The FERM Protein Yurt Is a Negative Regulatory Component of the Crumbs Complex that Controls Epithelial Polarity and Apical Membrane Size

Developmental Cell 11, 363–374, September, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.devcel.2006.06.001

The FERM Protein Yurt Is a Negative Regulatory Component of the Crumbs Complex that Controls Epithelial Polarity and Apical Membrane Size Patrick Laprise,1,4 Slobodan Beronja,1,4 Nancy F. Silva-Gagliardi,2 Milena Pellikka,1 Abbie M. Jensen,3 C. Jane McGlade,2 and Ulrich Tepass1,* 1 Department of Cell and Systems Biology University of Toronto Toronto, Ontario M5S 3G5 Canada 2 The Hospital for Sick Children Arthur and Sonia Labatt Brain Tumor Research Center and Department of Medical Biophysics University of Toronto Toronto, Ontario M5G 1X8 Canada 3 Department of Biology University of Massachusetts, Amherst Amherst, Massachusetts 01003

Summary The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Here, we show that binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. We also show that the mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. We propose that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation. Introduction The transmembrane protein Crumbs (Crb) is an essential component of a protein network that regulates epithelial polarization (for review, see Tepass et al. [2001]; Knust and Bossinger [2002], and Nelson [2003]). Drosophila Crb localizes to the apical membrane of epithelial cells where it acts as an apical determinant (Tepass et al., 1990; Wodarz et al., 1995). Drosophila embryos that lack Crb show several epithelial defects as they do not assemble a zonula adherens (ZA, the circumferential adherens junction of epithelial cells), lose epithelial tissue integrity, and show an increase in cell death (Tepass et al., 1990; Tepass and Knust, 1990; Tepass, 1996; Grawe et al., 1996). Crb also makes important contribu-

*Correspondence: [email protected] 4 These authors contributed equally to this work.

tions to the cellular morphogenesis of epithelial photoreceptor cells (PRCs) in the Drosophila compound eye. Crb controls ZA integrity during PRC morphogenesis, and later during PRC development, Crb acts as a positive regulator of apical membrane size. Finally, Crb is required for the survival of PRCs under light-stress condition (Pellikka et al., 2002; Izaddoost et al., 2002; Johnson et al., 2002). Crb overexpression leads to an expansion of the apical membrane (Wodarz et al., 1995; Pellikka et al., 2002), which raises the question of how Crb activity is normally attenuated so that the correct amount of apical membrane is generated and maintained. The human and mouse genomes encode three Crb orthologs (den Hollander et al., 1999, 2001; Pellikka et al., 2002; Makarova et al., 2003; van den Hurk et al., 2005). Mutations in human CRB1 were identified as a cause of severe inherited forms of retinal degeneration (retinitis pigmentosa and Leber congenital amaurosis) (den Hollander et al., 1999, 2001; Lotery et al., 2001). Analysis of Crb1 mutant mice showed a prominent fragmentation of the ZA (the outer or external limiting membrane) in retinas undergoing terminal differentiation as well as lightdependent retinal degeneration (Mehalow et al., 2003; van de Pavert et al., 2004). In addition, Crb1 mutant PRCs show smaller inner and outer segments, which are apical membrane subdomains. CRB3 was implicated in the assembly of tight junctions, epithelial polarity, and the formation of an apical cilium of normal length (Roh et al., 2003; Fan et al., 2004). Thus, Crb proteins have conserved functions in epithelial polarity and size regulation of apical membrane domains. All Crb proteins have a highly conserved short cytoplasmic tail that contains a PDZ domain binding (PDB) site at its C terminus and a juxtamembrane region that was predicted to act as a FERM (band 4.1, ezrin, radixin, moesin) domain binding (FDB) site (Klebes and Knust, 2000). Both binding sites are important for the function of Crb and CRB3 (Klebes and Knust, 2000; Izaddoost et al., 2002; Medina et al., 2002; Fogg et al., 2005). The PDZ binding site interacts with the adaptor PDZ protein Stardust (Sdt; Pals1/Mpp5 in mammals) that links Crb to the PDZ protein PATJ (Bachmann et al., 2001; Hong et al., 2001; Roh et al., 2002). Sdt/Pals1 and PATJ show functions similar to Crb or CRB3 (Tepass and Knust, 1993; Nam and Choi, 2003; Hong et al., 2003; Roh et al., 2003; Shin et al., 2005; Michel et al., 2005; Richard et al., 2006; Nam and Choi, 2006). Recently, it was suggested that the zebrafish gene mosaic eyes (moe) encodes a FERM protein that may interact with Crb proteins (Jensen and Westerfield, 2004). The phenotype of moe mutants is similar to the phenotype of nagie oko, a zebrafish ortholog of Drosophila sdt, and moe was implicated in tight junction formation similar to mammalian CRB3 (Wei and Malicki, 2002; Jensen and Westerfield, 2004). Here, we investigate the function of the FERM protein Yurt, the Drosophila ortholog of Moe, as a Crb interaction partner. yurt mutant embryos display defects in head morphogenesis and dorsal closure (Ju¨rgens et al., 1984; Hoover and Bryant, 2002), but the cellular

Developmental Cell 364

functions of Yurt and its potential role in epithelial polarity have not yet been analyzed. We show that Yurt binds to the cytoplasmic tail of Crb, an interaction we also observed between mammalian Crb and Yurt orthologs. Our data suggests that the interaction between Yurt and Crb is part of a negative-feedback loop that regulates epithelial polarity and apical membrane growth. Results Transient Colocalization of Yurt and Crb Our Yurt-specific antibodies detected multiple protein isoforms in embryonic lysates. Treatment with l phosphatase revealed four Yurt polypeptides that show various levels of phosphorylation (Figure 1A and Figure S1, see the Supplemental Data available with this article online). These isoforms have molecular weights of 135 kDa (Yurt-a), 106 kDa (Yurt-b), 101 kDa (Yurt-g), and 84 kDa (Yurt-d). Yurt-b is encoded by a transcript containing all known yurt exons as indicated by available cDNA clones and RT-PCR analysis (Figure 1C, not shown). Yurt-g and Yurt-d are alternative splice forms that lack sequences encoded by exon 6 or exon 9, respectively. Yurt-a is not yet fully characterized, but our mass-spectrometry analysis suggests that it contains the FERM domain as well as exons 7 and 8. Yurt-b, -g, and -d retain the FERM domain and a C-terminal PDB site and differ only in the variable region that lacks known protein domains (Figure 1C). All four Yurt polypeptides are differentially expressed (Figure 1B). Yurt-d is maternally provided, found in unfertilized eggs, and persists until midembryogenesis after which it declines. Yurt-a is first detected prior to gastrulation, accumulates at high levels during midembryogenesis, and subsequently declines. Yurt-b is first detected during gastrulation, while Yurt-g is initially seen at midembryogenesis, and these two isoforms accumulate toward the end of embryogenesis. Yurtb and Yurt-g are also the predominant isoforms in larvae and adult heads (Figure 1B). To determine whether all four isoforms are encoded by the yurt gene, we examined embryonic lysates that lack maternal and zygotic expression of yurt (yurt M/Z). For this analysis, two previously characterized mutations were used (yurtE15 and yurtE99) (Manseau et al., 1988; Hoover and Bryant, 2002). In addition, we generated new yurt deletion mutations (yurt75 and yurt65) by imprecise P element excision of EY01443 (Bellen et al., 2004), which is inserted in the yurt 50 UTR (Figure 1C). yurt M/Z mutants did not express detectable amounts of any of the Yurt isoforms (Figure 1D), indicating that these yurt alleles are protein null, and that all four isoforms are products of the yurt locus. Yurt is found in many tissues, in particular, all epithelia that express Crb (Figure S1). As reported previously (Hoover and Bryant, 2002), we found that Yurt associates with the plasma membrane, in particular, the basolateral membrane of epithelial cells. Enrichment of Yurt is detected at the furrow canal during cellularization (Figure 1E) and at the apical aspect of the lateral membrane during gastrulation (Figure 1F). The apical extent of Yurt distribution reaches the ZA (labeled with Armadillo [Arm]/b-catenin) at this stage but does not extend apical to it. In contrast, starting at stage 13, Yurt is de-

tected apical to the ZA in the marginal zone, a region that corresponds to the vertebrate tight junction and is enriched in apical determinants such as Crb (Figures 1G and 1H). Yurt is also found in the region of the ZA and the septate junction, and low levels were seen at the remaining basolateral membrane. We next examined the distribution of Yurt in PRCs to determine whether the transient colocalization of Yurt and Crb is a general feature of Yurt distribution. Crb and its binding partners Sdt and PATJ are found at the stalk membrane, one of two apical membrane domains of PRCs (Figure 1I) (Pellikka et al., 2002; Izaddoost et al., 2002; Hong et al., 2003). Yurt is associated with the basolateral membrane and the ZA but not detected at the apical membrane of PRCs up to 80% of pupal development (pd) (Figures 1J and 1K). At 85% pd, Yurt was first detected at the stalk membrane (Figure 1L). At 90% pd (Figure 1M) and in adults (not shown), Yurt is largely confined to the stalk membrane, and little if any Yurt is found at the basolateral membrane. Together, these results show that Yurt and Crb transiently colocalize at the marginal zone in mid-to-late stage embryos and at the stalk membrane in PRCs raising the possibility that these proteins interact directly. Crb Recruits Yurt to the Apical Membrane To find out whether the apical recruitment of Yurt requires Crb, we studied Yurt distribution in crb mutants. Results from crb mutant embryos were inconclusive due to the strong morphological defects in these embryos. However, crb mutant PRCs clearly failed to recruit Yurt to the stalk membrane and showed an enrichment of Yurt at the basolateral membrane (Figure 1N). To further examine the interactions between Yurt and Crb, we overexpressed either Yurt or Crb and analyzed the distribution of the other molecule. Overexpression of Yurt, facilitated by the UAS elements contained within the EY01443 P element, led to an accumulation of Yurt along the entire basolateral membrane and the apical junctional complex (data not shown). Ubiquitous overexpression of Yurt had no deleterious effect on viability or morphology, and the amount and distribution of Crb appeared normal. In contrast, overexpression of Crb from a UAS-crb transgene, which promotes excessive apical membrane formation and disrupts epithelial integrity (Wodarz et al., 1995), caused an abnormal accumulation of Yurt that codistributed with Crb (Figure 2A). Crb appears to specifically stabilize Yurt-b and affects the phosphorylation of Yurt-a (Figure S2). These findings indicate that Crb recruits Yurt to the apical membrane. Endogenous Yurt and Crb were found to coimmunoprecipitate (co-IP) from embryonic lysates of mid-tolate stage embryos (stages 12–17), but not from younger embryos (stages 6–10) (Figure 2B), which correlates with the lack of colocalization of Yurt and Crb at these stages (Figure 1F). GST pull-down experiments revealed that the FERM domain of Yurt is sufficient to mediate the interaction with Crb and that this interaction is specific as the FERM domain of Coracle, the Drosophila Band 4.1 ortholog (Fehon et al., 1994), did not interact with Crb (Figure 2C). Finally, Far-Western analysis indicated that the cytoplasmic domain of Crb fused to GST protein (Figure 2D), but not GST alone (not shown), can directly interact with the FERM domain of Yurt. These data

Yurt Negatively Regulates Crumbs Activity 365

Figure 1. Yurt Isoforms Expression and Localization (A) Overnight embryo lysate was treated with dual specificity l phosphatase prior to immunoblot analysis with Yurt-specific antibody (GP7). (B) Immunoblot showing the developmental expression profile of Yurt isoforms. (C) Schematics of the yurt genomic region, yurt transcripts, and the Yurt protein. Blue color indicates open reading frame. PDB, PDZ binding site; VR, variable region. (D) Yurt expression in yurt M/Z embryos compared to wild-type (WT) embryos. (E) During cellularization (stage 5), Yurt (red) is found in the apical and lateral membrane and enriched at the furrow canals marked by PATJ (green). (F) At stage 9, Yurt staining (red) is found at the lateral membrane and enriched just basal to the ZA labeled by anti-Arm antibody (green). (G–H) At stage 14, Yurt is enriched in the upper third of the lateral membrane, overlaps with the ZA (Arm staining in G0 ), and is found in the marginal zone where it colocalizes with Crb (H00 ). (I) Schematic of adult PRCs. ZA, zonula adherens. (J–K) At 75% pd, Yurt (red) is found at the basolateral membrane of PRCs and is enriched at the base of the ZA (Arm, blue) but is not detected at the apical stalk membrane marked by PATJ (green). (L) At 85% pd, Yurt (red) is detected at the basolateral membrane but also extends apical (arrowhead) beyond the ZA (Arm, blue) and partially overlaps with PATJ (green) in the stalk membrane. (M) At 90% pd, Yurt (red) is predominantly detected at the apical stalk membrane (PATJ, green). (N) crb11A22 mutant adult PRCs are identified by the loss of PATJ staining (green; marked by asterisks), and failed to recruit Yurt (red) to the stalk membrane. Instead, Yurt is enriched at the basolateral membrane. Scale bars: (E)–(H), 10 mm; (J)–(N), 5 mm.

suggest that direct binding to the Crb cytoplasmic tail of the FERM domain of Yurt is responsible for the Yurt Crb interaction in vivo. The Yurt Crb Interaction Is Conserved in Mammals The human (and mouse) genome encodes three Crb orthologs and two orthologs of Yurt and zebrafish Moe. The first Yurt/Moe ortholog is EHM2 (Shimizu et al., 2000), and the second human ortholog is annotated as EPB41L5 (erythrocyte protein band 4.1-like 5). We renamed EPB41L5 to Yurt/Mosaic eyes-like 1 (YMO1). To investigate whether the Crb Yurt interaction is conserved in mammals, we generated antibodies

against YMO1, CRB1 and CRB2 together with DNA constructs encoding full-length EHM2 and full-length and modified versions of YMO1 and CRB proteins (Figure 3A). YMO1 and CRB1 form a complex when expressed in HEK293 T cells (Figure 3D). This interaction is strongly reduced by point mutations in the FDB site of CRB1 (CRB1 AAA) (Figures 3A and 3D). The YMO1 CRB1 association is lost as a result of deleting the YMO1 FERM domain (Figure 3D). We also observed that YMO1 interacts with CRB2 and CRB3 in a FERM domain-dependent manner (Figures 3E and 3F). GST pulldowns confirmed the interaction between YMO1 and CRB proteins (Figures 3G and 3H) and showed that the

Developmental Cell 366

Figure 2. Yurt Interacts with Crb through Its FERM Domain (A) ptc-Gal4 UAS-crb embryo. Overexpression of Crb (green) leads to an increase in the levels of Yurt (red). (B) Coimmunoprecipitation of Crb using anti-Yurt antibody and wildtype embryos stages 6–10, 12–17, and an overnight (O/N) collection. (C) Lysate from an overnight embryo collection was subjected to GST pull-down with glutathione-agarose beads loaded with either GST, GST fused with the FERM domain of Yurt (GST-Yurt FERM), GST fused to a C-terminal portion of Yurt (GST-Yurt C TERM), or GST fused to the FERM domain of Coracle (GST-Cor FERM). Crb was specifically pulled down in the presence of the FERM domain of Yurt. (D) Far-Western using the FERM domain of Yurt as bait and GSTCrbintra as a probe shows a direct interaction between these two polypeptides. Scale bar: (A), 50 mm.

C-terminal PDB site in YMO1 is not required for the interactions between YMO1 and CRB1 or CRB2 (Figure 3G). The YMO1 CRB interaction appears to be specific as YMO1 does not interact with the cytoplasmic tail of syndecan-1 that also contains a predicted FDB site (Figure 3H) (Rapraeger, 2000). We observed similar interactions between EHM2 and CRB1, CRB2, and CRB3 (Figure S3). In the adult mouse retina, YMO1 is enriched apical to the ZA marked by Cadherin where it colocalized with Crb2 (Figures 3B and 3C) and was also more broadly distributed in PRCs and other cells of the retina. EHM2 showed no specific enrichment apical to the ZA in the neural retina (not shown). Taken together, these results suggest that the interaction between Yurt and Crb is a conserved feature of the Crb complex and that the mammalian Crb and Yurt orthologs can interact in all six possible combinations. Yurt Negatively Regulates Apical Membrane Formation We found that yurt M/Z mutants of four alleles (yurt65, yurt75, yurtE15, and yurtE99) displayed defects in epithelial polarity. yurt M/Z mutants showed first irregularities in the distribution of Crb and Arm after gastrulation when the germband is fully extended (stages 10/11) (Figures 4I

and 4J, not shown). By stage 12, Crb showed basolateral misdistribution in yurt M/Z mutant epithelia when epithelial tissue structure still appeared normal (Figures 4A, 4C, 4E, and 4F). During stages 13 to 15 severe defects in Crb distribution and in epithelial tissue organization were particularly apparent in the head and ventral ectoderm (Figures 4B, 4D, 4G, 4H, 4K, and 4L). Epithelial defects recovered to some extent toward the end of embryogenesis (not shown). Other epithelia including the dorsal epidermis exhibited milder defects in tissue structure in yurt M/Z embryos (Figures 4M and 4N and data not shown). In zygotic yurt mutants, abnormal localization of Crb and epithelial disruptions are confined to the head ectoderm (not shown). These results indicate that Yurt is required for the normal epithelial organization of the head and ventral ectoderm and to a lesser degree of other epithelia and that Yurt contributes to confining Crb to the apical membrane. The Crb binding partners PATJ and bH-Spectrin, similar to Crb, showed misdistribution in epithelia that exhibit multilayering in yurt M/Z mutants (Figures 5A, 5B, 5D, and 5E). Likewise, Arm (Figures 5G and 5H) or Bazooka (not shown) were either retained apically in yurt M/Z mutant epithelia that exhibit apparently normal structure or showed an irregular punctate distribution in multilayered cell clusters. In contrast to the cuticle of zygotic yurt mutants, cuticle in the head and ventral trunk of yurt M/Z mutants appeared abnormally infolded and convoluted (Figures 5J–5M). Histological sections confirmed that the ventral cuticle is highly infolded and occupies a larger surface area compared to wild-type (13.4 mm of cuticle per cell in yurt M/Z mutants [n = 39] versus 6.9 mm in wild-type [n = 62]) (Figures 5N and 5O), consistent with a larger apical, cuticle secreting membrane. The broader distribution of apical markers and the convoluted cuticle are defects similar to those seen in embryos that overexpress Crb (Wodarz et al., 1995) (Figures 5C, 5F, and 5I). We conclude that the apical membrane is enlarged in yurt M/Z mutants. Expanded apical membranes were also found in yurt mutant PRCs. At day 1 after eclosure, stalk membranes of yurt mutant cells are 2.5 mm in length compared to 1.9 mm in wild-type. Interestingly, stalk length defects are progressive: at day 7 after eclosure, stalks have a length of 2.7 mm and 3.6 mm after 14 days (Figures 6A–6D and 6I). Our previous work showed that overexpression of Crb leads to an expansion of the apical stalk membrane, and loss of Crb reduces stalk membranes to approximately 50% of their normal size (Pellikka et al., 2002) (Figures 6F and 6I). Consistent with the observed elongation of stalk membrane in yurt mutants, loss of Yurt did not cause a misdistribution or reduction of Crb, PATJ, and bH-Spectrin (Figure 6K, not shown), which promote stalk membrane growth (Pellikka et al., 2002; Richard et al., 2006). These findings suggest that Yurt is a negative regulator of stalk membrane length and that persistent Yurt activity is required to maintain normal stalk length. Yurt mutant eyes show normal external morphology. PRCs display mild defects in adherens junction positioning (Figure 6J) and in rhabdomere shape, including enlarged cross-section profiles and spilt rhabdomeres (Figure 6C). In contrast to crb mutants, no significant shortening in the proximodistal length of rhabdomeres

Yurt Negatively Regulates Crumbs Activity 367

Figure 3. YMO1 Interacts with All Three Human CRB Proteins (A) Schematic of the human CRB proteins and YMO1 and the mutant versions used. TM, transmembrane; FDB, FERM domain binding site; PDB, PDZ domain binding site. (B and C) Adult mouse retina labeled for YMO1 (green, B) and CRB2 (green, C) and pan-Cadherin antibodies (Cad, red). (D–F) Co-IP from lysates of HEK293T cells that were cotransfected with pairs of cDNA or a vector control as indicated. The expression level of proteins from the transfected cDNAs was monitored with whole-cell lysates (Lysate). (G–I) HEK293T cells were transfected with cDNAs encoding different proteins as indicated and lysed 24–48 hr later. Lysates were subjected to pull-down experiments with GST fused to the cytoplasmic tail of human CRB proteins. GST protein alone or fused to the cytoplasmic tail of human syndecan 1 (GST-hSyn) were used as negative controls. The expression levels were monitored with whole-cell lysate (Lysate), and Coomassie staining was used to evaluate the amount of GST fusion protein.

or a fragmentation of the ZA was observed in PRCs that lack Yurt. Loss of Crb and mouse Crb1 lead to light-dependent PRC degeneration (Johnson et al., 2002; van de Pavert et al., 2004). Similar to crb mutants and in contrast to wild-type, we observed massive PRC degeneration in yurt mutant eyes after 7 days of constant light exposure (Figure 6E), indicating that Yurt like Crb is required for PRC survival under light-stress conditions. Yurt Negatively Regulates Crb Function To examine the functional interactions between Yurt and Crb, we analyzed yurt crb double mutant ommatidia. PRCs that lack both Yurt and Crb display a crb-like phenotype and have stalk membranes that are reduced to

approximately 50% of their normal length (Figures 6F, 6H, and 6I). This genetic interaction is consistent with the view that yurt and crb act in one genetic pathway and, as the crb phenotype is epistatic over the yurt phenotype, that yurt acts upstream of crb. To further characterize the functional interactions between Crb and Yurt, we analyzed the phenotype of double mutant embryos. yurt mutations suppress the zygotic phenotype of the null allele crb11A22 and more dramatically of the hypomorphic allele crbS87-2 (Figures 7A and 7C–7E). Also, defects in junctional integrity and cellular organization of the epidermis are strongly rescued in double mutants as compared to crb mutant embryos (Figures 7G–7L). These results suggest that Yurt is a negative

Developmental Cell 368

Figure 4. Crb Is Mislocalized in yurt Mutant Embryos (A, B, E, and G) Crb distribution in a wild-type stage 11 (A and E) and stage 13 (B and G) embryo is restricted to the apical membrane. (C and F) Stage 12 yurt75 M/Z mutant stained for Crb showing ectopic localization of Crb in the basolateral membrane. (D and H) Crb distribution is apolar, and the epidermis and trachea are multilayered in a stage 13 yurt75 M/Z embryo. (I and J) Face-on view of Crb distribution in the ventral epidermis of a stage 11 wild-type (I) and yurt75 M/Z mutant (J) embryo. Crb distribution is irregular when Yurt is absent. (K–N) Crb distribution in the ventral (K and L) or lateral (M and N) epidermis of a stage 13 wild-type (K and M) and a yurt75 M/Z (L and N) embryo. Disruption of Crb distribution and tissue structure is largely confined to the ventral epidermis in a yurt75 M/Z embryo. Scale bars: (A)–(D), 100 mm; (E)–(H), 10 mm; (I)–(N), 10 mm.

regulator of Crb. The suppression of the embryonic crb11A22 phenotype by yurt mutations initially did not appear to be consistent with the notion that yurt acts through modulating crb activity as crb11A22 is considered a null allele, and previous work did not detect a maternal contribution of crb (crb11A22 M/Z embryos have the same phenotype as crb11A22 embryos) (Tepass and Knust, 1990). However, crb has a maternal component of expression as unfertilized eggs contain crb mRNA and protein (Figure S4). In contrast to the zygotic double mutants, embryos that derive from double mutant germline clones (yurt M/Z crb M/Z) show a phenotype similar to crb mutants that is characterized by the presence of cuticle grains and small cuticle vesicles (Figures 7A and 7B). This indicates that the suppression of the crb phenotype by yurt mutations requires residual crb activity. Taken together, our data are consistent with the conclusion that Yurt function in epithelial organization is mediated by a negative modulation of Crb activity. Yurt Does Not Cooperate with Lgl, Dlg, and Scrib in Suppressing Crb Function Our analysis of Yurt revealed several features that had also been reported for the basolateral determinants Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble

(Scrib). Yurt colocalizes with Lgl, Dlg, and Scrib at the basolateral membrane and yurt mutations suppress the crb phenotype similar to mutations in lgl, dlg, and scrib (Tanentzapf and Tepass, 2003; Bilder et al., 2003). This raises the possibility that yurt may cooperate with these genes in counteracting apical determinants such as Crb. However, we also note significant differences between yurt and lgl, dlg, and scrib. (1) Lgl, Dlg, and Scrib are required for epithelial apical-basal polarity in gastrulating embryos (e.g., Bilder and Perrimon, 2000), while Yurt is not (this work). (2) yurt M/Z crb M/Z embryos show a crb phenotype (Figure 7B) arguing that the suppression of crb mutant defects by yurt mutations (Figures 7C and 7E) requires residual crb activity. In contrast, the crb phenotype is suppressed in scrib M/Z crb M/Z embryos (with the same allele, crb11A22), and these embryos display a scrib mutant phenotype (Tanentzapf and Tepass, 2003). (3) yurt mutant imaginal discs did not show an overgrowth phenotype as seen in lgl, dlg, or scrib mutant discs (e.g., Bilder et al., 2000). To further explore potential interactions between yurt and lgl, dlg, and scrib, we studied double mutants and examined the distribution of Lgl and Dlg in yurt mutants. Double-mutant combinations between lgl, dlg, and scrib show striking positive interactions (Bilder et al., 2000). In contrast, no interaction

Yurt Negatively Regulates Crumbs Activity 369

Discussion

Figure 5. Epithelial Polarity Defects and Enlarged Apical Membranes in yurt Mutants (A and B) PATJ distribution in the ventral epidermis of a stage 13 wild-type (A) and yurt75 M/Z (B) embryo. (C, F, and I) PATJ (C), bH-Spectrin (F), and Armadillo (I) distribution in the ventral epidermis of a stage 13 embryo overexpressing Crb (da-GAL4, UAS-crb). (D and E) Distribution of bH-Spectrin in the ventral epidermis of stage a 13 wild-type (D) and yurt75 M/Z (E) embryo. (G and H) Arm distribution in the ventral epidermis of a stage 13 wild-type (G) and yurt75 M/Z (H) embryo. (J) Cuticle of a wild-type embryo. (K–M) Cuticle of a zygotic yurt75 mutant (K) and a yurt75 M/Z mutant (L and M). Arrows point to convoluted ventral cuticle. (N) Transmission EM of a stage 17 wild-type embryo. Arrow points to cuticle. (O) Transmission EM showing that the cuticle is highly infolded and extended in stage 17 yurt75 M/Z mutant embryos. Arrows point to cuticle. Scale bars: (A)–(I), 10 mm; (J)–(L), 100 mm; (M), 50 mm; (N) and (O), 5 mm.

was detected in double mutant combinations of yurt and dlg or lgl (Figure 7F and Figure S5). Furthermore, Lgl, Dlg, and Scrib show codependent membrane localization, whereas the localization of Dlg and Lgl was normal in yurt M/Z mutants (Figure S5). Together, these results suggest that Yurt acts independently of Lgl, Dlg, and Scrib to counteract Crb activity.

The data presented here support the hypothesis that the FERM protein Yurt is a negative regulatory component of the Crb complex that modulates Crb function in two distinct aspects of epithelial organization: epithelial apical-basal polarity and the regulation of apical membrane size (see Figure 7M). The conclusion that Yurt opposes Crb activity and does so as a component of the Crb complex is supported by several observations. (1) Yurt is recruited to the apical membrane by Crb and can bind directly to the FDB site in the cytoplasmic tail of Crb. (2) Loss of Yurt disrupts epithelial polarity and causes an enlargement of the apical membrane as seen when Crb is overexpressed. (3) yurt mutations suppress the crb mutant phenotype but only when residual Crb activity remains, suggesting that Yurt exerts its effects through Crb. A clear distinction between Crb function in apicalbasal polarity and in regulating the size of an apical membrane domain is seen in PRCs. Overexpression of Crb in late PRCs leads to an expansion of the apical stalk membrane without disrupting apical-basal polarity or the apical junctional complex (Pellikka et al., 2002), in contrast to most other epithelial cells (Wodarz et al., 1995; Klebes and Knust, 2000; Izaddoost et al., 2002). yurt mutant PRCs display longer stalk membranes similar to PRCs that overexpress Crb and unlike PRCs that lack Crb, which have shorter stalks. We also detected extended apical membranes in the late embryonic epidermis of yurt mutants. The function of Yurt as a negative regulator of apical membrane size appears to be conserved in vertebrates. We show that the Yurt orthologs YMO1 and EHM2 can bind to all three human CRB proteins and that YMO1 colocalizes with Crb2 in the inner segment of mouse PRCs. Moreover, zebrafish Crb proteins physically interact with the Yurt ortholog Moe, and loss of Moe causes larger apical membranes in fish PRCs (Y.-C. Hsu, J.J. Willoughby, A.K. Christensen, and A.M.J., unpublished data). We conclude that negative regulatory feedback within the Crb complex could be a general mechanism for the regulation of apical membrane size. Similar to yurt, mutations in genes encoding basolateral polarity proteins such as Lgl oppose the activity of apical polarity factors (Bilder et al., 2003; Tanentzapf and Tepass, 2003; Benton and St. Johnston, 2003; Plant et al., 2003; Betschinger et al., 2003; Yamanaka et al., 2003, 2006). We did not detect functional interactions between yurt and lgl or dlg, and although yurt and lgl, dlg, or scrib mutations suppress the crb mutant phenotype, remaining crb activity is required to ameliorate the crb mutant defects in the absence of Yurt but not in the absence of Scrib. These findings suggest that Yurt acts directly on Crb and not indirectly through a cooperation with basolateral determinants. Crb proteins promote the formation of apical membrane or apical membrane subdomains of Drosophila embryonic epithelial cells (Wodarz et al., 1995; Myat and Andrew, 2002), Drosophila PRCs (Pellikka et al., 2002), mammalian PRCs (Mehalow et al., 2003), and the apical cilium of mammalian kidney cells (Fan et al., 2004). The Crb binding partners Sdt, PATJ, and bHSpectrin also positively regulate apical membrane

Developmental Cell 370

Figure 6. Stalk Membrane Length Is Increased in yurt Mutant PRCs (A and B) Transmission EM of wild-type PRCs. Arrowheads point to ZAs and arrows to stalk membrane in (B), (D), (F), and (H). (C and D) yurt75 mutant PRCs show abnormal rhabdomere morphology and extended stalks. (E) yurt75 mutant PRCs show light-stress-dependent degeneration. (F) crb11A22 mutant PRCs have shortened stalks. (G and H) yurt75 crb11A22 double mutant PRCs show a crb-like phenotype with shortened stalk membranes. (I) Histogram showing the progressive increase in stalk length in yurt75 mutant PRCs at 1 day (d), 7 days, and 14 days after eclosure. Student’s t test showed significant differences between wild-type and mutants at p < 0.0001; yurt 1 day versus yurt 7 days, p < 0.05; yurt 7 days versus yurt 14 days, p < 0.0001. No significant difference was found between crb11A22 and yurt75 crb11A22 double mutants. Error bars represent standard deviation. (J) Basolaterally displaced adherens junctions (arrowheads; Arm, blue) are seen in yurt75 mutant PRCs marked by the absence of Yurt (red). (K) Crb (blue) and bH-Spectrin (bH-Spec; green) localize to the stalk membrane in yurt75 mutant PRCs. Arrowhead points to apical abnormalities occasionally detected in yurt75 mutant cells. Scale bars: (A), (C), (E), and (G), 2 mm; (B), (D), (F), and (H), 1 mm; (J) and (K), 10 mm.

formation (Pellikka et al., 2002; Hong et al., 2003; Richard et al., 2006). Crb forms a complex with bH-Spectrin, but the nature of this interaction has remained unclear (Pellikka et al., 2002; Medina et al., 2002). The FDB site in the Crb cytoplasmic tail is required to promote epithelial polarity and is important for the linkage of Crb to bH-Spectrin (Klebes and Knust, 2000; Medina et al., 2002). Moesin was suggested as a candidate for mediating this interaction as it can form a complex with Crb and bH-Spectrin (Medina et al., 2002). However, direct binding of Moesin to Crb has not been reported and the loss of Moesin causes defects that do not resemble the Crb loss- or gain-of-function phenotype. Moreover, Moesin does not colocalize with Crb at the stalk membrane (Karagiosis and Ready, 2004). Our data suggest that Yurt does not link Crb to bH-Spectrin, although Yurt can bind to the Crb FDB site. Loss of Yurt from PRCs does not cause bH-Spectrin to detach from the stalk as was reported for PRCs that lack Crb (Pellikka et al., 2002) and as would be expected if Yurt is the critical linker between Crb and bH-Spectrin. Moreover, association of bH-Spectrin with the stalk and loss of bH-Spectrin from the stalk in crb mutants occurs before Yurt becomes apparent at the stalk membrane. These results imply that Crb can interact with bH-Spectrin in a Yurt-independent manner. In addition to Yurt, the FDB site of Crb is likely to interact with a yet-unknown positive regulator of epithelial polarity and is therefore a critical site for the modulation of Crb complex activity.

Crb has a well-established role in epithelial apicalbasal polarity and contributes to the assembly of the ZA in Drosophila and the ZA and tight junction in mammalian epithelia (e.g., Tepass 1996; Mehalow et al., 2003; Fogg et al., 2005). In contrast to Crb and Sdt, which are required for epithelial polarity already in gastrulating embryos (Tepass and Knust, 1990, 1993, Tepass, 1996; Grawe et al., 1996; Muller and Wieschaus, 1996), Yurt becomes an essential component of the Crb complex later in epithelial development. Yurt localization appears to be confined to the basolateral membrane at early stages, and biochemical interactions between Yurt and Crb were not detected. The shift in the subcellular distribution of Yurt is unlikely to reflect the differential expression of the four Yurt isoforms. For example, the recruitment of Yurt to the stalk membrane does not correlate with an isoform shift as Yurt-b and Yurt-g are the predominant isoforms throughout pupal retinal development (data not shown). Thus, the interaction between Crb and specific Yurt isoforms is presumably regulated through modification of Yurt or Crb or both proteins. One attractive possibility is that phosphorylation of Yurt, which we document, is important for regulating the Crb Yurt interaction. The association of several other FERM domain proteins such as Merlin or Moesin with membrane proteins is regulated through phosphorylation, which causes a conformational change that allows the FERM domain to interact with transmembrane receptors (reviewed in Bretscher et al., 2002). Recent work showed that the phosphorylation

Yurt Negatively Regulates Crumbs Activity 371

Figure 7. Genetic Interaction between yurt and crb (A) Cuticle of a crb11A22 mutant embryo. (B) Cuticle of a crb11A22 yurt75 double M/Z mutant embryo. The crb mutant phenotype is epistatic over the yurt mutant phenotype. (C) Cuticle defects observed in a crb11A22 mutant embryo are ameliorated in a crb11A22 yurt75 zygotic double mutant embryo. (D and E) Cuticle defects observed in the crbS87-2 mutant embryo (D) are suppressed in a crbS87-2 yurt75 zygotic double mutant embryo (E). (F) Cuticle of a dlgm52 yurt75 zygotic double mutant embryo. Removal of zygotic dlg does not modify the yurt mutant phenotype. (G–I) Face-on view of Arm distribution in the ventral epidermis of a stage 13 wild-type (G), crb11A22 (H), and yurt75 crb11A22 (I) zygotic mutant embryo. (J–L) The defects in Cor distribution observed in a crbS87-2 mutant embryo (K) are suppressed in a crbS87-2 yurt75 zygotic double mutant embryo (L). (M) Model illustrating the functional interactions between Yurt and other polarity factors. Global regulatory feedback between apical and basolateral determinants controls apical-basal polarity, whereas local regulatory feedback between Yurt and other components of the Crb complex contributes to apical-basal polarity and to the regulation of apical membrane size. Scale bars: (A)–(F), 100 mm; (G)–(L), 10 mm.

of the juxtamembrane region of Crb by aPKC is important for Crb function (Sotillos et al., 2004). This modification could prevent premature recruitment of Yurt as a negative regulator of Crb activity during early epithelial development. It will be an important challenge to determine how the temporal and spatial interactions between Crb and Yurt are regulated. The negative regulation of the Crb/Sdt/PATJ/bHSpectrin complex by Yurt does not involve significant changes in the localization or levels of these proteins. To find out how Yurt interferes with Crb complex function will likely depend on elucidating the molecular mechanism of how Crb and its other binding partners cause cell polarization and apical membrane growth, which currently is not understood. Regulation of apical membrane size is a critical feature of epithelial development, which is required during epithelial polarization and in terminally differentiated cells to maintain functional apical domains such as the stalk membrane of PRCs or the apical cilia of epithelial cells. Figure 7M illustrates a model that summarizes the functional interactions between polarity proteins that we have explored in this study. Our analysis of Yurt emphasizes that the

Crb complex has two distinct functions in controlling epithelial cell architecture, the global regulation of apicalbasal polarity, and the local control of apical membrane size. We hypothesize that both levels of control involve Yurt-dependent negative feedback regulation that acts directly upon Crb activity. Global negative feedback occurs between apical and basolateral polarity proteins to set up apical versus basolateral membrane territories segregated by an apical junctional complex. Local negative feedback occurs among apical polarity proteins to restrict apical membrane growth. Apical membrane growth facilitated by the Crb complex without local suppression of its activity (that is, without involvement of Yurt) may occur early in epithelial development when the apical membrane shows net growth, while in more mature epithelial cells, homeostasis of apical-basal polarity and apical membrane size is achieved through Yurt-mediated local restriction of Crb complex activity. Experimental Procedures Drosophila Genetics yurt75 and yurt65 were generated by imprecise P element excision of EY01443 (P{EPgy2}yurtEY01443) (Bellen et al., 2004). yurt75 deletes

Developmental Cell 372

4.8 kb of genomic DNA including the 50 UTR and 1080 bp of the yurt ORF. yurt65 deletes 144 bp, including 36 bp of the predicted 50 UTR. yurtE15, yurtE99, crb11A22, and crbS87-2 were described previously (Tepass and Knust, 1990; Hoover and Bryant, 2002). Germline clones were generated with the FLP-DFS technique (Chou and Perrimon, 1996). Overexpression of Crb constructs or Yurt was achieved by crossing UAS-crb, UAS-crbintra, UAS-crbintra(A10A16), or EY1443 to ptc-GAL4 or da-GAL4 (Wodarz et al., 1995; Klebes and Knust, 2000). Clones in the retina were induced as described (Pellikka et al., 2002). Plasmid Constructs and Mutagenesis Expression constructs for human myc-tagged CRB1 and CRB3 were obtained from B. Margolis (Roh et al., 2002; Makarova et al., 2003). Mouse EHM2 cDNA was obtained from M. Tani (Shimizu et al., 2000). EHM2 and YMO1 were amplified by PCR and subcloned into pFlagCMV2 (Sigma). A partial human CRB2 cDNA encoding amino acids (aa) 351–1285 was subcloned in pFlagCMV2. The FDB site mutation of CRB1 (Y1398A; P1340A; E1405A) was generated by using QuikChange II site directed mutagenesis (Stratagene). YMO1 mutants lacking either the FERM domain or the PDB were generated by PCR and cloned into pFlagCMV2. GST-fusion proteins were expressed with the pGEX-4T1 vector (Clontech). The GSTcoracle FERM construct was provided by R. Fehon. Antibody Production Antibodies against Yurt aa 1081–1199 were generated in rats and guinea pigs. Rabbit polyclonal antibodies against human CRB1 and CRB2 were raised against aa 255–407 and aa 1060–1224 of these proteins, respectively. The YMO1 rabbit polyclonal antibody was raised against aa 669–731 of the mouse protein. Immunocytochemistry Fly embryos and mouse and fly retinas were fixed as previously described (Tepass et al., 1990; Tepass, 1996; Pellikka et al., 2002). Primary antibodies: guinea pig anti-Yurt (GP7), rat anti-Yurt (RA3), mouse monoclonal anti-Arm (N2-7A1; Developmental Studies Hybrydoma Bank [DSHB]), rat anti-Crb (Pellikka et al., 2002), rabbit anti-PATJ (Tanentzapf et al., 2000), rabbit anti-YMO1, rabbit antiCRB2 and pan-cadherin (CH-19, Sigma). Secondary antibodies were conjugated to Cy3, Cy5 (Jackson Immunoresearch Laboratories), or Alexafluor 488 (Molecular Probes). Immunoblotting and Phosphatase Assay Drosophila embryos were homogenized in 1% Triton X-100 buffer (Laprise et al., 2002). HEK293T cells were transfected with LipofectAMINE 2000 reagent (Invitrogen) according to manufacturer’s instructions. Cells were cultured at 37
C for 24–48 hr before lysis in 0.2% Triton X-100 buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, 100 mM sodium fluoride containing COMPLETE protease inhibitor tablet [Roche Applied Science]). SDS-PAGE and immunoblots were done as described (Laprise et al., 2002). Primary antibodies: guinea pig anti-Yurt (GP7), rat antiCrb (Pellikka et al., 2002), rabbit anti-bH-Spectrin (243, Thomas and Kiehart, 1994), mouse monoclonal anti-b-tubulin (E7, DSHB), antiGST clone B-14 (Santa Cruz Biotechnology), anti-FlagM2 (Sigma), Myc9E10 (DSHB), anti-CRB3 (Makarova et al., 2003), rabbit antiCRB1, rabbit anti-CRB2, rabbit anti-YMO1. Blots were visualized with the ECL system (Amersham Biosciences). l phosphatase treatment followed manufacturer’s instructions (New England Biolabs). Immunoprecipitations and GST Pull-Down Experiments Immunoprecipitation was achieved by incubating antibodies and protein A or G Sepharose beads with 1 mg of protein from embryo or HEK293 T cell lysates overnight at 4
C. For GST pull-down experiments, 1 mg of lysate was incubated with 10–20 mg of GST-fusion protein coupled to glutathione-Sepharose 4B beads (Amersham Biosciences) overnight at 4
C. After five washes with the respective lysis buffer, protein complexes were subjected to immunoblot analysis. Far-Western Analysis The FERM domain of Yurt (aa 39–272) was cleaved from a GST-YurtFERM fusion protein with thrombin (Amersham Biosciences) and was transferred to a nitrocellulose membrane after SDS-PAGE.

The membrane was saturated with a solution of 5% nonfat milk in PBS 0.05% Tween 20 (blocking solution) and then incubated overnight at 4
C with the GST-Crbintra fusion protein (Sotillos et al., 2004) at a dilution of 2 mg/ml in blocking solution. Extensive washes in PBS 0.05% Tween 20 were followed by immunoblot staining with anti-GST antibody. Electron Microscopy Embryos and adult eyes were processed for transmission electron microscopy as described (Tepass and Hartenstein, 1994; Pellikka et al., 2002). Light-Stress Experiment Newly eclosed wild-type flies and flies with yurt75 mutant eyes were kept in bright (800 lux) constant light for 7 days. Supplemental Data Supplemental Data including five figures are available at http://www. developmentalcell.com/cgi/content/full/11/3/363/DC1/. Acknowledgments We are grateful to Henry Hong for technical assistance. We thank R. Fehon, B. Margolis, S. Campuzano, B. Ganetzky, G. Thomas, the Bloomington Drosophila Stock Center, and the DSHB for reagents. We appreciate the critical discussion and comments on the manuscript by D. Godt and T. Harris. P.L. was supported by postdoctoral fellowships from the Fonds de la Recherche en Sante´ du Que´bec and the Canadian Institute for Health Research. N.F.S.-G was supported by a postdoctoral fellowship from The Foundation Fighting Blindness Canada. S.B. was supported by a predoctoral fellowship from the Vision Science Research Program, University of Toronto. This work was supported by a grant from the Canadian Institute for Health Research to U.T. and a grant from The Foundation Fighting Blindness Canada to C.J.M. U.T. is an investigator of the Canadian Institute for Health Research. Received: December 22, 2005 Revised: May 6, 2006 Accepted: June 1, 2006 Published: September 1, 2006 References Bachmann, A., Schneider, M., Theilenberg, E., Grawe, F., and Knust, E. (2001). Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature 414, 638–643. Bellen, H.J., Levis, R.W., Liao, G., He, Y., Carlson, J.W., Tsang, G., Evans-Holm, M., Hiesinger, P.R., Schulze, K.L., Rubin, G.M., et al. (2004). The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167, 761– 781. Benton, R., and St Johnston, D. (2003). Drosophila PAR-1 and 14–3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells. Cell 115, 691–704. Betschinger, J., Mechtler, K., and Knoblich, J.A. (2003). The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl. Nature 422, 326–330. Bilder, D., and Perrimon, N. (2000). Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403, 676–680. Bilder, D., Li, M., and Perrimon, N. (2000). Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113–116. Bilder, D., Schober, M., and Perrimon, N. (2003). Integrated activity of PDZ protein complexes regulates epithelial polarity. Nat. Cell Biol. 5, 53–58. Bretscher, A., Edwards, K., and Fehon, R.G. (2002). ERM proteins and merlin: integrators at the cell cortex. Nat. Rev. Mol. Cell Biol. 3, 586–599.

Yurt Negatively Regulates Crumbs Activity 373

Chou, T.B., and Perrimon, N. (1996). The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144, 1673–1679.

Manseau, L.J., Ganetzky, B., and Craig, E.A. (1988). Molecular and genetic characterization of the Drosophila melanogaster 87E actin gene region. Genetics 119, 407–420.

den Hollander, A.I., ten Brink, J.B., de Kok, Y.J., van Soest, S., van den Born, L.I., van Driel, M.A., van de Pol, D.J., Payne, A.M., Bhattacharya, S.S., Kellner, U., et al. (1999). Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12). Nat. Genet. 23, 217–221.

Medina, E., Williams, J., Klipfell, E., Zarnescu, D., Thomas, G., and Le Bivic, A. (2002). Crumbs interacts with moesin and beta(Heavy)spectrin in the apical membrane skeleton of Drosophila. J. Cell Biol. 158, 941–951.

den Hollander, A.I., Heckenlively, J.R., van den Born, L.I., de Kok, Y.J., van der Velde-Visser, S.D., Kellner, U., Jurklies, B., van Schooneveld, M.J., Blankenagel, A., Rohrschneider, K., et al. (2001). Leber congenital amaurosis and retinitis pigmentosa with Coats-like exudative vasculopathy are associated with mutations in the crumbs homologue 1 (CRB1) gene. Am. J. Hum. Genet. 69, 198–203. Fan, S., Hurd, T.W., Liu, C.J., Straight, S.W., Weimbs, T., Hurd, E.A., Domino, S.E., and Margolis, B. (2004). Polarity proteins control ciliogenesis via kinesin motor interactions. Curr. Biol. 14, 1451–1461. 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. Fogg, V.C., Liu, C.J., and Margolis, B. (2005). Multiple regions of Crumbs3 are required for tight junction formation in MCF10A cells. J. Cell Sci. 118, 2859–2869. Grawe, F., Wodarz, A., Lee, B., Knust, E., and Skaer, H. (1996). The Drosophila genes crumbs and stardust are involved in the biogenesis of adherens junctions. Development 122, 951–959. Hong, Y., Stronach, B., Perrimon, N., Jan, L.Y., and Jan, Y.N. (2001). Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature 414, 634–638. Hong, Y., Ackerman, L., Jan, L.Y., and Jan, Y.N. (2003). Distinct roles of Bazooka and Stardust in the specification of Drosophila photoreceptor membrane architecture. Proc. Natl. Acad. Sci. USA 100, 12712–12717. Hoover, K.B., and Bryant, P.J. (2002). Drosophila Yurt is a new protein-4.1-like protein required for epithelial morphogenesis. Dev. Genes Evol. 212, 230–238. Izaddoost, S., Nam, S.C., Bhat, M.A., Bellen, H.J., and Choi, K.W. (2002). Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature 416, 178–183. Jensen, A.M., and Westerfield, M. (2004). Zebrafish mosaic eyes is a novel FERM protein required for retinal lamination and retinal pigmented epithelial tight junction formation. Curr. Biol. 14, 711–717. Johnson, K., Grawe, F., Grzeschik, N., and Knust, E. (2002). Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration. Curr. Biol. 12, 1675–1680. Ju¨rgens, G., Wieschaus, E., Nu¨sslein-Volhard, C., and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Rouxs Arch. Dev. Biol. 193, 284–295. Klebes, A., and Knust, E. (2000). A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila. Curr. Biol. 10, 76–85. Knust, E., and Bossinger, O. (2002). Composition and formation of intercellular junctions in epithelial cells. Science 298, 1955–1959. Karagiosis, S.A., and Ready, D.F. (2004). Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis. Development 131, 725–732. Laprise, P., Chailler, P., Houde, M., Beaulieu, J.F., Boucher, M.J., and Rivard, N. (2002). Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation. J. Biol. Chem. 277, 8226– 8234. Lotery, A.J., Jacobson, S.G., Fishman, G.A., Weleber, R.G., Fulton, A.B., Namperumalsamy, P., Heon, E., Levin, A.V., Grover, S., Rosenow, J.R., et al. (2001). Mutations in the CRB1 gene cause Leber congenital amaurosis. Arch. Ophthalmol. 119, 415–420. Makarova, O., Roh, M.H., Liu, C.J., Laurinec, S., and Margolis, B. (2003). Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Pals1). Gene 302, 21–29.

Mehalow, A.K., Kameya, S., Smith, R.S., Hawes, N.L., Denegre, J.M., Young, J.A., Bechtold, L., Haider, N.B., Tepass, U., Heckenlively, J.R., et al. (2003). CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina. Hum. Mol. Genet. 12, 2179–2189. Michel, D., Arsanto, J.P., Massey-Harroche, D., Beclin, C., Wijnholds, J., and Le Bivic, A. (2005). PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells. J. Cell Sci. 118, 4049–4057. Muller, H.A., and Wieschaus, E. (1996). armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134, 149–163. Myat, M.M., and Andrew, D.J. (2002). Epithelial tube morphology is determined by the polarized growth and delivery of apical membrane. Cell 111, 879–891. Nam, S.C., and Choi, K.W. (2003). Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila. Development 130, 4363–4372. Nam, S.C., and Choi, K.W. (2006). Domain-specific early and late function of Dpatj in Drosophila photoreceptor cells. Dev. Dyn. 235, 1501–1507. Nelson, W.J. (2003). Adaptation of core mechanisms to generate cell polarity. Nature 422, 766–774. Pellikka, M., Tanentzapf, G., Pinto, M., Smith, C., McGlade, C.J., Ready, D.F., and Tepass, U. (2002). Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature 416, 143–149. Plant, P.J., Fawcett, J.P., Lin, D.C., Holdorf, A.D., Binns, K., Kulkarni, S., and Pawson, T. (2003). A polarity complex of mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl. Nat. Cell Biol. 5, 301–308. Rapraeger, A.C. (2000). Syndecan-regulated receptor signaling. J. Cell Biol. 149, 995–998. Richard, M., Grawe, F., and Knust, E. (2006). DPATJ plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye. Dev. Dyn. 235, 895–907. Roh, M.H., Makarova, O., Liu, C.J., Shin, K., Lee, S., Laurinec, S., Goyal, M., Wiggins, R., and Margolis, B. (2002). The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J. Cell Biol. 157, 161–172. Roh, M.H., Fan, S., Liu, C.J., and Margolis, B. (2003). The Crumbs3Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J. Cell Sci. 116, 2895–2906. Shimizu, K., Nagamachi, Y., Tani, M., Kimura, K., Shiroishi, T., Wakana, S., and Yokota, J. (2000). Molecular cloning of a novel NF2/ ERM/4.1 superfamily gene, ehm2, that is expressed in high-metastatic K1735 murine melanoma cells. Genomics 65, 113–120. Shin, K., Straight, S., and Margolis, B. (2005). PATJ regulates tight junction formation and polarity in mammalian epithelial cells. J. Cell Biol. 168, 705–711. Sotillos, S., Diaz-Meco, M.T., Caminero, E., Moscat, J., and Campuzano, S. (2004). DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila. J. Cell Biol. 166, 549– 557. Tanentzapf, G., and Tepass, U. (2003). Interactions between the crumbs, lethal giant larvae and bazooka pathways in epithelial polarization. Nat. Cell Biol. 5, 46–52. Tanentzapf, G., Smith, C., McGlade, J., and Tepass, U. (2000). Apical, lateral, and basal polarization cues contribute to the development of the follicular epithelium during Drosophila oogenesis. J. Cell Biol. 151, 891–904.

Developmental Cell 374

Tepass, U. (1996). Crumbs, a component of the apical membrane, is required for zonula adherens formation in primary epithelia of Drosophila. Dev. Biol. 177, 217–225. Tepass, U., and Hartenstein, V. (1994). The development of cellular junctions in the Drosophila embryo. Dev. Biol. 161, 563–596. Tepass, U., and Knust, E. (1990). Phenotypic and developmental analysis of mutations at the crumbs locus, a gene reuired for the development of epithelia in Drosophila melanogaster. Rouxs Arch. Dev. Biol. 199, 189–206. Tepass, U., and Knust, E. (1993). Crumbs and stardust act in a genetic pathway that controls the organization of epithelia in Drosophila melanogaster. Dev. Biol. 159, 311–326. Tepass, U., Theres, C., and Knust, E. (1990). crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell 61, 787–799. Tepass, U., Tanentzapf, G., Ward, R., and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35, 747–784. Thomas, G.H., and Kiehart, D.P. (1994). Beta heavy-spectrin has a restricted tissue and subcellular distribution during Drosophila embryogenesis. Development 120, 2039–2050. van de Pavert, S.A., Kantardzhieva, A., Malysheva, A., Meuleman, J., Versteeg, I., Levelt, C., Klooster, J., Geiger, S., Seeliger, M.W., Rashbass, P., et al. (2004). Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure. J. Cell Sci. 117, 4169–4177. van den Hurk, J.A., Rashbass, P., Roepman, R., Davis, J., Voesenek, K.E., Arends, M.L., Zonneveld, M.N., van Roekel, M.H., Cameron, K., Rohrschneider, K., et al. (2005). Characterization of the Crumbs homolog 2 (CRB2) gene and analysis of its role in retinitis pigmentosa and Leber congenital amaurosis. Mol. Vis. 11, 263–273. Wei, X., and Malicki, J. (2002). nagie oko, encoding a MAGUK-family protein, is essential for cellular patterning of the retina. Nat. Genet. 31, 150–157. Wodarz, A., Hinz, U., Engelbert, M., and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 67–76. Yamanaka, T., Horikoshi, Y., Sugiyama, Y., Ishiyama, C., Suzuki, A., Hirose, T., Iwamatsu, A., Shinohara, A., and Ohno, S. (2003). Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity. Curr. Biol. 13, 734–743. Yamanaka, T., Horikoshi, Y., Izumi, N., Suzuki, A., Mizuno, K., and Ohno, S. (2006). Lgl mediates apical domain disassembly by suppressing the PAR-3-aPKC-PAR-6 complex to orient apical membrane polarity. J. Cell Sci. 119, 2107–2118.