An alternative mode of epithelial polarity in the Drosophila midgut

Apical–basal polarity is essential for the formation and function of epithelial tissues, whereas loss of polarity is a hallmark of tumours. Studies in Drosophila have identified conserved polarity factors that define the apical (Crumbs, Stardust, Par-6, atypical protein kinase C [aPKC]), junctional (Bazooka [Baz]/Par-3), and basolateral (Scribbled [Scrib], Discs large [Dlg], Lethal [2] giant larvae [Lgl]) domains of epithelial cells. Because these conserved factors mark equivalent domains in diverse types of vertebrate and invertebrate epithelia, it is generally assumed that this system underlies polarity in all epithelia. Here, we show that this is not the case, as none of these canonical factors are required for the polarisation of the endodermal epithelium of the Drosophila adult midgut. Furthermore, like vertebrate epithelia but not other Drosophila epithelia, the midgut epithelium forms occluding junctions above adherens junctions (AJs) and requires the integrin adhesion complex for polarity. Thus, Drosophila contains two types of epithelia that polarise by fundamentally different mechanisms. This diversity of epithelial types may reflect their different developmental origins, junctional arrangement, or whether they polarise in an apical–basal direction or vice versa. Since knock-outs of canonical polarity factors in vertebrates often have little or no effect on epithelial polarity and the Drosophila midgut shares several common features with vertebrate epithelia, this diversity of polarity mechanisms is likely to be conserved in other animals.


Introduction
Most animal organs and tissues are composed of epithelial cells that adhere laterally to each other to form sheets that act as barriers between compartments. The formation of epithelial monolayers depends on the coordinated polarisation of each cell along its apical-basal axis, and this polarity underlies all aspects of epithelial biology [1,2]. For example, the function of epithelia as barriers to fluids and pathogens depends on the correct positioning of the occluding cell-cell junctions (septate junctions [SJs] in invertebrates and tight junctions in vertebrates), whereas the adhesion between cells depends on the lateral localisation of cadherindependent adherens junctions (AJs).
Much of our understanding of how epithelial cells polarise comes from studies of Drosophila melanogaster that have identified a conserved set of epithelial polarity factors that define different cortical domains along the apical-basal axis of the cell. The apical domain is specified by the transmembrane protein Crumbs, the adaptor protein Stardust, and the Par-6/atypical protein kinase C (aPKC) complex; the boundary between the apical and lateral domains is defined by Bazooka (Baz, Par-3 in other organisms), which positions the apicalmost lateral junction; and the rest of the lateral domain is marked by Scribbled (Scrib), Discs large (Dlg), and Lethal (2) giant larvae (Lgl) [3]. Null mutations in any of these factors disrupt epithelial polarity in the primary epithelium that forms from the cellular blastoderm of the Drosophila embryo and gives rise to most of the structures of the larva and adult [4][5][6][7][8][9][10][11]. Similarly, loss of any of these genes disrupts the secondary epithelium formed by the follicle cells that surround the developing oocyte [12][13][14]. In each tissue, Baz seems to play a pivotal role in positioning the apical AJs and in localising the apical factors, which then exclude Baz from the apical domain [15][16][17][18][19]. The identity of the apical and lateral domains is then maintained by mutual antagonism between apical and lateral factors [20,21]. The requirement for some of these factors becomes less stringent in polarised epithelia as they mature. For example, Crumbs is particularly important in epithelial tissues that are remodelling their cell junctions as they undergo morphogenetic rearrangements, and Scrib, Dlg, and Lgl are not required to maintain polarity in mid-embryogenesis, as the Yurt group of lateral proteins takes over the antagonism of the apical factors, although Scrib and Dlg are still required for the formation of the SJs [20][21][22][23][24].
Epithelial cells are thought to have evolved at the origin of multicellularity, as cells first started to adhere to each other to form sheets, suggesting that apical-basal polarity is an ancient invention that is controlled by a conserved mechanism [25]. In support of this view, all of the canonical epithelial factors in Drosophila are conserved in vertebrates and localise to the equivalent cortical domains [26][27][28][29][30][31][32][33]. Knock-downs or knock-outs of some of these factors impair polarity in certain contexts, such as in Xenopus embryonic blastomeres and in some cultured cell lines [34][35][36][37]. In most cases, however, knock-outs of canonical polarity factors have little or no effect on polarity or cause unrelated phenotypes. For example, mice homozygous for a null mutation in PAR-3 die in mid-gestation from a heart defect caused by a failure of the epicardial cell migration, but other embryonic epithelia appear to form normally [38].
Similarly, knock-out of PAR-3 in mouse mammary stem cells disrupts the morphogenesis of the mammary gland but not the ability of the cells to polarise [39,40]. Finally, Scrib functions as a planar cell polarity gene in mice but has no obvious effect on apical-basal polarity [41]. Although this lack of polarity phenotypes in mammals could be a result of redundancy between paralogues, it raises the possibility that at least some vertebrate epithelia polarise by different mechanisms from the model Drosophila epithelia. In support of this view, many mammalian epithelia require integrin adhesion to the basement membrane to orient their polarity, whereas the well-characterised Drosophila epithelia do not [42][43][44]. Furthermore, vertebrate epithelial cells have an inverted arrangement of junctions compared to insect epithelia: the apical junction in vertebrates is the occluding, tight junction, which forms at the apical/lateral boundary above a lateral zonula adherens (ZA), whereas insect cells form lateral SJs, below an apical ZA [45][46][47].
The possibility that the polarisation of some epithelia is independent of the canonical polarity system prompted us to ask if all Drosophila epithelia polarise in the same way. We therefore examined polarity in the Drosophila adult midgut epithelium, which is mainly absorptive rather than secretory and is endodermal in origin, unlike the well-characterised epithelia, which are secretory and arise from the ectoderm or mesoderm [48]. The adult midgut is a homeostatic tissue in which basal intestinal stem cells (ISCs in Fig 1F) divide to produce new cells that integrate into the epithelium to replace dying enterocytes (ECs), which are shed into the gut lumen. This has the advantage that one can generate homozygous mutant stem cell clones in heterozygotes at the late pupal or adult stage to produce clonal patches of mutant ECs in the adult midgut without disrupting the development of the tissue. Our results reveal that the polarisation of the midgut epithelium does not require any of the canonical polarity factors and has several features in common with vertebrate epithelia, making it a useful model for investigating alternative polarity pathways.

Results
The midgut is a typical epithelium with an apical brush border marked by F-actin ( Fig 1A) and phospho-Moesin (pMoe) (Fig 1B) as well as Myosin IA ( Fig 1E) and an apical domain marked by Myosin 7a (Fig 1C). However, our analysis led us to rediscover an interesting property of this epithelium: the smooth SJs, which form the occluding barrier to paracellular diffusion, form at the apical side of the lateral domain, above AJs, which are diffusely distributed over the lateral domain [49-52] (Fig 1D and 1F). This is the opposite way around compared to other Drosophila epithelia, in which the AJs condense to form a ZA around the apical margin of the cell, with the SJs, if present, positioned more basally in the lateral membrane. The organisation of junctions in the midgut therefore resembles the junctional arrangement in mammals, in which the occluding tight junctions form above the AJ.
In secretory epithelia, the Crumbs/Stardust complex defines the apical and marginal region and anchors the Par-6/aPKC complex in this domain [5,9,53]. Apical Crb and aPKC then exclude Baz/Par-3 to define the apical/lateral boundary by positioning the apical AJs [17][18][19]. This raises the question of whether these factors mark the same positions or the same structures when the junctions are reversed in the midgut.
Crumbs is not detectable in the adult midgut epithelium, as has previously been observed in the embryo [4] (Fig 2A). We used the mosaic analysis with a repressible cell marker (MARCM) technique [54] to generate positively marked clones in the adult midgut epithelium for null mutations in crumbs or stardust (crb 8F-105 , crb 11A22 ; sdt k85 ). The adults were then dissected 8 to 10 days after clone induction to allow the stem cells, which divide about once a day, to go through multiple divisions because this rules out any perdurance of wild-type proteins  tubP-GAL80 ts . Crb (green) localises to the BL but does not perturb SJ formation as revealed by Tsp2a (red). (D) Adult midgut epithelium expressing Baz-GFP under endogenous control (protein trap line), stained with anti-GFP (green) and anti-Baz (white). Baz/Baz-GFP is not detectable in ECs but is expressed in ISCs, in which it localises apically (white arrowhead). Cora (red) labels the SJ. (E) ECs homozygous mutant for baz 4 (marked by GFP, green). The apical brush border forms normally as revealed by staining for F-actin with phalloidin (red). (F) Ectopic expression of UAS-Baz-GFP in adult midgut ECs using MyoIA-GAL4; tubP-GAL80 ts has no effect on the formation of the apical SJ marked by Dlg (red). Baz-GFP localises to the apical domain and AJs (green expressed before the clones were induced. However, clones of crb and sdt null mutations give no obvious phenotypes (Fig 2B, S1A, S1D and S1E Fig). Overexpression of Crumbs expands the apical domain in other Drosophila epithelia [55]. By contrast, ectopic expression of Crumbs in the adult midgut epithelium does not affect EC polarity and the Crumbs protein does not localise apically, concentrating instead in the basal labyrinth (BL), an extensive set of tubular membrane invaginations from the EC basal surface (Fig 2C). Baz/Par-3 is also not detectable in ECs, although it is expressed in the ISCs, in which it localises apically as previously reported [56] (Fig 2D). MARCM clones homozygous for baz null alleles (baz 815-8 ; baz 4 ) occur at a similar frequency to wild-type clones and contain a normal number of cells, arguing against a role for Baz in asymmetric stem cell division, consistent with the view that these divisions are random and largely symmetric [57]. More importantly, baz null mutant ECs integrate into the epithelium and develop normal apical-basal polarity, in contrast to other epithelia in Drosophila (Fig 2E, S1B, S1C and S1F Fig). Baz-green fluorescent protein (GFP) localises apically and to the AJs when ectopically expressed in the midgut epithelium, consistent with binding to the Par-6/aPKC complex and to E-cadherin complexes ( Fig 2F). However, ectopic Baz expression has no effect on the polarity of the ECs or on the formation of an apical SJ.
Both aPKC and Par-6 are expressed in the midgut and localise apically, as they do in all other epithelia (Fig 3A and 3B). In most polarised cells, the apical localisation of the Par-6/ aPKC complex depends on Baz/Par-3, and in epithelia, this also requires Crumbs and Stardust [2]. Consistent with our observation that these proteins are absent from ECs, neither Baz nor Crb are required for the localisation of Par-6, indicating that the latter must be targeted apically by a distinct mechanism (S1A and S1B Fig). Surprisingly, the apical domain forms normally in par-6 Δ226 and aPKC k06043 mutant clones, and the morphology of the cells is unaffected (Fig 3C, S1H and S1J Fig). Although aPKC k06043 is considered a null allele, the corresponding P element insertion does not disrupt the shorter isoforms of aPKC. Thus, it is conceivable that some aPKC activity remains in aPKC k06043 homozygous mutant cells. We therefore used CRISPR to generate a complete null, aPKC HC , a frameshift mutation resulting in a premature stop codon and a truncation of aPKC at amino acid 409, which is located in the middle of the kinase domain (S1L Fig). Homozygous aPKC HC clones also show no phenotype, forming normal actin-rich brush borders and SJs, confirming that aPKC is dispensable for EC polarity (Fig 3D and 3E). Nevertheless, Par-6 is lost from the apical domain in aPKC k06043 clones and aPKC is not apical in par-6 Δ226 clones, showing that their localisations are interdependent ( Fig 3F and 3G). Thus, the Par-6/aPKC complex still marks the apical domain of the midgut epithelium but is not required for its formation or maintenance.
The . We therefore also examined the role of Yurt in the midgut. Yurt localises to the SJs, as it does in the embryo, but yurt mutant clones still polarise normally and form SJs that recruit Lgl (Fig 5). Finally, we examined Par-1, which localises laterally in other epithelia in which it plays a role in localising AJs through phosphorylation of Baz and in organising the  microtubule cytoskeleton [60,61]. Par-1 is expressed in the ISCs, in which it localises laterally but is not detectable in the ECs. Consistent with its lack of expression, par-1 9 mutant ECs show normal apical-basal polarity (S3A and S3B Fig). Thus, all the canonical epithelial polarity factors are dispensable for the polarisation of the midgut epithelium, even though Par-6, aPKC, Scrib, Dlg, Lgl, and Yurt are expressed and localise to equivalent positions to secretory epithelia.
The relationships between the lateral factors has been difficult to assess in other epithelia because mutants in scrib, dlg, and lgl give rise to round, unpolarised cells without an identifiable lateral domain [11]. We took advantage of the normal EC polarisation in these mutants to investigate the interdependence of their recruitment to the SJs. Neither Dlg nor Lgl are recruited to SJs in cells depleted of Scrib by RNAi (Fig 4B and S2D Fig). Scrib localises normally in dlg mutant clones, whereas both Scrib and Dlg localise normally to the SJs in lgl mutant clones (Fig 4C-4F). Thus, there is a simple hierarchical relationship between these factors in the midgut epithelium, in which Scrib is required to recruit Dlg, which is needed for Lgl localisation.
The surprising observation that none of the classical epithelial polarity factors are required for the apical-basal polarisation of the midgut epithelium raises the question of how polarity is generated and maintained. Given the similar junctional arrangement to mammalian epithelia, we addressed whether polarity in midgut ECs depends on integrin-dependent adhesion to the extracellular matrix, as it does in several mammalian epithelia [42][43][44]. Components of the integrin adhesion complex, such as the α-integrin Mew and the essential cytoplasmic adaptor proteins Talin (Drosophila Rhea) [63] and Kindlin (Drosophila Fit 1 [64]; Fit 2 is not detectable expressed in the midgut) are highly localised to the basal side of the midgut epithelium ( Fig  6A). The expression of two α-integrins and two β-integrins in the midgut complicates the genetic analysis of their function, so we focused on the cytoplasmic components of the integrin adhesion complex. Clones of cells homozygous for null alleles of rhea (rhea 79a and rhea B128 ) detach from the basement membrane and fail to polarise, forming irregularly shaped cells that do not form SJs or an apical domain ( Fig 6B). Most rhea mutant cells remain within the epithelial layer, below the SJs of the wild-type cells, probably because they do not form SJs themselves ( Fig 6B and S4A Fig). Despite their inability to polarise and integrate into the epithelium, the mutant cells still appear to differentiate: they become polyploid, the size of their nuclei is not significantly different from that of wild-type cells (rhea nuclear long axis: 6.0 μm ± 1.0 μm versus heterozygous cells: 6.9 μm ± 0.9 μm), and they express the marker for differentiating ECs, Pdm1 [65] (Fig 6B). Cells mutant for both Fit1 and Fit2 show a similar phenotype to Talin mutants. Mutant cells have normal nuclear dimensions (Fit1 Fit2 nuclear long axis: 6.6 μm ± 1.2 μm versus heterozygous cells: 6.9 μm ± 1.0 μm) and express Pdm1 but fail to localise apical markers or form SJs (Fig 6C and S4B Fig).
The ISCs lie beneath the epithelium and differentiating ECs must therefore integrate into the epithelium from the basal side, inserting between the SJs of the flanking ECs while maintaining an intact barrier. We therefore examined the effects of mutations in the core SJ components Tsp2a and Mesh [51,52]. More than 90% of mutant cells fail to integrate through the SJs of the neighbouring wild-type cells, and the clones form clusters on the basal side of the epithelium (Fig 7A, S3A and S4C Figs). Nevertheless, the mutant cells still appear to differentiate normally, as shown by their nuclear size (Tsp2a nuclear long axis: 6.0 μm ± 0.9 μm versus heterozygous cells: 6.6 μm ± 0.8 μm) and Pdm1 expression (S4D Fig). Wild-type cells start to  form an apical domain as they integrate, before they have a free apical surface, as shown by the enrichment of apical components such as Myo7a (arrowhead in Fig 7B). By contrast, apical markers only weakly localise in cells mutant for SJ components and never form a clear apical domain, even if the cells are extruded from the apical side of the epithelium (Fig 7B and 7C). Thus, the midgut epithelium appears to polarise in a basal to apical manner, in which adhesion to the ECM is required for the formation of the SJs and the SJs are needed for the formation of the apical domain (Fig 7D). An alternative mode of epithelial polarity in the Drosophila midgut

Discussion
Our results reveal that the intestinal epithelium polarises by a fundamentally different mechanism from other Drosophila epithelia, as none of the classical epithelial polarity genes are required for its apical-basal polarisation. This cannot be attributed to redundancy between paralogues, as might be the case in vertebrates, because all of the polarity factors are singlecopy genes in Drosophila. Thus, our observations provide strong evidence against the idea that there is a universal system for polarising epithelial cells. Nevertheless, a core set of the polarity factors (Par-6, aPKC, Scrib, Dlg, and Lgl) is expressed in the midgut epithelium, and these proteins localise to the equivalent positions to other Drosophila epithelia. Thus, they may still serve important functions in the midgut epithelium that are not essential for the overall apical-basal polarisation of the cells.
Given that all other epithelia in Drosophila use the canonical polarity pathway, our observations raise the question of why the midgut epithelium is different. This is unlikely to reflect the fact that the midgut is absorptive rather than secretory, as secretory cells in the midgut, such as the enteroendocrine (ee) cells and the acid-secreting copper cells, polarise in the same way as the ECs (S1J and S1K Fig). One key difference between the midgut epithelium and other epithelia is that it is the only epithelial tissue of endodermal origin in the fly, whereas all other epithelia are ectodermal or mesodermal. Thus, it is possible that endodermal epithelia are intrinsically different in the way that they polarise. In support of this view, it has recently been found that PAR-3, PAR-6, and aPKC are degraded in the invaginating endomesoderm of the Cnidarian Nematostella vectensis and are not required for this tissue to form an epithelium [67]. Thus, the difference between endodermal and ectodermal polarity systems may have evolved before the origin of Bilateria. The Drosophila midgut arises from the cellular blastoderm of the embryo and initially polarises in the same way as other embryonic epithelia before it undergoes an epithelial-to-mesenchymal transition (EMT) under the control of the endodermal GATA family transcription factor, Serpent [68]. Serpent drives EMT at least in part by inhibiting the transcription of crumbs and stardust and might therefore contribute to the switch in polarity mechanisms. However, Serpent is turned off as the midgut primordium migrates and is not expressed when the cells reform an epithelium to generate the midgut tube. Indeed, continued expression of Serpent blocks the cells from re-epithelising after migration. Thus, a pulse of Serpent expression may trigger the switch in polarity mechanisms, perhaps through downstream transcription factors, but Serpent itself prevents epithelial polarisation.
A second important difference between the adult midgut epithelium and other epithelia in Drosophila is its reversed arrangement of occluding junctions and AJs, with apical SJs forming above lateral AJs. In other Drosophila epithelia, Baz plays a key role in concentrating the AJs at the apical margin of the lateral membrane to form the ZA [69]. Thus, the absence of Baz in midgut ECs may contribute to the absence of an apical ZA. This cannot be the only factor making the midgut different, however, as ectopic expression of Baz in midgut ECs does not alter the position of the SJs. One reason why the SJs may have evolved to form above the AJs in the midgut is that this places the barrier to paracellular diffusion apically, thereby preventing the contents of the gut lumen from accessing the lateral sides of the cell, which is presumably important because the gut is full of digestive enzymes and potential pathogens. Other Drosophila epithelia that face the external environment-such as the epidermis, trachea, foregut, and hindgut-secrete an impermeable cuticle, which provides a protective covering to prevent pathogens from accessing the cell surface [70]. The development of the typical arthropod cuticular exoskeleton may therefore have freed these epithelia from the need to place their occluding junctions apically, allowing the apical positioning of the ZA.
The third important difference between the midgut epithelium and other epithelia is that the ECs polarise in a basal-to-apical direction as they integrate into the epithelium, whereas all other Drosophila epithelia polarise in an apical-to-basal direction. For example, the primary embryonic epithelium forms during cellularisation, as the furrow canals grow in from the apical surface of the embryo between the nuclei, while the follicle cells polarise in response to an apical cue from the germ line [14,71]. Thus, it is possible that the mechanism by which cells polarise depends on the order in which they generate the basal, lateral, and apical domains. Since the midgut is the only endodermal epithelium in Drosophila, the only epithelium with apical SJs, and the only epithelium that polarises from basal to apical, it is not possible to determine which of these characteristics underlies its alternative mechanism of cell polarisation, and this will require analysis in other organisms with a greater diversity of epithelial cell types.
Whatever the reason for the alternative polarity mechanism in the Drosophila midgut epithelium, its polarity is much more similar to that of well-characterised vertebrate epithelia than other Drosophila epithelia. Firstly, the midgut and vertebrate epithelia have apical occluding junctions above lateral AJs, whereas other Drosophila epithelia have a reversed arrangement of junctions. Secondly, the midgut epithelium does not require the canonical epithelial polarity factors that are essential in other Drosophila epithelia, and this also seems to be the case in some vertebrate epithelia, although this may be due to redundancy between paralogues. Thirdly, the midgut epithelium and a number of vertebrate epithelia depend on signals from the integrin adhesion complex to polarise correctly [42][43][44]. These similarities suggest that the Drosophila midgut epithelium may prove a better in vivo model for at least some types of vertebrate epithelia. It will therefore be important to determine whether vertebrates also contain distinct types of epithelia whose polarity is controlled by different factors.

Stock maintenance
Standard procedures were used for Drosophila husbandry and experiments. Flies were reared on standard fly food supplemented with live yeast at 25˚C. For the conditional expression of UAS responder constructs (e.g., RNAi) in adult flies, parental flies were crossed at 18˚C and the resulting offspring reared at the same temperature until eclosion. Adult offspring were collected for 3 days and then transferred to 29˚C to inactivate the temperature-sensitive GAL80 protein.
To generate MARCM or GFP-negative clones, flies were crossed at 25˚C and the resulting offspring subjected to heat shocks either as larvae (from L2 until eclosion) or as adults (5-9 days after eclosion). Heat shocks were performed at 37˚C for 1 h (twice daily). Flies were transferred to fresh food vials every 2-3 days and kept at 25˚C for at least 9 days after the last heat shock to ensure that all wild-type gene products from the heterozygous progenitor cells had turned over. For this study, all (midgut) samples were obtained from adult female flies.

Formaldehyde fixation
Samples were dissected in PBS and fixed with 8% formaldehyde (in PBS containing 0.1% Triton X-100) for 10 min at room temperature. Following several washes with PBS supplemented with 0.1% Triton X-100 (washing buffer), samples were incubated in PBS containing 3% normal goat serum (NGS, Stratech Scientific Ltd, Cat. #005-000-121; concentration of stock solution: 10 mg/ml) and 0.1% Triton X-100 (blocking buffer) for 30 min. This fixation method was only used for samples in which F-actin was stained with fluorescently labelled phalloidin, as phalloidin staining is incompatible with heat fixation.

Heat fixation
The heat fixation protocol is based on a heat-methanol fixation method used for Drosophila embryos [82]. Samples were dissected in PBS, transferred to a wire mesh basket, and fixed in hot 1X TSS buffer (0.03% Triton X-100, 4 g/L NaCl; 95˚C) for 3 s before being transferred to ice-cold 1X TSS buffer and chilled for at least 1 min. Subsequently, samples were transferred to washing buffer and processed for immunofluorescence stainings.

Immunofluorescence staining
After blocking, samples were incubated with the appropriate primary antibody/antibodies diluted in blocking buffer at 4˚C overnight. Following several washes, samples were incubated with the appropriate secondary antibody/antibodies either at room temperature for 2 h or at 4˚C overnight. Samples were then washed several times in washing buffer and mounted in Vectashield containing DAPI (Vector Laboratories) on borosilicate glass slides (No. 1.5, VWR International). All antibodies used in this study were tested for specificity using clonal analysis (MARCM) or RNAi.

Generation of endogenous EGFP-aPKC
Endogenously tagged aPKC with EGFP fused to the N-terminus was generated by CRISPRmediated homologous recombination. In vitro synthesised gRNA [91] to a CRISPR target approximately 60 nucleotides downstream from the aPKC start codon (target sequence GAA TAGCGCCAGTATGAACATGG) and a plasmid donor containing the ORF of EGFP as well as appropriate homology arms (1.5 kb upstream and downstream) were coinjected into nos-Cas9-expressing embryos (Bloomington #54591; also known as CFD2) [92]. Single F0 flies were mated to y w flies and allowed to produce larvae before the parent was retrieved for PCR analysis. Progeny from F0 flies in which a recombination event occurred (as indicated by PCR) were further crossed and analysed to confirm integration. Several independent EGFP-aPKC lines were isolated. Recombinants carry the EGFP coding sequence inserted immediately downstream of the endogenous start codon and a short linker (amino acid sequence: Gly-Ser-Gly-Ser) between the coding sequence for EGFP and the coding sequence for aPKC. Homozygous flies are viable and healthy.

Generation of aPKC[HC]
We used the CRISPR/Cas9 method [91] to generate a null allele of aPKC. sgRNA was in vitro transcribed from a DNA template created by PCR from two partially complementary primers: forward primer: 5 0 -GAAATTAATACGACTCACTATAggattacggcatgtgtaaggGTTTTAGAGC TAGAAATAGC-3 0 ; reverse primer: 5 0 -AAAAGCACCGACTCGGTGCCACTTTTTCAAG TTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3 0 . The sgRNA was injected into Act5c-Cas9 embryos [92]. Putative aPKC mutants in the progeny of the injected embryos were recovered, balanced, and sequenced. The aPKC HC allele contains a small deletion around the CRISPR site, resulting in one missense mutation and a frameshift that leads to stop codon at amino acid 409 in the middle of the kinase domain, which is shared by all isoforms (S1L Fig). The aPKC HC allele was subsequently recombined onto FRTG13 to generate MARCM clones. No aPKC protein was detectable by antibody staining in both midgut and follicle cell clones, and follicle cells homozygous for aPKC HC display a phenotype that is indistinguishable from that observed in follicle cells homozygous for the aPKC K06403 allele.

Quantification and statistical analysis
The proportions of rhea, Fit, and Tsp2a mutant cells inside the epithelial layer were calculated as follows: images were taken of different regions of several midguts containing MARCM clones stained with an apical marker. Cells that were above the neighbouring cells or had a clear apical domain were counted as 'cells NOT inside the layer', whereas cells without a detectable free apical surface were counted as 'cells inside the layer'. Data were analysed with Graphpad Prism software. The graph in S4A Fig shows the average percent of cells inside the layer ± SD%.
The nuclear long axis of rhea, Fit, and Tsp2a mutant clones was manually measured in 20 random mutant cells (stained with DAPI, excluding obvious ISCs) and 20 surrounding heterozygous cells using Fiji.