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Mechanisms of Development 87 (1999) 77±91 www.elsevier.com/locate/modo 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 0925-4773/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00141-0 78 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 100 mm, I 20 mm, J 2 mm. 82 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 100 mm, C 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 100 mm, C; G 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 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 50 mm, B; E 5 mm, C; F 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 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. References Baumgartner, S., Martin, D., Hagios, C., Chiquet-Ehrismann, R., 1994. Ten-m, a Drosophila gene related to tenascin, is a new pair-rule gene. EMBO J. 13, 3728±3740. Buznikov, G.A., Shmukler, Y.B., Lauder, J.M., 1996. From oocyte to neuron: do neurotransmitters function in the same way throughout development? Cell. Mol. Neurobiol. 16, 537±559. Campos-Ortega, J.A., Hartenstein, V., 1985. The Embryonic Development of Drosophila melanogaster, Springer, Berlin. Choi, D.-S., Ward, S.J., Messaddeq, N., Launay, J.-M., Maroteaux, L., 1997. 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells. Development 124, 1745±1755. Colas, J.-F., Launay, J.-M., Kellermann, O., Rosay, P., Maroteaux, L., 1995. Drosophila 5-HT2 serotonin receptor: coexpression with fushitarazu during segmentation. Proc. Natl. Acad. Sci. USA 92, 5441±5445. Colas, J.-F., Launay, J.-M., Maroteaux, L., 1999. Maternal and zygotic controls of serotonin biosynthesis are necessary for Drosophila germband extension. Mech. Dev. 87, 67±76. Costa, M., Sweeton, D., Wieschaus, E., 1993. Gastrulation in Drosophila: cellular mechanisms of morphogenetic movements. In: Bate, M., Martinez-Arias, A. (Eds.). The Development of Drosophila melanogaster, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 425± 465. Cowin, P., Burke, B., 1996. Cytoskeleton-membrane interactions. Curr. Opin. Cell. Biol. 8, 56±65. Hartenstein, V., Campos-Ortega, J.A., 1985. Fate-mapping in wild-type Drosophila melanogaster. I. The spatio-temporal pattern of embryonic cell divisions. Roux's Arch. Dev. Biol. 194, 181±195. Ip, Y., Maggert, K., Levine, M., 1994. Uncoupling gastrulation and mesoderm differentiation in the Drosophila embryo. EMBO J. 13, 5826± 5834. Irvine, K., Wieschaus, E., 1994. Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes. Development 120, 827±841. Jacob, Y., Sather, S., Martin, J., Ollo, R., 1991. Analysis of Kruppel control elements reveals that localized expression results from the interaction of multiple subelements. Proc. Natl. Acad. Sci. USA 88, 5912±5916. J.-F. Colas et al. / Mechanisms of Development 87 (1999) 77±91 Kandel, E.R., O'Dell, T.J., 1992. Are adult learning mechanisms also used for development? Science 258, 243±245. Keller, R., Shih, J., Sater, A., 1992. The cellular basis of the convergence and extension of the Xenopus neural plate. Dev. Dyn. 193, 199±217. Knust, E., Leptin, M., 1996. Adherens junctions in the Drosophila embryo: the role of E-cadherin in their establishment and morphogenetic function. Bioessays 18, 609±612. Launay, J.-M., Birraux, G., Bondoux, D., Callebert, J., Choi, D.-S., Loric, S., Maroteaux, L., 1996. Ras involvement in signal transduction by the serotonin 5-HT2B receptor. J. Biol. Chem. 271, 3141±3147. Leptin, M., 1995. Drosophila gastrulation: from pattern formation to morphogenesis. Annu. Rev. Cell Biol. 11, 189±212. Meinhardt, H., 1995. Dynamics of stripe formation. Nature 376, 722±723. Michael, D., Martin, K.C., Seger, R., Ning, M.-M., Baston, R., Kandel, E.R., 1998. Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of Aplysia. Proc. Natl. Acad. Sci. USA 95, 1864±1869. MuÈller, H.-A.J., Wieschaus, E., 1996. Armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134, 149±163. NuÈsslein-Volhard, C., Wieschaus, E., Kluding, H., 1984. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193, 267±282. Oda, H., Tsukita, S., Takeichi, M., 1998. Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation. Dev. Biol. 203, 435±450. Palen, K., Thorneby, L., Emmanuelsson, M., 1979. Effects of serotonin and serotonin antagonists on chick embryogenesis. Roux's Arch. Dev. Biol. 187, 89±103. 91 Peifer, M., Pai, L.M., Casey, M., 1994. Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. Dev. Biol. 166, 543±556. Peifer, M., 1998. Birds of a feather ¯ock together. Nature 395, 324±325. Preston, C.R., Engels, W.R., 1996. P-element-induced male recombination and gene conversion in Drosophila. Genetics 144, 1611±1622. Reynolds, E.R., O'Donnell, J.M., 1987. An analysis of the embryonic defects in Punch mutants of Drosophila melanogaster. Dev. Biol. 123, 430±441. Rickoll, W., Counce, S., 1981. Morphogenesis in the embryo of Drosophila melanogaster ± Germ band extension in the maternal-effect lethal mat(3)6. Roux's Arch. Dev. Biol. 190, 245±251. Rosay, P., Colas, J.-F., Maroteaux, L., 1995. Dual organisation of the Drosophila neuropeptide receptor NKD gene promoter. Mech. Dev. 51, 329±339. Stappert, J., Kemler, R., 1994. A short core region of E-cadherin is essential for catenin binding and is highly phosphorylated. Cell Adhes. Commun. 2, 319±327. Tepass, U., Hartenstein, V., 1994. The development of cellular junctions in the Drosophila embryo. Dev. Biol. 161, 563±596. Wieschaus, E., NuÈsslein-Volhard, C., 1986. Looking at embryo. In: Roberts, D. (Ed.). Drosophila, A Practical Approach, IRL, Oxford/ Washington DC, pp. 199±227. Wieschaus, E., Sweeton, D., Costa, M., 1991. Convergence and extension during germ band elongation in Drosophila embryos. In: Keller, R. (Ed.). Gastrulation, Plenum, New York, pp. 213±223. Williams, C.L., Hayes, V.Y., Hummel, A.M., Tarara, J.E., Halsey, T.J., 1993. Regulation of E-cadherin-mediated adhesion by muscarinic acetylcholine receptors in small cell lung carcinoma. J. Cell Biol. 121, 643± 654.