Transcription factors of the Grainy head (Grh) family are required in epithelia to generate the impermeable apical layer that protects against the external environment. This function is conserved in vertebrates and invertebrates, despite the differing molecular composition of the protective barrier. Epithelial cells also have junctions that create a paracellular diffusion barrier (tight or septate junctions). To examine whether Grh has a role in regulating such characteristics, we used an epidermal layer in the Drosophila embryo that has no endogenous Grh and lacks septate junctions, the amnioserosa. Expression of Grh in the amnioserosa caused severe defects in dorsal closure, a process similar to wound closure, and induced robust expression of the septate junction proteins Coracle, Fasciclin 3 and Sinuous. Grh-binding sites are present within the genes encoding these proteins, consistent with them being direct targets. Removal of Grh from imaginal disc cells caused a reduction in Fasciclin 3 and Coracle levels, suggesting that Grh normally fine tunes their epithelial expression and hence contributes to barrier properties. The fact that ectopic Grh arrests dorsal closure also suggests that this dynamic process relies on epithelia having distinct adhesive properties conferred by differential deployment of Grh.

During Drosophila development, the Grainy head (Grh) transcription factor is expressed in the epidermis and a subset of other epithelia that form strongly adhesive layers exposed to the external environment (e.g. trachea) (Bray and Kafatos, 1991; Hemphala et al., 2003; Uv et al., 1994). In the absence of grh, these epithelial cells have altered morphology and lose expression of enzymes that cross-link the apical extracellular matrix (cuticle) (Bray and Kafatos, 1991; Hemphala et al., 2003; Mace et al., 2005; Ostrowski et al., 2002). Similarly, in mice lacking the Grh-related gene GRHL3, the outer protective layer of the skin, the stratum corneum, is defective (Ting et al., 2005; Yu et al., 2006), and in Xenopus embryos, GRHL3 and GRHL1 are expressed in the outer cells and regulate the expression of keratins (Chalmers et al., 2006; Tao et al., 2005). Thus, Grh proteins have highly conserved roles in regulating terminal differentiation of robust protective epithelia.

In addition to regulating terminal differentiation per se, Grh might also have other functions in these epithelia. For example, at stages before the stratum corneum is formed, Grhl3 mutant mice have defects in re-epithelialisation following wounding (Stramer and Martin, 2005; Ting et al., 2005). They also have altered levels of many tight-junction-associated proteins, including occludins and claudins (Yu et al., 2006). Likewise, in Drosophila, grh expression commences prior to cuticle secretion and correlates with stages at which these epithelia acquire occluding junctions (septate junctions) (Tepass and Hartenstein, 1994). Nevertheless, Grh is dispensable for the establishment of basic barrier properties, because septate junctions are still present in grh-mutant tracheal cells (Hemphala et al., 2003). However, because the barrier characteristics of occluding junctions vary between epithelia (Furuse and Tsukita, 2006), the conserved expression of grh family proteins in the highly impermeable surface epithelia led us to investigate further whether Grh could directly regulate expression of epithelial junction components in Drosophila.

We began our investigations by expressing Grh ectopically in the amnioserosa (AS), a single-layered epithelium that has no septate junctions (Tepass and Hartenstein, 1994; Gorfinkiel and Martinez Arias, 2007), to determine whether Grh could convert this tissue into one with barrier epithelia characteristics. The AS is normally devoid of Grh expression and plays an important role in co-ordinating the fusion between the epidermal sheets during dorsal closure (Fig. 1A,B). From these studies, we uncovered a role for Grh in regulating expression of septate junction proteins, which we have further confirmed using loss-of-function mutations and by showing that the genes contain Grh-binding sites. Thus, in addition to co-ordinating expression of matrix proteins, Grh also regulates the intrinsic barrier properties of epithelia through its effects on components of cell junctions.

To test the role of Grh in regulating epithelial characteristics, we specifically expressed the epidermal splice forms (N/K) in the amnioserosa, an epithelial tissue normally devoid of Grh (using c381::Gal4 and G332::Gal4; Fig. 1C,F-H). This was sufficient to block dorsal closure (Fig. 1G,H), an effect previously seen with ubiquitous Grh overexpression (Attardi et al., 1993). The effects were most penetrant with c381::Gal4 (hereafter referred to as ASc381>grh) which resulted in 100% of embryos having dorsal holes at stage 17/hatching, when all wild-type embryos had completed dorsal closure (Fig. 1H; ASG332>grh resulted in >50% with dorsal holes). Defects were already evident earlier (stage 14-16). In ASc381>grh embryos, the amnioserosa was less contracted than wild type and contained cells with abnormal morphology. In addition, the epidermal edges failed to meet at the poles (Fig. 1G). Thus, expression of Grh disrupted the ability of amnioserosa cells to function in dorsal closure, suggesting that it altered their fundamental properties and/or perturbed their interactions with the epidermis.

Fig. 1.

Expression of Grh in the amnioserosa perturbs the progression and outcome of dorsal closure. (A,B) Dorsal closure: a membrane-associated Myc-tag labels amnioserosa at the stages indicated (lateral view; c381::Gal4). Arrows mark the edge of the epidermal sheets. (C,F) Grh expression in wild-type (C) and ASG332>grh (F) embryos (arrows indicate amnioserosa; stage 14, lateral view). (D,G) Phalloidin staining marks the cell outlines in dorsal view of wild-type (D) and ASc381>grh (G) stage-15 embryos; zippering has commenced at the poles in control but not in ASc381>grh embryos. Arrows mark the edge of the epidermal sheets. (E,H) Cuticles from hatching stage. ASc381>grh embryos (H) have a dorsal hole (arrows) and internal organs are extruded (asterisk).

Fig. 1.

Expression of Grh in the amnioserosa perturbs the progression and outcome of dorsal closure. (A,B) Dorsal closure: a membrane-associated Myc-tag labels amnioserosa at the stages indicated (lateral view; c381::Gal4). Arrows mark the edge of the epidermal sheets. (C,F) Grh expression in wild-type (C) and ASG332>grh (F) embryos (arrows indicate amnioserosa; stage 14, lateral view). (D,G) Phalloidin staining marks the cell outlines in dorsal view of wild-type (D) and ASc381>grh (G) stage-15 embryos; zippering has commenced at the poles in control but not in ASc381>grh embryos. Arrows mark the edge of the epidermal sheets. (E,H) Cuticles from hatching stage. ASc381>grh embryos (H) have a dorsal hole (arrows) and internal organs are extruded (asterisk).

Grh promotes expression of septate junction proteins

One explanation for the defects caused by Grh expression in amnioserosa cells is that these cells acquire epidermis-like characteristics, such as septate junctions (SJs) characteristic of conventional barrier epithelia. Proteins that localise to SJs include the FERM-domain protein Coracle, the immunoglobulin-family adhesion protein Fasciclin 3 (Fas3), the transmembrane protein Neurexin (Nrx), and the claudin-related proteins Sinuous (Sinu) and Megatrachea (Behr et al., 2003; Genova and Fehon, 2003; Lamb et al., 1998; Schulte et al., 2003; Tepass et al., 2001; Wu et al., 2004). In addition, the Discs large (Dlg)-Scribble-l(2)gal (Lgl) complex initially localises basolaterally and becomes incorporated into SJs (Knust and Bossinger, 2002). To investigate whether Grh regulates such components we examined their expression in ASc381>grh embryos. Expression of Fas3, Coracle and Sinu was strikingly upregulated in the amnioserosa of ASc381>grh embryos in comparison to control embryos (Fig. 2A-F,I-J′,K-M,O). Nrx and Atpα (Na/K-ATPase subunit) were more weakly upregulated (Fig. 2H,K and data not shown), and Dlg was upregulated in a patchy manner, although this effect was less penetrant (Fig. 2G,J″,N,P). Thus, levels of several different SJ proteins are increased by Grh expression in the amnioserosa. Of these, Fas3 was the earliest that could be detected.

In conventional epithelia, junctional proteins are localised to discrete domains in the lateral membrane (Knust and Bossinger, 2002). In ASc381>grh embryos, the sub-cellular localisation of SJ proteins was abnormal. Fas3, Coracle and Dlg were more diffuse than in wild type and frequently expanded along the apical and/or basal surface (Fig. 2M-P and data not shown). For example, Fas3 proteins were present in a more apical plane than the adherens junction component E-cadherin (Fig. 2O,O′), and Dlg was expanded throughout basal and apical regions (Fig. 2P,P′). Because E-cadherin itself was still localised at apical junctions in AS>grh embryos (Fig. 2N,P), the underlying apical/basal polarity appears unaffected. Thus, the altered distribution of Fas3, Coracle and Dlg suggests that Grh is sufficient to promote expression of SJ proteins, but not to ensure the correct organisation of these proteins within the apical-basal axis.

SJ proteins are reduced in grh-mutant cells

The upregulation of SJ proteins caused by ectopic Grh expression is complementary to the apical membrane expansion detected in grh loss-of-function mutants (Hemphala et al., 2003). However, SJ proteins (e.g. Coracle) are still present in the mutant tracheal and epidermal cells (Hemphala et al., 2003) (data not shown). Thus, Grh is apparently not essential for expression of SJ proteins, although it can clearly promote their expression ectopically. One way to reconcile these differences is if Grh fine-tunes the expression of such proteins to increase or strengthen lateral junctions in mature epithelia. We tested this by generating clones of grh-mutant cells in the wing imaginal disc, in which the juxtaposition of wild-type and mutant cells aids detection of subtle changes in expression levels. SJ proteins were still present in mutant wing disc cells, as they were in mutant tracheal cells. However, using this approach it was possible to detect a reduction in the levels of Fas3 and Coracle in cells lacking grh (Fig. 3A,B). This was most consistent for Fas3: the majority (11/16) of clones scored had a detectable reduction in Fas3. With Coracle, the effects were more variable, but 5/16 grh-mutant clones had subtle decreases in its levels. The fact that the effects were subtle and variable could be a consequence of timing, because we assayed the consequences of removing Grh at a relatively early stage in the maturation of these epithelia. Nevertheless, removal of Grh was not sufficient to compromise the barrier properties of the tracheal epithelia in the embryo, as measured by dextran exclusion experiments (Fig. 3E). Fluorescent dextrans injected into wild-type and grh-mutant embryos failed to enter the lumen of the trachea, indicating that they are unable to pass through the junctions. By contrast, when injected into mutant embryos in which SJs were compromised, dextran rapidly spread throughout the tracheal lumen. Thus, Grh is not essential for the establishment of SJs, although it can influence the levels of SJ proteins (at least in the wing disc) and is sufficient to promote their expression ectopically. These data suggest a model in which Grh in Drosophila elevates the expression of SJ proteins in a similar manner to the effects of GRHL3 in mice on claudins and occludins, proteins found in the analogous tight junctions (Yu et al., 2006). In neither animal is there complete loss of these proteins in the grh/Grhl3 mutants, but their levels and distribution are altered in a manner that could alter the robustness of an epithelial barrier.

Fig. 2.

Grh promotes expression of SJ components in the amnioserosa. Stage-14 control (A,C,E-I,M,N) and ASc381>grh (B,D,J-L,O,P) embryos stained as indicated. Dorsal views of whole embryos (A-D) or of amnioserosa (E-L, single confocal sections; J-J″ are individual channels from one embryo). (M-O) Transverse sections stained as indicated to reveal the apical/basal distributions. (M,O) Upregulated Fas3 (FasIII) in ASc381>grh embryos accumulates apically (green in O, white in O′; e.g. orange arrowheads). (N,P) Dlg is also upregulated in ASc381>grh embryos (green, P; white P′) and spreads along the apical surface (e.g. orange arrowheads). (M-P) White arrows mark lateral junction sites.

Fig. 2.

Grh promotes expression of SJ components in the amnioserosa. Stage-14 control (A,C,E-I,M,N) and ASc381>grh (B,D,J-L,O,P) embryos stained as indicated. Dorsal views of whole embryos (A-D) or of amnioserosa (E-L, single confocal sections; J-J″ are individual channels from one embryo). (M-O) Transverse sections stained as indicated to reveal the apical/basal distributions. (M,O) Upregulated Fas3 (FasIII) in ASc381>grh embryos accumulates apically (green in O, white in O′; e.g. orange arrowheads). (N,P) Dlg is also upregulated in ASc381>grh embryos (green, P; white P′) and spreads along the apical surface (e.g. orange arrowheads). (M-P) White arrows mark lateral junction sites.

Grh binds to target sites in fas3 and coracle genes

To investigate whether genes encoding SJ proteins could be direct targets of Grh, the coracle and fas3 genes were analysed for sequences that had good matches to a weighted matrix derived from known Grh-binding sites (Almeida and Bray, 2005) and that were conserved in the cognate genes from highly diverged drosophilids (D. pseudoobscura, D. virilis, D. mojavensis). There were two conserved matches to the Grh-binding-site consensus in the first intron of fas3 (fas3A, 5′-ACCGGTTT-3′; fas3B, 5′-ACCAGTTT-3′) and in the first intron of coracle [coraA, 5′-ACCAGTTT-3′ (–strand); coraB, 5′-ACCGGTTT-3′ (–strand)]. These four sites were recognised by Grh in vitro in a competition assay in which their binding affinities were compared with a high-affinity Grh target site, Gbe2, from the dopa decarboxylase gene (Uv et al., 1994) (Fig. 3C). Putative sites from fas3 and coracle significantly reduced binding to the labelled Gbe2 probe, and were even more effective than a similar excess of the cognate Gbe2 site, demonstrating that they are high-affinity binding sites for Grh. Thus, both fas3 and coracle have the potential to be direct targets of Grh.

To further test their potential for regulation by Grh, fragments encompassing the Grh-binding sites were inserted upstream of a minimal promoter fused to luciferase and expression was assayed in the presence and absence of Grh in transient transfection assays (Fig. 3D). In total, 3/4 fragments conferred Grh responsiveness (>2.5×) on the reporter. In addition, two fragments from sinu that encompassed putative Grh-binding sites [sinu1, 5′-ACCTGTTC-3′ (–strand); sinu2, 5′-TCCGGTTT-3′] were tested in the same assay, and sinu2 also showed a response to Grh. Together, these data suggest that the effect of Grh on SJs involves direct regulation of component-encoding genes.

Effects of Grh expression on adhesive properties of the amnioserosa and epidermis

Because ectopic expression of Grh has a profound effect on dorsal closure, we looked more closely at the morphology of ASc381>grh cells and the distribution of other adhesion complexes. In ASc381>grh embryos there were large variations in shape and size of amnioserosa cells and the contacts with the adjacent dorsal epidermis were dramatically different (Fig. 4C-F and data not shown). A subset of epidermal cells had expanded contact with an amnioserosa cell at the expense of their neighbours, which became bunched together (Fig. 4C″,D″,F). It appeared, therefore, that many Grh-expressing amnioserosa cells had maximised the contact with a single epidermal cell, rather than making contact with five to six cells, as in wild type. Ultimately, some amnioserosa cells appeared to lose contact with the epidermal cells.

The change in morphology in ASc381>grh embryos was accompanied by altered distribution of β-position-specific (βPS) integrins and E-cadherin (Fig. 4). In wild-type embryos, these co-localise to prominent dots at the interface between amnioserosa and epidermal cells and are present in overlapping domains associated with amnioserosa cell-cell contacts (Narasimha and Brown, 2004) (Fig. 4A,B). In ASc381>grh cells, βPS integrins appeared diffuse and frequently spread across the apical surface (Fig. 4C,C′). Furthermore, less βPS integrin accumulated at the interface between epidermal and AS cells (Fig. 4C′,D′); instead, it was concentrated in regions with bunched epidermal cell contacts and no longer localised with E-cadherin (Fig. 4D-D″). Thus, Grh perturbs other adhesive characteristics of amnioserosa cells. This could be an indirect consequence of the increase in SJ proteins, causing altered distribution of apical and basal adhesion receptors, or Grh could additionally regulate the expression levels of cadherin and integrins (Almeida and Bray, 2005). Whichever the mechanism, amnioserosa cells acquire altered adhesion properties with neighbouring epidermal cells, which could explain why dorsal closure is perturbed.

Fig. 3.

fas3 and coracle are potential targets of Grh. (A,B) Levels of Fas3 (FasIII; red, A,B; white, A′,B′) and Coracle (blue, A,B; white, A″, B″′) are subtly reduced in grh-mutant clones (identified by absence of GFP, green, in A,B). Clone boundaries are indicated by arrows. (C) Grh binds to sites from fas3 and coracle genes. Amount of Grh complexed with labelled Gbe2-binding sites in the absence (O) or presence of the indicated competitors (5× molar excess and 50× molar excess). N-box is an unrelated E(spl) target site. Lane 1 has no added protein; all other lanes contain GST-Grh bacterial extract. (D) Fold stimulation by Grh of luciferase reporter genes containing fragments spanning the indicated sites. 4×Gbe, positive control with oligomerised Gbe2 sites; hid1, negative control. (E) Rhodamine-conjugated dextran is excluded from the tracheal lumen (arrows) of wild-type (wt) and grh-mutant (grhB32/grhB32) embryos but not from SJ mutants (Atpα; SJ).

Fig. 3.

fas3 and coracle are potential targets of Grh. (A,B) Levels of Fas3 (FasIII; red, A,B; white, A′,B′) and Coracle (blue, A,B; white, A″, B″′) are subtly reduced in grh-mutant clones (identified by absence of GFP, green, in A,B). Clone boundaries are indicated by arrows. (C) Grh binds to sites from fas3 and coracle genes. Amount of Grh complexed with labelled Gbe2-binding sites in the absence (O) or presence of the indicated competitors (5× molar excess and 50× molar excess). N-box is an unrelated E(spl) target site. Lane 1 has no added protein; all other lanes contain GST-Grh bacterial extract. (D) Fold stimulation by Grh of luciferase reporter genes containing fragments spanning the indicated sites. 4×Gbe, positive control with oligomerised Gbe2 sites; hid1, negative control. (E) Rhodamine-conjugated dextran is excluded from the tracheal lumen (arrows) of wild-type (wt) and grh-mutant (grhB32/grhB32) embryos but not from SJ mutants (Atpα; SJ).

Fig. 4.

Grh expression results in altered cellular morphology and disrupts the distribution of integrins and cadherin. Dorsal views of control (A,B) and ASc381>grh (C-F) embryos stained with antibodies against βPS integrin (green, A-D; white, A′-D′) and E-cadherin (magenta, A-E; white, A″-D″,E′,F). (A-D) ASc381>grh cells have poor localisation and patchy accumulation of βPS integrin (e.g. arrows, C,C′), reduced βPS integrin levels at epidermal interface (arrowheads, C,C′,D,D′), reduced co-localisation between βPS integrin and E-cadherin (compare B with D), and some mislocalisation of E-cadherin (e.g. arrows C″). (E,F) Defects in the epidermis include bunching (orange arrows) and splaying (green arrows) of epidermal cells, and a detachment between the epidermis and the amnioserosa. (E) Nuclei are visualised with DAPI.

Fig. 4.

Grh expression results in altered cellular morphology and disrupts the distribution of integrins and cadherin. Dorsal views of control (A,B) and ASc381>grh (C-F) embryos stained with antibodies against βPS integrin (green, A-D; white, A′-D′) and E-cadherin (magenta, A-E; white, A″-D″,E′,F). (A-D) ASc381>grh cells have poor localisation and patchy accumulation of βPS integrin (e.g. arrows, C,C′), reduced βPS integrin levels at epidermal interface (arrowheads, C,C′,D,D′), reduced co-localisation between βPS integrin and E-cadherin (compare B with D), and some mislocalisation of E-cadherin (e.g. arrows C″). (E,F) Defects in the epidermis include bunching (orange arrows) and splaying (green arrows) of epidermal cells, and a detachment between the epidermis and the amnioserosa. (E) Nuclei are visualised with DAPI.

Concluding comments

The results we obtained from ectopic Grh expression have helped uncover functions that are not easily evident from loss-of-function experiments and suggest that Grh is normally involved in fine-tuning the expression levels of proteins, such as Fas3, Coracle, Sinu, Nrx and Dlg, which are involved in conferring robust barrier function on the epidermis. Given the observation that GRHL3 also fine-tunes the levels of junction proteins in mice (Yu et al., 2006), it appears that this represents a highly conserved aspect of Grh function. In addition, Grh might be intrinsic to the observed cross-talk between the extracellular matrix and junctional complexes (Tonning et al., 2005; Wang et al., 2006), because it plays a role in regulating both elements.

Grh transcription factors are also components in a conserved mechanism for wound healing, in part via their effect on extracellular matrix deposition/synthesis (Mace et al., 2005; Stramer and Martin, 2005). Our results suggest that regulation of cell junctions might also be important for epidermal `sealing'. They further suggest that differences in Grh levels or activity could regulate morphogenesis within an epithelium, as well as the ability of epithelia to adhere to one another, by influencing the levels and distribution of septate/tight junction proteins and other adhesion molecules. This could also explain the role of GRHL3 during neural tube closure in mice (Ting et al., 2003), an epithelial fusion event that shares features with dorsal closure.

Genetics

For ectopic expression of Grh, c381::Gal4 and G332-Gal4 drivers were combined with UAS::grhN/K, and embryos from 4- to 6-hour collections were aged at 25°C to enrich for stages 13-16. grh-mutant clones were induced (1 hour at 37°C) in larvae of the genotype hsFLP/w; FRT42D grhB32/FRT42D PcEGFP.

Immunochemistry

Whole-mount staining of embryos was performed according to standard procedures. Primary antibodies were monoclonal mouse anti-βPS (CF6G11, 1:3), anti-Fas3 (7G10 1:30), anti-Dlg (4F3, 1:100) and anti-Atpα (a5, 1:100), all obtained from the Developmental Studies Hybridoma Bank; rabbit anti-Dlg (1:500-1000), anti-Sinu (Wu et al., 2004); rat anti-E-cadherin [1:20 (Oda et al., 1994)]; guinea-pig anti-Coracle [1:1000 (Fehon et al., 1994)] and anti-Nrx (Baumgartner et al., 1996). Alexa-Fluor-488/568 (Molecular Probes) Cy2, Cy3 or Cy5 (Jackson Immunodiagnostics)-conjugated secondary antibodies were used at 1:200. Thick sections of fluorescently stained embryos were made as previously described (Narasimha and Brown, 2006).

Fluorescent images were acquired on a BioRad Radiance 2000 confocal microscope. Where projections are presented, sections were scanned at 0.2-0.5 micron steps. Note that, in Fig. 3A,B the GFP channel from a projection is superimposed on one optical section of the protein staining.

Target-site analysis, electrophoretic mobility-shift assays and luciferase assays

DNA sequence spanning the fas3, coracle and sinu genes was searched for a match to a weighted matrix derived from known Grh sites (Almeida and Bray, 2005) using Target-Explorer (Sosinsky et al., 2003). The UCSC vista browser http://pipeline.lbl.gov/cgi-bin/gateway2?bg=dm1 was used to determine conservation between D. melanogaster and other Drosophila species (Couronne et al., 2003). Positions of sites with respect to starting ATG in D. melanogaster are: fas3A, 31,097 bp downstream; fas3B, 34,852 bp downstream; coraA, 1169 bp upstream; coraB, 4402 bp upstream.

Electrophoretic mobility-shift assays (EMSAs) were carried out as described previously (Uv et al., 1994). Reactions contained 0.5 μl of a 1:10 dilution of bacterial extract containing Gst-P/E fusion protein, 20 femtomoles of 32P-labelled gbe2 double-stranded oligonucleotide (5′-CTAGCGATTGAACCGGTCCTGCGGT-3′; underlined oligonucleotides correspond to putative Grh-binding sites) and 100 fM (femtomoles) or 1 pM (picomole) of the following cold competitors where indicated: fas3A, 5′-CTAGATCGCAACCGGTTTGGGT-3′; fas3B, 5′-CTAGAGGGAACCAGTTTTGCCT-3′; coraA, 5′-CTAGAGCAAACTGGTTCAGCT-3′; coraB, 5′-CTAGAAAAAACCGGTTGTT-3′; and N-box, 5′-GATCAGCCACGAGCCACAAGGATTG-3′.

For luciferase assays, fragments encompassing the Grh-binding sites were amplified from genomic DNA by PCR and subcloned into a pGL3-min luciferase reporter vector containing the minimal hsp70 promoter. Details available on request. Resulting plasmids were transfected into Drosophila S2 cells with a renilla control plasmid in the presence or absence of a plasmids expressing Grh (pMT-Gal4 + UAS-GrhN). Transfection conditions and luciferase assays (Promega) were carried out as described previously (Nagel et al., 2005).

We thank Greg Beitel, Manzoor Bhat, Peter Bryant, Rick Fehon and Hiroki Oda for antibodies, and members of our labs for discussions. This work was supported by grants from the Medical Research Council (S.J.B.), the Wellcome Trust (N.H.B.) the Swedish Research Council (A.U.) and TIFR (M.N.).

Almeida, M. S. and Bray, S. J. (
2005
). Regulation of post-embryonic neuroblasts by Drosophila Grainyhead.
Mech. Dev.
122
,
1282
-1293.
Attardi, L. D., Von Seggern, D. and Tjian, R. (
1993
). Ectopic expression of wild-type or a dominant-negative mutant of transcription factor NTF-1 disrupts normal Drosophila development.
Proc. Natl. Acad. Sci. USA
90
,
10563
-10567.
Baumgartner, S., Littleton, J. T., Broadie, K., Bhat, M. A., Harbecke, R., Lengyel, J. A., Chiquet-Ehrismann, R., Prokop, A. and Bellen, H. J. (
1996
). A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.
Cell
87
,
1059
-1068.
Behr, M., Riedel, D. and Schuh, R. (
2003
). The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila.
Dev. Cell
5
,
611
-620.
Bray, S. J. and Kafatos, F. C. (
1991
). Developmental function of Elf-1: an essential transcription factor during embryogenesis in Drosophila.
Genes Dev.
5
,
1672
-1683.
Chalmers, A. D., Lachani, K., Shin, Y., Sherwood, V., Cho, K. W. and Papalopulu, N. (
2006
). Grainyhead-like 3, a transcription factor identified in a microarray screen, promotes the specification of the superficial layer of the embryonic epidermis.
Mech. Dev.
123
,
702
-718.
Couronne, O., Poliakov, A., Bray, N., Ishkhanov, T., Ryaboy, D., Rubin, E., Pachter, L. and Dubchak, I. (
2003
). Strategies and tools for whole-genome alignments.
Genome Res.
13
,
73
-80.
Fehon, R. G., Dawson, I. A. and Artavanis-Tsakonas, S. (
1994
). A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene.
Development
120
,
545
-557.
Furuse, M. and Tsukita, S. (
2006
). Claudins in occluding junctions of humans and flies.
Trends Cell Biol.
16
,
181
-188.
Genova, J. L. and Fehon, R. G. (
2003
). Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function in Drosophila.
J. Cell Biol.
161
,
979
-989.
Gorfinkiel, N. and Martinez Arias, A. (
2007
). Requirements for adherens junction components in the interaction between epithelial tissues during dorsal closure in Drosophila.
J. Cell Sci.
120
,
3289
-3298
Hemphala, J., Uv, A., Cantera, R., Bray, S. and Samakovlis, C. (
2003
). Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling.
Development
130
,
249
-258.
Knust, E. and Bossinger, O. (
2002
). Composition and formation of intercellular junctions in epithelial cells.
Science
298
,
1955
-1959.
Lamb, R. S., Ward, R. E., Schweizer, L. and Fehon, R. G. (
1998
). Drosophila coracle, a member of the protein 4.1 superfamily, has essential structural functions in the septate junctions and developmental functions in embryonic and adult epithelial cells.
Mol. Biol. Cell
9
,
3505
-3519.
Mace, K. A., Pearson, J. C. and McGinnis, W. (
2005
). An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.
Science
308
,
381
-385.
Nagel, A. C., Krejci, A., Tenin, G., Bravo-Patino, A., Bray, S., Maier, D. and Preiss, A. (
2005
). Hairless-mediated repression of notch target genes requires the combined activity of Groucho and CtBP corepressors.
Mol. Cell. Biol.
25
,
10433
-10441.
Narasimha, M. and Brown, N. H. (
2004
). Novel functions for integrins in epithelial morphogenesis.
Curr. Biol.
14
,
381
-385.
Narasimha, M. and Brown, N. H. (
2006
). Confocal microscopy of Drosophila embryos. In
Cell Biology: A Laboratory Handbook
(ed. J. E. Celis), pp.
77
-86. San Diego: Academic Press.
Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M. (
1994
). A Drosophila homolog of Cadherin associated with Armadillo and essential for embryonic cell-cell adhesion.
Dev. Biol.
165
,
716
-726.
Ostrowski, S., Dierick, H. A. and Bejsovec, A. (
2002
). Genetic control of cuticle formation during embryonic development of Drosophila melanogaster.
Genetics
161
,
171
-182.
Schulte, J., Tepass, U. and Auld, V. J. (
2003
). Gliotactin, a novel marker of tricellular junctions, is necessary for septate junction development in Drosophila.
J. Cell Biol.
161
,
991
-1000.
Sosinsky, A., Bonin, C. P., Mann, R. S. and Honig, B. (
2003
). Target Explorer: an automated tool for the identification of new target genes for a specified set of transcription factors.
Nucleic Acids Res.
31
,
3589
-3592.
Stramer, B. and Martin, P. (
2005
). Cell biology: master regulators of sealing and healing.
Curr. Biol.
15
,
R425
-R427.
Tao, J., Kuliyev, E., Wang, X., Li, X., Wilanowski, T., Jane, S. M., Mead, P. E. and Cunningham, J. M. (
2005
). BMP4-dependent expression of Xenopus Grainyhead-like 1 is essential for epidermal differentiation.
Development
132
,
1021
-1034.
Tepass, U. and Hartenstein, V. (
1994
). The development of cellular junctions in the Drosophila embryo.
Dev. Biol.
161
,
563
-596.
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R. (
2001
). Epithelial cell polarity and cell junctions in Drosophila.
Annu. Rev. Genet.
35
,
747
-784.
Ting, S. B., Wilanowski, T., Auden, A., Hall, M., Voss, A. K., Thomas, T., Parekh, V., Cunningham, J. M. and Jane, S. M. (
2003
). Inositol- and folate-resistant neural tube defects in mice lacking the epithelial-specific factor Grhl-3.
Nat. Med.
9
,
1513
-1519.
Ting, S. B., Caddy, J., Hislop, N., Wilanowski, T., Auden, A., Zhao, L. L., Ellis, S., Kaur, P., Uchida, Y., Holleran, W. M. et al. (
2005
). A homolog of Drosophila grainy head is essential for epidermal integrity in mice.
Science
308
,
411
-413.
Tonning, A., Hemphala, J., Tang, E., Nannmark, U., Samakovlis, C. and Uv, A. (
2005
). A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea.
Dev. Cell
9
,
423
-430.
Uv, A. E., Thompson, C. R. and Bray, S. J. (
1994
). The Drosophila tissue-specific factor Grainyhead contains novel DNA-binding and dimerization domains which are conserved in the human protein CP2.
Mol. Cell. Biol.
14
,
4020
-4031.
Wang, S., Jayaram, S. A., Hemphala, J., Senti, K. A., Tsarouhas, V., Jin, H. and Samakovlis, C. (
2006
). Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea.
Curr. Biol.
16
,
180
-185.
Wu, V. M., Schulte, J., Hirschi, A., Tepass, U. and Beitel, G. J. (
2004
). Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control.
J. Cell Biol.
164
,
313
-323.
Yu, Z., Lin, K. K., Bhandari, A., Spencer, J. A., Xu, X., Wang, N., Lu, Z., Gill, G. N., Roop, D. R., Wertz, P. et al. (
2006
). The Grainyhead-like epithelial transactivator Get-1/Grhl3 regulates epidermal terminal differentiation and interacts functionally with LMO4.
Dev. Biol.
299
,
122
-136.