Complex Interactions between GSK3 and aPKC in Drosophila Embryonic Epithelial Morphogenesis

Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.


Introduction
Epithelial structures are generated when groups of cells respond to signals defining their fate and organization. One example occurs during the late stages of Drosophila epidermal development. Here signaling prods groups of cells to undergo dramatic morphological changes to form elongated, rectangular cells that secrete actin-based hairs called denticles. Other groups, following different signals, form irregular cells that do not generate denticles [1,2,3]. These cells, therefore, translate extracellular signals into morphogenetic changes allowing a close examination of how signaling may influence polarity [4,5].
Generally, cells in epithelia tend to pack together in roughly hexagonal structures [6]. In contrast to this simple array, morphogenesis in the late epidermis leads to a subset of cells taking on a rectangular, organized morphology. This organization, within the plane of the tissue, depends on the asymmetric distribution of adherens junctions, apical-basal polarity determinants and cytoskeletal components [4,5,7,8,9,10,11]. This tissue is patterned by a variety of signaling pathways including Wnt signaling [12,13]. The involvement, however, of apical-basal polarity proteins suggests a non-canonical Wnt signal, especially since aPKC has been shown to function in non-canonical Wnt signaling [5,14,15,16,17].
We investigated the function of aPKC in planar organization of the Drosophila embryonic epidermis. aPKC was enriched at the dorsal/ventral margins of epidermal cells. This distribution was regulated by the Wnt pathway component Glycogen Synthase Kinase 3 (GSK3 or Zw3). Through a genetic approach, we propose a role for GSK3 in linking signaling, to polarity and adhesion.

Results and Discussion
The apical polarity protein aPKC is asymmetrically distributed in epidermal cells During the final stages of embryonic development, epidermal cells undergo a morphogenetic change that transforms a disorganized epithelium into a structured, aligned, and organized epidermis. The rectangular cells go on to secrete actin-based denticles in a regular pattern (Fig. 1A, D). The great surprise, however, was the finding that this process requires the asymmetric distribution of apical-basal determinants and adherens junctions within the plane of the epithelium while maintaining perfect apical-basal polarity [4,5,9,10,11]. As the process begins, polarity determinants are asymmetrically distributed within the rectangular cells; the baso-lateral components are enriched on the anterior/ posterior (A/P) cell margins directly opposite to apical polarity determinants, which are enriched on the dorsal/ventral (D/V) cell margins ( Fig. 1 B and C). Adherens junctions co-localize with apical determinants at D/V cell margins (Fig. 1C) [5,7,8,10].
We focused on the apical kinase aPKC because it is a key regulator of polarity and is enriched at D/V margins (Fig. 1B) [18]. We investigated its role in morphogenesis by looking at aPKC mutant embryos, however we did not observe a strong phenotype in denticle formation or alignment. In aPKC mutants there was only a mild effect on denticles (null or amorphic aPKC mutant, Fig. 1F), and similarly overexpression of aPKC showed little effect ( Fig. 1G compare to Fig. 1E) [19,20]. This is likely due to an incomplete loss of aPKC protein in mutant embryos due to maternal RNA loading ( Fig. 1H shows aPKC protein present in aPKC null zygotic mutants even at late stages); however, we were unable to analyze maternal mutants at late stages as they disintegrate during early gastrulation processes [18]. In order to overcome the weakness of the phenotype, we turned to GSK3 mutants as loss of this kinase can enhance aPKC levels and activity [8].
GSK3 regulates the asymmetric distribution of aPKC aPKC regulates adhesion, and its protein levels are regulated by GSK3 phosphorylation and ubiquitin-mediated proteasomal degradation [8]. Based on this, we investigated whether GSK3's regulation of aPKC affects epidermal morphogenesis. Although various targets for GSK3 have been proposed [21], the clearest phenotype in loss-of-function GSK3 mutant embryos is the ectopic activation of canonical Wnt signaling causing all epithelial cells to switch to the naked cell fate [22]. These embryos lack pattern and obvious polarity, their cells do not secrete denticles, and all examined markers appear uniform (null or amorphic GSK3 maternal and zygotic mutant, Fig. 2 A-A'''). In order to assess the effects of GSK3 deletion in denticle-producing cells, we restored denticles by attenuating canonical Wnt signaling with a downstream mutation in armadillo (arm). Our genetic system utilized two arm mutations: arm F1a blocks most Wnt function, whereas arm XM19 blocks Wnt signals completely [23,24]. In the stronger arm XM19 mutant, patterning was abolished, all cells had a similar shape, secreted a denticle, and the planar distribution of all markers was uniform around cell margins (Fig. 2 B-B'''). In the weaker arm F1a mutant, some patterning was maintained, and cell shape changes To clarify whether markers were asymmetrically distributed, we quantified the fluorescent signals of Arm, aPKC and Dlg in mutant embryos and compared it to wild-type embryos (Fig. 3). These results suggested that although patterning is similar, cell shapes and asymmetry of markers appeared more disrupted in the arm F1a , GSK3 double mutants when compared to arm F1a alone.

Distinct roles of aPKC in apical-basal polarity and morphogenesis
Although the arm mutant phenotypes were not considerably altered by loss of GSK3, the mild phenotype of the arm F1a , GSK3 double mutant provided a sensitized background in which we could further assess the role of aPKC in morphogenesis. We introduced aPKC null, zygotic mutations into both arm F1a , GSK3 (hypomorphic arm and null GSK3 both maternal and zygotic mutants) and arm F1a mutants. arm F1a ; aPKC double mutants maintained some patterning, and while cell shapes were somewhat disrupted, cells appeared to align into rows ( Fig. 4A-B). However, in the arm F1a , GSK3; aPKC triple mutant, denticle and cell alignment were significantly disrupted (Fig. 4C). Specifically, proper cell shapes and cell alignment were lost, and instead of organization into even rows, cells curved and formed denticle swirls. Together, our results point to GSK3 and aPKC regulating cellular alignment in the epithelium.
aPKC expression in GSK3 mutants leads to embryos with severe apical-basal defects [8]. It was therefore not possible to assess denticle organization in these embryos. Denticle development did, however, proceed in embryos only missing the maternal dose of arm F1a and GSK3 Fig. 5A). In this mutant, we expressed aPKC DN , a gain-of-function form of aPKC where the N-terminal domain normally used for binding to Par-6 and restricting the localization and activity of aPKC is deleted [25]. These embryos displayed randomly oriented denticles (Fig. 5B, phenotype was observed in 16/115 (25% Expected)), instead of the relatively uniform pattern seen in embryos without the activated aPKC DN transgene (Fig. 5A). We examined these defects further by looking at the localization of phospho-Tyrosine (pTyr), a convenient marker of denticle precursors, and Arm to highlight cell-cell junctions ( Fig. 5D-F, pTyr is present in all cells, but forms puncta in denticle producing cells). Again, similar to the mutants discussed above, we found that cells secreting denticles were not rectangular or aligned in regular rows (Fig. 5F, schematic in Fig. 5C). This effect was specific to GSK3 mutants as neither expression of aPKC DN in otherwise wild-   type embryos, nor in arm F1a mutant embryos showed an effect on denticle organization (Fig. 1G and not shown). Therefore, by differentially altering the levels of aPKC we find that aPKC performs multiple functions in cell polarity. One caveat is that since in aPKC DN the Par-6 binding region is deleted, it is not restricted to the apical compartment through Par-6 binding, therefore the effect we observe may occur in another cellular compartment.

aPKC refractive to GSK3 phosphorylation has morphogenesis defects
To further investigate the interaction between aPKC and GSK3, we searched aPKC for GSK3 consensus phosphorylation sites and found two putative target residues. We constructed a transgene of aPKC (aPKC AA ) carrying point mutations at two predicted GSK3 consensus sites (S330A and T422A). In order to test if these residues are phosphorylated by GSK3, we expressed Drosophila aPKC and aPKC AA in HeLa cells. Following immunoprecipitation with a V5 affinity tag antibody, we conducted kinase assays with the two forms of the protein and recombinant GSK3. Though there is a low level of autophosphorylation with both proteins (as seen in lanes 2 and 4 where there is no GSK3 added), the wild-type version of aPKC is phosphorylated by GSK3, whereas aPKC AA is not (compare lane 3 to lane 5, Fig. 6A). Western blotting showed that comparable amounts of aPKC AA and aPKC WT protein were present in the HeLa cell extracts (Fig. 6B).
Since the aPKC AA protein is refractive to GSK3 phosphorylation, we hypothesized that expression of aPKC AA should be similar to that of endogenous aPKC protein in the absence of GSK3 kinase activity. Wild-type aPKC appears in a striped pattern due to GSK3-mediated degradation [8], but expressed aPKC AA protein does not form stripes (Fig. 6I). To test if this expression pattern is an artifact of overexpression, we also expressed wild-type aPKC under identical conditions, and observed striped expression of aPKC (Fig. 6L). Co-staining with E-cadherin reveals aPKC upregulation in the rectangular cells (Fig. 6M-N). The lack of striped aPKC AA suggests it is refractive to down-regulation by GSK3 [8] Taken together, these data point to aPKC being a target of GSK3.
In order to test if these aPKC residues are required for polarity, we expressed aPKC AA in wild-type embryos. aPKC AA did not cause any apparent apical-basal phenotype, but in contrast to aPKC DN (Fig. 6C), aPKC AA did cause denticle alignment defects (Fig. 6D). We next expressed aPKC AA in embryos lacking aPKC function. Drosophila embryos with a loss-of-function allele of aPKC develop normally through early stages of embryogenesis, though many die before hatching [19,20]. As shown in Fig. 6E, denticle organization defects due to loss of aPKC are mild, and are rescued by expression of both wild-type aPKC (Fig. 6F) and aPKC DD (Fig. 6G). However, expression of aPKC AA leads to some denticle orientation defects (Fig. 6H), although the severity is lower than that observed for GSK3 mutants (Fig. 5B). This is likely due to the mismatched nature of our genetic backgrounds with maternal and zygotic mutants used, but taken together the results suggest a role for aPKC phosphorylation in epithelial morphogenesis.

A genetic model
Previous studies have suggested roles for apical-basal polarity components in several planar polarity processes and stem cell divisions [26,27,28,29,30]. Our findings define roles for polarity determinants in planar polarity and epidermal cell morphogenesis. Apical polarity proteins like aPKC are polarized and establish domains on the D/V cell margins leading to an upregulation of adherens junctions, and exclusion of the basal-lateral determinants [4,5]. This is consistent with the current model of apical-basal polarity, which posits that apical components lead to the localization of junctions and compete with the basal-lateral components to establish independent domains within a cell [31,32,33]. We show that such domains are established within the plane of the epithelium across many cells, thus raising the possibility that this co-opted apical-basal polarity mechanism regulates morphogenesis in this tissue. For the exclusion model to work properly the levels of apical and basal determinants must be tightly regulated. Therefore, GSK3 plays a crucial role in maintaining the levels of aPKC, although the symmetry-breaking event remains unclear. Future experiments will have to explain how polarity is established; nevertheless, the interaction between polarity, adhesion and the Wnt pathway may have implications for how cancer cells escape tissues during metastasis or are maintained asymmetrically as stem cells [34].
The major caveat of these experiments pertains to our attempt to address these issues in vivo. To accomplish this, we must use complex genetic approaches. This is not a problem for genes with only one function, however here we have concentrated on GSK3a gene with myriad functions. For example, one recent study identified 42 direct phosphorylation targets [21]. In order to study GSK3's interaction with aPKC, we blocked GSK3's best-studied function in canonical Wnt signaling, but this leaves many others. Despite this caveat, our findings are most simply explained through a role for GSK3in regulating epithelial morphogenesis through its interaction with aPKC. We cannot, however, exclude other explanations as we have not examined the roles of other GSK3 targets.

Crosses and expression of UAS constructs
Maternally mutant eggs were generated by the dominant female sterile technique [35]. Oregon R was used as the wild-type strain. Please see Flybase for details on mutants used (flybase.bio.indiana. edu). aPKC k06403 is a P-element insertion that behaves like a null mutation [19,20]. Other mutants used were the amorphic zw3 M11-1 , the partial loss-of-function arm F1a (mutation leads to Arginine 394 being mutated to a Histidine) that reduces Arm's affinity toward TCF, and the strong hypomorph arm XM19 that contains a stop codon at the end of repeat twelve (stop codon introduced after amino acid 680), deleting the entire C-terminal region preventing Wg signaling [24]. For expression experiments, the armadillo and daughterless-GAL4 drivers were used.
New aPKC constructs: we mutated two consensus GSK3 (Ser330 and Thr422 according to the Drosophila aPKC coding sequence) sites in aPKC to alanines using double strand mutagenesis (QuickChange, Stratagene), and cloned this otherwise full-length aPKC construct into pUASt vector using Gateway technology (Invitrogen). The UAS-aPKC DN consisting of amino acids 180-606 [25] was used for genetic crosses.
All X-chromosome mutants use FRT 101. The following crosses were conducted:

Western Blotting
Embryos were lysed in extract buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10% Glycerol, Complete Mini Protease, Sigma). The extracts were separated by 7.5% SDS-PAGE, and blotted as described in Peifer et al. (1994). Extracts were normalized using the BCA assay (Novagen). Overnight embryo collections were used to make extracts for Western blots. Kinase assays were performed on both recombinant proteins and proteins immunoprecipitated from embryonic extracts. Recombinant proteins were either purchased from Cell Signaling Technologies (GSK3b and PKCf) or prepared from bacterial lysates [40]. aPKC phosphorylation was assayed as described in [27].
HeLa cell transfections were performed by standard methods. aPKC full length and aPKC AA were recombined into pDEST40 a vector with 6XHis and V5 tags as C-terminal fusions (Invitrogen). Proteins were immunoprecipitated with the V5 antibody (Invitrogen), and subjected to kinase assays as above.

Fluorescence Quantification
The intensity of fluorescent staining was measured similar to the procedure used in Harris and Peifer, 2007 [20]. Mean intensity was calculated using Image J software (NIH) for lines that were 1.5-3.0 microns in length at the dorsal-ventral and anteriorposterior edges of cells in denticle-producing rows 2-5. In addition, mean intensity was measured within the cytoplasm of each cell. This background measurement was subtracted from the measurements at the dorsal-ventral and anterior-posterior edges and the ratio between these corrected measurements (dorsalventral over anterior-posterior) was calculated. If there was no difference in intensity between edges, we expect a ratio of 1. The ratios were graphed on a logarithmic scale to allow for positive and negative values in the presentation. For measurements 3 to 5 embryos and 10 to 40 cells per embryo were used to obtain intensities.