Fasciclin2 (Fas2) and Discslarge (Dlg) localize to the basolateral junction (BLJ) of Drosophila follicle epithelial cells and inhibit their proliferation and invasion. To identify a BLJ signaling pathway we completed a genomewide screen for mutants that enhance dlg tumorigenesis. We identified two genes that encode known BLJ scaffolding proteins, lethal giant larvae (lgl) and scribble (scrib), and several not previously associated with BLJ function, including warts (wts) and roughened eye (roe), which encode a serine–threonine kinase and a transcription factor, respectively. Like scrib, wts and roe also enhance Fas2 and lgl tumorigenesis. Further, scrib, wts, and roe block border cell migration, and cause noninvasive tumors that resemble dlg partial loss of function, suggesting that the BLJ utilizes Wts signaling to repress EMT and proliferation, but not motility. Apicolateral junction proteins Fat (Ft), Expanded (Ex), and Merlin (Mer) either are not involved in these processes, or have highly spatio-temporally restricted roles, diminishing their significance as upstream inputs to Wts in follicle cells. This is further indicated in that Wts targets, CyclinE and DIAP1, are elevated in Fas2, dlg, lgl, wts, and roe cells, but not Fat, ex, or mer cells. Thus, the BLJ appears to regulate epithelial polarity and dynamics not only as a localized scaffold, but also by communicating signals to the nucleus. Wts may be regulated by distinct junction inputs depending on developmental context.

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EPITHELIA are composed of apical–basal polarized cells that contact each other and surrounding tissues through intercellular junctions. A universal theme is that each type of epithelial junction is organized by a transmembrane receptor bound to a cytoplasmic scaffolding protein, thus enabling assembly of a supermolecular complex of proteins within the cellular cortex at sites of intercellular contact (KNUST 2002). Epithelial junctions are relatively stable in stationary epithelia, but they undergo extensive remodeling when epithelia transform into migratory cells during embryonic development, tissue repair and regeneration, and pathological conditions such as cancer (GRUNERT et al. 2003; SHOOK and KELLER 2003; SZAFRANSKI and GOODE 2004; LARUE and BELLACOSA 2005; THIERY and SLEEMAN 2006; SZAFRANSKI and GOODE 2007). Understanding how epithelial junctions are used for epithelial maintenance and epithelial remodeling during normal development may allow targeting tumor invasion without perturbing normal tissue homeostasis.

We use the Drosophila follicular epithelium as a simple in vivo model to gain insight into the cellular and molecular events that initiate and drive epithelial invasion (GOODE et al. 2005). A Drosophila female has a pair of ovaries. Each ovary has 30–40 tubes called ovarioles in which egg development progresses (a sequence of maturing Drosophila egg chambers within an ovariole is shown in Figure 2A). Each egg chamber is composed of 16 germ cells: 15 nurse cells and 1 oocyte (SPRADLING 1993). A monolayer epithelium of follicle cells surrounds the germ cells. The follicle cells proliferate during the early stages of oogenesis [stages 1–6 (s1–s6); Figure 2A], to keep pace with rapidly growing germ cells. Once follicle cell proliferation ceases, differentiation begins, setting the stage for two migration events during midoogenesis. A small cluster of cells that reside in the anterior epithelium, called border cells (BC), undergo a partial epithelial-mesenchymal transition (EMT), break free from adjacent epithelial cells, and migrate posterior between the nurse cells to the oocyte (Figure 2A) (NIEWIATOMSKA et al. 1999; STARZ-GAIANO and MONTELL 2004; SZAFRANSKI and GOODE 2007). At the same time, most follicle cells that surround the nurse cells move posterior to encapsulate the oocyte (GOODE et al. 2005).

View larger version (47K): In this window In a new window Download PPT slide   FIGURE 2.— Characterization of dlg enhancer phenotypes. A–L are organized horizontally left to right by increasing dlg strength (X chromosome, dlg genotype), and vertically top to bottom by increasing dlg enhancer strength (autosome, enhancer genotype). Strings of maturing egg chambers that are representative of the indicated X chromosome; autosome genotype are shown (arrows point to tumors). (A) Control egg chambers of genotype dlgsw/dlghf; Gla/+ appear wild type. Stage 4 (s4) to s9 egg chambers are evident. Each egg chamber has 16 germ cells, 15 anterior nurse cells, and a posterior oocyte. A follicular epithelium surrounds each egg chamber. A small cluster of cells called border cells exit the anterior epithelium at stage 9 and migrates between the nurse cells to the oocyte. (B) Control egg chambers of genotype dlgip20/dlghf; Dr/+. They have small tumors that invade the germ line starting as early as stage 2 of oogenesis (arrows). (C) Control egg chambers of genotype dlglv55/dlghf; Dr/+. They have intermediate-size tumors that invade more frequently than in dlgip20/dlghf; Dr/+ animals (see text). (D, G, and J) All dlg enhancers cause tumors in dlgsw/dlghf animals that typically do not have tumors (compare to control, A). (E, H, and K) All dlg enhancers increase the frequency and size of tumors in dlgip20/dlghf animals. Whereas tumors are observed in 66% of dlgip20/dlghf ovarioles, they are observed in 100% of dlgip20/dlghf; enhancer/+ ovarioles, even for the weakest enhancers. (F, I, and L) In dlglv55/dlghf; enhancer/+ animals, the tumors become increasingly larger and more aggressive in appearance with increasing dlg enhancer strength, often appearing to consume the entire egg chamber in the most severe cases (L).

 
Each cell within the follicular epithelium has five junctions (TANENTZAPF et al. 2000; SZAFRANSKI and GOODE 2007). An apical junction connects to the germ line, while a basal junction connects to the basement membrane that surrounds each egg chamber. Three lateral junctions interconnect each follicle cell. From the apical to the basal, they are the apicolateral junction (also called the tight junction in vertebrates), the adherens junction, and the basolateral junction (BLJ). Five proteins that localize to the BLJ, Fasciclin2 (Fas2), Neuroglian (Nrg), Discslarge (Dlg), Scribble (Scrib), and Lethal-giant-larvae (Lgl), are conserved in structure and localization in vertebrate epithelia (KNUST and BOSSINGER 2002). Fas2 is a transmembrane protein of the immunoglobulin superfamily (IgSF) (GOODMAN et al. 1997). Dlg is a scaffolding protein composed of three PDZ domains, an SH3 domain, a membrane protein 4.1 binding site, and a GuK domain (WOODS et al. 1996). Dlg creates scaffolds by multimerizing at the BLJ and by using its multiple binding domains to recruit specific combinations of signaling, adhesion, scaffolding, and cytoskeletal molecules (FUNKE et al. 2005). For example, Fas2 directly binds to Dlg PDZ1+2. Nrg, another IgSF member, appears to be connected to the Fas2–Dlg–Lgl scaffold only indirectly through the actin–spectrin cortex (WEI et al. 2004; SZAFRANSKI and GOODE 2007). Scrib, another multi-PDZ domain scaffolding protein, binds to the Dlg GuK domain (MATHEW et al. 2002). Lgl is also a BLJ protein because it precisely colocalizes with Fas2 and Dlg in normal follicle cells or when Fas2 is expressed ectopically (SZAFRANSKI and GOODE 2004), but the intermolecular interactions that bind Lgl to the Fas2–Dlg scaffold remain to be defined.

During normal egg chamber development, Fas2 is expressed at high levels in early proliferating follicle cells. Then, when follicle cells stop dividing and prepare to migrate, Fas2 levels dramatically decrease and Fas2 appears to be completely shut off in border cells, reflecting its motility repressing function. Dlg, Lgl, and Nrg, which also repress movement, are expressed ubiquitously in all follicle cells throughout oogenesis (SZAFRANSKI and GOODE 2007). Thus spatiotemporal patterns of BLJ activity appear to be tightly controlled by spatiotemporal patterns of Fas2 expression, which represses cellular movement until midoogenesis. At that time, Fas2 loss in anterior follicle cells causes a dynamic rearrangement of epithelial junctions that is essential for border cells to undergo partial EMT. The partial EMT involves the process of membrane lateralization, which is constituted by a loss of apical and basal junctions and a redistribution of BLJ proteins around the circumference of the border cells (SZAFRANSKI and GOODE 2007). The EMT is partial because the border cells retain apicolateral and adherens junctions, which provide the intercellular contacts that help to sculpt the stereotypical rosette pattern of the border cell cluster (NIEWIADOMSKA et al. 1999; SZAFRANSKI and GOODE 2007).

Mutation of Fas2, dlg, lgl, or Nrg during early oogenesis causes follicle cells to lose epithelial polarity, undergo EMT, and to invade into the germ line, which in many ways resembles normal border cell migration (GOODE et al. 2005; SZAFRANSKI and GOODE 2007) (see Figure 2). The EMT in these Fas2, dlg, lgl, or Nrg tumor-like cells appears to be more complete than the partial EMT that defines border cell differentiation, because in addition to a lateralized distribution of BLJ proteins, apicolateral and adherens junction proteins also become diffusely redistributed in a lateralized pattern that overlaps with BLJ proteins (SZAFRANSKI and GOODE 2007). Thus, the tumor cells do not form a rosette, but rather invade as streams and clusters of cells that appear disorganized compared to border cells. The importance of Fas2, Nrg, Dlg, and Lgl in regulating intercellular contacts and membrane and cytoskeletal dynamics, suggests that they suppress invasive tumorigenesis via contact inhibition of proliferation and movement (SZAFRANSKI and GOODE 2004).

To migrate, border cells must turn on EMT and motility promoting transcription factors such as Stat and Slbo (MONTELL 2003). Fas2 and dlg tumor cells appear to bypass this normal migration mechanism by prematurely derepressing EMT and a motility pathway that acts through Rac1 (SZAFRANSKI and GOODE 2007). In Fas2 and dlg border cells, derepression of the motility pathway causes faster migration (SZAFRANSKI and GOODE 2004) and in early follicle cells, causes an increased incidence of tumor invasion (SZAFRANSKI and GOODE 2007). Detailed genetic analysis of Fas2 and dlg indicates that derepression of both the EMT and motility pathways are necessary, but neither is sufficient for early tumor invasion. Whereas complete loss of dlg causes EMT and epithelial invasion, some partial loss-of-function dlg mutants cause follicle cells to accumulate in stratified layers without invading (GOODE et al. 2005). In both cases follicle cells have the lateralized signature characteristic of EMT, indicating that EMT is not sufficient for invasion (SZAFRANSKI and GOODE 2007). Likewise, derepression of motility is necessary but not sufficient for tumor invasion, because some partial loss-of-function Fas2 and dlg mutants cause faster border cell migration, but without early epithelial invasion. In these mutants, EMT in the early epithelium remains repressed, so tumor formation and subsequent invasion remain blocked (SZAFRANSKI and GOODE 2007). Thus, the BLJ controls two pathways, one that represses EMT, the other motility, and these pathways function together to repress invasive tumorigenesis (Figure 1A).

View larger version (29K): In this window In a new window Download PPT slide   FIGURE 1.— Summary of the dlg enhancer screen. (A) Model of the BLJ. Transmembrane Fas2 and scaffolding Dlg repress tumor growth and invasion (SZAFRANSKI and GOODE 2007). They repress three genetically separable activities, EMT, motility, and proliferation. EMT results in membrane lateralization (see text for DISCUSSION). (B) Cross scheme to identify dlg enhancers on the second and third chromosomes. A dominant marker (DM) gene, typically Gla on the second chromosome, or Dr on the third chromosome, was used to follow segregation of mutant chromosomes. The Balancer on the first chromosome was FM7a, on the second chromosome was Cyo, and on the third, TM3, Sb. In the first cross, v+Y is an insertion onto the Y chromosome of a fragment of the X chromosome that covers the dlg locus [10B6-10]. v+Y is introduced into the background of each mutant (cross 2, +/v+Y; mutant/DM) to rescue dlghf lethality in dlghf/v+Y; mutant/DM flies in cross 3. The third cross was completed at 18°, the dlghf/dlgip20 permissive temperature, to bypass dlg lethality. Experimental and control animals were recovered at 18°, then shifted to 25° for 72 ± 2 hr. The same scheme was used to construct dlghf/dlgsw; mutant/+ and dlghf/dlglv55; mutant/+ animals (see text). (C) The screen was completed in three phases. In the primary screen, we used a panel of deletions that uncover most autosomal regions (Bloomington deficiency kit, http://flybase.bio.indiana.edu/). In the secondary screen, we further tested each region that enhanced dlg invasion with additional deletions, to confirm or rule out the enhancing interaction and to refine the position of the interacting locus. Finally, we tested mutations located within the delineated locus to identify the enhancing gene. (D) Schematic summary of the primary screen. The figure shows 178 deficiencies assayed in the primary screen. The deficiencies uncover the genetic regions indicated by the width of the boxes. Deficiencies that scored 100% invasion are solid boxes. Deficiencies that did not enhance invasion are open boxes. The cytological limits of each enhancer locus are shown (the breakpoints are defined by the minimal region that must include the enhancing gene as determined by the pattern of overlap between all enhancing deficiencies). The gene identified as the dlg enhancer at each locus is shown.

 
In addition to repressing EMT and movement, BLJs also inhibit follicle cell proliferation. However, overproliferation does not appear to be essential for invasion, since small clusters of Fas2 and dlg cells that resemble a border cell rosette can completely delaminate from the follicular epithelium of early egg chambers and migrate to the oocyte (GOODE et al. 2005; SZAFRANSKI and GOODE 2007). These Fas2 and dlg tumor clusters indicate that the tumor cells are actively migrating, not merely pushed between germ cells as a result of excessive proliferation.

Nonetheless, the most typical pattern of Fas2 and dlg tumor invasion is large streams of cells that remain attached at their point of origin to the follicular epithelium, as the leading edge of the stream moves toward the oocyte (GOODE et al. 2005; see Figure 2). The tumor stream appears to be fed by follicle cell proliferation because invasion of even very large tumor streams typically does not result in a gap in the native epithelium depleted of follicle cells or cause dramatic epithelial thinning (Figure 2; GOODE et al. 2005). In contrast, follicle cells deprived of EGF signals results in epithelial thinning and gaps completely void of follicle cells (GOODE et al. 1996b). Further, BrdU labeling experiments and direct cell counts indicate that excessive proliferation generates the large numbers of cells needed to create dlg tumor streams (see Figure 2) (GOODE and PERRIMON 1997; GOODE et al. 2005). However, overproliferation is insufficient for invasion, as indicated by some hypomorphic dlg mutants that cause EMT and overproliferation, and some hypomorphic Fas2 mutants that cause derepression of motility and overproliferation, neither of which results in tumor invasion (GOODE et al. 2005; SZAFRANSKI and GOODE 2007). Thus, overproliferation is not essential for invasion, but facilitates the process by continually providing new cells that feed the invasive tumor stream. In summary, Fas2, Dlg, and Lgl repress proliferation, EMT, and motility (Figure 1A). Both EMT and motility are necessary, but neither are sufficient for tumor invasion, they cooperate to promote motility. In contrast, proliferation merely facilitates invasion by continually providing new cells for tumor growth.

There is considerable evidence that vertebrate orthologs of Fas2, Dlg, Lgl, and Nrg are also important for suppressing epithelial invasion in mammals and humans (MATSUMINE et al. 1996; FOGAR et al. 1997; ROESLER et al. 1997; HOOVER et al. 1998; PERL et al. 1999; HUANG et al. 2003; SCHIMANSKI et al. 2005; ARLT et al. 2006; GRIFONI et al. 2007; SZAFRANSKI and GOODE 2007). Further, mammalian and human orthologs of dlg and lgl have been shown to rescue the corresponding fly mutants (GRIFONI et al. 2004). Thus, dissecting the mechanisms by which BLJ proteins suppress epithelial invasion in Drosophila may improve our understanding of the mechanisms by which metastasis is initiated during human cancer progression.

Here we aimed at identifying a signaling pathway that acts downstream of the BLJ to inhibit invasive tumorigenesis. We completed a genomewide screen to find loci that caused a dosage-sensitive enhancement of dlg invasive tumorigenesis. We identified two genes, lgl and scrib, that encode scaffolding proteins previously demonstrated to be in a BLJ complex with Dlg, thus providing proof of principle of the screen's efficacy. In addition, we identified three new genes not previously linked to BLJ function, warts (wts), roughened eye (roe), and ebi. wts encodes a serine–threonine kinase that acts as a tumor suppressor in imaginal disc tissues. wts shared several phenotypic similarities with scrib, and enhanced Fas2, dlg, and lgl invasion as strong or stronger than scrib. The unique subset of phenotypes that scrib, wts, and roe share with Fas2, dlg, and lgl indicate that they act specifically in the EMT and proliferation branches of a BLJ pathway and not in the motility-repressing branch. Further, apicolateral junction proteins Fat (Ft), Expanded (Ex), and Merlin (Mer) either are not involved in these processes or have highly spatiotemporally restricted roles, diminishing their significance as upstream inputs to Wts in follicle cells. This is further indicated in that Wts targets, CyclinE and DIAP1, are elevated in Fas2, dlg, lgl, wts, and roe cells, but not in Fat, ex, or mer cells. We propose that Fas2–Dlg–Lgl signaling through Wts to Roe defines a novel BLJ signaling pathway that represses EMT and proliferation. Thus, the BLJ appears to regulate epithelial polarity and dynamics not only by acting as a localized scaffold, but also by communicating signals to the nucleus. Wts appears to be regulated by distinct junctional inputs depending on developmental context.

Stocks and culture conditions:

The following dlg alleles were used in this study: dlg^hf, dlg^sw, dlg^ip20, dlg^lv55, dlg^m35, and dlg^m52 (WOODS and BRYANT 1989, 1991). For the screen and subsequent assays, temperature-sensitive, heteroallelic combinations of dlg were reared at 18° (Figure 1B). Experimental (dlg; Deficiency/+) and control (dlg; Balancer/+) animals were collected within 12 hr of eclosion. The adults were then shifted to the restrictive temperature (25°) for 72 hr to induce tumor invasion (GOODE and PERRIMON 1997). Other crosses were performed at 25°.

Second and third chromosome deficiency kits were obtained from the Bloomington Stock Center (http://flybase.bio.indiana.edu/). For gene identification, a search was completed in FlyBase (http://www.flybase.org/) for all gene mutations within the cytological interval defining the locus. All mutations within and just outside the cytological interval that were linked to a known transcription unit and/or that were lethal or female sterile were tested for enhancement of dlg invasion (see RESULTS for further information). Most mutations described in the text were obtained from the Bloomington Stock Center. Other mutants were obtained from the following labs: scrib mutants (Bilder lab), lgl⁴ and lgl^ts3 (Knoblich lab), and wts^x1 (Halder lab).

For clonal analysis, experimental animals were generated by crossing w; dlg^m52 FRT¹⁰¹/FM7 (GOODE and PERRIMON 1997) and w; Fas2^B112 FRT¹⁰¹/FM7 to hsFLP, lacZ, FRT¹⁰¹ (SZAFRANSKI and GOODE 2004), and yw; lgl⁴ FRT 42D/CyO (MANFRUELLI et al. 1996), w; FRT82B scrib⁶⁷³/TM6 (BILDER and PERRIMON 2000), w; FRT82B wts^x1/TM3 (UDAN et al. 2003), and FRT82B roe² p^p/TM3 (generated from Ki¹ roe² p^p/TM3, see http://www.flybase.org/) to the appropriate FRT GFP chromosome stocks (see http://www.flybase.org/). Late third-instar larvae and early pupae were heat-shocked as described (SZAFRANSKI and GOODE 2004). The following UAS lines were used: UAS-dlg²⁴⁰ (HOUGH et al. 1997), UAS-scrib (MATHEW et al. 2002), UAS-lgl (HUTTERER et al. 2004), UAS-wts³⁷ (LAI et al. 2005), UAS-roe (ST PIERRE et al. 2002), and UAS-ebi (FlyBase). GR1 Gal4 (MANSEAU et al. 1997) is expressed in all follicle cells (SZAFRANSKI and GOODE 2007).

Quantification of tumor invasion:

For the screen, 200 ovarioles were scored for the presence of invasive tumors. The percentage of ovarioles with an invasive tumor was determined. Percentage of invasion positively correlates with severity of tumor invasion in individual egg chambers and with the earliest stage at which tumor invasion is most predominant (Figure 2 and see text). The percentage-of-invasion score provided a simple, reproducible assay to quantify the severity of tumor invasion during the screen. The same assay was used for detailed characterization of some gene interactions (Figure 3A).

View larger version (24K): In this window In a new window Download PPT slide   FIGURE 3.— Quantitative analysis of dlg enhancement and suppression. (A) Frequency of dlgsw/dlghf; enhancer/+ tumor invasion. lgl causes the highest frequency, scrib and wts an intermediate frequency, and ebi and rn the lowest frequency of invasion. All enhancements were significant (**P < 0.01). (B) For all dlgip20/dlghf; enhancer/+ genotypes invasion occurs in 100% of ovarioles (Figure 2). To compare the strengths of enhancement we determined the average tumor size (MATERIALS AND METHODS). Removal of one copy of lgl (dlgip20/dlghf; lgl/+) causes tumors that are four to five times the size of dlgip20/dlghf tumors, while removal of one copy of ebi, rn, scrib, or wts causes tumors that are roughly twice larger (**P < 0.01). (C) Suppression of dlglv55/dlghf tumorigenesis by dlg enhancer overexpression. The Gal4-UAS system (BRAND and PERRIMON 1993) was used to drive overexpression of each UAS-dlg enhancer transgene with GR1-Gal4, which is expressed in all follicle cells throughout oogenesis (SZAFRANSKI and GOODE 2007). dlglv55/dlghf tumorigenesis was most significantly suppressed by dlg, lgl, and wts (**P < 0.01) and to a lesser but still significant degree by scrib and roe (*P < 0.05). ebi showed no significant suppression.

 
However, the percentage-of-invasion assay was inadequate for comparing Fas2 and dlg tumors, or invasive vs. noninvasive Fas2 tumors (Figures 3 and 4). For these experiments, we completed measurements to determine the average tumor size. To control for differences in tumor size due to potential differences in egg chamber size, we normalized the score as follows. A Zeiss Axioplan-2 microscope equipped with a Hamamatsu ORCA digital camera was used to capture a cross-sectional image at the center of 20–30 stage 5–8 follicles for each genotype. We then used Axiovision 3.1 (Carl Zeiss Vision) to measure (1) the area that includes all tumor cells, and (2) the egg chamber area. Dividing the average tumor area by the average egg chamber area normalized the average tumor area. The normalized, average tumor area is referred to as the average tumor size (Figures 3 and 4).

View larger version (30K): In this window In a new window Download PPT slide   FIGURE 4.— Genetic interaction of dlg enhancers with Fas2null. (A) Fas2 tumors invade into the germ line (arrows). (B) Fas2; wts/+ tumors are larger, but are typically noninvasive (arrows). All Fas2; enhancer/+ animals have the same pattern of noninvasive tumors. Occasionally a few isolated cells that have broken from the main tumor are observed between the germ cells (arrowheads). (C) Tumor scores for Fas2; enhancer/+ egg chambers. All dlg enhancers enhanced Fas2 6- to 10-fold, with the exception of scrib, which showed a 2-fold enhancement (**P < 0.01; *P < 0.05). (D) Suppression of Fas2 tumorigenesis by overexpression of dlg enhancers. Overexpression was accomplished as described in Figure 3C. All but ebi showed significant suppression of Fas2 tumorigenesis.

 

Immunofluoresence and imaging:

Ovaries were fixed for 20 min in 4% formaldehyde in PBS. For the screen, ovarioles were teased apart using tungsten needles to keep the egg chambers within each ovariole together, so that tumor invasion could be scored as percentage of ovarioles with tumors (see above). Egg chambers were visualized by staining with Alexa⁴⁸⁸ phalloidin (1:10; Molecular Probes, Eugene, OR). The following primary antibodies were used: rabbit anti-β-gal (1:2000; Cappel), rabbit anti-PH3 (1:1000; Upstate Biotechnology, Cleveland), mouse anti-BrdU (1:250; GE Healthcare), mouse anti-CycE (1:1000; Helena Richardson lab) (RICHARDSON et al. 1995), and mouse anti-DIAP1 (1:200; Bruce Hay lab) (YOO et al. 2002). All images were captured using a Zeiss LSM510 laser scanning confocal microscope and processed using Adobe Photoshop.

For BrdU labeling experiments, ovaries were dissected into Shields and Sang M3 Insect Medium (Sigma, St. Louis), then transferred to the same media containing 0.5 mg/ml BrdU labeling solution (Sigma), and incubated for 1 hr. The ovaries were then fixed and processed for PI and BrdU staining as previously described (GOODE and PERRIMON 1997). To determine differences in the number of BrdU⁺ cells in clones, confocal miscroscopy was used to obtain a cross section from follicles that had an approximately equal area of clone vs. nonclone cells. The total number of propidium iodide- and BrdU-labeled cells in the clone vs. nonclone areas was ascertained. From this, the ratio of BrdU to propidium iodide positive cells was calculated. Ten or more egg chambers were assayed per genotype. The same procedure was used to measure PH3 and CycE.

Statistical analysis:

SPSS v12.0 for Windows was used for all statistical analyses. Error bars were calculated as the standard error of the mean. P-values were calculated by applying an independent sample t-test.

dlg enhancer screen:

Our goal was to identify a BLJ signaling pathway that controls epithelial tumorigenesis and invasion. We chose to screen for enhancers of dlg tumor invasion because Dlg is the predominant scaffolding protein that organizes the BLJ, and because dlg invasion is simple to score. To identify a genetic background to screen for enhancers of dlg invasive tumorigenesis, we characterized three dlg mutant combinations, dlg^hf/dlg^sw, dlg^hf/dlg^ip20, and dlg^hf/dlg^lv55. dlg^hf is a temperature-sensitive allele that permits dlg^hf/dlg^ip20 and dlg^hf/dlg^lv55 animals to develop at 18°, thus bypassing dlg lethality at 25° (dlg^hf/dlg^sw animals are viable at 25°) (GOODE and PERRIMON 1997). Once dlg^hf/dlg^ip20 and dlg^hf/dlg^lv55 animals reached adulthood, they were shifted to 25° for 48 ± 6 hr to analyze their ovarian phenotypes. To quantify the strength of invasion in dlg^hf/dlg^sw, dlg^hf/dlg^ip20, and dlg^hf/dlg^lv55 animals, we scored the percentage of ovarioles with invasive tumors (MATERIALS AND METHODS). Approximately 0.5% of ovarioles from dlg^hf/dlg^sw developed very tiny tumors, most appearing wild type (Figure 2A). Sixty-three percent of ovarioles from dlg^hf/dlg^ip20 mutants had small- to intermediate-sized invasive tumors, most originated by stages 4–6 (Figure 2B). Ninety-nine percent of ovarioles from dlg^hf/dlg^lv55 mutants had large invasive tumors that originated predominantly during stages 1–3 (Figure 2C). dlg^sw, dlg^ip20, and dlg^lv55 had increasingly large carboxyl-terminal deletions of the Dlg GuK domain (WOODS and BRYANT 1989, 1991), thus the severity of invasive tumorigenesis correlates with increasing GuK truncation. On the basis of these data, we reasoned that the weak- to intermediate-strength phenotypes observed in dlg^hf/dlg^ip20 ovaries would be readily modifiable, and thus ideal for a screen.

We used an F₃ cross scheme to construct dlg^hf/dlg^ip20; Deficiency/+ animals (Figure 1B). Although the F₃ cross scheme is relatively laborious to execute, the benefit of the scheme is that enhancement of invasive tumorigenesis was assayed relatively directly, as opposed to lethality, polarity, proliferation, or another phenotype that may or may not be important for invasion. For the primary screen we assayed the Bloomington deficiency kit (Figure 1C) (MATERIALS AND METHODS). The kit uncovers 80% of the second and third chromosomes (Figure 1D), which corresponds to 64% of the Drosophila genome. We identified 18 nonoverlapping deficiencies that increased the fraction of ovarioles with egg chambers having tumors from 63 to 97–100% (MATERIALS AND METHODS).

To determine which of these 18 loci contain an enhancer of dlg invasive tumorigenesis, we completed a secondary screen with additional deficiencies that overlap these candidate loci (Figure 1C). Ten of the 18 loci were ruled out because overlapping deficiencies scored <80%. In addition, we did not further test the region defined by Df(2L)Kr14 (60F2; 60F5), which scored 97%, because overlapping deficiencies were not available. As summarized below, at the remaining 7 loci overlapping deficiencies and mutations scored 100%. The results indicate that we could reliably identify a dlg enhancer in the primary screen when 100% of ovarioles in dlg^hf/dlg^ip20; deficiency/+ ovaries had egg chambers with invasive tumors.

To identify the enhancer genes within the seven confirmed deficiency intervals, all available lethal, female sterile, and visible mutations within each region were screened (MATERIALS AND METHODS). Candidate enhancer genes were identified according to the following criteria: (1) gene mutations failed to complement all deficiencies defining the locus, (2) both tumor penetrance and size were quantitatively similar to deficiencies defining the locus, (3) more than one mutant allele of each enhancer gene enhanced invasion, and (4) enhancement was observed in three dlg backgrounds, dlg^hf/dlg^sw, dlg^hf/dlg^ip20, and dlg^hf/dlg ^lv55. As described below, we used these criteria to identify a dlg enhancer gene at five of the seven enhancing loci.

Identification of dlg enhancers:

A summary of the screen that led to identification of five dlg enhancing genes is presented below. The genes are listed in order of strength with which they enhanced dlg tumorigenesis. The quantity of enhancement was assayed in several dlg backgrounds to help verify that the strength of functional interdependence between dlg and each dlg-enhancer was a consistent trait. The relative strength of the interactions is important because it provides an initial indicator of the relative participation of each interacting gene in dlg pathways. For example, a strong interaction might indicate that the gene is involved in all three dlg pathways that repress proliferation, EMT, and motility (see Introduction) (Figure 1A). A moderate or weak interaction may indicate that the gene is required either predominantly in only one or two dlg pathways or partially in all three pathways. These putative distinctions were verified by subsequent analysis.

lgl:

Nine overlapping deficiencies, spanning from 21A1 to 21B4 (Df(2L)PM4, Df(2L)net18, Df(2L)PM44, Df(2L)net62, Df(2L)PM51, Df(2L)TE21A, Df(2L)PMG, Df(2L)PM45, and Df(2L)net-PMF), caused 100% of dlg^hf/dlg ^ip20 ovarioles to have one or more egg chambers with tumors. A null mutation of lgl, lgl⁴ (MANFRUELLI et al. 1996), failed to complement all of these deficiencies. lgl⁴, but not mutations in five other genes uncovered by these deficiencies, enhanced dlg^hf/dlg^ip20 invasion with similar penetrance and expressivity compared to deficiencies (Figures 2K and 3, A and B). To confirm and characterize enhancement in more detail, we tested both the deficiencies and the lgl⁴ mutation in dlg^hf/dlg^sw egg chambers, which typically appear like wild type (Figure 2A). dlg^hf/dlg^sw; Df(2L)PM44/+, dlg^hf/dlg^sw; Df(2L)PMG/+, and dlg^hf/dlg^sw; Df(2L)PM45/+ animals produce tumors in 100, 91, and 72% of ovarioles, respectively, comparable to the 80% observed in dlg^hf/dlg^sw; lgl⁴/+ animals (Figure 2J). Further, all three deficiencies and lgl⁴ dramatically enhanced dlg^hf/dlg^lv55 invasive tumorigenesis, causing tumors that appear to engulf the entire egg chamber, the strongest phenotype observed in the screen (Figure 2L).

To further test lgl, we assayed the temperature-sensitive mutant lgl^ts3, and the hypomorphic mutant lgl^KG05323 (MANFRUELLI et al. 1996). Importantly, lgl^ts3 produced tumors in 45% of dlg^hf/dlg^sw ovarioles at the restrictive temperature, 29°, but no tumors were found at 25°, the permissive temperature. The temperature conditional enhancement of dlg invasion provides strong confirming evidence that lgl is the responsible dlg enhancer at this locus. Further, lgl^KG05323 enhanced invasion in all three dlg backgrounds. The penetrance in dlg^hf/dlg^sw egg chambers was less than the null mutant lgl⁴ (16% of ovarioles compared to 80%), as expected for a partial loss-of-function mutation.

We conclude that enhancement of dlg invasion by several lgl alleles, and the similar enhancing scores of lgl⁴ compared to deficiencies of the locus, in multiple dlg genetic backgrounds, indicates that lgl is the gene primarily responsible for enhancement at this locus. Consistent with this, Lgl colocalizes with Dlg at the BLJ, and lgl has been placed in a dlg functional network on the basis of similarity of phenotype in several tissues, a common requirement for suppressing tumorigenesis, and genetic interactions with dlg (BILDER et al. 2000; ALBERTSON and DOE 2003; SZAFRANSKI and GOODE 2007). lgl was the strongest dlg enhancer that we observed, suggesting that it is involved in all three dlg functions of repressing proliferation, EMT, and motility (Figure 1A), borne out in subsequent analysis (see below). Identification of lgl as an invasion enhancer provided strong evidence for the utility of the screen and indicated that additional components of a dlg functional network could be identified using this approach.

wts:

This region was identified in the primary screen by Df(3R)tll-g/+, which caused 100% of ovarioles to have egg chambers with invasive tumors. Two small overlapping deficiencies, Df(3R)Exel8194 and Df(3R)Exel9020, confirmed and refined the position of this putative enhancer locus to 100A4–100A5 (PARKS et al. 2004). These two deficiencies caused 37 and 30% of dlg^hf/dlg^sw ovarioles to have tumors, respectively. Df(3R)Exel9020 removes 38.5 kb (PARKS et al. 2004). The only genes deleted by this deficiency are zfh1, which encodes a zinc-finger homeodomain protein involved in mesodermal patterning (POSTIGO et al. 1999), and warts (wts), a tumor suppressor gene (JUSTICE et al. 1995). Two wts mutations, but not zfh1, enhanced invasive tumorigenesis in all three dlg backgrounds (Figure 2, G–I, M, and N). These two mutations failed to complement all three deficiencies defining this region, demonstrating that wts was the dlg enhancer at this locus. The null mutant wts^x1, and the partial loss-of-function allele wts^P4, caused 48 and 35% of dlg^hf/dlg^sw ovarioles to have tumors, comparable to deficiencies (Figure 3A). The strength of the wts interaction with dlg^hf/dlg^sw was about half that of lgl, which was further verified in the dlg^hf/dlg^ip20 and dlg^hf/dlg^lv55 backgrounds (Figure 3B and data not shown). The weaker interaction of wts with dlg when compared to lgl indicates that wts is required either in all three dlg pathways, but to a lesser degree than lgl, or in only one or two dlg pathways essential for repressing invasive tumorigenesis (Figure 1A). The wts locus encodes a kinase that is a potent tumor suppressor in imaginal tissues (JUSTICE et al. 1995), suggesting that it may relay signals from the BLJ. Below we present experiments to test this hypothesis, and to determine in which dlg pathways wts is involved in.

scrib:

This region was identified by Df(3R)Tl-1, which caused 100% of dlg^hf/dlg^ip20 ovarioles to have egg chambers with invasive tumors. No overlapping deficiencies were available. Df(3R)Tl-1 caused 32% of dlg^hf/dlg^sw ovarioles to have invasive tumors and strongly enhanced tumor invasion in dlg^hf/dlg^lv55 egg chambers, with penetrance and phenotypic patterns most similar to wts (Figure 3, A and B). To identify the enhancer gene, six lethal mutations in five genes were analyzed. scrib⁶⁷³ and scrib¹ (BILDER et al. 2000), but not mutations in the other genes, enhanced invasive tumorigenesis in all three dlg backgrounds (Figures 2, G–I and 3, A and B). scrib⁶⁷³ and scrib¹ caused invasion in 27 and 30% in dlg^hf/dlg^sw ovarioles, much like Df(3R)Tl-1 (Figure 3A), and both mutations failed to complement Df(3R)Tl-1. The similar invasion score of Df(3R)Tl-1 and scrib mutations, in all three dlg backgrounds, indicates that scrib is the dlg enhancer gene at this locus. Consistent with this, scrib alone has ovarian phenotypes that resemble dlg (BILDER et al. 2000). Further, dlg, lgl, and scrib genetically interact, indicating that they are components of functional network (BILDER et al. 2000; PENG et al. 2000; ALBERTSON and DOE 2003). However, our analysis indicates that scrib is not functionally equivalent to lgl in regards to dlg function, since the tumor scores for scrib were roughly a third to a half of those observed for lgl (Figure 3, A and B). Thus, as for wts, the weaker interaction of scrib with dlg when compared to lgl indicates that scrib is required either for all three dlg pathways, but to a lesser degree than lgl, or in only one or two dlg pathways essential for repressing invasive tumorigenesis (Figure 1A). Scrib is a scaffolding protein that has four PDZ domains and 16 leucine-rich repeats and colocalizes with Dlg and Lgl in BLJs (BILDER and PERRIMON 2000). Thus, along with lgl, identification of scrib provided proof of principle that the screen was successfully identifying crucial components of a BLJ network.

roe:

Three overlapping deficiencies, Df(3R)dsx2M, Df(3R)dsx29, and Df(3R)dsx37, caused 100% of dlg^hf/dlg^ip20 ovarioles to have egg chambers with invasive tumors. These deficiencies are predicted to remove sequences in region 84D8–84E1. Df(3R)dsx2M and Df(3R)dsx37 caused 3% of dlg^hf/dlg^sw ovarioles to have tumors and strongly enhanced dlg^hf/dlg^lv55 invasion. To identify the enhancer gene, five mutations in four genes were analyzed. roe¹ and roe² (ST PIERRE et al. 2002), but not mutations in the other genes, enhanced invasion in all three dlg backgrounds (Figures 2, J–L and 3, A and B), and failed to complement all three deficiencies, indicating that roe is the gene responsible for enhancement at this locus. roe¹ and roe² caused 15 and 9% of dlg^hf/dlg^sw ovarioles to have tumors, three to five times greater than Df(3R)dsx2M and Df(3R)dsx37. roe¹ and roe² may cause greater enhancement compared to the deficiencies if another gene resides within the deficiencies that acts as a dlg suppressor; alternatively, there may be a difference in genetic backgrounds. By several criteria, roe was a mild enhancer compared to lgl, scrib, and wts (Figure 3A and see below), but by other assays appeared similar to scrib and wts (Figure 3B and see below). Further analysis indicates that like wts and scrib, roe does not act in all branches of BLJ signaling (see below) (Figure 1A). roe encodes a Krüppel family of zinc-finger transcription factors (ST PIERRE et al. 2002), which combined with its functional overlap with wts and scrib in controlling EMT and proliferation, suggests that it might be a target for BLJ signals in the nucleus. However, the strength of the interaction was not as strong as for wts and scrib, indicating that it may be only one of several targets.

ebi:

One deficiency, Df(2L)al, caused 100% of dlg^hf/dlg^ip20 ovarioles to have egg chambers with invasive tumors. No overlapping deficiencies were available to confirm or refine this locus. However, Df(2L)al caused 8% of dlg^hf/dlg^sw ovarioles to have tumors, and significantly enhanced dlg^hf/dlg^lv55 invasion, with penetrance and phenotypic patterns that resemble those seen at other mildly enhancing loci (Figures 2, D–F, and 3, A and B). Df(2L)al is predicted to remove sequences from 21B8–21D1. We examined 15 lethal mutations in 14 genes in this region. Two ebi mutations, ebi^k16213 and ebi^WKS.24 (TSUDA et al. 2002), failed to complement Df(2L)al and both enhanced invasion in all three dlg backgrounds (Figures 2, D–F, and 3, A and B). The comparable enhancement of ebi and Df(2L)al, in multiple dlg genetic backgrounds, indicates that ebi is the gene primarily responsible for enhancement at this locus. ebi encodes for a nuclear ubiquitin ligase implicated in Notch signaling (TSUDA et al. 2002). Thus, like Roe, Ebi may be another target for BLJ signals in the nucleus. The strength of the ebi interaction with dlg was comparable to roe, but we did not observe any ebi ovarian phenotypes (see below), suggesting that ebi may only participate in one of three BLJ pathways.

Two additional dlg enhancer loci:

Df(2L)ed1 removes sequences between 24A2 and 24D4 and caused 100% of dlg^hf/dlg^ip20 ovarioles to have egg chambers with invasive tumors (Figure 1D). No overlapping deficiencies were available to confirm or refine this locus. Df(2L)ed1 did not cause invasion in dlg^hf/dlg^sw egg chambers, but significantly enhanced invasion in the dlg^hf/dlg^lv55 background. To identify the enhancer gene at the 24A2; 24D4 locus, mutations in cappuccino, a gene required for cell polarity, were assayed (MANSEAU and SCHÜPBACH 1989). No enhancement of the dlg phenotype was detected.

The other enhancing locus was located between 90D2 and 90D6 (Figure 1D), defined by overlapping deficiencies Df(3R)RD31 and Df(3R)DG4. Df(3R)RD31 caused 2% of dlg^hf/dlg^sw ovarioles to have egg chambers with invasive tumors, but Df(3R)DG4 showed no effect. Both deficiencies caused 100% of dlg^hf/dlg^ip20 and dlg^hf/dlg^lv55 ovarioles to have tumors, with similar phenotypes to those observed for other loci. One lethal mutation was examined in the 90D2–90D6 region, but no enhancement was observed. Thus, we were unable to identify the enhancing gene at this or the Df(2L)ed1 locus, both of which were the weakest enhancers observed in the screen. For these reasons we did not analyze these loci further.

Fas2 tumorigenesis is also enhanced by lgl, scrib, wts, roe, and ebi:

Fas2 is a transmembrane protein that precisely colocalizes with Dlg in the BLJ via binding of its intracellular domain to Dlg PDZ1+2 (SZAFRANSKI and GOODE 2007). Our functional analysis suggests a model in which Fas2 intercellular interactions mediate contact inhibition of proliferation and movement by regulating Dlg protein conformations in the cellular cortex, which may in turn determine which factors associate with the BLJ and thus become active. To obtain additional evidence that lgl, scrib, wts, roe, and ebi function in a BLJ pathway, we tested if they also enhance Fas2 phenotypes. For these experiments we used Fas2^null, which causes invasive tumors (Figure 4A). Fas2^null completely eliminates Fas2, so it might be expected that lgl, scrib, wts, roe, and ebi would not enhance Fas2^null if they function in the same pathway as Fas2. However, because Dlg has multiple binding domains, putatively binding many proteins, it seemed more likely that Fas2 regulates only a fraction of Dlg activity, and thus Fas2 null mutations are functionally equivalent to a dlg hypomorph. To test this, we compared the average tumor size for Fas2^null to the weak- to intermediate-strength mutant combination dlg^hf/dlg^ip20 (see MATERIALS AND METHODS). Fas2^null scored 7 (Figure 4C) and dlg^hf/dlg^ip20 scored 50 (Figure 3B). The data confirm that Fas2 can only account for a relatively small portion of Dlg tumor suppressing function. Thus, Fas2^null tumorigenesis is a reasonable assay to measure enhancement by lgl, wts, scrib, roe, or ebi.

When one copy of lgl, scrib, wts, roe, and ebi is removed in Fas2^null egg chambers, the resulting tumors are significantly larger compared to Fas2^null alone (Figure 4B, C). lgl caused an 10-fold increase in Fas2^null tumor size, wts a 7- to 8-fold increase, ebi and roe a 6- to 7-fold increase, and scrib a modest 2-fold increase (Figure 4C). We were surprised that scrib enhanced Fas2 tumors 3- to 5-fold less than lgl, wts, roe, or ebi, so we repeated the experiment several times with scrib⁶⁷³, as well as with an additional allele, scrib¹, with the same result. Another surprising finding was that whereas Fas2^null alone often caused invasive tumors (Figure 4A), removal of one copy of lgl, wts, scrib, roe, or ebi caused all tumors to become predominantly noninvasive (Figure 4B). We consider possible insights into BLJ function that these two surprising findings suggest in the DISCUSSION.

We conclude that lgl was the strongest Fas2 enhancer, and with the exception of scrib, the remaining enhancers showed a similar, but less severe increase in tumor size compared to lgl, much like the trend of enhancement observed for dlg in dlg^hf/dlg^ip20 egg chambers (Figure 3B). The data thus further indicate that lgl, scrib, wts, roe, and ebi are critical for mediating BLJ suppression of invasive tumorigenesis.

lgl invasive tumorigenesis is unmasked in interactions with Fas2, dlg, scrib, wts, and roe:

Lgl is another protein that is localized to the BLJ and that suppresses epithelial tumorigenesis (SZAFRANSKI and GOODE 2007). To obtain further evidence that Fas2, dlg, scrib, wts, and roe function in a BLJ network, we tested if they also genetically enhance lgl. Approximately 10% of lgl tumors are invasive, while the remaining 90% have a noninvasive phenotype in which follicle cells pile up at the poles of the egg chamber (Figure 5), resembling dlg hypomorphic mutant combinations (Figure 6). The relatively low frequency of invasive tumors in lgl mutants suggests that the branch of the BLJ pathway that represses cellular motility (Figure 1A) is not as strongly derepressed as in dlg mutants. This is puzzling because lgl loss causes faster border cell migration, much like Fas2 and dlg loss (SZAFRANSKI and GOODE 2004), thus suggesting the conflicting view that the branch of the BLJ pathway that represses motility has been disrupted in lgl mutants. An important observation that might help to rectify this discrepancy is that for Fas2 and dlg, loss of the motility-repressing branch of the BLJ pathway alone is not sufficient for tumor invasion, but must be lost together with EMT for invasion to occur (Figure 1A) (SZAFRANSKI and GOODE 2007). Thus, lgl tumors may have a latent invasive capacity, but fail to express it more fully because some aspect of EMT crucial for movement remains repressed.

View larger version (73K): In this window In a new window Download PPT slide   FIGURE 5.— Fas2, dlg, scrib, wts, and roe enhance lgl. (A) At 25° all lgl4/lglts3 egg chambers appear wild type. (B) At 29° all lgl4/lglts3 ovarioles have one or more egg chambers with tumors. Approximately 90% of the tumors accumulate at the poles (arrows). Approximately 8% of the tumors are invasive (I). (C) dlgm52/+; lgl4/lglts3 egg chambers develop tumors at the 25° permissive temperature. Approximately 60% of these tumors are invasive (arrow, I). (D) At 29° most dlgm52/+; lgl4/lglts3 egg chambers have noninvasive tumors that are larger than the tumors in lgl4/lglts3 (arrows, compare to B). However, invasive tumors are observed at a low frequency, as for lgl4/lglts3. (E) Fas2/+; lgl4/lglts3 egg chambers develop tumors at 25°. One hundred percent of these tumors are invasive (I) and some break away from the epithelium and migrate much like a border cell cluster (arrow). (F) Fas2/+; lgl4/lglts3 egg chambers at 29° causes noninvasive tumors that are typically larger than the tumors in lgl4/lglts3 at 29° alone (arrows, compare to B). However, invasive tumors are observed at a low frequency, as in lgl4/lglts3 egg chambers. (G) lgl4/lglts3; wtsX1/+ egg chambers develop invasive tumors at 25°. Approximately 40% of tumors are invasive (arrow). (H) lgl4/lglts3; wtsX1/+ egg chambers at 29° causes noninvasive tumors that are larger than the tumors in lgl4/lglts3 at 29° alone (arrows). However, invasive tumors are observed at a low frequency, as in lgl4/lglts3 egg chambers. scrib and roe showed a similar pattern of interactions (I).

 

View larger version (66K): In this window In a new window Download PPT slide   FIGURE 6.— Clonal analysis of wts, scrib, roe, ft, and mer; ex. Egg chambers are stained with phalloidin to reveal actin in cellular cortexes (red). (A) Tumor cells accumulate in stratified layers at the anterior and posterior poles of dlgm35/dlgip20 egg chambers, but they do not invade. The tumor cells delaminate toward the germ line and have a rounded, mesenchymal-like morphology. (B–J) Clonal analysis of roe, wts, scrib, ft, and mer; ex. Cells without lacZ or GFP (green) are homozygous mutant for the indicated mutations. (B) roe2 causes small, noninvasive tumors that delaminate toward the germ line (arrow). Most tumors accumulate around the oocyte, but occasionally at the anterior of the egg chamber as well. More typically, anterior roe2 cells remain in the epithelium but have a rounded morphology (arrowheads), like tumor cells that have completely delaminated. (C and D) wtsX1 and scrib673 tumors are similar to dlgm35/dlgip20 tumors. (E–H) Analysis of BLJ proteins -Spectrin and Dlg in wts and scrib follicle cells. -Spectrin and Dlg are localized to the BLJ in the native epithelium (arrows). -Spectrin and Dlg accumulate around the circumference of wts and scrib tumor cells (arrowheads). This lateralized phenotype is indicative of EMT (see text). (I and J) Clones of ft or mer; ex double mutant cells do not develop tumors.

 
To test this, we asked if removal of one copy of dlg would cause lgl tumors to become invasive. We used animals heterozygous for the temperature-sensitive mutant lgl^ts3 and the null mutation lgl⁴ (MANFRUELLI et al. 1996). At the 25° permissive temperature, lgl⁴/lgl^ts3 egg chambers were morphologically indistinguishable from wild type. At the 29° restrictive temperature, mostly noninvasive tumors accumulated at the poles of lgl⁴/lgl^ts3 egg chambers (Figure 5, A and B). The phenotype was similar to the null mutant lgl⁴ (not shown). When one copy of dlg was removed in lgl⁴/lgl^ts3 egg chambers (dlg^m52/+; lgl⁴/lgl^ts3) at the 25° permissive temperature, invasive tumors frequently developed (Figure 5, C and I). No tumors were observed in the control FM7/+; lgl⁴/lgl^ts3 egg chambers from their sisters. When we completed the same experiment at the lgl restrictive temperature (29°), we found the tumors were larger than lgl⁴/lgl^ts3 tumors, but surprisingly were mostly noninvasive, much as for lgl⁴/lgl^ts3 alone (Figure 5I).

The data thus confirms that lgl cells have a latent invasive capacity. However, support for the hypothesis that lgl is continuing to repress a function crucial for EMT, thus preventing lgl cells from becoming invasive, is less clear. If the latter were true, we would expect to observe invasive tumors at both the lgl permissive and restrictive temperatures. Instead, dlg enhances lgl⁴/lgl^ts3 tumor size at the restrictive temperature, without enhancement of invasion. One possibility is that lgl loss alone may cause a critical imbalance between the three BLJ pathways that repress proliferation, EMT, and motility (Figure 1A), which favors proliferation and EMT, while at the same time precluding the choice to migrate. In contrast, at the lgl permissive temperature, all three pathways are close to a threshold level, so that removal of one copy of dlg allows all three BLJ pathways to operate together.

To further test if lgl cells have a latent invasive capacity, we asked if Fas2 and scrib would also enhance lgl⁴/lgl^ts3. As for dlg^m52, removal of one copy of Fas2 or scrib caused de novo invasive tumors at the lgl⁴/lgl^ts3 permissive temperature, but noninvasive tumors predominated at the restrictive temperature (Figure 5, E and F), further indicating that strong loss of lgl somehow precludes expression of a latent invasive capacity in lgl cells. The number of tumors observed in Fas2^null/+; lgl⁴/lgl^ts3 egg chambers was 10% of that observed in dlg^m52/+; lgl⁴/lgl^ts3 egg chambers, as expected because Fas2^null behaves like a dlg hypomorph (see previous section). However, in contrast to dlg and scrib, 100% of Fas2^null/+; lgl⁴/lgl^ts3 tumors were invasive, with some breaking away from the epithelium and migrating much like a border cell cluster (Figure 5, E, F, and I). The cluster phenotype is especially interesting because Fas2 is the only epithelial junction protein that we have found to be lost in the anterior epithelium at the time of border cell migration, a morphogenetic switch that is essential for permitting border cell movement (SZAFRANSKI and GOODE 2007). Thus Fas2 homophilic adhesion may be especially important for promoting follicle cell attachments that inhibit cluster delamination.

Since removal of one copy of Fas2, dlg, or scrib causes de novo invasive tumor formation in lgl⁴/lgl^ts3 animals at the permissive temperature, we reasoned that this would be an excellent assay to test if wts and roe are truly components of a BLJ network. We were particularly interested in wts, because by several assays it enhanced Fas2 and dlg as strong, or stronger than, scrib (Figures 3B and 4C). Further, wts encodes a kinase known to be a potent tumor suppressor in other tissues (JUSTICE et al. 1995). Thus one possibility was that Wts relays signals from the BLJ to Roe in the nucleus that are important for suppressing epithelial invasion. If this was true, then removal of one copy of wts or roe would be expected to cause de novo tumor formation in lgl⁴/lgl^ts3 egg chambers at the permissive temperature. The data supported this hypothesis. Removal of one copy of wts or roe caused invasive tumors in lgl⁴/lgl^ts3 animals at the permissive temperature (Figure 5E), and gave similar results to removal of one copy of Fas2, dlg, or scrib at the 29° restrictive temperature (Figure 5F).

The de novo generation of invasive lgl tumors by removal of one copy of scrib, wts, and roe are especially meaningful because analysis described below indicates that scrib, wts, and roe are required to repress EMT and proliferation but not motility. The data thus support the model whereby lgl cells have a latent invasive capacity that is masked by a crucial EMT function that continues to be expressed in lgl cells, but is unmasked when scrib, wts, or roe are reduced. Further, enhancement of Fas2, dlg, and lgl tumorigenesis by wts/+ and roe/+, as well as de novo formation of invasive tumors in dlg^hf/dlg^sw; wts/+, lgl^ts3/lgl⁴; wts/+, dlg^hf/dlg^sw; roe/+, and lgl^ts3/lgl⁴; roe/+ egg chambers, is consistent with the hypothesis that Wts acts to relay signals from the BLJ to Roe in the nucleus that are important for suppressing invasive tumorigenesis.

Fas2 and dlg are suppressed by overexpression of lgl, scrib, wts, and roe:

If the lgl, scrib, wts, roe, and ebi function in a BLJ pathway, then we might expect that their overexpression would have the opposite impact on Fas2 and dlg tumorigenesis as removal of one copy. To test this, we used the UAS-Gal4 system (BRAND and PERRIMON 1993) to overexpress lgl, scrib, wts, roe, and ebi in Fas^null and in an intermediate dlg tumor background, dlg^hf/dlg^lv55 (Figures 2C and 4A). We used GR1 Gal4 to drive UAS enhancer gene expression in all follicle cells (SZAFRANSKI and GOODE 2007). We found that overexpression of all of the dlg enhancer genes except ebi suppressed Fas^null and dlg^hf/dlg^lv55 tumorigenesis (Figures 3C and 4D). UAS lgl and UAS wts were the strongest suppressors, suppressing at a level comparable to rescue by UAS dlg (Figure 3C). The results further support that lgl, scrib, wts, and roe function in a BLJ network with Fas2 and dlg to suppress tumorigenesis. ebi may function in a parallel pathway, or perhaps in only one of the three branches of the BLJ pathway (Figure 1A).

Clonal analysis:

Previous work has shown that dlg, lgl, and scrib have similar ovarian tumor phenotypes, which combined with genetic interaction data, suggested that they functionally cooperate to control polarity and proliferation (BILDER et al. 2000; PENG et al. 2000; ALBERTSON and DOE 2003; SZAFRANSKI and GOODE 2007). However, as described in the above analysis, lgl interacts with dlg stronger than scrib, suggesting that scrib either is not required in all three dlg pathways (Figure 1A) or is required to a lesser degree in all three pathways compared to lgl. Similar reasoning applies to wts, roe, and ebi. To help distinguish these alternatives, we used clonal analysis to compare the cell and tissue phenotypes of Fas2, dlg, and lgl to scrib, wts, roe, and ebi. We found that wts and scrib egg chambers had a very similar phenotype to dlg mutants that specifically disrupt the Dlg SH3 and GuK domains. Supernumerary follicle cells accumulate in stratified layers at the poles of the egg chamber, starting as early as stage 2 of oogenesis (Figure 6, A–D). Like dlg and scrib cells, wts cells often delaminate toward the germ line and have gross aberrations in epithelial polarity (Figure 6, A–D). The genetic interactions (previous sections), and similarities between the dlg and scrib phenotypes when compared to wts, indicate that these genes are functionally interdependent. However, complete loss of Fas2 or dlg not only causes overproliferation and EMT, but also invasion of follicle cells between germ cells (Figures 2 and 4), a phenotype never observed in wts or scrib clones. Thus scrib and wts appear to be crucial for the proliferation and EMT branches of the BLJ pathway, but not for repressing motility.

A similar but less severe tumor phenotype was observed in roe egg chambers. Small roe tumors accumulate predominantly at the posterior of the egg chamber, whereas in the anterior epithelium follicle cells become rounded, but do not delaminate from the epithelium (Figure 6B). We observed no morphological defects in ebi egg chambers (not shown). The clonal analysis data are thus consistent with the genetic interactions. lgl was the strongest enhancer and also caused the strongest tumor phenotype (Figure 5B and data not shown). scrib and wts were intermediate enhancers and had less severe phenotypes compared to complete loss of dlg and lgl (Figure 6, C and D). roe and ebi were mild enhancers and caused the weakest and no follicle phenotypes, respectively (Figure 6B and data not shown). These quantitative differences are most simply explained by the involvement of lgl in all three dlg pathways controlling epithelial invasion, whereas scrib, wts, and roe are only required in two branches, those that repress EMT and proliferation.

We have previously shown that both the EMT and motility branches of the BLJ pathway must be compromised for Fas2 and dlg mutant cells to invade (Figure 1A, see Introduction) (SZAFRANSKI and GOODE 2007). Thus, since neither scrib nor wts appears to be required for repressing motility (confirmed for border cell analysis, see below), the data suggest that the mechanism by which scrib and wts enhance dlg invasive tumorigenesis may be by increasing EMT. To further test if scrib and wts act in a BLJ pathway crucial for repressing EMT, we asked if their tumor cells have not only lost epithelial polarity, but have also become lateralized, a process characteristic of EMT (SZAFRANSKI and GOODE 2007). Lateralization is associated with a distribution of BLJ proteins around the circumference of the tumor cells, a step that not only precedes tumor invasion, but also appears to be essential for BC movement (SZAFRANSKI and GOODE 2007). We thus analyzed localization of BLJ proteins Dlg and -Spectrin in scrib and wts cells. In scrib and wts follicle cells that have not become tumorous, Dlg and -Spectrin are localized to the BLJ, as in wild-type follicle cells. In contrast, in tumorous scrib and wts follicle cells, Dlg and -Spectrin were localized around the circumference of the cell (Figure 6, E–H), as in dlg and Fas2 tumor cells (SZAFRANSKI and GOODE 2007). Thus, scrib and wts follicle cells appear to undergo an EMT lateralization process, similar to Fas2 and dlg tumor cells.

In summary, the wts, scrib, and roe phenotypes are very similar to the noninvasive phenotype associated with dlg mutations that specifically disrupt the Dlg SH3 or GuK domains (Figure 6A). In these dlg mutants, repression of motility remains largely intact (SZAFRANSKI and GOODE 2007). Thus, the specificity of the noninvasive wts, scrib, and roe phenotypes suggests that they act in a genetically separable dlg pathway that represses EMT and proliferation, but not motility (see Figure 1A and Introduction). Further support for this hypothesis is presented below.

Ft, ex, and mer; ex follicle cells do not have early wts phenotypes:

Several recent reports that use Drosophila wing and eye tissue as an experimental paradigm have provided strong evidence that the apicolateral junction provides upstream input that regulates Wts signaling. In particular, three apicolateral proteins that function as tumor suppressors appear to play an important role in Wts signaling. One is Fat (Ft), a transmembrane molecule of the Cadherin family (HARIHARAN 2006; WILLECKE et al. 2006). The other two are cytoplasmic adapter proteins, Expanded (Ex) and Merlin (Mer) (EDGAR 2006; HAMARATOGLU et al. 2006). The prevailing model is that activation of Ft at the cell surface recruits Ex, which together with Mer, activates Wts signaling (EDGAR 2006). Nonetheless, the genetic evidence indicates that Ft-Ex-Mer cannot account for all of the upstream inputs to Wts (HAMARATOGLU et al. 2006). For example, ft and ex overgrowth phenotypes are not as robust as for wts, and unlike wts, ft and ex are not required to promote developmentally regulated apoptosis (HARVEY and TAPON 2007). We thus considered the possibility, on the basis of several pieces of our data, that the BLJ may be an important upstream activator of Wts signaling in follicle cells: (1) wts was one of only a handful of genes isolated in our screen that enhanced dlg tumorigenesis, (2) wts interacted with dlg and lgl as strong, or stronger than scrib, which encodes for a known component of the BLJ, and (3) clonal analysis demonstrated that wts has a similar tumor phenotype to dlg, lgl, and scrib.

To determine the relative importance of the apicolateral junction vs. the BLJ to Wts signaling in follicle cells, we asked if ft, ex, or mer had wts-like follicle cell phenotypes. We analyzed egg chambers using the null mutants ft^G-rv (WILLECKE et al. 2006), mer⁴, ex^e1, and the double null mer⁴; ex^e1 (HAMARATOGLU et al. 2006), and compared their phenotype to wts^x1. ft egg chambers were indistinguishable from wild type throughout oogenesis (Figure 6I and data not shown), while ex, mer, and mer; ex egg chambers appear wild type through stage 7 of oogenesis (Figure 6J and see below for analysis of midoogenesis phenotypes). The data thus appear to exclude, or indicate a minor role for Ft, ex, and mer in wts processes during early oogenesis, when wts is required to repress proliferation and EMT (Figure 6). The data outlined in the previous section thus strongly suggest that the BLJ provides the upstream input to Wts during early oogenesis, a hypothesis we test more extensively below.

Comparison of Fat, ex, mer, and Fas2, dlg, lgl, scrib, wts, roe, and ebi midoogenesis defects:

Stage 7 marks the beginning of midoogenesis, when follicle cells stop proliferating and start to differentiate. As the follicle cells differentiate from stages 7 to 10, they enter an endoreplication cycle that increases their ploidy from 2N to 8N or greater, evident as an increase in follicle cell nucleus size (SPRADLING 1993). Notch (N) is required for the proliferation to endoreplication switch (SHCHERBATA et al. 2004). In N mutants, supernumerary follicle cells accumulate that have a conspicuously small nucleus during stages 7–10, indicating that they have failed to differentiate and enter the endoreplication cycle.

Two recent reports show that ex and wts have a small nucleus phenotype similar to the N phenotype (supplemental Figure 1, A and B) (MEIGNIN et al. 2007; POLESELLO and TAPON 2007). The phenotype is restricted predominantly to posterior cells, but is also sometimes observed in anterior cells, even though all ex or wts follicle cells are mutant (supplemental Figure 1, A and B). We found that neither Ft nor mer expressed the small nucleus phenotype and found that wts expressed the small nucleus phenotype with higher penetrance and expressivity compared to ex (supplemental Figure 1, A and B) (MEIGNIN et al. 2007; POLESELLO and TAPON 2007). A similar difference in penetrance and expressivity between wts and ex is observed for tumor growth in eye, wing, and other imaginal disc-derived tissues (HAMARATOGLU et al. 2006; HARVEY and TAPON 2007). The difference in imaginal tissues appears to result from functional redundancy between Ex and Mer, both membrane protein 4.1 family members. Whereas mer or ex alone cause relatively mild overgrowth phenotypes in imaginal tissue, mer; ex double mutant overgrowth is much more dramatic and is quantitatively similar to wts (HAMARATOGLU et al. 2006; HARVEY and TAPON 2007). To test if Mer-Ex functional redundancy may also be important in follicle cells, we analyzed mer; ex double mutant clones. The small nucleus phenotype was observed in 90% of ex clones (n = 16), but only 30% of mer; ex clones (n = 14), suggesting that Mer may have an opposing role to Ex in regulating this aspect of follicle cell differentiation. Although further work will be needed to test this hypothesis, we can conclude that for the endocycle switch in follicle cells, Wts appears to be regulated in a fundamentally different manner than in imaginal tissues, both in the lack of a requirement for Ft, and in the differing roles of Ex and Mer.

The ex small nucleus phenotype is not as penetrant as the wts phenotype, suggesting that additional regulatory inputs must be needed for Wts activation during midoogenesis. To test if the BLJ may also regulate Wts at this time, we asked if Fas2, dlg, lgl, scrib, roe, and ebi also have a small nucleus phenotype. We observed this phenotype for all of these mutants (supplemental Figure 1, C–G; data for ebi not shown), consistent with the notion that the BLJ is also important for regulating Wts during midoogenesis. The phenotype was highly penetrant for all mutants except Fas2, indicating that although Fas2 appears to be involved, it may not be the predominant BLJ ligand that regulates the endocycle switch.

To further analyze the putative roles of the apicolateral vs. BLJ in Wts regulation at midoogenesis, we examined an additional wts phenotype. The wts small nucleus phenotype is typically observed in posterior populations of follicle cells that accumulate in two or more disorganized layers, in contrast to the ordered monolayer of follicle cells characteristic of wild type (supplemental Figure 1B) (MEIGNIN et al. 2007; POLESELLO and TAPON 2007). The same disorganized phenotype is observed in ex, and to a much lesser degree in mer follicle cells (MACDOUGALL et al. 2001; MEIGNIN et al. 2007; POLESELLO and TAPON 2007), but in both mutants the phenotype is typically much less expressive compared to wts (supplemental Figure 1A and data not shown). Further, as for the small nucleus phenotype, there is no dramatic increase in expressivity of the disorganized epithelium phenotype in mer; ex double mutants, further indicating an absence of Mer-Ex synergism in follicle cells, and further indicating that they are not likely to collaborate in Wts activation by the same mechanism as in other tissues. In contrast, in Fas2, dlg, lgl, scrib, roe, and ebi mutants, the disorganized epithelium is observed with an expressivity equal to or greater than that seen for wts, consistent with the importance of the BLJ in regulating Wts during midoogenesis (supplemental Figure 1, B–G; data for ebi not shown). Thus, the data are consistent with the BLJ playing an important role in Wts regulation during both early and midoogenesis, but as follicle cells differentiate at midoogenesis, we suggest that Wts in addition may receive input from Ex to regulate the endocycle switch and possibly from both Mer and Ex to maintain epithelial integrity. To further test this model we compared the role of the BLJ and apicolateral junctions in positioning the germinal vesicle.

Mer and Ex, like Wts, are required in follicle cells at stages 7–8 of oogenesis for sending a signal to the oocyte that permits migration of the germinal vesicle from the posterior to the presumptive anterior–dorsal corner of the oocyte (MACDOUGALL et al. 2001; MEIGNIN et al. 2007; POLESELLO and TAPON 2007). In mer, ex, and wts mutants the germinal vesicle fails to migrate, causing it to be mislocalized at the posterior pole of the ooctye starting as early as stage 8. However, the phenotype is not observed in Ft egg chambers and is much more penetrant in wts compared to mer or ex (MEIGNIN et al. 2007; POLESELLO and TAPON 2007) (this study). In addition, as for the small nucleus and epithelial integrity defects, we see no dramatic enhancement in the germinal vesicle defects in mer; ex double mutants, further indicating that Fat, Mer, and Ex do not have the same collaborative role in Wts activation in follicle cells as they do in other tissues. Since the germinal vesicle defect is observed in 40% of mer; ex double mutants, but in >80% of wts egg chambers (supplemental Figure 1), there must be additional inputs to Wts in controlling this process. We thus analyzed the germinal vesicle defect in BLJ mutants. We observed this phenotype in Fas2, dlg, scrib, but only rarely in lgl, and not in roe or ebi egg chambers (supplemental Figure 1, H and I). The data are thus consistent with a role for the BLJ junction in regulating Wts signaling that is important for germinal vesicle localization, through a branch of the BLJ signaling in which Lgl and Roe play no role or a relatively minor role.

Our data suggest that during midoogenesis Ex and Mer cannot regulate Wts in the same manner as in other tissues and most importantly cannot account for all if any input to Wts. Further, Ex and Mer are not likely to be involved in Wts regulation during early oogenesis, because wts tumors are observed starting at stage 3, whereas Ex and Mer phenotypes are not observed until after stage 7. Rather, our data are most consistent with the BLJ playing the predominant role in Wts regulation throughout oogenesis, with Ex playing an accessory role in Wts regulation starting at stage 7. Additional experiments described below further test the relative importance of components of the BLJ vs. the apicolateral junction in regulating Wts signaling. Further, our data do not exclude the possibility that in follicle cells Ex may function independently of the apicolateral junction. For example, Ex is membrane protein 4.1 family member, and membrane protein 4.1 binds and colocalizes with hDlg in human cells (LUE et al. 1994). This suggests the possibility that Dlg may function independently of Ex during early oogenesis, then collaborate with Ex at midoogenesis to increase activation of Wts important for driving differentiation.

wts, scrib, roe, and mer, but not ft or ex, are required for border cell migration:

Fas2, dlg, and lgl all cause invasive tumors as well as faster border cell migration. The faster border cell migration phenotype is caused by derepression of a branch of the BLJ motility pathway that is also essential for tumor invasion (SZAFRANSKI and GOODE 2007). In contrast, in dlg mutants that specifically disrupt the Dlg SH3 and/or GuK domains, such as dlg^ip20/dlg^m35, noninvasive tumors predominate (Figure 6A), and border cells migrate only slightly faster than wild type (SZAFRANSKI and GOODE 2007). Thus, the branch of the BLJ pathway that represses motility appears to be only slightly derepressed in dlg^ip20/dlg^m35 cells.

The noninvasive phenotypes of scrib, wts, and most roe tumors are similar to dlg^ip20/dlg^m35 (Figure 6), suggesting that they act predominantly within a branch of the BLJ pathway that is essential for repressing EMT and proliferation. To further test if scrib, wts, and roe are required in a branch of BLJ signaling essential for repressing motility, we analyzed their border cell migration phenotypes. In contrast to Fas2, dlg, and lgl, which cause faster migration, we found that scrib, wts, and roe border cells have dramatically delayed, or completely blocked movement (Figure 7). In combination with their noninvasive tumor phenotypes, the data further indicate that <