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 AS^c381>grh embryos. Expression of Fas3, Coracle and Sinu was strikingly upregulated in the amnioserosa of AS^c381>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 AS^c381>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.

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Fig. 2.

Grh promotes expression of SJ components in the amnioserosa. Stage-14 control (A,C,E-I,M,N) and AS^c381>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 AS^c381>grh embryos accumulates apically (green in O, white in O′; e.g. orange arrowheads). (N,P) Dlg is also upregulated in AS^c381>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 AS^c381>grh cells and the distribution of other adhesion complexes. In AS^c381>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 AS^c381>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 AS^c381>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.

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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 (grh^B32/grh^B32) embryos but not from SJ mutants (Atpα; SJ^–).

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Fig. 4.

Grh expression results in altered cellular morphology and disrupts the distribution of integrins and cadherin. Dorsal views of control (A,B) and AS^c381>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) AS^c381>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 grh^B32/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).