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RESEARCH ARTICLE

Cadherin switching during the formation and differentiation of theDrosophila mesoderm – implications for epithelial-to-mesenchymal transitions

Gritt Schafer1,*, Maithreyi Narasimha2,*, Elisabeth Vogelsang1 and Maria Leptin1,`

ABSTRACT

Epithelial-to-mesenchymal transition (EMT) is typically accompanied

by downregulation of epithelial (E-) cadherin, and is often additionally

accompanied by upregulation of a mesenchymal or neuronal (N-)

cadherin. Snail represses transcription of the E-cadherin gene both

during normal development and during tumour spreading. The

formation of the mesodermal germ layer in Drosophila, considered

a paradigm of a developmental EMT, is associated with Snail-

mediated repression of E-cadherin and the upregulation of N-

cadherin. By using genetic manipulation to remove or overexpress

the cadherins, we show here that the complementarity of cadherin

expression is not necessary for the segregation or the dispersal of the

mesodermal germ layer in Drosophila. However, we discover

different effects of E- and N-cadherin on the differentiation of

subsets of mesodermal derivatives, which depend on Wingless

signalling from the ectoderm, indicating differing abilities of E- and N-

cadherin to bind to and sequester the common junctional and

signalling effector b-catenin. These results suggest that the

downregulation of E-cadherin in the mesoderm might be required

to facilitate optimal levels of Wingless signalling.

KEY WORDS: Drosophila, Gastrulation, Cell adhesion, b-catenin,

Wingless signalling

INTRODUCTIONThe organisation and dynamics of many tissues in multicellular

animals rely on cell–cell adhesion mediated by the conserved

cadherin superfamily of transmembrane proteins. The highest

density of cadherins in a mature epithelium is found at theultrastructurally distinct adherens junctions. Classic cadherins

mediate adhesion between cells through molecular bridges that

are formed through homophilic interactions of the extracellularcadherin domains (Nagafuchi et al., 1987; Nose et al., 1988),

although heterotypic interactions can also occur (Nose et al.,

1988). The cytoplasmic and transmembrane domains of

cadherins serve as platforms for protein interactions andmediate links both to the cytoskeleton and to signalling

pathways. Both linkages rely on interactions with b-catenin

(also known as Armadillo in Drosophila), a protein that binds to

cadherin through its cytoplasmic domain. In vivo, the link to the

cytoskeleton requires a-catenin (Desai et al., 2013; Kwiatkowskiet al., 2010; Pacquelet and Rørth, 2005; Sarpal et al., 2012),although biochemical studies have indicated that a-catenin caneither be bound to actin or b-catenin, but not to both (Drees et

al., 2005). The link to signalling pathways relies on interactionsof cadherins with both b-catenin and p120-catenin, and also oninteractions of the cadherin extracellular domain (Oda and

Takeichi, 2011).

There are more than 20 classic cadherins, and they share a

similar gross organisation of extracellular and cytoplasmicdomains. They are grouped into subtypes, such as the E-cadherin and the N-cadherin subfamilies, based on sequence

similarities and their expression in specific tissues. In general,they show preferential binding to cadherins of their own type,with limited binding to other types. Early evidence for suchpreferential binding came from the ‘sorting’ observed in mixtures

of cells expressing different cadherins or different amounts of thesame cadherin. Such studies led to the suggestion that differentialadhesion mediated by the extracellular cadherin domains was

responsible for the sorting of cells (Nose et al., 1988; Steinbergand Takeichi, 1994). Recent studies have revealed thatdifferential binding to the cytoskeleton, mediated by the

cytoplasmic domain of the cadherin molecule, and thecorresponding differences in tension, rather than sequencesimilarity of the extracellular cadherin domains, might underlie

cell sorting (Krieg et al., 2008; Maıtre et al., 2012).

Different members of the cadherin family show distinct, tissue-specific patterns of expression. A striking conservation of these

expression patterns across species is observed for the epithelialand neuronal expression of E- and N-cadherin, respectively(Takeichi, 1988). In the early Drosophila embryo, two members

of the class I subtype of cadherins are expressed. The geneencoding E-cadherin, shotgun (shg), is transcribed in the futureectoderm, whereas N-cadherin is expressed in the mesoderm.

This complementary expression pattern is determined by thetranscription factors that control mesodermal development. Twistactivates N-cadherin expression in the mesoderm, whereas Snail

represses E-cadherin expression (Oda et al., 1998). This mode oftranscriptional regulation, most notably the repression of E-cadherin by Snail, has also been demonstrated in epithelialtumour cells undergoing the epithelial-to-mesenchymal transition

(EMT) (Batlle et al., 2000; Cano et al., 2000). The possibility thatqualitative differences in adhesion might underlie the segregationof tissues during morphogenesis was first suggested by

Holtfreter’s observation that cells dissociated from germ layerssort out in vitro (Townes and Holtfreter, 1955). A switch in thetype of cadherin expressed, for example from E-cadherin to N-

cadherin, has been observed during several biological processes,

1Institute of Genetics, University of Cologne, Zulpicher Strasse 47a, 50674Cologne, Germany. 2Department of Biological Sciences, Tata Institute ofFundamental Research, Colaba, Mumbai 400005, India.*These authors contributed equally to this work

`Author for correspondence (mleptin@uni-koeln.de)

Received 31 July 2013; Accepted 9 January 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1511–1522 doi:10.1242/jcs.139485

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including gastrulation [in the frog (Nandadasa et al., 2009),chicken (Hatta and Takeichi, 1986) and fly (Oda et al., 1998)],

formation of the primitive streak (Nakagawa and Takeichi, 1995),tumorigenesis [e.g. melanoma (Li et al., 2001)] and in someEMTs (Wheelock et al., 2008). Indeed, Snail also controls thetranscriptional downregulation of E-cadherin in EMT associated

with tumorigenesis (Yang and Weinberg, 2008), and is regardedas a bona fide EMT inducer (Nieto, 2011). Whether such a switchin cadherin types is functionally meaningful for tissue

morphogenesis (for example to segregate the mesoderm fromthe ectoderm), or whether it is necessary to drive changes in cellbehaviour (such as the cell shape changes required for cell

migration) is unclear. In addition, although the downregulation ofE-cadherin is necessary for the transition from epithelial tomesenchymal cells, neither its precise mode of regulation nor the

role of N-cadherin in this process are well understood.In addition to interacting with the cytoskeleton, cadherins can

mediate cell signalling. Two signalling pathways that areinfluenced by cadherins include the Wnt and FGF signalling

pathways. The former relies on the relocalisation of b-catenin, acomponent of the adherens junction, to the nucleus in response tochanges in the levels of E-cadherin (Heasman et al., 1994; Sanson

et al., 1996). Wnt signalling in turn can influence E- and N-cadherin levels through its regulation of the levels of thetranscription factors Snail and Twist (Yang and Weinberg,

2008). Although the Snail promoter has been found to containbinding sites for LEF, the Wingless signalling effector in Xenopus

(Vallin et al., 2001), no effect on Snail expression was found

upon constitutive activation of Wingless signalling in vitro (Kimet al., 2002). One way in which cadherins are connected with theFGF signalling pathway, which is essential for neurite outgrowthin cultured neurons, is through interactions between the FGF

receptor and the extracellular domain of N-cadherin (Cavallaroand Christofori, 2004; Williams et al., 2001). Another connectionbetween cadherins and FGF signalling, observed in frogs, chicks

and mice, relies on FGF signalling for induction or maintenanceof the expression of genes of the Snail family, resulting indownregulation of E-cadherin (Buxton et al., 1997). Consistent

with this, mice lacking the FGF receptor FGFR1 fail to undergoan EMT at gastrulation (Ciruna and Rossant, 2001). In thisrespect, FGF functions in a manner analogous to that of Twist inthe Drosophila mesoderm. The binding of p120-catenin to

cadherin also influences signalling through the Rho GTPases,key regulators of cytoskeletal organisation and adhesion, whoserole seems to be to ensure the stability of junctional complexes

(Castano et al., 2007). Whether different cadherins engagesignalling pathways differently is also unclear, although thereare reports that different isoforms of Armadillo (Drosophila b-

catenin) might engage E- and N- cadherins (Loureiro and Peifer,1998).

In the work we describe below, we investigate the relevance

of the complementary patterns of expression of E- and N-cadherin during Drosophila gastrulation. In contrast to theprevailing view that the segregation of the mesoderm from theectoderm is controlled by developmentally regulated differential

adhesion that is driven by the two types of cadherin in theectoderm and mesoderm (Oda and Tsukita, 1999), we find noearly requirement for differential adhesion mediated by the two

cadherin types, either for the epithelial-to-mesenchymal-liketransition or for segregation between the ectoderm andmesoderm. Instead, our results argue that the differential

effects on Wingless signalling downstream of the two

cadherins are responsible for the influence of these cadherinson late mesoderm differentiation.

RESULTSZygotic loss of function of either E-cadherin or N-cadherinhas no consequences for mesoderm morphogenesisDrosophila E-cadherin (Tepass and Hartenstein, 1994) isexpressed at low levels throughout the early blastoderm embryofrom maternally provided mRNA. The zygotic transcription of

the gene is activated at high levels in the cellularising blastodermstage embryo, but remains inactive in the future mesodermthrough repression by the transcription factor Snail (Oda et al.,

1998). Thus, the ectoderm expresses high levels of E-cadherin,whereas the mesoderm continues to express the lower maternallevel that remains detectable until after the mesoderm has

invaginated. N-cadherin (encoded by CadN) is expressed in theearly mesoderm under the control of Twist, and is later expressedin neuronal tissues (Iwai et al., 1997). The protein encoded by theneighbouring CadN2 locus has a partially redundant function in

photoreceptor morphogenesis (Prakash et al., 2005).Although the consequences of the loss of function of E-cadherin

and N-cadherin in Drosophila have been described previously

(Dottermusch-Heidel et al., 2012; Iwai et al., 1997; Oda et al., 1993;Prakash et al., 2005; Tepass and Hartenstein, 1994), the preciseroles of these proteins in the earliest stages of mesoderm

morphogenesis have not been studied in detail. We therefore re-analysed mutant embryos during gastrulation for subtlemorphological defects. To delete all zygotic N-cadherin

transcribed from both the CadN and CadN2 loci (Hill et al.,2001), we used Df(2L)E48, a deficiency that uncovers a smallregion containing both genes [see Materials and Methods; Fig. 1B(Walsh and Brown, 1998)] (Nick Brown, personal communication).

These experiments confirmed that neither the (zygotic) loss of E-cadherin (shg) nor the loss of the N-cadherin (CadN, CadN2) genesresulted in defects in the formation of the ventral furrow, the

segregation of the germ layers or spreading of the mesodermal celllayer (data not shown). This deficiency allowed normaldevelopment and mesoderm differentiation beyond the end of

gastrulation to the fully extended germband stage (Fig. 1A,B).

Changing the complementary cadherin gene expressionduring early embryogenesisEmbryos that are homozygous for either of the mutationsdiscussed above lack zygotic expression of either E-cadherin orN-cadherin, but in each case the other cadherin is still present. It

is plausible that differential expression of the remaining cadherinmight be sufficient to keep the cell populations apart duringmesoderm spreading, in the same manner as qualitative or

quantitative differences in cadherin expression lead to the sortingof cell populations in vitro (Miyatani et al., 1989; Nagafuchiet al., 1987; Ninomiya et al., 2012; Nose et al., 1990; Steinberg

and Takeichi, 1994) and in vivo (Godt and Tepass, 1998;Gonzalez-Reyes and St Johnston, 1998; Hayashi and Carthew,2004). We therefore analysed mesodermal development inembryos lacking both zygotic E-cadherin and N-cadherin.

These mutant embryos contain only the maternally supplied E-cadherin, which is ubiquitously expressed at low levelsthroughout the whole embryo and is uniformly expressed in

ectoderm and mesoderm (Oda et al., 1998). Such embryosdevelop normally during gastrulation and form a ventral furrow(data not shown). In addition, later stages of mesoderm

development, including mesoderm spreading, were not

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significantly affected. Staining with antibodies against Even-skipped (Eve) or Fasciclin 3 (Fas3), which highlights either clusters

of heart and muscle precursor cells in the dorsal mesoderm (PCs;Fig. 1C–F) or the developing visceral mesoderm (VM; Fig. 1G,H)(Tepass and Hartenstein, 1994), respectively, showed that zygotic

cadherins are not necessary for early mesoderm development orsubsequent differentiation. In particular, the complementaryexpression of the cadherins was not required for an early

morphogenetic function during gastrulation. The results alsosuggested that adhesion provided by maternal E-cadherin issufficient to sustain mesoderm morphogenesis.

Effects of increased levels of E-cadherin on invaginationand spreadingIt is possible that the maintenance of reduced levels of E-cadherin

is crucial for mesoderm morphogenesis, and hence that there is aneed to counter the increase in E-cadherin levels that is normallyseen in the ectoderm during gastrulation. This assumption fits

with the importance of Snail-mediated transcriptional repressionfor mesoderm morphogenesis (Seher et al., 2007). To test thishypothesis, we overexpressed E-cadherin using the full-length E-

cadherin trangenes UAS cadh5 and UAS cadh5/9, and either amaternal-Gal4 or a twist-Gal4 driver both in wild-type embryosand in embryos lacking CadN. We initially investigated whetherhigh levels of E-cadherin per se are detrimental for mesoderm

development (even in the presence of N-cadherin), whereas thesecond set of experiments tested whether N-cadherin might be

necessary to counteract the potentially negative effects of high E-cadherin expression in the mesoderm.

In blastoderm stage embryos, E-cadherin localises to thedeveloping adherens junctions at a sub-apical position in theblastoderm epithelium. With the onset of cell shape changes in

the mesoderm, the adherens junctions disassemble and re-localiseat the most apical point of the cell–cell contact (Kolsch et al.,2007). This post-transcriptional modulation of E-cadherin is also

indirectly controlled by Snail (Kolsch et al., 2007), at least inpart through repression of the Bearded gene (Chanet andSchweisguth, 2012), a feature also seen during sea urchingastrulation (Wu and McClay, 2007). However, E-cadherin is

not restricted to this apical point, but is present throughout thelateral cell membranes, in decreasing concentrations towards thebasal end of the cell (Mathew et al., 2011). Armadillo does not

mirror this distribution, but is highly concentrated only at themost apical position (Mathew et al., 2011) (Fig. 2D).

In embryos expressing E-cadherin under the control of twist-

Gal4, the overexpressed E-cadherin becomes detectable at thetime of ventral furrow formation (Fig. 2A–C). In addition to araised level along the lateral membranes, an atypical

accumulation of E-cadherin is seen in the perinuclear region(Fig. 2B9,C9, arrowhead), probably representing protein that istrapped in the secretory pathway (Pacquelet and Rørth, 2005),suggesting that there is a limit on the amount of protein that can

be transported to the plasma membrane. Higher levels ofmembrane-associated E-cadherin are also seen by the time the

Fig. 1. Phenotypes of CadN and E-cad (shg)mutants. (A,B) Control and CadN mutant embryosstained for Twist (Twi, brown) and N-cadherin (N-cad,blue). (C–H) Stage-11 embryos of the indicatedgenotypes stained for Even-skipped (Eve, blue) andN-cadherin (brown) (C–F) or Fasciclin3 (Fas3, blue)and N-cadherin (brown) (G,H). Eve is expressed in 11segmental pericardial-cell clusters (PC) in the dorsalmesoderm. Fas3 is expressed in the visceralmesoderm (VM). Control embryos are from aDf(2L)E48/CyO collection of embryos [homozygousfor either the balancer or Df(2L)E48/CyO].

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mesoderm has invaginated. At this point, when a significantdifference in E-cadherin expression between the ectoderm and the

mesoderm becomes evident in wild-type embryos because of thetranscriptional upregulation of E-cadherin in the ectoderm, themesoderm retains the early low level of expression. A strong

signal is seen at the apical adherens junction and along the lateralcell membranes. The ectopic expression is also detectable in thecells neighbouring the ventral furrow that do not invaginate (i.e.

the mesectoderm and the adjacent ventral ectoderm), because theearly Twist expression domain extends into this area. Likematernally provided E-cadherin in the wild-type embryo (Clarket al., 2011), the ectopically expressed E-cadherin (Fig. 2F9)

persists after the invaginated mesoderm has dispersed intoindividual cells. In addition, unlike endogenous E-cadherin,which is localised mainly at the interface between mesodermal

and ectodermal cells and at the mesodermal cell membrane thatfaces the yolk (Fig. 2E9), ectopic E-cadherin is localised alongthe entire mesodermal cell membrane once the mesodermal cell

layer has become established (Fig. 2F9).In wild-type mesodermal cells, some N-cadherin localises to

the plasma membrane, but it is more abundant in a punctate

pattern in the cytoplasm (Fig. 2E). When N-cadherin isoverexpressed throughout the blastoderm, it is also mostly seenin the cytoplasm in ectodermal cells and does not colocalise withE-cadherin (supplementary material Fig. S1A). In embryos

expressing high levels of E-cadherin in the mesoderm, themembrane localisation of N-cadherin seemed to be further

reduced (Fig. 2G), whereas the punctate cytoplasmic patternwas unaffected or slightly enhanced (supplementary material Fig.

S1C). Thus, the plasma membrane localisation of cadherins mightbe subject to tight regulation, and it seems that E-cadherin is moreefficiently localised to the plasma membrane than is N-cadherin.

The absence of defects during gastrulation shows that highlevels of E-cadherin in the mesoderm do not affect cellsegregation between mesoderm and ectoderm, and that the

transcriptional downregulation of E-cadherin is not a prerequisitefor the mesoderm to lose its epithelial state or for the creation of asingle-cell layer. This is unexpected, given the widespreadconservation of the regulation of EMT (Nieto, 2011), and

indicates that a different mechanism or a combination ofmechanisms must contribute to the mesodermal EMT.

A redundant role for E-cadherin downregulation duringmesodermal EMT?The FGF signalling pathway is necessary for mesoderm spreading

and migration (Michelson et al., 1998; Vincent et al., 1998;Wilson et al., 2005). In the absence of FGF, which is expressed inthe ectoderm (Gryzik and Muller, 2004; Stathopoulos et al.,

2004), the mesodermal cells fail to extend filopodia in thedirection of the source of FGF (Clark et al., 2011). As soon asthe mesoderm has invaginated, cell division occurs, with therounding up of cells contributing to the dispersal of the mesoderm

and the dissolution of adherens junctions. When both mitosisand FGF signalling are blocked (in dof, stg double mutants),

Fig. 2. Mis-expression of E-cadherin in theembryonic mesoderm. (A–C) Cross sectionsthrough the ventral furrow in stage-6 control(A) and E-cadherin overexpressing(B,C) embryos stained for E-cadherin (E-cad,red) and Neurotactin (Nrt, green, to visualise cellmembranes), and with DAPI (blue).Overexpressed E-cadherin is seen both at itsnatural subcellular location at the adherensjunctions and ectopically, in the cytoplasm nearthe nucleus (arrowhead in B,C). (D) Single-pixelfluorescence intensity measurements ofE-cadherin and Armadillo along the lateralmembranes (5 mm from apical to basal) in control(twi-G4, grey) and E-cadherin-overexpressingembryos (twi-G4..cadh5, black). (E,F) Detailshowing the mesodermal cell layer apposed tothe ectoderm in stage-10 control embryos (E) andin embryos overexpressing E-cadherin in themesoderm (F), stained for N-cadherin (green)and E-cadherin (red) to reveal their subcellularlocalisation. (G) The fluorescence intensity ofE-cadherin and N-cadherin on the mesodermalmembrane traced from the end closest to theectoderm (exterior) to the end at the yolk surface(interior). Scale bars: 10 mm. Error barsrepresent s.e.m.

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E-cadherin retains its junctional localisation in the mesoderm fora longer time than in the wild type, and the epithelial structure

of the invaginated tube also persists for longer. Nonetheless,the mesodermal cells eventually spread out on the ectoderm(Clark et al., 2011). Thus, neither mitosis nor FGF signalling nortranscriptional downregulation of E-cadherin seem to be

necessary to allow the mesodermal EMT.To test whether the three processes act redundantly to facilitate

mesoderm dispersal, we overexpressed E-cadherin in the

mesoderm of dof, stg double-mutant embryos. We observed nofurther exacerbation of the mesodermal defects at early stages(Fig. 3B,C). Despite the abnormally high levels of E-cadherin in

such embryos, the mesodermal cell layer nevertheless flattenedout on the underlying ectoderm. However, although the transitionfrom an epithelial tube to a cell layer occurred both in dof, stg andin dof, stg, twi-Gal4..E-cadherin embryos, the invaginated

epithelial tube retained its epithelial structure and round cross-section for longer in the mutants than in the wild type (Fig. 3A–C; see also Clark et al., 2011), and the establishment of

contact between ectoderm and mesoderm was also delayed(supplementary material Fig. S2C–E). The cells on the ventralside of the invaginated tube eventually established a close

apposition to the ectoderm, but their shapes were abnormal. Thecell mass subsequently lost its round structure and flattenedagainst the ectoderm, spreading sideways, but, apart from the

reduced number of cells, this stage was similar in appearance towild-type embryos (Fig. 3F). The cells eventually covered mostof the ectoderm in many cases, but the more dorsally located cellsfailed to reach the ectoderm, so that two layers of mesodermal

cells persisted, and only in rare cases was a single layerestablished. In many embryos, the inner dorsal cell layer seemedto disintegrate (supplementary material Fig. S2H–K), suggesting

that the ectoderm might also provide a trophic signal.In summary, even when a high level of E-cadherin is

maintained, and FGF signalling and mitosis are blocked, the

mesodermal cells are able to spread out and form a new cell layer.Although indirect effects of Snail on E-cadherin localisation anddynamics are not addressed by these experiments, the resultsshow that Snail-mediated transcriptional repression of E-cadherin

is not essential for the mesodermal EMT. If it is important at allfor E-cadherin to be downregulated, it is more likely that the need

lies elsewhere or that downregulation is accomplished post-transcriptionally.

Effect of high levels of E-cadherin onmesoderm differentiationWe did detect abnormalities in embryos overexpressing E-cadherin at later stages. At the extended germband stage, the

pattern of expression of Eve in mesodermal derivatives wasaltered. At this stage, Eve is expressed both in the segmentalneuronal precursors and in clusters of cells in the dorsal

mesoderm that contain the precursors for pericardial cells andthe dorsal muscle DA1. The neuronal pattern of Eve expressionwas normal in embryos expressing high levels of E-cadherin in

the mesoderm, but the staining for the mesodermal clusters wasabsent or drastically reduced (Fig. 4A,B; supplementary materialFig. S3A–D). The Eve-positive cell clusters arise in a dorsally

Fig. 3. Overexpression of E-cadherin in a dof, stg double mutant. (A–C) Cross sections of control (twi-Gal4) and dof, stg double-mutant embryos eitheralone or in combination with E-cadherin overexpression in the mesoderm (twi-Gal4»cadh5, dof, stg). Embryos are stained for E-cadherin (E-cad) andNeurotactin (Nrt), as indicated, showing the mesodermal cells after completion of epithelial invagination. (D–F) In control (D), dof, stg mutant (E) and dof, stg

mutant embryos overexpressing E-cadherin (F), the mesoderm flattens out on the ectoderm.

Fig. 4. Mesodermal differentiation in embryos mis-expressing E-cadherin. Control (A,C,E) and E-cadherin-overexpressing embryos (in themesoderm, B,D,F) stained for Eve (stage 11; A,B) to mark pericardial-cellclusters (PC), stained for Mef2 (stage 16; C,D) to mark somatic, visceral andcardiac muscle nuclei or assayed for bagpipe (bap) expression (E,F) to markthe visceral mesoderm (E,F).

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located band of the mesoderm that receives differentiation signals

from the overlying ectoderm (Lee and Frasch, 2000; Lockwoodand Bodmer, 2002), and that also contains precursors for othermesodermal derivatives, such as the somatic dorsal muscles. When

we stained older embryos of the same genotype for Mef2, a markerof these cells, we found that they were also reduced or absent(Fig. 4C,D). Thus, the dorsal mesoderm apparently fails to

differentiate properly in embryos overexpressing E-cadherin.This could be due to either the failure of the mesodermal celllayer to spread sufficiently far to reach the region in which it canreceive the appropriate signals, or its inability to receive or respond

to a differentiation signal. We excluded a third possibility, namely,that cells were unresponsive because they were dying, by stainingthe embryos with antibodies against activated caspase3, which did

not reveal abnormal cell death (data not shown).To investigate the first hypothesis, we focused on the region in

which the Eve-positive cells should arise, to see whether the

mesodermal cell layer was present or absent in these regions.Fig. 5 shows an embryo in which only one Eve-positivepericardial cell cluster is detectable (Fig. 5A). We analysed

optical sections at a level just below the ectoderm. The sectionsreveal the mesoderm underneath the ectoderm from the ventral(top) to the dorsal (bottom) region in each of the selectedsegments (Fig. 5C–E). It is evident that the mesoderm in each

segment extends equally far dorsally, and, in particular, that in the

two segments lacking Eve-staining, mesodermal cells are present

in the region in which the Eve-positive cells should arise. Z-sections confirm the presence of two cell layers at the positionsindicated in Fig. 5C–E. Thus, the mesodermal cell layer in these

embryos has extended as far dorsally as it does in normalembryos, and mesoderm is present in the region where it canreceive ectodermal differentiation signals. The defect must

therefore be an inability to receive or interpret the signals fromthe ectoderm. An additional morphological defect in olderembryos (supplementary material Fig. S3E–G) might be causedby mesectodermal cells with abnormally high levels of E-

cadherin failing to internalise.

High levels of E- but not N-cadherin perturb Wingless-dependent mesoderm differentiationOverexpression of E-cadherin in the ectoderm has been shown tointerfere with the ability of the cells to respond to the Wingless

signal (Sanson et al., 1996). The excess of E-cadherin is thoughtto bind to and reduce the pool of b-catenin (Armadillo) that isavailable to transmit the Wingless signal to the nucleus. We

tested whether high levels of E-cadherin in the mesodermaffected the distribution of Armadillo or other proteins associatedwith E-cadherin (Fig. 6A,B,E,F; supplementary material Fig. S4).In cells overexpressing E-cadherin, we found that the level of

Armadillo at the plasma membrane was strongly increased

Fig. 5. Mesodermal spreading in embryosoverexpressing E-cadherin in the mesoderm.Embryos expressing Cadh5/9 in the mesoderm usingthe twist-Gal4 driver stained for Even-skipped (Eve,green) and Armadillo (red), and stained with DAPI(blue). (A) Overview of a whole stage-11 embryo. (B,B9)Higher magnification of three hemisegments in theboxed region in A. The regions selected for highermagnification analysis are outlined in B. (C–E) Singleoptical planes from the imaged stack of the regionshown in B. The focal plane lies within the ectoderm atthe top of the image and in the mesoderm at the bottomof the image. The white lines mark the positions at whichthe yz- and xz-sections in C9–E9 and C0–E0 were taken.(C9–E9) The white dashed line highlights the mesoderm.Scale bars: 10 mm.

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(Fig. 6D). The Armadillo-interacting protein a-catenin alsoshowed increased levels (supplementary material Fig. S4A).Another E-cadherin-associated protein, p120-catenin, which is

barely detectable or absent in wild-type mesodermal cells,showed no increase after overexpression of E-cadherin(supplementary material Fig. S4B). This suggests that over-

expression of E-cadherin interferes with the normal participationof Armadillo in Wingless signalling, and with the differentiationof Wingless-dependent mesodermal cell types (Baylies et al.,

1995; Wu et al., 1995). To further test the notion that E-cadherininterferes with mesoderm differentiation because of its effect onArmadillo signalling, rather than its function in adhesion, weexpressed a form of E-cadherin that can mediate adhesion, but

that does not have an Armadillo-binding site in its intracellulardomain. This construct, E-cadDb/aCat, can mediate all E-cadherin and b-catenin functions at adherens junctions, including

cell sorting, epithelial integrity and cell migration (Pacquelet andRørth, 2005), suggesting that the sole function of the b-catenin-binding domain at cell junctions is to facilitate the interaction

with a-catenin. In contrast to full-length E-cadherin, over-expression of E-cadDb/aCat in the mesoderm had no effect onthe differentiation of the pericardial cells, or any other aspect of

mesoderm development, thus identifying the Armadillo-bindingdomain as a key mediator of the effects of E-cadherin onWingless signalling (Fig. 6L).

If the loss of Eve-positive heart precursor cells is due to theinability of the dorsal mesoderm to respond to Wingless, onemight expect the cell population to be transformed to the

alternative Wingless-independent mesodermal fate. The heartprecursors arise in the sloppy-paired domain (which includes thewingless expression domain) of the dorsal mesoderm; the cells in

the neighbouring domain differentiate into visceral mesoderm,which is characterised by expression of the transcriptionfactor Bagpipe (Bap). The expression domain of bap increases

when wingless signalling is reduced (Riechmann et al., 1997).Consistent with this, overexpressing E-cadherin in the mesodermresulted in a substantial expansion of the bap expression domainin the dorsal mesoderm (Fig. 4E,F). Thus, high levels of E-

cadherin do lead to the same fate transformation as the loss ofWingless.

If the reason for downregulation of E-cadherin by Snail in the

mesoderm is to allow efficient Wingless signalling, then N-cadherin might be expected not to have negative effects onWingless signalling. We first tested this in the ectoderm, where

E-cadherin has previously been shown to be able to abrogateWingless signalling (Sanson et al., 1996). Expression of E-cadherin using the nullo-Gal4 driver led to the stabilisation of

Armadillo at cell membranes, and efficiently downregulated theexpression of engrailed (Fig. 6K), a Wingless target. By contrast,expression of N-cadherin using the same driver had no effect on

Fig. 6. Distribution of b-catenin (Armadillo) inmesodermal cells overexpressing E-cadherin.Sections of late-stage-10 control (A,E) and E-cadherin-overexpressing embryos (B,F) stainedfor E-cadherin (E-cad, green) and Armadillo(Arm, red) as indicated. Dashed boxes in A9 andB9 indicate the areas that are enlarged in E and F,respectively. (C,D) Section of a late-stage-10control (C) and an N-cadherin-overexpressingembryo (D) stained for N-cadherin (N-cad, grey).(C) The fluorescence intensity was adjusted inPhotoshop so that the endogenous N-cad signalis visible. (G–I) Staining for Armadillo (red) inmesodermal cells overexpressing E-cadhDB/acat(G), N-cad (H) or E-cadhDCyt/N-cadh (I). Scalebar: 10 mm. (J,K) Stage-11 embryos expressingUAS-Ncadh (J) or UAS-Cadh5/9 (K) under thecontrol of nullo-Gal4, stained for Engrailed (En,green). (L) Quantitation of Eve-positive cellclusters in stage-11 embryos of the indicatedgenotypes. Overexpression of the full-lengthcadherin transgenes (cadh5, cadh 5/9) anddominant-negative Tcf produced significantreductions (P,0.001) in the number of clusters;n§10 embryos in each case. Error barsrepresent s.e.m.

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engrailed expression (Fig. 6J). Thus, high levels of E-cadherinbut not N-cadherin interfere with Wingless signalling in the

ectoderm.Similarly, the overexpression of N-cadherin in the mesoderm

had no effect on the differentiation of any of the dorsalmesodermal derivatives (Fig. 6L). Its overexpression led to

increased recruitment of Armadillo to the puncta in thecytoplasm, rather than an enrichment at the plasma membranes(Fig. 6H). Thus, the mesoderm can tolerate increased levels of N-

cadherin expression but not increased levels of E-cadherin. Thisprovides the first experimental demonstration that N-cadherindiffers from E-cadherin in its ability to modulate Wingless

signalling, a property that might rely on the different affinities ofN- and E-cadherin for Armadillo that have been demonstratedbiochemically in the nervous system (Loureiro and Peifer, 1998).

Further support for this conclusion comes from the finding that itis specifically the intracellular domain of E-cadherin that isresponsible for the interference with Wingless signalling.Overexpression of a chimeric construct in which the

extracellular domain of E-cadherin is fused to the cytoplasmicdomain of N-cadherin (E-cadhDCyt/N-cadh) had no effect onmesoderm differentiation (Fig. 6L).

The physiological effects of expressing the various cadherinscould either be due to biochemical differences in their actions, orto the fact that they are expressed at different levels. A

comparison of the levels of overexpression that were achievedby the various constructs, by using an antibody against theextracellular domain of E-cadherin, revealed that cadh5, cadh5/9

and E-cadhDCyt/N-cadh were expressed at similar levels. E-cadDb/aCat was detectable at lower levels but was, nonetheless,sufficient to sustain the movement of border cells in ovaries(Pacquelet and Rørth, 2005). The levels of overexpressed N-

cadherin also significantly exceeded the endogenous level(Fig. 6C,D), and, like E-cadherin overexpression, overexpressionof N-cadherin caused Armadillo recruitment and stabilisation

(Fig. 6H).The difference in the effect of E-cadherin and N-cadherin

could be due to either the difference in their subcellular

localisation (junctional vs cytoplasmic), or a biochemicaldifference in the intracellular domain. The chimeric protein E-cadhDCyt/N-cadh localised primarily at the plasma membraneand was able to stabilise Armadillo at the junctions (Fig. 6I). This

demonstrates that the increased recruitment of Armadillo to theplasma membrane is not sufficient to cause suppression ofWingless signalling. Instead, N-cadherin might recruit a different

sub-pool of Armadillo as shown previously in a different context(Loureiro and Peifer, 1998).

Curiously, in apparent contradiction to this conclusion, the

effect of overexpressing E-cadherin in the mesoderm wasweakened if N-cadherin levels were lowered by half or if itwas removed completely (Fig. 7C–F). In other words, although

N-cadherin competes less efficiently for Armadillo whenoverexpressed, its reduction counteracts the sequestering ofArmadillo by high levels of E-cadherin in the mesoderm(Fig. 7G,H). We discuss possible explanations for this paradox

below, but opposite effects of N-cadherin and E-cadherin havealso been documented in the context of eye morphogenesis(Mirkovic and Mlodzik, 2006).

In summary, the complementarity of cadherin expression in theectoderm and mesoderm is not needed for early mesodermmorphogenesis. Instead, the transcriptional downregulation of E-

cadherin by Snail might be required because of the potential for

high levels of E-cadherin to interfere with cell differentiation in

the mesoderm.

DISCUSSIONMost morphogenetic movements depend on the dynamic

regulation of cell–cell and cell–substrate adhesion (Bulgakovaet al., 2012). In the work described here, we found no discernibledifference in the role of the two cadherins in mediating the

morphogenesis of the mesoderm, and therefore no role for theswitch between E- and N-cadherin in the context of gastrulation.Instead, the failure to downregulate E-cadherin interfered with

correct mesoderm differentiation, a process known to depend onWingless signalling.

On the requirement of the cadherin switchWe confirm that there is no requirement for either zygotic E-cadherin or N-cadherin for the formation of the ventral furrow.These results differ from observations in other models, including

chicken (Nandadasa et al., 2009), and from previous suggestionsthat the switch is necessary in the fly (Oda et al., 1998). In bothflies and mice, the early effects of the loss of E-cadherin function

[maternal E-cadherin in flies and zygotic in mice (Larue et al.,1994; Riethmacher et al., 1995; Tepass et al., 1996)] preclude ananalysis of gastrulation in the complete absence of E-cadherin.

Fig. 7. Overexpression of E-cadherin in CadN-deficient embryos. Stage-11 embryos of the indicated genotypes were stained for Even-skipped(Eve, blue) and N-cadherin (N-cad, brown) (A–F), and E-cadherin (E-cad,green) and Armadillo (Arm, red) (G,H) to visualise the effects on thepericardial-cell clusters and on Armadillo localisation, respectively. Scalebars: 10 mm.

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Drosophila gastrulation and the EMTOne motivation for the investigations presented here was the

resemblance of Drosophila gastrulation to the EMT, and theobservation of cadherin switching in other EMT settings. Asclassically defined, an EMT is associated with the loss of cellcontacts and the consequent dissociation of cells from the sheet,

a process mediated by the action of the ‘EMT inducers’, Snail,Zeb and Twist, and which includes transcriptional repression ofE-cadherin as a key molecular signature. Cancer metastasis and

the formation of the neural crest are thought to representconventional EMTs by these criteria (Nieto, 2009; Nieto, 2011).Although the loss of cell contacts mediated by the

downregulation of E-cadherin is one requirement for an EMT,others, including cytoskeletal reorganisation and matrixremodelling to facilitate a change in cell shape, motility and

migration are also necessary. Another contribution to motilityduring an EMT is the cadherin switch. N-cadherin is expressedprimarily by mesenchymal and neural tissues, and promotesmotility (Iwai et al., 1997; Li et al., 2001; Nandadasa et al., 2009).

Its upregulation is a feature of some, but not all, EMTs (Nieto,2011), and might result in the modulation of adhesion strengththat is necessary for motility.

Drosophila gastrulation converts a stable epithelium into amore mesenchymal tissue. This relies on several functions of thetranscription factor Snail, evident from the fact that the loss of E-

cadherin is insufficient to rescue the effects of a Snail mutant (ourunpublished observations). Unlike a classical EMT, however,single cells do not delaminate from the sheet during Drosophila

gastrulation (Thiery et al., 2009), and the resulting mesodermremains as a layer. Whereas cell shape changes that are driven byapical constriction require the tethering of the actin cytoskeletonto adherens junctions (Dawes-Hoang et al., 2005), the changes

that accompany spreading, dispersal and migration of themesoderm are less well understood, but are thought to rely onthe loosening of cell contacts for the acquisition of motility. In

neuronal growth cones, N-cadherin interacts, through itsextracellular domain, with the FGF receptor (Cavallaro andChristofori, 2004; Williams et al., 2001). The latter is also a key

regulator of mesoderm migration in Drosophila. Whereas FGFsignalling is downstream of the transcription factors Twist andSnail in the Drosophila mesoderm, it is upstream of Snail invertebrate gastrulation and in the regulation of EMT (Ciruna and

Rossant, 2001), and seems to function in a manner analogous toTwist in its requirement for the maintenance of snail expression.Despite these intriguing parallels, we found that the complete loss

of N-cadherin has no effect on invagination.Another possibility is that the switch is a redundant or permissive

mechanism that facilitates other processes that allow the

mesodermal cells to disperse and form the mesodermal cell layer.FGF receptor signalling in the mesoderm is needed for the efficientand even distribution of the mesodermal cells over the ectoderm

(Bae et al., 2012; Wilson et al., 2005). FGF signalling mediates theclose apposition of the basal side of the newly invaginatedmesodermal epithelium against the ectoderm, and, subsequently,the filopodia-mediated motility of the cells away from the site of

invagination. At the same time, the mesodermal cells undergo awave of mitoses, which might help the dispersal of the cells (Clarket al., 2011). However, as shown previously, inhibiting mitosis and

FGF signalling does not prevent the formation of a mesodermal celllayer (Clark et al., 2011). Here, we show that a cell layer is stillformed even when the level of E-cadherin is increased in addition to

the inhibition of mitosis and FGF signalling.

Our results suggest that gastrulation can be thought of as acollective EMT. The mesoderm collectively loses its epithelial

character and polarity, and acquires motility that is directionaland involves adhesion to the substrate. This might necessitate thatthe cells maintain some contact with one another, rather than loseall contact. Consistent with this, E-cadherin in the mesoderm is

not completely lost from the mesodermal cells. Although thezygotic transcription of E-cadherin is upregulated in the ectodermand this upregulation is repressed in the mesoderm, the

maternally provided E-cadherin persists in the mesoderm (Clarket al., 2011; this study). This might also explain the lack of strongeffects of cadherin overexpression on gastrulation.

An alternative possibility is that EMT in the mesoderm isaccomplished by mechanisms other than the transcriptionaldownregulation of E-cadherin expression. Indeed, in addition to

changes in the levels of E-cadherin, changes in localisation of E-cadherin also accompany mesoderm morphogenesis. Thetranscription factor Snail is required for an early change in thelocation of the adherens junctions from a subapical to an apical

position in the invaginating epithelium, although a low level ofprotein is distributed over a broader domain in the lateral cellmembranes (Kolsch et al., 2007; Mathew et al., 2011). The

adherens junctions are subsequently dismantled after theinvagination is completed, cells lose their apico-basal polarity,and E-cadherin is detected over the whole cell surface. As the

mesodermal cell layer is established, E-cadherin becomesconcentrated at the interface with the ectoderm and on the cellsurface facing the yolk. Experimentally overexpressed E-cadherin

shows a broadly similar behaviour, with some additional stainingin the perinuclear region, presumably as a result of saturating thesecretory system or the adherens junctions. These findingssuggest that changes in cell adhesion mediated by mechanisms

other than the transcriptional downregulation of E-cadherin mightbe responsible for the post-invagination behaviour of themesoderm. They further strengthen the idea that multiple events

occurring downstream of Snail contribute to the EMT.Mechanisms that do not rely on Snail or on the transcriptional

repression of E-cadherin can also contribute to EMT. For

example, the EMT in the Drosophila posterior midgut is drivenby Serpent-mediated transcriptional repression of the apicalmembrane regulator Crumbs, which in turn influences thesubcellular localisation of E-cadherin (Campbell et al., 2011).

Although EMT, as classically defined, necessitates the loss ofpolarity and the transcriptional downregulation of E-cadherin bythe EMT transcription factors (Snail, Twist and Zeb), a precise

understanding of the full range of mechanisms that are involved isstill missing. Analysis of gene regulatory networks in the seaurchin also suggests that multiple networks accomplish distinct

features of the EMT (Wu and McClay, 2007), and Snail mightmediate only a subset of these.

Cadherins and mesoderm differentiationThe most striking phenotype observed in the mesoderm uponoverexpression of full-length E-cadherin is the failure of thepericardial cells to differentiate, and an expansion of visceral

mesoderm precursors. The overexpression of N-cadherin had noeffect, highlighting qualitative differences between the twocadherins in their ability to influence differentiation. Mesoderm

differentiation relies on combinatorial signalling (Bate, 1990;Baylies et al., 1998). PC-cell specification depends on Winglesssignalling (Bate and Rushton, 1993), which can be modulated by

adhesion through Armadillo and can influence adhesion

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qualitatively and quantitatively (Jaiswal et al., 2006; Sansonet al., 1996; Ulrich et al., 2005). The overexpression of E-

cadherin abrogates Wingless signalling in the Drosophila wing,through sequestration of Armadillo (Sanson et al., 1996).Whereas overexpressed full-length E-cadherin effectivelyrecruited Armadillo to the membrane in the mesoderm and

ectoderm, N-cadherin overexpression was less efficient at doingso. Instead, overexpression of N-cadherin resulted in anobservable increase in the cytoplasmic pool of Armadillo.

Three observations are intriguing: (1) the inability ofoverexpressed N-cadherin to influence Wingless signalling, (2)the fact that the concomitant removal of N-cadherin can rescue

the wingless phenotype produced by E-cadherin overexpressionand (3) the insufficiency of junctional Armadillo recruitment asan explanation for the wingless phenotype. The first observation

suggests that higher levels of N-cadherin are necessary todownregulate Wingless signalling or that N-cadherin is lessefficient than E-cadherin at sequestering Armadillo. Both of theseexplanations might rely on the predominant subcellular

localisation of both N-cadherin and the recruited Armadillo(cytosolic rather than membrane-associated). However, ourexperiments with the chimaeric cadherins reveal that the

junctional localisation of overexpressed E-cadherin and theconsequent junctional recruitment of Armadillo are notsufficient to account for the downregulation of Wingless

signalling by E-cadherin. It is also possible that the bindingaffinities of Armadillo to E-cadherin and N-cadherin aredifferent. A third possibility is that the effects of the two

cadherins on Wingless signalling are driven by distinctmechanisms. N-cadherin has been reported to antagoniseWingless signalling through its interaction with the Wnt co-receptor LRP, which targets b-catenin for degradation (Hay et al.,

2009). This mechanism requires the localisation of N-cadherin atthe membrane, which in the Drosophila mesoderm is low. Theseresults suggest that differences between E- and N-cadherin in

their ability to recruit, bind and retain Armadillo – bothqualitative differences (different pools or isoforms) andquantitative differences (different binding strengths) – might

underlie their effects on Wingless signalling. Taken together,these possibilities also provide an explanation for why the lossof N-cadherin rescues the phenotype produced by theoverexpression of E-cadherin: this might result from the

reduced downregulation of Wingless signalling in the absenceof N-cadherin. A distinct form of Armadillo binds to N-cadherinin the nervous system (Loureiro and Peifer, 1998). Whether the

mesoderm also expresses different forms of Armadillo or otherlinker proteins remains unknown. In addition, the differentbiochemical properties of CadN and CadN2 that are suggested by

differences in their length and sequence need to be examinedexperimentally.

The regulation of cadherins during morphogenesisAlthough we have focused on the expression and localisation ofE-cadherin and N-cadherin, many other post-transcriptionalmodes of regulation affect cell behaviours during

morphogenesis. Regulation of localisation through the activityof the Rho1 GTPase accompanies apical constriction during theinvagination of the ventral furrow [causing more apical

localisation of adherens junctions (Dawes-Hoang et al., 2005;Kolsch et al., 2007)]. The formation of epithelial folds seems torely solely on the progressively basal shift of the adherens

junctions, driven by the polarity regulator Bazooka and Rap1

(Wang et al., 2012; Wang et al., 2013). Junction remodelling bylocalised endocytosis underlies cell intercalation during

germband extension (Levayer et al., 2011). Differences in cellbehaviour can also result from modulating the link with thecytoskeleton (Roh-Johnson et al., 2012). In addition, the effectiveincrease in adhesion upon E-cadherin overexpression in the

mesoderm might be modulated by the membrane traffickingpathways that either facilitate the degradation of E-cadherin orcontrol its incorporation into the membrane secretory pathway

(Langevin et al., 2005; Lohia et al., 2012). Indeed, the low levelsof p120-catenin in the mesoderm might be part of a mechanismthat ensures E-cadherin degradation. It is possible that one or

more of these mechanisms also operate in the mesoderm andserve as additional controls that ensure that there is no net changein functional adhesion upon cadherin overexpression, explaining

the lack of effects of overexpression of E-cadherin on earlymesoderm morphogenesis. By contrast, signalling is moresensitive to small changes in the level of E-cadherin.

ConclusionsIn summary, our results rule out qualitative differences in E- andN-cadherin as a mechanism for the segregation of germ layers.

They also rule out a role for the transcriptional repression of E-cadherin by the transcription factor Snail in the spreading andmigration of the mesoderm. Our results suggest that N-cadherin

does not have a role in early mesoderm morphogenesis, and theyuncover striking differences in the ability of E- and N-cadherin toinfluence Wingless signalling that might rely on differences in the

interactions of these cadherins with Armadillo. The regulation ofadhesion by other mechanisms might be responsible for allowingmesoderm cell dispersal. A detailed understanding of the relatedcell biology will be of great interest.

MATERIALS AND METHODSDrosophila stocks and crossesAll Drosophila stocks were cultured under standard conditions at 22 C or

25 C. The mutant alleles shgIG29, shgIH81 and NCadM19 were obtained from

Ulrich Tepass, Tadashi Uemura and Elisabeth Knust, respectively. The

deficiency Df(2L)E48, originally isolated as an allele of cassowary (listed

as cassowaryE48 in Flybase), was initially identified in a screen for adhesion

mutants (Walsh and Brown, 1998). The following Bloomington

deficiencies were used to map Df(2L)E48: Df(2L)TW119, Df(2L)M36F-

S5 and Df(2L)TW137. Homozygous mutant embryos were identified by the

absence of N-cadherin staining. We used meiotic recombination to generate

the triple cadherin mutant [Df(2L)E48 shgIG29] on the second chromosome.

The driver lines mat-Gal4, nullo-Gal4, twi-Gal4 and en-Gal4 were

used to drive the expression of UAS-cadh5/9 (Sanson et al., 1996), UAS-

cadh5 (Sanson et al., 1996), UAS-Ncadh (Iwai et al., 1997), UAS-arm3

(Sanson et al., 1996), UAS-arm23 (Sanson et al., 1996), UAS-E-CadDb/

aCat (Pacquelet and Rørth, 2005), UAS-E-cadDCyt/N-cad (Pacquelet

and Rørth, 2005) and UAS-TCFDN (van de Wetering et al., 1997).

To drive the ectopic expression of E-cadherin in N-cadherin-null

mutants, we recombined Df(2L)E48 with twi-Gal4 on the second

chromosome and established a Df(2L)E48/CyO; UAS-Cadh5/9 line.

Meiotic recombination was used to generate the twi-Gal4, Df(2L)E48,

shgIG29 line on the second chromosome. We used homologous

recombination to recombine UAS-cadh5 to a dof1 stg double-mutant

chromosome and established a twi-Gal4; dof1 stg line.

Immunohistochemistry, immunofluorescence and in situhybridisationEmbryos were fixed in equal amounts of 3.7% formaldehyde in PBS and

heptane for both immunohistochemistry and in situ hybridisation. For

immunohistochemistry, fixed embryos were stained for Twist (1:1000,

Siegfried Roth), Even-skipped [1:2500, Developmental Studies Hybridoma

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Bank (DSHB)], Fasciclin3 (1:5, DSHB) and N-cadherin (DN-Ex8; 1:25,

DSHB). Biotinylated secondary antibodies (anti-rabbit-IgG, anti-mouse-

IgG and anti-rat-IgG, Dianova, Germany) and Vectastain ABC Elite kit

(Vector Laboratories) were used for immunohistochemical detection with

diaminobenzidine (DAB). Embryos were mounted in Araldite for imaging.

Heat-fixed embryos (fixative: 0.4% NaCl2, 0.03% Triton X-100) were

used for fluorescence staining for E-cadherin (DCAD2, 1:50), Armadillo

(1:100), a-catenin (1:100), p120-catenin (1:100), N-cadherin (DN-Ex8,

1:20), Even-skipped (1:1000) and Neurotactin (1:5000, DSHB).

Fluorescently labelled secondary antibodies were from Dianova

(1:200). DNA was stained with 49,6-diamidino-2-phenylindole (DAPI).

Embryos were embedded in Fluoromount G (SouthernBiotech), and

cross-sectioning was performed with a 27G injection needle.

Imaging and quantificationImaging was performed on a Zeiss Meta Confocal 510 (CECAD Imaging

facility) with the following objectives: Plan-Neofluar 206/0.5, Plan-

Neofluar 406/1.3 Oil DIC, Plan-Apochromat 636/1.4 Oil DIC.

Immunohistochemical staining was analysed by using a Zeiss Axioplan

microscope. Image stacks and optical sections were processed in

Volocity (PerkinElmer) and ImageJ (NIH). Fluorescence intensity

measurements were performed on the original images using Volocity

and ImageJ. A single-pixel-width line was hand drawn along the plasma

membrane. The plasma membrane was identified by E-cadherin staining.

Excel was used for statistical analyses. Statistical significance was

determined by using the Student’s t-test.

AcknowledgementsWe thank Nick Brown for informing us about the existence of CadN2 and forproviding Df(2L)E48 before the Drosophila genome annotation was published;Pernille Rørth, Eli Knust, Tadashi Uemura, Uli Tepass, Benedicte Sanson, theDSHB and Bloomington Stock Center for antibodies and fly stocks; AstridSchauss and the CECAD Imaging facility for help with imaging. We are grateful toNick Brown, Angela Nieto, Carien Niessen, Doris Wedlich and members of theLeptin laboratory for critical comments on the manuscript.

Competing interestsThe authors declare no competing interests.

Author contributionsG.S., M.N. and E.V. carried out the experimental work. G.S., M.N. and M.L. wrotethe manuscript. All authors were involved in the documentation and discussion ofthe results, and in the planning of the experimental approaches.

FundingThis work was funded by Deutsche Forschungsgemeinschaft [grant numberSFB572 to M.L.] and the Tata Institute of Fundamental Research (TIFR) (to M.N.).

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139485/-/DC1

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