Crumbs acts via the FERM-domain protein Expanded to regulate Salvador/Warts/Hippo signaling in Drosophila

Summary

Background

Altered expression of apicobasal polarity factors is associated with cancer in vertebrates and tissue overgrowth in invertebrates, yet mechanisms by which these factors affect growth regulatory pathways are not well defined. We have tested the basis of an overgrowth phenotype driven by the Drosophila protein Crumbs (Crb), which nucleates an apical membrane complex that functionally interacts with the Par6/Par3/aPKC and Scrib/Dlg/Lgl apicobasal polarity complexes.

Results

We find that Crb-driven growth is dependent upon the Salvador-Warts-Hippo (SWH) pathway and its transcriptional effector Yorkie (Yki). Expression of the Crb intracellular domain elevates Yki activity and this correlates in tissues and cultured cells with loss of Expanded (Ex), an apically localized SWH component that inhibits Yki. Reciprocally, loss of crb elevates Ex levels, although this excess Ex does not concentrate to its normal location at apical junctions. The Ex-regulatory domain of Crb maps to the juxtamembrane FERM-domain binding motif (JM), a cytoskeletal interaction domain distinct from the PDZ-binding motif (PBM) through which Crb binds polarity factors. Expression of Crb-JM drives Yki activity and organ growth with little effect on tissue architecture, while reciprocally Crb-PBM produces tissue architectural defects without significant effect on Yki activity.

Conclusions

These studies identify Crb as a novel SWH regulator via JM-dependent effects on Ex levels and localization, and show that discrete domains within Crb may allow it to integrate junctional polarity signals with a conserved growth pathway.

Introduction

Mutations in any of three Drosophila melanogaster genes required for the maintenance of apicobasal polarity - Discs large (Dlg), lethal giant larvae (lgl), and scribble (scrib) - result in disorganized overgrowth of epithelial tissues [1]. The discoveries that homologs of these genes are downregulated in cancer and targeted for inactivation by mammalian tumor viruses [2] have further advanced the hypothesis that defective epithelial polarization can drive ectopic cell proliferation and tissue overgrowth in metazoans. The mechanisms by which Drosophila Dlg, Lgl and Scrib restrict cell proliferation are not well understood, although it is assumed that they are an extension of their more primary roles in cell polarization.

The polarization of Drosophila epithelial cells is controlled by functional interactions between membrane-associated protein complexes [1]. In the embryonic ectoderm, the Dlg/Scrib complex localizes to the basolateral cell membrane and functionally antagonizes the apical Par6/Par3/aPKC (Par) complex. The Par complex recruits a second apically localized complex, composed of the Crumbs (Crb), Patj and Pals1/Stardust (Sdt) proteins, which represses activity of the Scrib complex. Mutations in Dlg, lgl or scrib lead to membrane ‘apicalization’ in which the Crb complex spreads ectopically into the basolateral membrane. Overexpression of Crb in discs leads to a similar spreading phenotype and produces overgrowth in a manner overtly similar to that seen in Dlg, scrib, or lgl mutants [3, 4]. Certain endocytic mutants that block Crb turnover also cause disc overgrowth [4, 5], although the role of Crb in this phenotype is not clear. Intriguingly, a crb transgene encoding the transmembrane region and the small 37 amino acid intracellular tail is oncogenic when expressed in discs [3, 4], arguing that the Crb intracellular tail contains a growth regulatory domain. The Crb cytoplasmic tail has two recognized motifs, one that links to the spectrin and actin cytoskeleton and another that interacts with polarity regulatory factors such as Sdt, Patj, and Par6 [6]. The ability of Crb to drive tissue growth is thus either due to a previously described role for one of these motifs, or to a previously unrecognized growth regulatory domain embedded within the Crb cytoplasmic tail.

Here we show that the Crb cytoplasmic tail drives organ growth via the Salvador-Warts-Hippo (SWH) pathway, a conserved signaling network that restricts cell proliferation and promotes apoptosis by regulating the Yorkie (Yki) transcriptional co-factor [7]. The activities of the core SWH proteins are controlled by inputs from a variety of ‘non-core’ peripheral regulators that are required for growth inhibition by the SWH pathway, and in some cases render SWH responsive to different upstream inputs, including those from planar cell polarity pathways, morphogen gradients, and adhesion molecules [8]. We show that Crb restricts accumulation of Expanded [9], a FERM-domain protein that localizes to the apical-membrane and acts as a ‘peripheral’ regulator of the SWH pathway [10]. Moreover, this Ex-regulatory function maps to a small motif within the Crb cytoplasmic tail that coincides with juxtamembrane FERM-binding motif, which is distinct from the domain through which Crb binds polarity factors. These studies identify the Crb protein as a novel regulator of the SWH pathway via its effects on Ex, and suggest that discrete domains within Crb may allow it to simultaneously regulate apical polarity and tissue growth.

Results

The intracellular domain of crb requires yki to drive tissue growth

Previous studies have shown that engrailed-Gal4 (en-Gal4) driven expression of either a full-length UAS-crb transgene or one encoding only intact transmembrane and intracellular domains (UAS-crbⁱ) [3] produces wing overgrowth [4]. Because the en>crb genotype led to significant embryonic lethality (data not shown), the en>crbⁱ genotype was used to model the cell-autonomous effect of the Crb intracellular tail on wing development.

en>crbⁱ larval wing discs show posterior domain overgrowth and tissue disorganization (Fig. 1D) [see also 4]. en>crbⁱ adult wings also show an enlarged posterior compartment and cuticular defects (Fig. 1A). Quantification of posterior compartment size relative to the entire wing produces a value we termed the posterior compartment ratio (PCR) that is elevated in en>crbⁱ animals (Fig. 1F). To understand the basis for this PCR phenotype, chromosomal deficiencies and selected alleles of pro-growth genes were screened for their ability to dominantly reduce en>crbⁱ wing phenotypes. Multiple alleles of pro-growth members of the SWH growth regulatory pathway, including the transcriptional co-factor yorkie (yki), the microRNA bantam (ban), and the transcription factor scalloped (sd), dominantly suppressed the en>crbⁱ PCR phenotype (Figs. 1B,F). These alleles did not modify PCR of control wings, indicating their effects on en>crbⁱ wings do not reflect dosage-sensitive roles in wing growth (Fig. 1F). Co-expression of the Sav protein, which antagonizes Yki [7], also suppressed growth of larval and adult en>crbⁱ wings (Fig. 1C,E-F). Alleles of Tor, rheb, rolled/ERK, stat92E, and Akt1 did not modify the en>crbⁱ PCR phenotype (data not shown), indicating that crbⁱ-driven growth is specifically sensitive to the dose of SWH pathway components.

Figure 1. Overgrowth driven by the crbⁱ transgene is sensitive to the dose of SWH pathway genes.

Images and overlays of (A) transgenic en>crbⁱ and control en>+, (B) en>crbⁱ and en>crbⁱ,yki^B5/+, (C) en>crbⁱ and en>crbⁱ,sav wings. (D-E) Phalloidin-FITC staining of en>crbⁱ and en>crbⁱ,sav larval wings. (F) PCR in the indicated genotypes (minimum 10 wings per genotype; * p<0.05 compared to en>crbⁱ wings).

crbⁱ expression elevates Yki activity in developing epithelia

Consistent with the pattern of genetic interactions between crbⁱ and SWH alleles, the expression of multiple Yki targets [7] is elevated in en>crbⁱ larval wing cells (Fig 2A-D). The ex-lacZ and diap1-lacZ enhancer traps, which respectively report Yki-dependent transcription of the expanded (ex) and Drosophila Inhibitor of Apoptosis-1 (diap1) genes, are elevated in the posterior domain of en>crbⁱ wing discs (Fig. 2A-A′,C-C′). The bantam-GFP reporter, whose expression inversely correlates with activity of the Yki-target and pro-growth miRNA bantam (ban), is also reduced in the posterior domain of en>crbⁱ wing discs (Fig. 2B-B′). Wg protein levels, which are normally repressed by the SWH pathway in the proximal wing hinge, are elevated in the corresponding area of en>crbⁱ wing discs (Fig. 2D). In parallel, the en>crbⁱ genotype increased the amount of Yki that co-localizes with a DNA marker relative to the cortical pattern of Yki among cells in the anterior wing pouch (Fig. 2F-F″). Finally, crbⁱ-driven disc growth is also not associated with a dramatic shift in cell cycle phasing (Fig. 2E) or cell size (data not shown), suggesting that crbⁱ may promote balanced increases in the rates of cell growth and division as observed in core SWH mutants [7]. Thus the ability of multiple SWH pathway alleles to modify the en>crbⁱ phenotype correlates with elevated Yki activity and with SWH-like phenotypes in en>crbⁱ larval wing discs.

Figure 2. crbⁱ elevates Yki-activity.

α-β-gal staining or GFP fluorescence in wing discs carrying (A) ex-lacZ, (B) ban-GFP, or (C) diap1-lacZ in the background of (A,B,C) en>+ or (A′,B′,C′) en>crbⁱ. Arrows in A′ and C′ highlight elevated ex-lacZ and diap1-lacZ expression. (D) α-Wg stain in en>crbⁱ wings discs (posterior = right of dashed line). (E) FACS-analysis of en>GFP (black) and transgenic en>crbⁱ,GFP (blue) wing discs. (F) Co-staining for Yki (blue) and HP1 (nuclei; red) in en>crbⁱ,GFP discs (posterior = right of dotted line).

The Notch receptor interacts with the SWH pathway in certain tissues [11-13] and is regulated by crb in certain developmental contexts [14]. The effect of Notch heterozygosity on en>crbⁱ PCR could not be reliably measured due to significant wing-notching (data not shown). However expression of crbⁱ had no significant effect on the Notch reporter E(spl)mβ-CD2 reporter [15] (Fig. S1,B-C) or expression of the Wg and Cut proteins at the dorsal/ventral margin of the wing (data not shown). Notch protein localization and levels in wing cells were also unaffected by expression of crbⁱ (Fig. S1,G). Clonal loss of crb in the eye disc also did not affect E(spl)mβ-CD2 expression (Fig. S1,D). Thus, the crbⁱ-induced changes in Yki activity do not coincide with detectable changes in Notch abundance, localization, or transcriptional induction of a Notch pathway reporter.

crbⁱ downregulates Ex protein levels in wing disc cells

The localization of Crb to the apical membrane and apicolateral junctions of disc epithelial cells [16] suggests that expression of crbⁱ might affect the activity of SWH proteins that also localize to these same domains. No significant alterations were noted in the levels and localization of either Fat or Merlin (Mer), two apically localized SWH regulators, in en>crbⁱ wing discs (Fig. S1,E-F). Moreover, the Fat reporters dachs-V5 (data not shown) and four jointed-lacZ [17] are unaltered in en>crbⁱ wing discs (Fig. S1,H-I), and RNAi-knockdown of the palmitoyltransferase approximated (app), which is required for the overgrowth of ft mutant cells [18], did not suppress the crbⁱ-driven enlarged wing phenotype (Fig. S1,J). Thus crbⁱ does not appear to control wing growth via a ft-dependent pathway. By contrast, the apical SWH regulator Ex is depleted from the apical membrane of crbⁱ-expressing cells (Fig. 3A-A″′). In parallel, expression of en>crbⁱ leads to a drop in overall levels of Ex detected by immunoblot analysis (Fig. 3D). The adherens junction (AJ) protein Armadillo was unaffected by crbⁱ expression (Fig. S1,A-A′), indicating that the effect on Ex is not due to a general loss of AJ complexes in en>crbⁱ cells.

Figure 3. crbⁱ downregulates Ex levels.

(A-A′″) Lateral section of en>crbⁱ,GFP wing disc co-stained for Dlg (red) and Ex (blue). Dotted line denotes A:P boundary. (B) Images and overlays of en>crbⁱ and en>crbⁱ,ex wings. (C) PCR in the indicated genotypes. (D) Immunoblot of Ex in en>+ and en>crbⁱ wing discs. Arrowhead denotes Ex based on comigration with overexpressed Ex (not shown) (* = non-specific band). Lower panel is α-β-tub loading control. (E) Immunoblot of HA-Ex in Crb^8F105-expressing cells treated with the MG132 (lane 4) or chloroquine (lane 5). (F) Corresponding α-HA, α-VSV-G, and α-β-tub immunoblots of S2 cells expressing HA-Ex from the pAct-HA-Ex plasmid (lanes 2-4), and VSV-G-tagged forms of either crbⁱ (lane 3) or crb^8F105 (lane 4) from the pMT plasmid. (G) Lateral images of Ex:GFP in the posterior region of the wing pouch in the indicated genotypes.

As transcription of ex-lacZ is elevated in en>crbⁱ discs, crbⁱ thus appears to promote post-transcriptional down-regulation of Ex. A similar effect occurs in S2 cells expressing Ex from the constitutively expressed pAct plasmid [10]: Ex levels are reduced following induction of a crb transgene expression from the inducible pMt plasmid [19] (Fig. 3E-F). Re-expression of Ex from a UAS-ex transgene is also sufficient to revert the en>crbⁱ PCR phenotype (Fig. 3B,C). In view of this link between Crb overexpression and Ex loss, it is notable that the array of phenotypes produced by crbⁱ overexpression are quite similar and those associated with the ex alleles in the intact organism (Fig. S2), including large wings lacking posterior cross veins, small eyes with reduced numbers of ommatidia [9], a delay in morphogenetic furrow (MF) progression in the ventral portion of the eye disc and an ectopic MF produced dorsally [20], and an increase in the number of interommatidial cells in the pupal eye disc [10]. Thus, while it is likely that the intracellular tail of Crb has effects on additional cellular pathways, these phenotypic similarities indicate that a significant subset of the effects of Crbⁱ activity on developing tissues may be mediated via Ex.

The Crb juxtamembrane domain regulates Ex

The 37 amino acid intracellular tail of Crb contains two functional motifs that are conserved across Crb proteins in multiple species: (i) the 15-amino acid juxtamembrane FERM-binding motif (JM), which mediates a direct interaction with the FERM-domain protein Yurt, and indirectly interacts with DMoesin (DMoe) and β_H-spectrin to link Crb to the underlying actin/spectrin cytoskeleton, and (ii) the C-terminal PDZ-binding motif (PBM), which is composed of the last 4 residues of the Crb tail (ERLI) and directs interactions with Sdt and Patj to form a polarity regulatory module commonly referred to as the Crb complex [6]. In order to better understand the mechanism whereby crbⁱ downregulates Ex and activates Yki, several previously utilized crb transgenes (Fig. 4J) that inactivate either the JM domain or the PBM within the Crbⁱ protein [21] were assessed their ability to (i) increase wing size, (ii) activate Yki signaling, and (iii) eliminate apical Ex. Overexpression of a construct lacking the PBM, but maintaining the JM, increases PCR among adult wings to a similar degree as the intact crbⁱ transgene (Fig. 4B,G). Expression of crb-JM also depletes apical Ex (Fig. 4I) and increases Yki activity as detected by the ex-lacZ reporter, particularly in the pouch region of the wing disc (Fig. 4B′). crb-JM thus phenocopies crbⁱ in its effects on Ex and on SWH pathway activity. By contrast, a construct containing the PBM and inactivating mutations within the JM (crb-PBM) does not increase PCR or significantly elevate ex-lacZ expression (Fig. 4C-C′,G), and has no effect on apical Ex (Fig. 4H). Expression of crb-PBM did disrupt organization of the disc epithelium (Fig. S4,C-D) and wing morphology (Fig. 4C-C′) in a way not observed with crb-JM, indicating that the failure of the crb-PBM transgene to affect Ex is not due to a general lack of biological activity. A construct lacking intact JM and PBM domains (crbΔΔ) had no effect on wing size, structure or ex-lacZ (Fig. 4D-D′).

Figure 4. The Crb-JM controls Ex levels and Yki activity.

Paired light microscopic (A-F) and confocal images of α-β-gal staining to detect activity of the ex-lacZ transgene (A′-D′,F′) or diap1-lacZ (E′) in the indicated genotypes. (G) PCR values in the indicated genotypes. (*p<0.05 compared to en>crbⁱ). α-Ex (blue) staining in (H) en>crb-PBM,GFP and (I) en>crb-JM,GFP wing discs. Dotted line marks the A:P boundary. (posterior = right). Cartoon of crb transgenes; signal peptide (SP), myc tag (Myc), transmembrane domain (TM), juxtamembrane FERM-binding motif (JM), PDZ-binding motif (PBM), and amino acid substitutions are indicated [adapted from 40].

A similar link between the Crb JM domain and Ex was observed in S2 cells. Expression of either VSV-G-tagged Crbⁱ or VSV-G-tagged Crb^8F105, which contains a stop codon that prevents the translation of the last 23 amino acids (including the PBM) while preserving much of the JM [19], is sufficient to downregulate co-expressed Ex (Fig. 3E-F). Treatment with the proteasome inhibitor MG132 was able to partially reverse this effect, whereas treatment with the lysosomal inhibitor chloroquine did not (Fig. 3E). In parallel experiments in intact wing discs, crbⁱ is able to deplete levels of an Ex:GFP fusion protein [22] (Fig. 3G). Genetic reduction of proteasome activity with a dominant-negative allele of the proteasomal subunit Pros2β [23] also elevates levels of Ex:GFP levels in normal wing disc cells and partially restores Ex:GFP levels in discs that also express crbⁱ (Fig. 3G). Expression of crbⁱ did not stimulate endolysosomal routing of Ex:GFP as measured by the effect of treatment with the lysosomal inhibitor chloroquine on Ex:GFP localization (Fig. S3). Thus, it appears that Ex protein levels are antagonized by the proteasome, and that blocking proteasome activity can retard the effect of Crbⁱ on Ex, although the rescue of crbⁱ PCR by UAS-ex (see Fig. 3B) suggests that this mechanism cannot completely overcome the ability of overexpressed Ex to rescue PCR. Moreover, as in disc cells, the PBM domain is dispensable for the down-regulation of Ex in S2 cells, while constructs that retain the JM also retain the ability to regulate Ex.

Effects of crb loss on Ex protein

The effect of crbⁱ on Ex suggests that under physiologic conditions crb might be required to restrict accumulation of Ex protein. Consistent with this, cells homozygous for the crb^11A22 allele, which reduces endogenous Crb protein to background levels (Fig. 5A-A′), show elevated Ex levels (Fig. 5B-C) with no change in ex-lacZ expression (Fig. 5D). A similar effect occurs in crb^11A22 wing clones (Fig. 5K). Levels and localization of the Dlg, Arm, and Mer protein are not affected by the crb^11A22 allele (Fig. S1,K-M), confirming that the effect of crb loss on Ex is fairly specific. Optical sections through the apical and basal planes of the eye disc indicate that while a portion of the excess Ex in crb^11A22 localizes apically, a portion also drops more basally (Fig, 5B′,C′). Lateral sections through crb^11A22 eye and wing clones confirm that Ex accumulates in a linear manner along what appears to be the basolateral membrane of cells (Fig. 5I-I′, J-J′). Since the excess Ex that accumulates in core SWH mutants remains at the apical domain [e.g. 10]), this Ex ‘basal spreading’ phenotype is not a secondary effect of elevating Ex levels in crb cells, but rather appears to reflect a role for crb in localizing Ex.

Figure 5. Effect of crb loss on Ex in disc cells.

Confocal images of crb^11A22 (A-D, I-K), crb^8F105 (E-F), or crb^Y10AP12AE16A (G-H) clones in the eye (A-I) or wing (J-K) stained for Crb (A,E,G), Ex (B,C,F,H-K). (B) and (C) are apical and basal planes of the same disc. Arrowheads in (I) denote excess Ex in crb^11A22 cells that fails to localize apically. Disc in (I) is imaged through apical portion of epithelium; disc in (J) is imaged through entire epithelium. (D) α-β-gal staining to detect activity of the ex-lacZ transgene in crb^11A22 eye clones.

Site specific alleles support a role for the JM domain in the Ex-inhibitory role of crb: cells homozygous for the crb^8F105 allele, which lacks the C-terminal PBM and the preceding 19 amino acids but maintains a largely intact JM [24], show a more mild effect on Ex levels than the crb^11A22 allele (Fig. 5F-F′). This weaker phenotype could be due to a role for the PBM region in regulating the Ex-regulatory function of the JM, or to the loss of additional sequences between the JM and PBM that affect protein stability or function. The crb^8F105 allele has been reported to reduce Crb levels and alter Crb protein distribution in embryonic epithelial cells [25] but no differences in Crb protein were detected in crb^8F105 clones (Fig. 5E-E′). To more finely map the sequences within Crb that are required to restrict Ex in vivo, a genomic crb allele carrying three missense mutations in the JM region (crb^Y10AP12AE16E) [26] was tested for its effect on Ex. While the crb^Y10AP12AE16E allele has no obvious effect on Crb levels or localization (Fig. 5G-G′), it did elevate Ex levels in cells (Fig. 5H-H′). Thus amino acids in the Crb JM are both necessary and sufficient to restrict Ex levels in vivo.

Modification by Crb-interacting factors

The Crb JM interacts directly with the FERM-domain protein Yurt [27] and indirectly with the FERM-domain protein DMoe [19], both of which are structurally related to Ex. Although no evidence was found of a stable interaction between the Crb and Ex (see Discussion), genetic data suggest that the DMoe plays a role in the link between Crb and SWH activity. RNAi knockdown of DMoe [28] in en>crbⁱ discs suppressed both enlarged PCR (Fig. 4F,G) and ex-lacZ expression (Fig. 4F′), but did not rescue the drop in endogenous Ex induced by crbⁱ (Fig. S4,A) and had no independent effect on Ex:GFP (Fig. S4,B). Although this effect could be due to a non-specific effect of DMoe loss, the ability of DMoe-IR to uncouple Ex loss from Yki activation in crbⁱ-expressing cells argues that DMoe may play a more specific role downstream of Ex in a Crb/Ex pathway, or that DMoe acts in a parallel pathway that converges on Yki.

The Crb cytoplasmic tail is phosphorylated by the Drosophila atypical protein kinase C (DaPKC), a component of the Par complex that regulates polarity and endocytosis [29]. These modifications are thought to be involved in the ability of Crb to in turn participate in the establishment of epithelial polarity. As in these prior studies, a dominant-negative DaPKC transgene effectively suppressed the cuticular disorganization of en>crbⁱ wings (Fig 4E). However, it did not change en>crbⁱ PCR (Fig. 4G), rescue the drop in Ex protein (Fig. S4,C-C″), or prevent induction of diap1-lacZ (Fig. 4E′). By contrast, a transgene encoding a dominant-negative form of the GTPase Cdc42 (dn-cdc42) [30], whose effectors include the Crb- and aPKC-interactor Par6, was an efficient suppressor of en>crbⁱ posterior compartment growth (Fig. 4G). This variable effect could be due to differences in transgene strength or to functional differences between aPKC and cdc42, as observed in other studies [31]. Notably, the dn-cdc42 allele is able to suppress the crb-JM PCR phenotype (Fig. S4,F), indicating either that Cdc42 can act on the Crb tail independent of the PBM, or that Cdc42 acts by a distinct pathway to control wing size.

Effect of excess Ex in crb mutant cells

As transgenic overexpression of ex is growth-suppressive [22, 32], the excess Ex in crb mutant cells might be predicted to reduce Yki activity. However, RNAi knockdown of crb do not suppress organ overgrowth driven by a UAS-yki:YFP transgene [33] but rather enhanced them (Fig. S5,A-C). Expression of diap-lacZ, a sensitive readout of Yki activity [34], is also elevated in crb^11A22 cells posterior to the MF (Fig. 6A); DIAP1 protein shows a similar pattern in crb^11A22 and crb^Y10AP12AE16A clones (Fig. 6B-C). crb^11A22 and crb^Y10AP12AE16A cells also display a clonal growth advantage in the eye relative to both control and crb^8F105 chromosomes (Fig. S5,D-G). Thus, mutations in the JM are sufficient to deregulate DIAP1 levels and to confer a growth advantage in vivo. Moreover, the crb^11A22 allele acts as a dominant enhancer of the increased wing-size phenotype associated with the ex⁶⁹⁷ hypomorphic allele (Fig. 6D-E), arguing that at a genetic level crb normally promotes ex activity, and that the effect of crb loss on Ex levels and localization may compromise signaling through the SWH pathway.

Figure 6. crb alleles interact with ex and elevate DIAP1 expression posterior to the furrow.

Images of (A-B) crb^11A22 or (C) crb^Y10AP12AE16A clones (lacking GFP) in larval eye discs stained with (A) α-β-Gal to detect diap1-lacZ (red) or (B-C) α-DIAP1 (red). Arrowheads denote position of the MF. (posterior = left). Dotted line in (A) highlights a crb clone that projects posterior to the MF and expresses elevated diap1-lacZ. (D) Optical overlay and (E) size adult wings of the indicated genotypes. (* p<1.5×10^-7 relative to ex⁶⁹⁷ wings).

Discussion

Crb as dual regulator of polarity and growth

Crb nucleates apical membrane formation in the embryonic epidermis and other epithelial cell types in Drosophila. It exerts these effects primarily through two motifs in its intracellular tail: the C-terminal PBM and the juxtamembrane FERM-binding motif (JM). We find that Crb also acts as a peripheral regulator of the SWH pathway in larval discs via a previously unappreciated role for the JM domain in controlling levels of the Ex protein. Known peripheral regulators of the SWH pathway modulate signaling in response to upstream inputs including planar cell polarity pathways, morphogen gradients, and adhesion molecules [8]. Our data extend this theme by suggesting that Crb may serve as an interface between apicobasal polarity signals and the SWH pathway. Overexpression and loss of crb have opposing effects on Ex levels, and sequences in the Crb JM are both necessary and sufficient to control Ex in vivo. The ability of the Crb-JM to deplete Ex from cells suggests that mutations in endocytic genes that block Crb turnover and produce dramatic tissue disorganization and overgrowth (e.g. avl and ept/tsg101) [4, 5] may elicit their phenotypes in part via effects on Ex and SWH signaling. Indeed, vps25 mutants have been shown to downregulate Ex levels [35]. A more complete analysis of the role of Crb and Ex in endocytic tumor mutants is required to understand this link more fully.

The link between crb loss and diap1 expression is at present not clear. Since localization has been suggested to be an important determinant of Ex function [22], our finding that a portion of the excess Ex found in crb cells is displaced basally suggests that the function of this fraction of Ex may be somehow altered or compromised. Prior work showing that loss of the tumor suppressor ft can also mislocalize Ex and compromise its function [36-38] provides precedent for this type of effect, but does not provide insight into what aspect of Ex function might be affected by crb loss. Moreover, since ft alleles elicit far stronger effects on Yki activity than do crb alleles, the consequences of Ex defects in each background would appear to be quite different. Future analysis of the effect of crb loss on the biochemical properties and subcellular localization of Ex may provide insight into this issue.

Ex stability appears to correlate inversely with expression of the Crb JM region, which is known to interact with FERM-domain proteins that are structurally similar to Ex. Attempts to detect a physical interaction between Ex and the Crb intracellular tail using multiple techniques have not been successful (BSR and KHM; unpub.). While this does not preclude a Crb:Ex complex, it does suggest that Crb controls Ex via unidentified intermediates. The modular structure of the Crb protein raises the possibility that factors that interact with the intracellular and extracellular portions of the intact protein may modulate the JM-dependent regulation of Ex. If so, then Crb-dependent changes in Ex levels might couple SWH activity to both changes in intracellular signals as cells begin to polarize their membranes, and to variations in extracellular adhesion during developmental tissue morphogenesis and wound repair. Given the ability of SWH pathway alleles to confer a proliferative advantage in cell competition scenarios [39], it may be that crb that plays a more significant role as a SWH regulator in these types of regenerative and homeostatic growth regulatory programs. Major goals of future studies will therefore be to identify the precise mechanistic details of how Crb controls Ex, and how the intrinsic Ex-regulatory activity of the JM domain is linked to other functional domains of the Crb molecule.

The differential effect of the Crb-JM and Crb-PBM on growth and tissue architecture appears to conflict with a requirement for the JM in rescue of polarity defects in crb mutant embryos [21]. However, this is not without precedent [40] and may be may be explained by the well-documented differences between the roles of Crb in the embryonic epidermis and disc epithelium: loss of Crb disrupts the polarity of embryonic ectoderm and compromises tissue integrity [16], whereas loss of Crb in larval discs has minimal effect on the architecture, polarity, or organization of undifferentiated cells [41].

crb as a growth suppressor

In addition to the circumstantial evidence of a growth advantage conferred by crb alleles,recent work has shown that reduced expression of the murine crb3 gene, a homolog of Drosophila crb, can promote tumorigenicity of kidney epithelial cells and relieve contact inhibition [42]. A pro-proliferative effect of crb loss might seem at odds with recent work showing that loss of crb did not detectably alter overgrowth driven by wts inactivation [43, 44]. In these studies crb was analyzed as a downstream target of the SWH pathway; the data here show that it is also upstream of Ex. Loss of wts is thus predicted to be epistatic to the effects of crb loss on Ex. Rather, our data showing a upregulation of diap1 expression in crb clones indicate that crb alleles might enhance the effects of wts loss, although this would in all likelihood have little effect in the background of wts loss. Rather crb alleles may be more likely to synergize with mutations in other peripheral regulators of the SWH pathway such as mer, which functions redundantly to ex [10, 45].

Multiple polarity links to SWH activity

The link between Crb and Ex reinforces emerging links between polarity control and the SWH pathway. The polarity gene Discs large (Dlg) suppresses tumor growth of ovarian follicle cells via a pathway involving warts but independent of ex, ft, and mer [46]. The polarity factor Scribble can also interact with the Fat2 protein in the developing zebrafish kidney nephron [47]. In an accompanying study to the work presented here, Grzeschik et. al. have found that loss of Drosophila lgl can activate diap1-lacZ; significantly, this occurs without a loss of Ex protein (Fig. S6). In view of the link uncovered here between crb and Ex levels in imaginal epithelial cells, it would thus seem that multiple mechanisms link polarity and the SWH pathway, and that multiple links can exist between apicobasal polarity factors and SWH activity even within a single cell type.

Experimental Procedures

Genetics

Crosses performed at 25°C unless noted. Larval wing discs were harvested from animals kept at 20°C during embryogenesis. Animals were maintained at 20°C for adult wing analysis. Alleles used: UAS-myc-crb^intra; UAS-Ex:GFP; UAS-sav; yki^B5; ban^l(3)05967; sd^ETX4; ex⁶⁹⁷; th^jc58; fj-lacZ; dpp-lacZ; ban-GFP; UAS-ex; FRT82B,crb^11A22; FRT82B,crb^8F105; crb^Y10P12AE16A; UAS-aPKC^CAAX-DN; UAS-cdc42^N17.3; UAS-moe^IR-327-775; UAS-yki-YFP; UAS-crb^IR-1 and UAS-crb^IR-2 (VDRC #39178 and #39177); lgl⁴; E-spl(m)ß-CD2; UAS-app^IR; UAS-crb^JM (also UAS-Myc-Intra^ΔERLI); UAS-crb^PBM (also UAS–Myc–Intra^Y10A/E16A); UAS-crb^ΔΔ (also UAS–Myc–Intra^{Y10A/E16A/ΔERLI}); eyFLP;ubi-GFP,FRT80B; eyFLP;ubi-GFP,FRT82B; eyFLP;Act>CD2>Gal4;Rps17⁴,FRT80B; eyFLP;tub-Gal4;FRT80B,tub-Gal80; w;UAS-GFP,UAS-crbⁱ;FRT80B.

Cell Culture & FACS

S2 cells were cultured under standard conditions. Constructs used: pAc5.1-HA-Ex (G. Halder), pMT-VSV-G-crb-intra and pMT-VSV-G-crb-8F105 (A. Le Bivic). Transfected cells were analyzed 24-36 hours post-transfection (Cellfectin II, Invitrogen); where appropriate, CuSO₄ (0.5mm) was added for the final 12 hours. MG-132 (Sigma) was used at 50μM. Discs were treated with 100μM chloroquine (Sigma) as described previously [48]. Trypsin-dissociated discs were stained with 20 μM DRAQ-5 (Biostatus Limited), analyzed on a BD-LSR II cytometer via a 755 nM laser with a 780/60 nM BP collection filter, and analyzed on FlowJo (TreeStar).

Immunohistochemistry & Immunoblotting

Immunostaining, confocal microscopy, and immunoblotting performed as described previously [49]. Antibodies: mouse α-β-gal 1:1000 (Promega); mouse α-Wg 1:800 (DSHB); rabbit α-yki 1:1000 (K. Irvine); mouse α-HP1 1:20 (DSHB); 1:500 rat α-Crb (H. Bellen); mouse α-Dlg 1:20 (DSHB); rabbit α-GFP (Molecular Probes); guinea pig α-Ex 1:5000 (R. Fehon); rabbit α-Ex 1:200 (K. Irvine); goat α-ß-tub 1:10000 (Santa Cruz); mouse α-HA 1:1000 (Sigma); goat α-VSV-G 1:1000 (Bethyl Labs); mouse α-Arm 1:20 (DSHB); mouse α-DIAP1 1:50 (B. Hay); mouse α-rat CD2 1:100 (Research Diagnostics, Inc.); mouse-α-Notch 1:10 (DSHB, clone 9C6); guinea pig α-Mer 1:7500 (R. Fehon), rabbit α-Fat 1:200 [50] and rat α-Elav 1:200 (DHSB); Alexa-488 phalloidin, 1:100, and YOYO-1, 1:20,000 (Molecular Probes).

Wing/Eye Measurements

Eyes/wings were imaged on a Leica DFC500 CCD camera and quantified with Adobe Photoshop. Posterior compartment ratio (PCR) = posterior compartment size/total wing size.