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A novel protein complex, Mesh–Ssk, is required forseptate junction formation in the Drosophila midgut

Yasushi Izumi, Yuichi Yanagihashi and Mikio Furuse*Division of Cell Biology, Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku,Kobe 650-0017, Japan

*Author for correspondence (furuse@med.kobe-u.ac.jp)

Accepted 25 June 2012Journal of Cell Science 125, 4923–4933� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.112243

SummarySeptate junctions (SJs) are specialized intercellular junctions that restrict the free diffusion of solutes through the paracellular route in

invertebrate epithelia. In arthropods, two morphologically different types of SJs have been reported: pleated SJs and smooth SJs (sSJs),which are found in ectodermally and endodermally derived epithelia, respectively. However, the molecular and functional differencesbetween these SJ types have not been fully elucidated. Here, we report that a novel sSJ-specific component, a single-pass

transmembrane protein, which we term ‘Mesh’ (encoded by CG31004), is highly concentrated in Drosophila sSJs. Compromised mesh

expression causes defects in the organization of sSJs, in the localizations of other sSJ proteins, and in the barrier function of the midgut.Ectopic expression of Mesh in cultured cells induces cell–cell adhesion. Mesh forms a complex with Ssk, another sSJ-specific protein,and these proteins are mutually interdependent for their localization. Thus, a novel protein complex comprising Mesh and Ssk has an

important role in sSJ formation and in intestinal barrier function in Drosophila.

Key words: Drosophila, Midgut, Epithelial cell, Smooth septate junction

IntroductionEpithelia play important roles as barriers that separate distinct

compartments within the body. To accomplish these functions,

epithelial cells have specialized intercellular junctions, designated

as occluding junctions, that restrict the free diffusion of solutes

across the cellular sheets through the paracellular pathway. In

vertebrates, tight junctions (TJs) act as occluding junctions in

all epithelia, including endothelial cells. These barrier/channel

properties are determined primarily by membrane proteins of the

claudin family (Anderson and Van Itallie, 2009; Angelow et al.,

2008; Furuse, 2010).

In contrast to vertebrates, the epithelial cells of invertebrates

generally lack TJs (although a few exceptions have been reported).

Instead, they possess different membrane specializations, called

septate junctions (SJs), which perform the role of occluding

junctions (Lane et al., 1994a; Tepass and Hartenstein, 1994). In

ultrathin-section electron microscopy, SJs are observed as parallel

plasma membranes between adjacent cells with ladder-like septa

spanning the intermembrane space. Morphological variants of SJs

exist across the invertebrate phyla and some animals are reported

to possess multiple types of SJs specific to different types of

epithelial cells (Lane et al., 1994b; Green and Bergquist, 1982).

However, the molecular architectures of these SJ types are largely

unknown. In arthropods, two major classes of SJs have been

described, based on morphological appearance: pleated SJs (pSJs)

are observed in ectodermally derived epithelia and glia, while

smooth SJs (sSJs) are found mainly in the endodermally derived

midgut epithelium (Lane et al., 1994a; Tepass and Hartenstein,

1994). The outer epithelial layer of the proventriculus (OELP)

and the Malpighian tubules also possess sSJs, although

developmentally they originate from the ectoderm. The major

criteria distinguishing these two types of SJs are the arrangement of

the septa visualized in negatively stained membrane preparations

and the appearance of intramembrane particles observed in freeze-

fracture images. The septa in pSJs form regular undulating rows but

those in sSJs are arranged in regularly spaced parallel lines. This

structural difference seems to reflect differences in the molecular

architecture of sSJs and pSJs. Among these two SJ types, the

molecular and functional properties of pSJs have been extensively

analyzed in Drosophila ectodermal epithelia. The molecular

components of Drosophila pSJs include: the transmembrane

proteins, Neurexin IV (Baumgartner et al., 1996), Neuroglian

(Banerjee et al., 2010), Gliotactin (Schulte et al., 2003), Contactin

(Faivre-Sarrailh et al., 2004), Fasciclin III (FasIII) (Woods et al.,

1997), and Lachesin (Llimargas et al., 2004); an Na+/K+ ATPase

(Paul et al., 2003); and the cytoplasmic proteins, Coracle (Cora)

(Lamb et al., 1998), Discs large (Dlg) (Woods et al., 1996), Lethal

(2) giant larvae (Lgl) (Bilder et al., 2000), Scribble (Scrib) (Bilder

and Perrimon, 2000), and Varicose (Wu et al., 2007). Among them,

it was recently reported that Dlg is unlikely to be a core pSJ

component (Oshima and Fehon, 2011). The vertebrate homologs of

Neurexin IV, Neuroglian Contactin and Cora are concentrated at the

paranodal junctions (PJs) of vertebrate myelinated axons, which

possess ladder-like structures similar to those observed in SJs (Bhat,

2003). Thus, the molecular organization and morphology of pSJs are

similar to that of PJs. However, three claudin-like proteins,

Megatrachea (Behr et al., 2003), Sinuous (Wu et al., 2004) and

Kune-kune (Kune) (Nelson et al., 2010), have been identified as

functional pSJ components, suggesting that pSJs also have some

common features with TJs.

Morphological and physiological studies have suggested that

sSJs function to restrict or regulate the diffusion of solutes

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through the paracellular pathway (Skaer et al., 1987) but detailed

molecular and genetic analyses of sSJs are lacking. In Drosophila,

Ankyrin, a/b-spectrin, FasIII (Baumann, 2001) and Dlg (Maynard

et al., 2010) are localized at the apicolateral region of midgut

epithelial cells, and Lgl is localized at the sSJs of the

proventriculus (Strand et al., 1994).

Recently, we identified a novel protein with four membrane-

spanning domains, Snakeskin (Ssk), which specifically localizes at

sSJs and is required for the organization and function of sSJs

(Yanagihashi et al., 2012). Here, we identify a previously

uncharacterized putative membrane protein, which we have

named ‘Mesh’. Mesh specifically localizes at sSJs, induces cell–

cell adhesion in cultured cells, and is required for the formation

and function of sSJs in Drosophila. We also found that Mesh and

Ssk display mutually dependent localizations at sSJs and form a

complex with each other. Therefore, we conclude that Mesh acts

together with Ssk to organize sSJs.

ResultsMesh is a candidate for a novel sSJ-associated membrane

protein

To further identify sSJ-specific molecules, we generated

monoclonal antibodies (mAbs) in rats against an sSJ-containing

membrane fraction obtained from the midgut of silkworm (Bombyx

mori) fifth-instar larvae, and finally isolated two mAb clones that

specifically recognized the apical region of the lateral membrane

of midgut epithelial cells, where sSJs occur (Fig. 1A).

Immunoprecipitation of the midgut membrane fraction with these

mAbs identified a ,80 kDa protein (Fig. 1B). Mass spectrometry

revealed this protein to be silkworm BGIBMGA009402-PA

(supplementary material Fig. S1). The primary structure of this

protein contains a domain characteristic of a transmembrane-

spanning segment close to the C-terminus, a signal peptide, a NIDO

domain, an Ig-like E set domain, an AMOP domain, a vWD domain,

and a sushi domain (supplementary material Fig. S1). These

extracellular domains are found in cell adhesion proteins playing

important roles in cell–cell and/or cell–matrix adhesion (Bork et al.,

1994; Ciccarelli et al., 2002; Colombatti et al., 1993; Ichinose et al.,

1990; Mayer et al., 1998). To further investigate the function of this

protein in Drosophila, we looked for its Drosophila ortholog by

database searching and found the CG31004 gene (Fig. 1D;

supplementary material Fig. S1), which is located on the right arm

of the third chromosome. We named CG31004 protein ‘Mesh’ for

its immunofluorescence staining images in Drosophila midgut (see

below). Proteins characterized by similar domain compositions exist

in other invertebrates, including Caenorhabditis elegans (K03H1.5)

and sea urchins (LOC580458). In vertebrates, the mouse Susd2/Svs-

1 ortholog is the sole protein containing the AMOP, vWD, and sushi

domains (supplementary material Fig. S1), suggesting that Susd2/

Svs-1 is a vertebrate ortholog of Mesh (Sugahara et al., 2007).

The Flybase predicts that Mesh transcripts are translated into

three isoforms with different C-terminal cytoplasmic regions

Fig. 1. Mesh is a candidate for a novel sSJ-localizing protein. (A) Immunofluorescence staining of a frozen section of silkworm larval midgut using a mAb of

hybridoma clone 75. The signals were observed in the lateral regions of the epithelial cells. Arrows indicate the apex of the lateral plasma membrane. Basal

membranes are delineated by dots. An asterisk indicates the lumen of the midgut. Scale bar: 50 mm. (B) The membrane fractions of silkworm larval midguts (+) or

control buffer (2) were subjected to immunoprecipitation with mAb of clone 75. The immunoprecipitate was separated on a 12% SDS-polyacrylamide gel, and

the gel was stained with Coomassie Brilliant Blue. Mass spectrometry revealed a protein of relative molecular mass of 80,000 Da (asterisk) to be silkworm

BGIBMGA009402-PA. (C) Physical map of genomic region containing the mesh gene in Drosophila. Three kinds of the splicing variants are predicted in Flybase.

The piggyBac (pBac{WH}meshf04955) was inserted into the coding sequence of mesh transcripts as shown in the figure. Gray bar: untranslated regions of the mesh

transcript. Black boxes: coding sequences of the mesh transcripts. (D) Schematic representation of Mesh structure. The three Mesh isoforms share a large

extracellular region and differ in the cytoplasmic region. The domains in the extracellular region and the piggyBac insertion in the protein are shown. The Mesh

protein is hypothesized to be cleaved at the GDPH proteolytic site in the vWD domain. TM, putative transmembrane domain.

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(Fig. 1C,D). A piggyBac insertion, pBac{WH}CG31004f04955 is

located in the region shown in the schematic drawing of the mesh

gene and the protein (Fig. 1C,D). Embryos homozygous for the

meshf04955 chromosome hatched into first-instar larvae but died at

this stage. Df(3R)Excel6218 or Df(3R)tll-e, both of which lack the

mesh locus, failed to complement the lethality of meshf04955. The

lethality of meshf04955 homozygotes was rescued by precise

excision of pBac{WH}CG31004f04955 and the expression of a

mesh-RNAi using the 48Y-GAL4 driver at 25 C caused lethality at

the first-instar larval stage (data not shown), demonstrating that the

lethality is attributable to the piggyBac insertion in the mesh

gene. In addition, transheterozygotes for meshf04955 and

Df(3R)Excel6218 showed identical phenotypes to the phenotype

of meshf04955 homozygotes (see below; also supplementary

material Fig. S4A,B), and expression of Mesh with a 48Y-GAL4

driver in meshf04955 homozygotes rescued their phenotype

regarding sSJ organization (Fig. 3C9,F9). We confirmed that

meshf04955 eliminated the immunostaining of Mesh and that the

expression of a UAS-mesh in meshf04955 rescued the Mesh staining

(Fig. 3B,C,E,F). Taken together, these observations indicate that

mesh is an essential gene and that meshf04955 is a null or strong

loss-of-function allele of mesh.

Mesh localizes at sSJs in Drosophila

To determine the expression pattern and subcellular localization

of Mesh in Drosophila, anti-Mesh antibodies were generated

against the C-terminal cytoplasmic region. Western blot analysis

revealed that Mesh was mainly detected as a protein of relative

molecular mass 90,000 in embryos, in third-instar larvae, and in

extracts of S2 cells expressing Mesh (supplementary material

Fig. S2A). Mesh-PA/PB consists of 1431 amino acids with a

calculated molecular mass of 162,400, suggesting that the protein

is processed at a specific region. Indeed, higher-molecular-mass

bands (,200,000) were detected (supplementary material Fig.

S2A), and a putative GDPH cleavage site, an autocatalytic

proteolysis site in some mucins that cleaves between GD and PH

residues (Hollingsworth and Swanson, 2004), is located in the

vWD domain (a.a. 827–830 of Mesh-PA/PB) (Fig. 1D).

Immunofluorescence microscopic analyses revealed that the

expression of Mesh protein was first observed in the

endodermally derived tissues at embryonic stage 12 (Fig. 2A).

In late-stage embryos and third-instar larvae, Mesh was

expressed in the midgut, OELP and Malpighian tubules

(Fig. 2A–E), but was not expressed in the foregut and hindgut

(Fig. 2B,D,E; supplementary material Fig. S2C), demonstrating

Fig. 2. Mesh localizes to sSJs. (A) Double immunofluorescence staining of wild-type embryos using anti-Mesh (green) and anti-Dlg (red) antibodies. The

expression of Mesh protein was first observed in the endodermally derived epithelial cells at embryonic stage 12 (arrow). At stage 16, Mesh was exclusively

expressed in the midgut, the OELPs and the Malpighian tubules. Dlg was expressed in both endodermally and ectodermally derived epithelial cells.

(B–E) Antibody-stained wild-type third-instar larvae analyzed in the anterior midgut (B), the middle midgut (C), the posterior midgut (D) and the Malpighian

tubules (E) using anti-Mesh antibody. Mesh was expressed in the midgut, the OELPs and the Malpighian tubules and was localized at cell–cell contact regions in

their epithelial cells. Mesh signals were not detected in the foregut (B) and hindgut (E). (F) Immunoelectron microscopy of wild-type first-instar larval midguts

using anti-Mesh antibody. Immunolabels were detected at the bicellular contacts where the septa were observed. F9 is an enlarged view of F. (G) Antibody-stained

stage-16 embryos showing the proventriculus, which includes the boundary between the ectodermally derived foregut and endodermally derived midgut. Embryos

were double-stained for Mesh (G) and Kune (G9) as markers for sSJs and pSJs, respectively. The weak Kune expressions in the OELP are indicated by open

arrows in G9. G0 shows the merged image, in which dots delineate basal membranes of epithelial cells. The boundary cells (asterisk) expressing both Mesh

(arrows) and Kune (arrowheads) are identified. Scale bars: 100 mm (A, B–E); 500 nm (F); 5 mm (G–G0).

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that the expression of Mesh is specific for tissues bearing sSJs.

The immunoreactivities of these antibodies were diminished in

mesh mutant embryos and first-instar larvae, indicating the

specificity of our anti-Mesh antibodies (supplementary material

Fig. S2B,D). The expression pattern, timing, and subcellular

localization of Mesh correspond with those of Ssk, a previously

identified sSJ-specific protein (supplementary material Fig. S3A;

Fig. 3A–A90,D–D90). To confirm Mesh localization at sSJs, we

carried out immunoelectron microscopy using anti-Mesh

antibody. As shown in Fig. 2F, immunolabels were detected at

Fig. 3. Mesh is required for the localization of sSJ

components. (A–R) Immunofluorescence microscopic

analysis was performed in the OELPs and the anterior

midguts of first-instar larvae. In wild-type OELPs (A,G) and

midguts (D,I), Mesh was concentrated in the apicolateral

region of bicellular contacts and colocalized with Ssk

(A9, A90, D9 and D90). Dlg (A0, D0, G0 and I0), Lgl

(G9,I9), Cora (K,L) and FasIII (O,P) localized at the

apicolateral region of bicellular contacts where Mesh

colocalized with Dlg and Lgl (A90, D90, G90, I90). In

meshf04955, Ssk was mislocalized to apical and basolateral

membrane in the OELP (B9) and the midgut (E9). Dlg was

localized at the apicolateral region (B0, E0 H0 and J0). Lgl

was distributed along the lateral membrane in the meshf04955

OELP (H9) and midgut (J9) with partial concentration in the

apicolateral region (J9, arrowheads). Cora was observed at

the apicolateral region, but it spread into more basolateral

membrane regions in the meshf04955 OELP (M) and was

distributed to the cytoplasm in midgut epithelial cells (N). In

the meshf04955 OELP, FasIII was localized at the apicolateral

region (Q) and to the apical membrane (Q, arrowheads), and

it was observed as large aggregates in the apicolateral region

of the midgut (R). Expression of the UAS-mesh construct

with 48Y-GAL4 rescued Mesh (C,F) and Ssk

(C9,F9) localization in meshf04955 larvae. Scale bars: 5 mm.

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bicellular contacts where septa were observed, indicating thatMesh specifically localizes at sSJs in larval midgut epithelial

cells. Expression of Mesh was also observed in the apicolateralregion of epithelial cells in the adult midgut, OELP, andMalpighian tubules (supplementary material Fig. S3B),

indicating that Mesh is a component of sSJs in Drosophila

from the embryo through to adulthood.

Cells at the foregut–midgut boundary possess both pSJsand sSJs

Epithelia derived from ectoderm and endoderm possess pSJs and

sSJs, respectively, raising an intriguing question of how the SJs attheir boundary are organized. The specific localization of Meshat sSJs enabled us to investigate this issue. Stage-16 embryos

were double-stained with antibodies to Mesh and Kune asmarkers for sSJs and pSJs, respectively. Their localizationswere closely examined in the proventriculus, which includes

the boundary between the ectodermally derived foregut andendodermally derived midgut. As shown in Fig. 2G, weidentified boundary cells expressing both Mesh and Kune(Fig. 2G–G0, asterisk). In these cells, Kune localized at the

apicolateral membrane on the foregut side and Mesh localized onthe midgut side (Fig. 2G0), suggesting that individual boundarycells possess both pSJs and sSJs depending on which cells they

are adjacent to.

Mesh is required for proper localization of sSJ components

As described above, the mesh mutant animals hatched into thefirst-instar larvae, but died within 1 day. However, sSJs are not

completed until late stage 17 (Tepass and Hartenstein, 1994).Therefore, in the present study, we analyzed sSJ formation in thefirst-instar larvae. We focused on the OELP and the anteriormidgut epithelial cells because sSJ organization is clearest in

these columnar cells. Several pSJ proteins including Dlg, Lgl,and FasIII have been reported to localize at the apicolateralregion of bicellular contacts in Drosophila midgut (Baumann,

2001; Maynard et al., 2010; Strand et al., 1994). We confirmedthat these proteins all colocalized with Mesh in the apicolateralregion of wild-type OELP and midgut epithelial cells

(Fig. 3A0,D0,G9,G0,I9,I0,O,P; and data not shown). We alsochecked whether other pSJ proteins localize at sSJs andobserved that Cora colocalized with Mesh at the apicolateralregion (Fig. 3K,L; and data not shown). These results indicate

that Dlg, FasIII, Lgl, and Cora, at least, are both sSJ componentsand pSJ components.

To examine the role of Mesh in the molecular organization ofsSJs, we analyzed the subcellular localization of the sSJ proteinsin mesh mutants. Ssk was mislocalized to the apical andbasolateral membranes of the OELP and midgut epithelial cells

in mesh mutant larvae (Fig. 3B9,E9), and was often observed asaggregates in the cytoplasm (Fig. 3E9; supplementary materialFig. S4B). The intensity of Ssk signals in the apical membrane

was much higher than that of the basolateral membrane. Incontrast, Dlg was still localized at the apicolateral region(Fig. 3B0,E0,H0,J0), indicating that Mesh is not required for the

localization of Dlg. Moreover, the polarized distribution of Dlg inmesh mutants suggests that Mesh does not have a significant rolein specifying the apical-basal polarity of either OELP or midgut

epithelial cells. Lgl was distributed along the lateral membranewith partial concentration in the apicolateral region in the mesh

mutant OELP and midgut epithelial cells (Fig. 3H9,J9). Cora was

observed in the apicolateral region in the wild-type OELP and

midgut epithelial cells but it was spread more in the basal

direction in the mesh mutant OELP and was distributed

throughout the cytoplasm of the midgut epithelial cells

(Fig. 3M,N). In the mesh mutant OELP, FasIII was localized in

the apicolateral region but also mislocalized to the apical

membrane (Fig. 3Q). In contrast, it was observed as large

aggregates in the apicolateral region of the midgut epithelial cells

(Fig. 3R). Expression of the UAS-mesh construct with 48Y-

GAL4 rescued Ssk localization (Fig. 3C9,F9). In addition, mesh-

RNAi (12074-R1 generated by NIG-FLY) induced by 48Y-GAL4

on meshf04955/+ backgrounds decreased the level of Mesh at sSJs

and caused mislocalization of Ssk in the midgut epithelial cells

(supplementary material Fig. S5). Taken together, these results

indicate that Mesh determines the proper localization of several

sSJ proteins.

Mesh is required for proper sSJ organization

To further characterize the nature of the sSJ defect in mesh

mutants, ultrastructural analysis of the first-instar larvae was

performed. In wild-type midgut epithelial cells, typical sSJs were

observed at cell–cell contacts (Fig. 4A, brackets). In mesh

mutants, which were transheterozygotes for meshf04955 and

Df(3R)Excel6218, large gaps between the lateral membranes of

adjacent epithelial cells were frequently observed compared with

the wild-type (Fig. 4B,C, asterisks). However, a few septa were

still observed at the cell–cell contacts of the mesh mutant midgut

epithelial cells (Fig. 4B,C, brackets). pSJs in the mesh mutant

Fig. 4. Mesh is required for sSJ organization. Transmission electron

microscopy of wild-type (A) and meshf04955/Df(3R)Exel6218 (B,C) first-

instar larval midguts. In wild-type midgut, the typical sSJs were observed at

the bicellular contacts (A, brackets). In meshf04955/Df(3R)Exel6218 midguts,

large gaps between the lateral membranes of adjacent epithelial cells were

frequently observed (B,C, asterisks). A few septa were still observed at the

bicellular contacts of the meshf04955/Df(3R)Exel6218 midgut (B,C, brackets).

(D,E) In contrast to the sSJs, pSJs in the epidermis were intact in meshf04955/

Df(3R)Exel6218 midguts (E, bracket), as seen in the wild-type (D, bracket).

Scale bars: 500 nm.

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epidermis (Fig. 4E, bracket) were indistinguishable from those in

the wild-type (Fig. 4D, bracket). These results indicate that Mesh

is specifically required for proper sSJ organization.

Mesh is involved in the barrier function of the midgut

epithelium

We speculated that Mesh is involved in the barrier function of the

midgut epithelium. However, mesh mutant larvae fail to form the

three-layered structure of the proventriculus (supplementary

material Fig. S4D), although the structure is formed correctly

in stage-16 embryos, suggesting that mesh mutant animals cannot

maintain the proper structure of the proventriculus. Consistent

with this observation, colored yeast fed to mesh mutant larvae did

not accumulate in the gut, whereas it was observed throughout

their gut in wild-type larvae (data not shown). This phenotype

hampered the dye permeability assay used to examine the

integrity of the paracellular barrier, by feeding with a fluorescent

dye tracer. To overcome this problem, we generated mesh weak

loss-of-function conditions using mesh-RNAi (12074-R1) induced

by 48Y-GAL4 on meshf04955 heterozygous backgrounds. Uninduced

control first-instar larvae (UAS-mesh-RNAi/meshf04955) and

mesh-RNAi-induced first-instar larvae on wild-type (48Y-GAL4

.UAS-mesh-RNAi/+) or meshf04955 heterozygous (48Y-GAL4

.UAS-mesh-RNAi/meshf04955) backgrounds were fed fluorescent-

labeled dextran of 10 kDa and observed by confocal microscopy. In

control larvae, the midgut was well contrasted, with the fluorescent

tracer confined within the midgut (Fig. 5, upper panel). In contrast,

the tracer was detected in various parts of the body cavity in mesh-

RNAi-expressing meshf04955 heterozygous larvae (Fig. 5, middle

panel), indicating leakage of the tracer from the lumen of the

midgut. These observations indicate that Mesh is required for the

barrier function of the midgut epithelium in Drosophila. However,

we did not observe significant leakage of the tracer on the mesh-

RNAi-induced wild-type background (Fig. 5, lower panel),

suggesting insufficient RNAi-mediated suppression of Mesh on

the wild-type background.

Localization of Mesh to sSJs depends on Ssk but not on

Dlg, Lgl, Cora and FasIII

Since Ssk was mislocalized in mesh mutant sSJs, we next

investigated whether the localization of Mesh would be affected

by suppression of Ssk. As described in our previous report

(Yanagihashi et al., 2012), animals expressing ssk-RNAi with the

48Y-GAL4 driver exhibited a reduction in Ssk expression, while

those homozygous for Df(3L)ssk showed no expression of Ssk in

the midgut epithelial cells (Fig. 6B9,C9). In these cells, Mesh no

longer localized to the apicolateral region but was distributed

diffusely and formed some aggregates in the cytoplasm

(Fig. 6B,C). Thus, Mesh and Ssk are mutually dependent on each

other for their proper localization; Mesh is required for the

accumulation of Ssk at sSJs, and Ssk is required for the

translocation of Mesh from cytoplasm to sSJs. As observed in

mesh mutants, Lgl (Fig. 6E), Cora (Fig. 6G) and FasIII (Fig. 6I)

were mislocalized in Ssk-deficient cells. However, Dlg was still

localized at apicolateral region (Fig. 6B0,C0). Taken together, these

results suggest that Mesh acts together with Ssk to organize sSJs.

Next, we investigated the localization of Mesh in dlg, lgl, cora

and fasIII null mutants. In dlgm52 and lgl4 zygotic mutants, Mesh

and Ssk accumulated at the apicolateral region in the OELP and

midgut epithelial cells (data not shown), suggesting that Dlg and

Lgl are not required for the maintenance of sSJs. Since the

maternally supplied Dlg and Lgl are thought to be adequate for the

establishment of cell polarity and sSJs organization, we examined

the phenotype of dlgm52 and lgl4 maternal/zygotic mutant sSJs.

When eggs from wild-type animals were allowed to develop for

24 h at 25 C, they hatched into first-instar larvae and their midguts

developed a tube-like structure. In contrast, dlgm52 and lgl4

maternal/zygotic mutants exhibited a hypertrophied midgut

phenotype (Manfruelli et al., 1996). In the midgut epithelial cells

of these mutants, Mesh and Ssk accumulated in the apicolateral

region with faint leakage to the lateral membrane (Fig. 6J,K). In

fasIIIE25 mutants, Mesh was localized to the apicolateral region in

the midgut epithelial cells (Fig. 6L). Since cora5 mutant animals

Fig. 5. Mesh is required for barrier functions in midgut. Dye permeability assays of larvae with a weak mesh loss-of-function achieved by using mesh-RNAi

(12074-R1) induced by 48Y-GAL4. Uninduced control first-instar larvae (UAS-mesh-RNAi/meshf04955, upper panel) and mesh RNAi-induced first-instar larvae on

wild-type (48Y-GAL4 .UAS-mesh-RNAi/TM6B, lower panel) or meshf04955 heterozygous (48Y-GAL4 .UAS-mesh-RNAi/meshf04955, middle panel)

backgrounds were fed Alexa-Fluor-555-labeled 10 kDa dextran. In the control and mesh-RNAi-induced wild-type larvae, the midgut was defined clearly, with the

fluorescent tracer confined within the midgut (upper and lower panels). In contrast, the tracer was detected in various parts of the body cavity in mesh-RNAi-

expressing meshf04955 heterozygous larvae (middle panel). In the right panels, the background signals of green fluorescence excited by 488-nm laser irradiation

were used to trace the larval shape. GFP signals (arrow) are derived from the TM6B Ubi-GFP balancer in the mesh-RNAi-induced wild-type background larvae

(48Y-GAL4 .UAS-mesh-RNAi/TM6B). The images were taken in the same visual field. Scale bar: 100 mm.

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fail to hatch into larvae, we observed Mesh localization in the

stage-16 OELP, by which time Mesh as well as Cora had

accumulated in the apicolateral region of the wild-type (Fig. 6M,

data not shown). As shown in Fig. 6N, Mesh was localized to the

apicolateral region of the stage-16 OELP in cora5 mutants. Takentogether, these results indicate that the accumulation of Meshwithin the apicolateral region of the plasma membrane depends on

Ssk, but not on Dlg, Lgl, Cora or FasIII.

Mesh forms a complex with Ssk

Mesh and Ssk were mutually dependent for their localization at sSJs

(Figs 3,6), raising the possibility that Mesh is physically associatedwith Ssk. When the embryonic and larval extracts of Drosophila

were subjected to immunoprecipitation with anti-Mesh antibodies,Ssk coprecipitated with Mesh (Fig. 7A,B). Consistently, Mesh

coprecipitated with Ssk during immunoprecipitation fromembryonic extracts with anti-Ssk antibodies (Fig. 7C). NeitherMesh nor Ssk was precipitated by the pre-immune sera (Fig. 7A–C).

These results indicate that Mesh forms a complex with Ssk in vivo.

Mesh mediates the cell–cell adhesion

To investigate the possible role of Mesh as a cell–cell adhesion

molecule, we transfected Drosophila S2 cells with a Mesh–EGFPexpression vector and carried out an aggregation assay toexamine their adhesive properties. When S2 cells expressing

Mesh–EGFP were co-cultured with those expressing mCherry,only Mesh–EGFP-expressing cells formed the cell aggregation(Fig. 8A–C). Since S2 cells appear to lack endogenous Mesh

(supplementary material Fig. S2A), this experiment shows thatMesh expression leads to cell aggregation in a homophilicmanner. Furthermore, Mesh accumulated at cell–cell contact

regions between two cells expressing Mesh–EGFP (Fig. 8E,F,arrows). These results suggest that Mesh organizes sSJs bymediating cell adhesion via its homophilic interaction.

Fig. 7. Mesh forms a complex with Ssk. Mesh co-immunoprecipitated with

Ssk. The embryonic (A) and larval (B) extracts were subjected to

immunoprecipitation (IP) with anti-Mesh antibodies. Mesh was

immunoprecipitated with anti-Mesh antibodies, but not with pre-immune

serum (A,B, upper panel). The immunoprecipitates of Mesh contained Ssk

(A,B, lower panel). (C) Ssk immunoprecipitates from embryonic extracts also

contained Mesh (upper panel).

Fig. 6. Ssk is required for sSJ localization of Mesh.

Immunofluorescence microscopic analyses were performed

for the anterior midguts of the first-instar larvae or the

embryos. (A,D,F,H) In control (UAS-ssk-RNAi/TM6B, ssk-

RNAi-uninduced larvae) midguts, Mesh was concentrated in

the apicolateral region of bicellular contacts (A,A90). Ssk

(A9,A90), Dlg (A0,A90), Lgl (D), Cora (F) and FasIII (H) were

localized at the apicolateral region of bicellular contacts in

control midguts. (B,C,E,G,I) The first-instar larvae

expressing ssk-RNAi with the 48Y-GAL4 driver (48Y-

GAL4.UAS-ssk-RNAi/TM6B) exhibited a reduction in Ssk

expression in the midgut (B9). In these cells, Mesh was

distributed diffusely and formed aggregates in the cytoplasm

(B). In Df(3L)ssk midguts, in which Ssk signals were not

observed (C9), Mesh was distributed diffusely and formed

aggregates in the cytoplasm (C). Dlg was localized at the

apicolateral region in the ssk-RNAi (B0) and Df(3L)ssk

(C0) midguts. Lgl (E), Cora (G) and FasIII (I) were

mislocalized in the ssk-RNAi midguts. (J,K) In dlgm52 (J) and

lgl4 (K) maternal/zygotic mutants, Mesh was accumulated in

the apicolateral region of the midgut. (L) In fasIIIE25 mutants,

Mesh was localized to the apicolateral region of the midgut.

(M,N) In cora5 mutants, Mesh was localized to the

apicolateral region of the stage-16 OELP (N), as seen in the

wild-type (M). Scale bar: 5 mm.

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DiscussionWe have identified a novel membrane-spanning protein, Mesh,

which is specifically localized at sSJs and has cell adhesion

activity. Mesh is required for the formation of sSJs and

paracellular diffusion barriers in the Drosophila midgut. This

study, together with the recent identification of Ssk (Yanagihashi

et al., 2012), whose interaction with Mesh was shown in the

present study, provides a key starting point for understanding

sSJs, which must play crucial roles in the gut and renal functions

of arthropods, at the molecular level.

Implication of Mesh in the ultrastructure of sSJs

Electron microscopic observations have shown that sSJs and pSJs

can be distinguished morphologically. Obliquely sectioned pSJs

and sSJs are visualized as regular undulating rows and regularly

spaced parallel lines, respectively (Lane et al., 1994b), while both

types of SJs have ladder-like structures in the intermembrane

space. Of the two sSJ-specific integral membrane proteins, Ssk is

unlikely to be the structural element of the septa in sSJs, because

its extracellular loops are both too short (25 and 22 a.a.,

respectively) to bridge the intercellular space. In contrast, Mesh

induces cell–cell adhesion, implying that it may be one of the

components of the septa observed in ultrathin section electron

microscopy. Faint ladder-like structures were still observed in the

mesh mutants, suggesting that other membrane proteins also

contribute to the septal structures. FasIII is such a candidate

because it shows cell–cell adhesion activity (Snow et al., 1989) and

was still distributed to the apicolateral region, as well as the apical

region, in the mesh mutants (Fig. 3Q,R). However, fasIII null

mutant flies are viable (Whitlock, 1993) and both Mesh and Ssk are

normally localized at their sSJs, indicating that FasIII is dispensable

for sSJ formation. FasIII may provide robustness to the Mesh–Ssk-

mediated sSJ organization via its cell–cell adhesion activity.

SJ-boundary cells at foregut–midgut boundary

The issue of how SJs are organized in cells at the boundary

between pSJ- and sSJ-bearing epithelia is intriguing. Interestingly,

we observed boundary cells in which the pSJ marker Kune and sSJ

marker Mesh were concentrated in the anterior and posterior

regions, respectively, of the apicolateral membranes. This result

suggests that individual cells possess both pSJs and sSJs depending

on the orientation of their plasma membranes. The proventriculus

is originally derived from ectoderm (Tepass and Hartenstein,

1994). However, the OELP bears sSJs and expresses Mesh and

Ssk, suggesting that the OELP has both ectodermal and

endodermal characters. In fact, we observed weak Kune

expression in the OELP but not in the midgut (Fig. 2G0).

Therefore, the boundary cell may have the ability to form either

sSJs or pSJs according to the SJ type of adjacent cells. The

occurrence of such ‘SJ-boundary cells’ seems to be crucial because

they connect the ectodermally and endodermally derived epithelia

into a tandem tube while maintaining the continuity of the

paracellular barrier. However, we cannot completely exclude the

possibility that small amounts of pSJs and sSJs are also contained

in the sSJs on the midgut side and pSJs on the foregut side of the

SJ-boundary cells, respectively, to form hybrid junctions.

Interdependency between Mesh and Ssk for their sSJ

localization

Our analyses of Mesh and Ssk have clarified their interaction,

interdependency in their localizations, and requirements for the

organization and barrier function of sSJs, suggesting that Mesh–

Ssk is a key system for sSJ formation. In mesh mutants, Ssk failed

to localize at sSJs, but mislocalized to the apical and basolateral

plasma membrane domains. In ssk-RNAi and Df(3L)ssk fly, Mesh

no longer localized at the sSJs, but was distributed in the

cytoplasm. Ssk may translocate Mesh from the cytoplasm to sSJs

or to the plasma membrane. However, how the Mesh–Ssk complex

recognizes and localizes to sSJ regions remain elusive. Mesh

expression in S2 cells leads to cell aggregation without Ssk

expression, suggesting that there is a mechanism by which Mesh

translocates to the cell membrane and induces cell–cell adhesion

independently of Ssk in S2 cells. Detailed analysis of the dynamics

of Mesh–Ssk distribution will shed light on the mechanisms of sSJ

formation and the sorting systems for sSJ proteins.

A complicated hierarchy among sSJ components

By using Mesh and Ssk as specific markers for sSJs, we

confirmed that Dlg, Lgl and FasIII localize at sSJs in the larval

OELP and midgut epithelial cells. In addition, we found that Cora

is also concentrated into sSJs. Among these proteins that are

generally known as pSJ components, Lgl, Cora and FasIII were

mislocalized in mesh mutants and ssk-RNAi lines. On the other

hand, Lgl, Cora and FasIII were not required for the localization

of Mesh and Ssk at the apicolateral membrane. These

observations imply a possible hierarchy in the molecular

constituents of sSJs; Mesh-Ssk might act as a platform for the

assembly of Lgl, Cora and FasIII in endodermal epithelia. Such a

feature in sSJs is in sharp contrast to that in pSJs where each

molecular component is interdependent. Mutations in most of the

genes encoding pSJ-associated proteins result in disruption of the

barrier function and mislocalization of other pSJ proteins (Fehon

Fig. 8. Ectopic expression of Mesh induces cell-cell adhesion in

S2 cells. S2 cells transfected with the expression vectors for Mesh–

EGFP (A,C) and mCherry (B,C) were co-cultured. The S2 cell

aggregations were formed in Mesh–GFP-expressing cells

(A,C, arrow) but not in mCherry-expressing cells (B,C). (C) The

brightfield image was merged with the images of A and B.

(D–F) Mesh–EGFP (E,F, arrow) but not EGFP (D) accumulated at

the cell–cell contact region. Scale bars: 50 mm (A–C); 5 mm (D–F).

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et al., 1994; Baumgartner et al., 1996; Behr et al., 2003; Genovaand Fehon, 2003; Paul et al., 2003; Schulte et al., 2003; Faivre-Sarrailh et al., 2004; Llimargas et al., 2004; Wu et al., 2004; Wuet al., 2007; Nelson et al., 2010).

Interestingly, in mesh mutants and ssk-RNAi lines, Dlg stilllocalized at the apicolateral region of the OELP and midgut

epithelial cells, although sSJs were disrupted at the ultrastructurallevel. Furthermore, Mesh and Ssk were distributed to theapicolateral region in dlg mutants, suggesting that Mesh-Ssk

and Dlg are independent in their localizations. This is consistentwith a recent report that Dlg is probably not a core pSJcomponent (Oshima and Fehon, 2011). Nevertheless, a functional

relationship exists between Dlg and Lgl in determining cellpolarity in ectodermally derived epithelia. Therefore, in theabsence of Mesh and Ssk, Dlg may be unable to function properlybecause of an inadequate level of Lgl in the apicolateral regions.

In fact, dlgm52 and lgl4 maternal/zygotic mutants exhibited asimilar hypertrophied midgut phenotype (data not shown),suggesting that these proteins may function together in

endodermal epithelia, as well as in ectodermal epithelia (Bilderet al., 2003; Tanentzapf and Tepass, 2003).

The functions of Dlg, Lgl, Cora and FasIII at sSJs remainunknown. Dlg may act together with Lgl to regulate the apical-basal polarity in the early stage of epithelial development. In the

late developmental stage, compensation mechanisms for the Dlgfunction may rescue the apicolateral localization of Mesh, as notedin ectodermally derived epithelial cells of dlgm/z and lglm/z mutants(Bilder et al., 2003; Tanentzapf and Tepass, 2003). As larval

midgut sSJs are completed at the end of embryogenesis (stage 17)in Drosophila (Tepass and Hartenstein, 1994), the organization ofsSJ may not be influenced by early polarity defects of dlgm/z and

lglm/z mutants. Alternatively, Dlg and Lgl may be important for theregulation of the epithelial cell shape change that induces themidgut tube-like structure (Manfruelli et al., 1996). In ectodermally

derived epithelia, Cora acts together with Yurt to regulate theapicobasal polarity (Laprise et al., 2006; Laprise et al., 2009). Thus,Cora and a Yurt-like molecule may function together to organize

sSJs and/or to regulate the endodermal epithelial polarity.

Homologous proteins of Mesh in vertebrates

Homologous proteins, characterized by similar extracellular domainsto Mesh, are present in vertebrates (e.g. mouse Susd2/SVS-1),

implying that this family of proteins shares functions conservedacross species. Mouse Susd2/SVS-1 has been suggested as a tumor-reversing gene product, because it inhibited the growth of cancer cell

lines (Sugahara et al., 2007). Susd2/SVS-1 was distributed in theapical membrane of the epithelial cells in renal tubules and bronchialtubes, suggesting that it does not contribute to the cell–cell adhesionand/or paracellular barrier function in vertebrate epithelial cells.

However, expressing Susd2/SVS-1 in HeLa cells induces the cellaggregation (Sugahara et al., 2007), implying that this protein familyconserves the cell–cell adhesion activity. Further studies of the

functions of Mesh–Susd2/SVS-1 family proteins in vertebrates andin invertebrates will lead to a better understanding of the conservedphysiological functions in these proteins and of the evolution of

intercellular junctions across species.

Materials and MethodsFly stocks and geneticsThe fly strains meshf04955, Df(3R)Exel6218, fasIIIE25, lgl4 and 48Y-GAL4, wereobtained from the Bloomington Stock Center, and the mesh-RNAi strains, 12074-R1 was obtained from NIG-FLY. We also used the strains dlgm52 (a gift from P. J.

Bryant), cora5 (a gift from R. G. Fehon), UAS-ssk-RNAi and Df(3L)ssk(Yanagihashi et al., 2012). Germline clones of lgl4 and dlgm52 were made by theFLP-DFS technique (Chou and Perrimon, 1992). For the phenotype rescueexperiment, pUAST vectors (Brand and Perrimon, 1993) containing mesh wereconstructed and a fly strain carrying this construct was established. 48Y-GAL4,which drives GAL4 expression in the anterior and posterior midgut primordiumfrom embryonic stage 10 (Martin-Bermudo et al., 1997), was used to express UAS-mesh in meshf04955 for the rescue experiment.

Membrane fraction from silkworm midgut

The membrane fraction was prepared from midguts of silkworm 5th-instar larvaeaccording to the method described previously (Yanagihashi et al., 2012).

Production of monoclonal antibodies and identification of the antigens

Rat mAbs against membrane fractions of silkworm fifth-instar larval midguts weregenerated as described previously (Yanagihashi et al., 2012). For identification ofthe antigens, the membrane fractions (,500 mg) were centrifuged at the maximumspeed in a microcentrifuge for 20 min and the pellet was resuspended in 500 ml oflysis buffer [25 mM Tris-HCl, pH 8, 27.5 mM NaCl, 20 mM KCl, 25 mMsucrose, 10 mM EDTA, 10 mM EGTA, 1 mM DTT, 10% (v/v) glycerol, 1% NP40and protease inhibitor cocktail (Nakarai, Kyoto, Japan)] for 30 min at 4 C. Thelysates were centrifuged at the maximum speed for 20 min, and the supernatantswere used for immunoprecipitation with protein G sepharose (GE Healthcare)conjugated with the mAbs. The sepharose preparations were incubated with thesupernatants for 4 h at 4 C and were washed five times in lysis buffer. Boundproteins were separated by SDS-PAGE and analyzed by Coomassie Brilliant BlueG-250 (Wako) staining. Mass spectrometry analyses of the tryptic peptide massdata were carried out by the Integrated Center for Mass Spectrometry (KobeUniversity Graduate School of Medicine). The resulting tryptic peptide mass datawere matched against the NCBInr database using the Mascot program.

Production of polyclonal Abs

A region of the Mesh PA/PB protein (amino acids 1211–1431) was cloned intopGEX-6P (GE Healthcare) to produce a GST-fusion protein. The proteins wereexpressed in Escherichia coli. Polyclonal antibodies were generated in rabbits(995-1 and -2) and rats (8002) by MBL (Nagoya, Japan).

Immunohistochemistry

Embryos were fixed with 3.7% formaldehyde in PBS for 20 min. Larvae weredissected in Hanks’ Balanced Salt Solution and fixed with 3.7% formaldehyde inPBS with 0.4% Triton X-100. The following antibodies were used: rabbit and ratanti-Mesh, rabbit anti-Ssk (6981-1; 1:1000) (Yanagihashi et al., 2012), rabbit anti-Kune (1:1000) (Nelson et al., 2010), mouse anti-Dlg 1:50 [Developmental StudiesHybridoma Bank (DSHB)], mouse anti-coracle C615.16 1:50 (DSHB), mouse anti-FasIII 1:20 (DSHB), rabbit anti-Lgl 1:1000 (provided by F. Matsuzaki, RIKENCDB). Alexa Fluor 488-conjugated (Invitrogen), and Cy3- and Cy5-conjugated(Jackson ImmunoResearch Laboratories) secondary antibodies were used at 1:400.Samples were mounted in Vectashield (Vector Laboratories). Images were acquiredwith a confocal microscope (model TCS-SPE; Leica) with its accompanyingsoftware using HC PLAN Apochromat 206NA 0.7 and HCX PL Apochromat636NA 1.4 objective lens (Leica). Images were processed with Adobe PhotoshopH.

Electron microscopy

First-instar larvae of wild-type or mesh mutants were dissected and fixed overnight at4 C with a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 Mcacodylate buffer (pH 7.4). The specimens including the midguts were prepared asdescribed previously (Yanagihashi et al., 2012). For immunoelectron microscopy,first instar larvae were dissected and fixed for 2 h at room temperature with 4%paraformaldehyde in 0.1 M sodium phosphate buffer (PB) (pH 7.4). The specimenswere washed three times with 50 mM glycine in PB and incubated with 0.1% saponinin PB. After blocking with 10% normal goat serum in PB for 1 h, they were incubatedfor 2 days at 4 C with anti-Mesh antibody (995-2; 1:1000) diluted in the blockingsolution. After six washes with PB, the specimens were incubated for 2 h with asecondary antibody that had been conjugated with both the 1.4 nm NANOGOLDparticles (1:100; Nanoprobes, Inc.), followed by six washes. The specimens werefixed for 15 min with 2.5% glutaraldehyde in PB, washed with 50 mM glycine in PB,and again four times with 50 mM HEPES, pH 5.8 for 15 min. Signals were silver-enhanced by use of an HQ-silver kit (Nanoprobes, Inc.) for 14 min in the dark. Afterthorough washing with distilled water, they were fixed with 0.5% osmium oxide inPB for 1.5 h on ice and washed again with distilled water. Subsequently thespecimens were embedded with Epon 812. The ultrathin sections (50–100 nm) werestained doubly with 4% hafnium (IV) chloride and lead citrate, and observed with aJEM-1011 electron microscope (JEOL) at an accelerating voltage of 80 kV.

Co-immunoprecipitation and western blotting

Wild-type fly embryos and third-instar larvae were mixed with a 5-fold volume oflysis buffer [25 mM Tris-HCl pH 8, 27.5 mM NaCl, 20 mM KCl, 25 mM

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Sucrose, 10 mM EDTA, 10 mM EGTA, 1 mM DTT, 10% (v/v) glycerol, 0.5%NP40 and protease inhibitor cocktail from Sigma] and homogenized using a pestlefor 1.5 ml microfuge tubes. The method for immunoprecipitation was essentiallythe same as described above. Anti-Mesh (995-1 and -2) and anti-Ssk (6981-1and -2) antibodies were used for the immunoprecipitation and theimmunocomplexes were separated by SDS-PAGE, transferred to polyvinylidenedifluoride membranes and probed with an anti-Ssk (6981-1; 1:1000) and an anti-Mesh (995-1; 1:1000) antibodies.

Cell culture and aggregation assay

For the ectopic expression of Mesh in S2 cells, Mesh–EGFP and mCherry(Clontech) cDNA were subcloned into pMT-V5His (Invitrogen) and EGFP cDNAwas subcloned into pAC-V5His (Invitrogen). S2 cells were cultured at 25 C inSchneider medium containing 10% fetal bovine serum and antibiotics. DNAs(pAC-EGFP, pMT-mCherry, and pMT-Mesh-EGFP) were transfected into cellsusing the Effectene kit (Qiagen), and cells were cultured for 2 days beforeimmunostaining or aggregation assay. To induce the expression from pMT vectors,copper sulfate (final concentration: 500 mM) was added to the culture medium at24 h after transfection. For immunostaining, the cells were transferred ontoconcanavalin A-coated coverslips and incubated for 2 h before fixation withmethanol-acetone (1:1) for 10 min at 220 C. After washing with PBS with 0.05%Tween 20 (PBST), the fixed cells were blocked with 10% calf serum in PBST.Samples were then incubated with anti-GFP antibody (Roche) for 30 min at roomtemperature, followed by incubation with Alexa Fluor 488-conjugated secondaryantibody (Invitrogen) for 30 minutes. After washing with PBST, cells wereembedded in Fluorsave (Calbiochem). For aggregation assay, the cells were gentlydissociated by repeated pipetting and the cell concentrations were readjusted withcell culture medium to 16106 cells/ml. The cells were shaken at 100 rpm on arotation platform at room temperature. Aggregation of the cells was analyzed after2 h. Images were captured with a camera (ORCA-AG; Hamamatsu Photonics)mounted to a microscope (IX71; Olympus) with UPlanSApo 206NA 0.75objective lens (Olympus) using IP Lab (ver. 3.9.5r3) acquisition software (BDBiosciences).

Dye-feeding experiments

Embryos (1–15 h after laying) were put on yeast paste containing Alexa FluorH555-labeled dextran (MW 10,000 Invitrogen) to feed newly hatched larvae. After10–15 h, first-instar larvae were washed with water. Images were acquired with aconfocal microscope (model TCS-SPE; Leica) and its accompanying softwareusing an HC PLAN Apochromat 206NA 0.7 objective lens (Leica). Images wereprocessed with Adobe PhotoshopH.

AcknowledgementsWe are grateful to S. Yonemura, A. Nagafuchi, and all the membersof Furuse laboratories for helpful discussions. We also thank F.Matsuzaki for the antibody and the fly stocks, and R. G. Fehon, P. J.Bryant, the Bloomington Stock Center, the Drosophila GeneticResource Center at Kyoto Institute of Technology and the fly stocksof National Institute of Genetics (NIG-Fly) for fly stock.

FundingThis work was supported in part by grants from the Japan Society forthe Promotion of Science (JSPS) [grant number 09009170 to Y. I.];Takeda Science Foundation (to M. F. and Y. I.); Hyogo Science andTechnology Association (to Y. I.); and by the ‘‘Funding Program forNext Generation World Leading Researchers (NEXT Program)’’ ofJSPS, initiated by the Council for Science and Technology Policy[grant number LS084 to M. F.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.112243/-/DC1

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