versus endoderm invagination. (G) Embryos at stage 7 and (H) at
stage 8 show a persistent mass of ectodermal cells in the dorsal KruÈppel domain. Scale bars ˆ 100 mm.
lethality. However, the lethality is enhanced when Kr-UT1
gastrulae are heat-shocked in conditions which do not affect
the wild-type control (not shown).
The delay between anterior and posterior movements is
revealed by SEM in embryos where the mesoderm starts
closing anteriorly and pole cells, positioned outside at the
front of the endoderm invagination, can also be observed
(Fig. 7F). Later (at stage 8), the ectodermal cells located in
the dorsal KruÈppel domain do not appear to involute into the
normal dorsal transverse folds (Fig. 7G,H). Instead this
domain seems to form barrier such that the migrating
front of the germband is slightly slowed. In conclusion,
the local gap-like persistence of 5-ht2Dro also triggers a
global decoordination of germband extension with apparent
local inhibition of ectoderm movements.
2.8. Adherens junction distribution is regulated by 5-ht2Dro
signalling
Changes in ectoderm cohesion have been shown to coincide with the onset of cell intercalation. Also they are
accompanied by modi®cations of cellular apical shape
(from round to hexagonal and oblong) due to passive
stretching in the direction of the ectodermal extension
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
85
dillo in the regulation of E-cadherin-dependent cell adhesive properties (Cowin and Burke, 1996), we analysed the
fraction of this protein associated with E-cadherin at the
membrane. Confocal microscopy analysis of heat-®xed
Df(3R)HTRI homozygous embryos (at stage 7) reveals
that the membrane-associated Armadillo, rather than being
located as in normal ectodermal cells at their apex (within a
length of less than 2 mm), is distributed along their apical
side (over more than 4 mm in length) (Fig. 11). These results
reveal in 5-ht2Dro-null stage-7 embryos a reduction of apical
projections and an altered apico-basal localisation of junctional structures, presumably due to a delay in their apical
concentration.
3. Discussion
Fig. 8. Morphological analysis of germband extension in targeted Kr-Gal4UAS-5-ht2Dro cDNA by time-lapse video recording of wild-type (WT) and
of transgenic Kr-UT1 (Kr-5-HT2Dro) embryos. Matched images observed by
time-lapse video of germband extension are shown at various time
expressed in minutes and seconds, the zero time point de®ned as the initiation of extension movements and shown in Fig. 9. These embryos are
typical of at least three independent recordings. This set of pictures reveals
a slight delay in the germband extension. (see http://www1-igbmc.ustrasbg.fr/Maroteaux for full video sequence.)
movements (Hartenstein and Campos-Ortega, 1985; Costa
et al., 1993). Ultrastructural observations of wild-type
embryos have also revealed that junctions in ectodermal
cells become apically concentrated from stage 6 to stage 7
(Tepass and Hartenstein, 1994). Strikingly, we observed
that in homozygous Df(3R)HTRI embryos at apparent
stage 7 (with respect to cephalic furrow and mesodermal
invagination) most ectodermal cells have a round apex
presenting only few intercellular connecting structures.
This is similar to the ectodermal cells in control stage-6
embryos. Thus, the progression in ectoderm cohesion,
which normally occurs between stage 6 and 7, is impaired
in 5-ht2Dro null embryos (Fig. 10).
At gastrulation stage, E-cadherin constitutes the adhesive
part of the adherens junction structures, acting by homophilic and calcium-dependent interactions (Oda et al., 1998).
Junction clustering in the zonula adherens is dependent
upon association of the E-cadherin cytoplasmic tail to the
cortical actin cytoskeleton. Given the pivotal role of Arma-
We report that embryos homozygous for the de®ciency
Df(3R)HTRI, which removes the 5-ht2Dro receptor gene,
show abnormal gastrulation movements (Fig. 2). Unlike
embryos homozygous for the partially overlapping de®ciency Df(3R)HTR6 which leaves the 5-ht2Dro gene intact
(Fig. 1), the homozygous Df(3R)HTRI embryos show
delayed morphogenetic movements with incomplete germband extension and die at the end of embryogenesis or at the
early ®rst instar larval stage. The participation of genes
other than 5-ht2Dro within the 50 kb of DNA deleted in
Df(3R)HTRI cannot be completely ruled out. However, in
this region, the two transposon insertions, j7E8 and j5DI, are
not embryonic lethal. Also, no signal could be detected by in
situ hybridisation with embryos at stage 7 or younger, using
genomic probes from this region, except those containing
the receptor locus. These data, together with the fact that a
similar phenotype is observed (i) in the Y32 transgenic
strain which expresses the 5-ht2Dro antisense cDNA (Fig.
3) and is depleted for 5-ht2Dro binding sites (Table 2), and
(ii) in embryos homozygous for the punctual Pu rWE67 allele
of the Pu locus that are defective for 5-HT synthesis (Colas
et al., 1999), argue strongly that defective 5-HT2 signalling
is the main contributor of this phenotype. Furthermore, the
restricted ectopic expression of 5-HT2Dro cDNA in the KrUT1 strain which is also able to generate other gastrulation
defects (Fig. 4) also argues for the involvement of defective
5-HT signalling in extension abnormalities. Together, these
data strongly support the notion that during Drosophila
gastrulation, 5-HT2Dro signalling plays a key role in proper
convergent extension of ectoderm.
Germband extension in Drosophila has interesting similarities with convergent extension movements during vertebrate neurulation (Wieschaus et al., 1991; Keller et al.,
1992). In chicken embryos at the neurulation stage, 5-HT
antagonists affect cell movements in the neuroepithelium
(Palen et al., 1979). In mice, our group has reported that
treatments of neurulating embryos with 5-HT2B antagonists
induce defects in neural tube closure (Choi et al., 1997).
Furthermore, 5-HT may also function in the differentiated
86
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
Fig. 9. Morphological analysis of germband extension in targeted Kr-Gal4-UAS-5-ht2Dro cDNA gain of function embryos. Flat projection of the unrolled
surface of the video recorded embryos shows that in the Kr-UT1 embryos, the mesodermal invagination initiate ®rst anteriorly and apparently prematurely and
then about 7 min later anteriorly. The global movement is only slightly impaired, the initial delay is compensated by a late increase in cell speeds. However, it
reveals that dorsal contraction start just before the anterior initiation of movements. The KruÈppel region of 5-ht2Dro overexpression is marked on the top and
seems to be responsible for this difference in invagination timing. The nomenclature used is that of Fig. 3. Scales: vertical for the total time, 60 min for control
(WT) and 55 min for Kr-UT1 (Kr-5-HT2Dro), horizontal for the AP position on the embryo, total length 1000 mm.
nervous system, where remodelling of synapses by the regulation of cell adhesion molecules has been implicated in
learning and memory (Michael et al., 1998). Thus, our
observations support the hypothesis of Kandel and O'Dell
(1992) that molecular mechanisms involved in embryonic
development, can also function in the adult brain. Combined
with the present report, these observations suggest that the
embryonic functions of the 5-HT receptor observed in
Drosophila may also be applicable to other species and in
other physiological contexts.
3.1. 5-HT2Dro mediates segmentation gene functions in
germband extension
In Drosophila, the rapid phase of germband extension,
known to result exclusively from cell intercalation within
the ectoderm, is ultimately dependent upon the establishment of a striped expression pattern of pair-rule genes
(Irvine and Wieschaus, 1994). The 5-HT2Dro receptor ful®ls
the requirements to be able to accomplish the immediate
morphogenetic functions of pair-rule genes during ectoderm
extension. Namely 5-HT2Dro has (i) a peak of activity at
stage 7, (ii) an expression pattern of seven stripes and (iii)
an ectodermal restricted expression (Colas et al., 1995).
Although the greatest alteration in 5-HT2Dro expression has
been observed in the pair-rule mutant even-skipped (eve), all
of the gap or pair-rule mutants but none of the segment
polarity genes tested, altered its expression (Colas et al.,
1995).
Previous observations have shown that germband extension initiates by a dorsal pulling movement which (i) occurs
in acellular preblastoderm embryos, (ii) slightly precedes
the onset of ectodermal elongation, and (iii) is unaltered
in segmentation mutants. This suggests that pair-rule functions are not required for this movement (Irvine and
Wieschaus, 1994). In 5-HT2Dro null embryos, the dorsal pulling appears to be independent of 5-HT2Dro. In these embryos,
extension movements occur but consistently at a slower
speed. Furthermore, we observed some cellularised individuals that initiate but quickly reverse germband extension.
Independently, the Irvine's report indicated that, in the
strongest segmentation mutants, such as eve, the ectodermal
cell intercalation ®rst begins 10 min after the formation of
the cephalic furrow and the germband extension slows or
reverses after 15±25 min (Irvine and Wieschaus, 1994).
These common characteristics of extension movements
strongly suggest that, during germband extension, 5HT2Dro is one of the effectors for the morphogenetic action
of segmentation genes.
3.2. Extrinsic contribution of ectoderm to endodermal and
mesodermal invaginations
In 5-HT2Dro null embryos, the dorsal shift of the epithelial
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
87
Fig. 10. 5-ht2Dro-dependent modi®cations of cellular junctions during germband extension. The morphology of ectodermal epithelium observed in SEM for (A±
C) control embryos appears abnormal in (D±F) 5-ht2Dro null genotyped embryos. Apical shape of ectodermal cells is hexagonal oblong in (B) but round in (E).
Intercellular apical projections (microvilli) are dense in (C) but sparse in (F). Scale bars: …A; D† ˆ 50 mm, …B; E† ˆ 5 mm, …C; F† ˆ 2 mm.
cells supporting the pole cells appears delayed, consistent
with these support cells initiating their movement too late
with respect to endoderm pulling. This suggests that the
receptor's function in the ectoderm involves support cell
movement. The failure of some posterior cells to invaginate
into posterior midgut had been previously reported for the
pair-rule mutants, such as eve, that show signi®cantly
reduced germband extension (Irvine and Wieschaus,
1994). Synchronisation of the endoderm invagination
(which is internalised by an independent pull) with the ectoderm elongation (which pushes the cells supporting the pole
cells), is therefore required for pole cells to be correctly
centred in the proctodeal invagination, and is dependent
on the 5-HT2Dro receptor function.
Forces that drive ectoderm elongation are independent
of mesodermal cells, since germband extension appears
in fully dorsalised and lateralised embryos (Ip et al.,
1994; Leptin, 1995). Nevertheless, as shown by Costa
et al. (1993), germband anchoring to ventral cells is
required to orient elongation. The same authors have
also suggested that the intercalation of the ectoderm
within the lateral cells may be necessary for complete
closure of the ventral furrow. Our experiments consistently show that both 5-HT2Dro loss of function and
local gap-like ectopic expression of the receptor trigger
defects in closure of the ventral midline. Therefore, we
conclude that 5-HT2Dro coordinates movements of ectoderm convergence with the preceding ventral furrow
invagination thus enabling complete fusion of midventral
cells.
88
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
Fig. 11. Distribution of adherens junction-associated Armadillo protein at stage 7 after heat-®xation. Immunostaining detects, by confocal microscopy,
membrane-associated Armadillo in control embryos (WT) where subcortical Armadillo is localised at cell contact point and this localisation is strong in a
single apical focal plane (leftmost panel). Confocal reconstitution of the epithelial thickness of the lateral side of this embryo shows the concentration of the
membrane associated Armadillo strictly at the apical side. In 5-ht2Dro null retarded embryos (Df(3R)HTRI), subcortical Armadillo is localised at cell contact
point and this localisation appear more diffuse in a single apical focal planes (left panel). Confocal reconstitution of the epithelial thickness of the lateral side of
this embryo shows that the membrane associated Armadillo staining is distributed over a wide subapical area at the ectodermal cell side. The position of a cell
in the epithelial layer is marked as a dotted square, apical pole (Ap) at the right-hand side and basal pole (Ba) at the left-hand side. All embryos are anterior to
the left and dorsal to the top. Scale bars ˆ 5 mm.
3.3. 5-HT-signalling can regulate cell adhesion
How can 5-HT2Dro affect cell intercalation during germband extension? Within the early Drosophila gastrula,
maternal and zygotic E-cadherin and catenins are ubiquitously distributed (Knust and Leptin, 1996; Oda et al.,
1998). Modulation of adhesive properties within the timescale of cell intercalation requires regulatory mechanisms
faster than can be achieved by transcriptional regulation. In
5-HT2Dro null embryos, the absence of apical ectodermal
projections parallels the lack of an apical concentration of
adherens junctions (Figs. 10 and 11). These observations
indicate that 5-HT signalling may control the redistribution
of pre-existing junctional elements and imply that it can also
generate adhesive constraints. As a consequence, the 5HT2Dro stripped pattern would generate parasegmental adhesive differences necessary for cell intercalation. Modulation
of adhesive strength by clustering of spot adherens junctions
at the apex in zonula adherens is regulated by phosphorylation of intracellular portions of cadherin and/or b -catenin
(Peifer et al., 1994; Stappert and Kemler, 1994; Cowin and
Burke, 1996). Receptors of the 5-HT2 subfamily signal
through the trimeric G protein Gq (Launay et al., 1996).
In tumour cells, adhesion mediated by cadherins can be
regulated by receptors coupled to Gq (Williams et al.,
1993). In Drosophila, the link between 5-HT2Dro and cell
movements is, therefore, likely to rely on its control of Ecadherin-cytoskeleton association. Preliminary transmission electron microscopy data con®rm the abnormal distribution of adherens junction reported here by confocal
microscopy studies.
3.4. Why does elimination of the segmented 5-HT2Dro
expression lead to a non-segmented phenotype?
According to the model proposed by Irvine and
Wieschaus (1994), intercalation depends upon the establishment of stripes of pair-rule gene products. When these
stripes are widened or eliminated, either by pair-rule mutations, mutations in genes that regulate pair-rule gene expression, or ubiquitous expression of eve, then ectodermal cell
intercalation and germband extension is reduced. Although
the postulated adhesive molecules remain to be identi®ed,
the ability of this model to explain the effects of embryonic
patterning mutations on germband extension make it attractive. In its simplest form, this model for cell intercalation
requires only two types of adhesive cells, which would be
distributed in alternate segments. This model implied that (i)
within stripes, relative strengths of adhesion among cells
must be equal, (ii) cells of different stripes must differ quantitatively in their strength of adhesion, and (iii) defects
resulting from alterations of the segmented pattern may
not necessarily be segmented. Furthermore, it has been
reported that within a single tissue, cells expressing an identical cadherin can, on the basis of the expression level of this
protein, segregate into distinct populations (reviewed by
Peifer, 1998).
Our data indicate that both lack and excess of misexpressed 5-HT2Dro receptor induce abnormal germband extension accompanied by a change in the subcellular distribution
of cellular junctions. By inducing local gap-like ectopic 5HT2Dro expression, subtle desynchronised movements
restricted to the transgene overexpression domain and loss
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
of ectodermal plasticity are observed in the dorsal region of
the embryo. This corresponds to the initial location of the
KruÈppel domain, a region in which the receptor is normally
only weakly expressed (Fig. 7A±D). This phenotype is
reminiscent of that previously reported for embryos expressing eve ectopically, which also display a reduced germband
extension (Irvine and Wieschaus, 1994). Combined, these
data suggest that the alternate distribution of differentially
dosed receptor is required to generate normal germband
extension and is consistent with 5-HT signalling as a
mechanism generating parasegmental differences in adhesion necessary for cell intercalation. Since the defects do not
appear to have a segmented pattern, one has to assume that
5-HT signalling is involved in generating segmental differences in the wild-type Drosophila embryo, although direct
evidence for an alternate distribution of cell adhesive structures has not yet been reported. The identi®cation of direct
intracellular targets of 5-HT signalling which control cell
adhesion should constitute an important contribution to the
understanding of how extracellular signals control cell
movements.
4. Experimental procedures
89
described (Rosay et al., 1995). Two to six independent
transgenic lines were selected for each construct.
4.4. Embryo observation and staging
The embryos were laid on an agar plate, observed at 258C,
60% humidity and covered with oil 3S (Voltalef, Prolabo).
Embryos were staged using morphological criteria according to Campos-Ortega and Hartenstein (1985) and to
Wieschaus and NuÈsslein-Volhard (1986) with the modi®cations of Irvine and Wieschaus (1994). Brie¯y, embryos were
timed according to the formation of the cephalic furrow
which precedes germband extension by 5 min and to the
initial ventral movements of cells. Digital time-lapse videos
of embryos (1 image per 15 s) were recorded under bright®eld illumination and synchronised using similar criteria.
After recording, the embryos were followed for 24 h and
genotyped. DNA from a single recorded embryo was used
for PCR-genotyping. Computer video image analysis was
performed using software developed by J.-L.V. At least
three similar recordings from independent experiments
were used for phasing and speed calculations. Video recordings corresponding to Figs. 2 and 4 are available at http://
www1-igbmc.u-strasbg.fr/Maroteaux.
4.1. Genomic clones
The genomic clones shown in Fig. 1A were obtained from
EDGP (FORTH, Heraklion, Greece) (cosmids) and from
BDGP (EMBL, Heidelberg, Germany) (Bacteriophage P1).
4.2. Fly stocks
The following ¯y stocks were used: l(3)j3A4; l(3)10112;
ms(3)07735; l(3)j4D1; l(3)01456; l(3)02733. l(3)j5D1;
l(3)j7E8; l(3)02466; #19/1; Df(2R)F36; Pu rWE67;
Df(3R)110; Kr-Gal4.
4.3. Transgenic constructs
The full-length 5-ht2Dro cDNA was inserted into the
EcoRI±BglII restriction sites of the pCaSpeR-hs or
pUAST expression vector. The later was used to generate
the UT1 strain. This strain has been crossed with the KrGal4 strain which expresses Gal4 under KruÈppel transcriptional regulatory sequences (Jacob et al., 1991) to obtain,
after recombination, the Kr-UT1 strain. This strain overexpresses the 5-ht2Dro cDNA with the gap gene KruÈppel pattern
and is homozygous viable.
A 5-ht2Dro cDNA sequence which includes the N-terminus, the extracellular region and the ®rst transmembrane
region of 5-ht2Dro has been obtained by polymerase chain
reaction (PCR)-ampli®ed exon 1 from genomic DNA (1.1
kb). It has been used to generate the antisense 5-ht2Dro
construct by insertion in the reverse orientation into the
EcoRI±BglII sites of the pCaSpeR-hs (Y32) expression
vector. P-element transformation of ¯ies was performed as
4.5. Heat shock
Heat-shock conditions that minimally affect the control
embryos and maximise the lethality speci®cally induced by
the transgene were selected (Fig. 3A). 2 h 35 min±3 h 25
min old embryos collected at 258C, 60% humidity were
heat-shocked in tap water (378C) at 2 h 40 min for precisely
8 min and stopped in tap water (258C) at 2 h 48 min for 1
min. After slight drying and covering with oil, the embryos
showing cephalic furrow formation (stage 6) were eliminated until 3 h 15 min in order to select for cellularised
embryos aged between 2 h 40 min and 3 h 00 min during
the heat shock.
4.6. Cuticle analysis and lethality statistics
The embryonic lethality was evaluated after 24 to 48 h
(258C) and the non-hatched embryos expressed as the
percent of the total (500±1000 embryos; n . 3). Each
morphologically distinct type of embryo has been PCRgenotyped. For pictures, the cuticles of dead embryos
were prepared as described (NuÈsslein-Volhard et al., 1984).
4.7. Genetic experiments
Df(3R)HTRE was generated by X-ray mutagenesis of the
l(3)j3A4 chromosome. Oriented deletions (Df(3R)HTRI,
Df(3R)HTR6) have been produced from the l(3)j4D1 transposon insertion using the male recombination technique
(Preston and Engels, 1996).
90
J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91
4.8. In situ hybridisation and immunohistological
genotyping of embryos
Whole-mount embryos were prepared as described by
Colas et al. (1995) and hybridised (428C) to the digoxigenin
labelled 56E12 cosmid or a 5-ht2Dro cDNA clone (Fig. 1A).
For genotype determination, blue balancer chromosomes,
CyO P(twi-lacZ), TM3 Ser P(twi-lacZ) and TM3 P(hblacZ) were used. Before scanning microscopy analysis,
embryos were genotyped by immunostaining with polyclonal anti b -galactosidase (b -Gal) primary antibody and a
peroxidase-coupled secondary antibody. Embryos were
sorted on the basis of the strong b -Gal signal driven in
head of gastrulae by the hunchback promoter gene.
4.9. Genotyping by PCR
For genotype determination, blue balancer chromosomes,
CyO P(twi-lacZ), TM3 Ser P(twi-lacZ) and TM3 P(hb-lacZ)
were used. After collection (from 3 to 48 h, if not histolysed), DNA was extracted, and 1/10 of the DNA from a
single embryo was ampli®ed using regular PCR ampli®cation conditions for 38 cycles. Amplimers were selected from
the b -Gal sequence and from the balancer driver promoter
(hunchback or twist). DNA not ampli®ed with the balancer
amplimers were considered as homozygous for a de®ciency
if they can also be ampli®ed with amplimers of a gene
outside but not with primers inside the de®ciency under
the same conditions. This technique allowed us to genotype
unambiguously all individual embryos or larvae morphologically selected and presented here.
4.10. Scanning electron microscopy
Immunologically genotyped embryos were re®xed overnight (48C) by 2.5% glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2; 2 mM CaCl2, washed 30 min in the same
buffer. After post-®xation for 1 h (48C) in 1% osmium tetroxide, embryos were dried with a critical point-drying apparatus, mounted on aluminium stubs coated with palladiumgold using a cold sputter-coater and observed with a Philips
XL-20 microscope.
4.11. Heat ®xation and confocal microscopy
`Hard-boiled embryos' adapted from MuÈller and
Wieschaus (1996) were incubated overnight (48C) with
monoclonal anti-Armadillo antibody (1/15 dilution) then
with Cy3-conjugated goat anti-mouse secondary antibody
(1:500, Jackson ImmunoRes. Lab.) and mounted in 5%
propylgalate in glycerol. A confocal Leica microscope
was used. Image reconstruction was performed using software developed by J.-L.Vonesch.
Acknowledgements
We are indebted to I.S. Kiamos, M. Mlodzik, the Droso-
phila stocks centres of Bloomington (USA) and Umea
(Sweden), A. Spradling, P. Deak, J. Roote, M. Akam, and
S. DiNardo for providing ¯y strains, N. Perrimon for
pUAST plasmids, M. Peifer, S. Oda and S. Takeichi, for
antibodies, and M. Peifer for heat-®xation procedure. We
wish to acknowledge S. Wasser and P. Rosay for initial
participation in the work, N. Messaddeq and M. Digelmann
for help with electron microscopy and M.-L. Nullans for
excellent technical assistance. We thank Dr M. Bellard,
N.S. Foulkes and A. Giangrande for critical reading of the
manuscript, and Dr S. Birman, E. Borrelli, P. Heitzler, and
Professor H. JaÈckle for helpful discussions. This work has
been supported by funds from the Centre National de la
Recherche Scienti®que, the Institut National de la Sante et
de la Recherche MeÂdicale, the HoÃpital Universitaire de
Strasbourg, and the Universite Louis Pasteur, and by grants
from the European Community, the MinisteÁre de l'Enseignement SupeÂrieur et de la Recherche, the Fondation
pour la Recherche MeÂdicale, the Association FrancËaise
contre les Myopathies, the Association pour la Recherche
contre le Cancer, and the Ligue Nationale contre le Cancer.
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