discs large 1


DEVELOPMENTAL BIOLOGY

Embryonic

DLG is localized in an apical belt of the lateral cell membrane, at the position of the septate junction (Woods, 1991). Localization is to the cytoplasmic face of salivary gland cells (Woods, 1996).

Asymmetric cell division is important in generating cell diversity from bacteria to mammals. Drosophila neuroblasts are a useful model system for investigating asymmetric cell division because they establish distinct apical-basal cortical domains, have an asymmetric mitotic spindle aligned along the apical-basal axis, and divide unequally to produce a large apical neuroblast and a small basal daughter cell (GMC). Discs large (Dlg), Scribble (Scrib) and Lethal giant larvae (Lgl) tumour suppressor proteins regulate multiple aspects of neuroblast asymmetric cell division. Dlg/Scrib/Lgl proteins show apical cortical enrichment at prophase/metaphase, and then have a uniform cortical distribution. Mutants have defects in basal protein targeting, a reduced apical cortical domain and reduced apical spindle size. Defects in apical cell and spindle pole size result in symmetric or inverted neuroblast cell divisions. Inverted divisions correlate with the appearance of abnormally small neuroblasts and large GMCs, showing that neuroblast/GMC identity is more tightly linked to cortical determinants than cell size. It is concluded that Dlg/Scrib/Lgl are important in regulating cortical polarity, cell size asymmetry and mitotic spindle asymmetry in Drosophila neuroblasts (Albertson, 2003).

Larval

The distribution of proteins in the apico-lateral cell junctions in Drosophila imaginal discs was examined. The subcellular distribution of these proteins in normal and mutant proliferating cells was analyzed with marker antibodies and confocal microscopy. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous actin, labeled the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin III (FasIII) and Coracle (Cor) labeled the more basal septate junction. The junctional proteins labeled by these antibodies underwent specific changes in distribution during the cell cycle. A loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions (Woods et al., 1996).

The current study was expanded to examine the effects of mutations in other genes that also cause imaginal disc overgrowth. Based on staining with PY and Dlg antibodies, the apico-lateral junctional complexes appear normal in tissue from the hyperplastic overgrowth mutants fat facets, discs overgrown, lethal (2) giant discs and warts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lethal (2) giant larvae show abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signaling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signaling (Woods, 1997).

In third instar larval muscles, Dlg is concentrated primarily at type I neuromuscular junctions (NMJs). Closer examination reveals evenly distributed, weak immunoreactive spots at the muscle surface. In addition, a subcortical immunoreactive network, localized to the same range of optical sections as the muscle nuclei, was observed. Both types of extrasynaptic staining are specific to Dlg since they are not observed in mutant flies that express extremely low levels of a truncated form of Dlg (dlgX1-2 mutants) (Thomas, 2000).

To determine whether Dlg localization at extrasynaptic regions might represent intermediate trafficking steps, Dlg expression in muscles was analyzed during development. A highly sensitive anti-Dlg antibody (anti-DlgPDZ antibody) was used to determine the time course of Dlg expression at extrasynaptic regions. In stage 16 embryos, Dlg immunoreactivity is apparent in the ventral nerve cord, but virtually absent in both presynaptic termini and body wall muscles. At stage 17, after the initial formation of synaptic boutons, Dlg is detected at sites of contact between nerves and muscles. Double labeling with the neuron-specific anti-HRP antibody confirms that this immunoreactivity is presynaptic and that Dlg is still absent from the postsynaptic junctional region. At the same stage, however, extrasynaptic Dlg immunoreactivity becomes detectable, both as spots distributed throughout the muscle surface and as a subcortical network. Thus, expression of Dlg at distinct extrasynaptic sites clearly precedes its concentration at the postsynaptic membrane (Thomas, 2000).

A continuous shift of Dlg immunoreactivity from extrasynaptic to synaptic sites is observed during larval development. By the first instar stage, when Dlg begins to accumulate at the postsynaptic junctional membrane, extrasynaptic Dlg immunoreactivity at both the muscle membrane and the subcortical network is strong. By the second instar stage, synaptic Dlg localization is very strong, whereas extrasynaptic Dlg localization is significantly reduced. In third instar larvae, Dlg is almost exclusively localized at synapses and only a little Dlg can still be detected at the surface and subcortical region (Thomas, 2000).

The developmental analysis suggests a stepwise postsynaptic targeting of Dlg. To determine which domains of Dlg are required for distinct targeting steps, the subcellular distribution of FLAG-epitope-tagged Dlg deletion variants upon targeted expression in body wall muscles was studied. For this, advantage was taken of the GAL4-UAS expression system, using the GAL4 strain C57 as a muscle-specific activator. To evaluate the possible influence of endogenous Dlg on the localization of transgenic Dlg variants, expression of each construct was targeted in dlgX1-2 mutants (Thomas, 2000).

In all constructs, the last carboxy-terminal 40 amino acids of Dlg were replaced by the FLAG epitope. The importance of the carboxyl terminus for the synaptic localization of another MAGUK, PSD-95, is controversial. Several observations have demonstrated, however, that carboxy-terminal FLAG tagging has no detectable effect on the subcellular localization of Dlg. The hypomorphic allele dlg1P20 gives rise to a truncated protein lacking the same carboxy-terminal amino acids. The distribution of this gene product appears indistinguishable from the wild type. Upon targeted expression in muscles, Dlg-FLAG becomes enriched postsynaptically around type I boutons. Prominent FLAG immunoreactivity is also detected extrasynaptically at both the surface and the subcortical network. The extrasynaptic immunoreactivity in Dlg-FLAG-expressing flies is clearly stronger than in the wild type. However, a very similar increase in extrasynaptic immunoreactivity is observed upon expression of transgenic, non-tagged Dlg with an intact carboxyl terminus. This suggests that overexpression per se rather than the carboxy-terminal truncation is responsible for increased extrasynaptic localization of Dlg-FLAG (Thomas, 2000).

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). A new morphogenetic/cellular mechanism for disc eversion has been uncovered. Imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. The JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells (Pastor-Pareja, 2004).

One hallmark of epithelial cells is their distinct apico-basal cell polarity. This polarity depends on a set of intercellular connections, which encircle epithelial cells at the border of the apical and basal-lateral membrane domains. The cells in insect epithelial tissues are interconnected by zonula adherens (ZAs), which function in both cellular adhesion and signaling. DE-cadherin is the major constituent of the ZAs in a complex with Armadillo (Arm, ß-catenin) and Dalpha-catenin. In addition, epithelia of flies and other invertebrates exhibit septate junctions, which are located basally to the ZAs. Septate junctions prevent diffusion through the pericellular space and are functionally equivalent to vertebrate tight junctions (Pastor-Pareja, 2004).

All imaginal disc cells at the third instar larval stage presented ZAs in an apical belt. During disc eversion, however, it was found that ZAs components delocalize from the free edges of the PS cells, remaining cytoplasmic at the edges of the perforations arising through the PS/larval bilayer and in those PS cells leading the spreading of the discs over the larval tissues. As a consequence, ZAs are lost in these cells. Moreover, septate junction components, such as Coracle and Disc Large are also found to be missing from the membranes of leading front cells (Pastor-Pareja, 2004).

The loss of apico/basal polarity and adhesion of the PS cells during disc eversion is reminiscent of an epithelial-mesenchymal transition (EMT), as described for mesoderm and neural crest cells in vertebrates, and for the acquisition of the invasive phenotype in carcinomas (Pastor-Pareja, 2004).

In summary, the evagination of imaginal disc can be divided into the following sequential steps: (1) an overall positional change of the imaginal discs leading to the confrontation and apposition of the PS and the larval epidermis; (2) a regulated modulation (PEMT) of PS cells, which involves the downregulation of their cell-cell adhesion systems and allows them to move into their local neighborhood and invade the larval epithelium; (3) the fenestration of the peripodial/larval bilayer and the formation of an unbound peripodial leading front, which will direct imaginal spreading by planar cell intercalation, and (4) a bulging of the imaginal tissue (Pastor-Pareja, 2004).

Once the hole is opened, the planar intercalation of PS cells ensures that, first in the hole and later in the leading front, all four dorsal, ventral, anterior, and posterior compartments of the wing disc are represented. This mechanism also guarantees the maintenance of a continuous epithelial barrier (Pastor-Pareja, 2004).

N-glycosylation requirements in neuromuscular synaptogenesis

Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:alpha-3-D-mannoside-beta1,2-N-acetylglucosaminyl-transferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid N-glycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre- and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)gl] scaffolds. It is concluded that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis (Parkinson, 2013).

This study began with the hypothesis that disruption of synaptomatrix N-glycosylation would alter trans-synaptic signaling underlying NMJ synaptogenesis (Dani, 2012). MGAT1 loss transforms the synaptomatrix glycan environment. Complete absence of the HRP epitope, α1-3-fucosylated N-glycans, is expected to require MGAT1 activity: key HRP epitope synaptic proteins include fasciclins, Neurotactin and Neuroglian, among others. This study shows that HRP epitope modification of the key synaptogenic regulator Fasciclin 2 is not required for stabilization or localization, suggesting a role in protein function. However, complete loss of Vicia villosa (VVA) lectin reactivity synaptomatrix labeling is surprising because the epitope is a terminal β-GalNAc. This result suggests that the N-glycan LacdiNAc is enriched at the NMJ, and that the terminal GalNAc expected on O-glycans/glycosphingolipids may be present on N-glycans in this synaptic context. Importantly, VVA labels Dystroglycan and loss of Dystroglycan glycosylation blocks extracellular ligand binding and complex formation in Drosophila, and causes muscular dystrophies in humans. This study shows that VVA-recognized Dystroglycan glycosylation is not required for protein stabilization or synaptic localization, but did not test functionality or complex formation, which probably requires MGAT1-dependent modification. Conversely, the secreted endogenous lectin MTG is highly elevated in Mgat1 null synaptomatrix, probably owing to attempted compensation for complex and hybrid N-glycan losses that serve as MTG binding sites. MTG binds GlcNAc in a calcium-dependent manner and pulls down a number of HRP-epitope proteins by immunoprecipitation (Rushton, 2012), although the specific proteins have not been identified. It will be of interest to perform immunoprecipitation on Mgat1 samples to identify changes in HRP bands. Importantly, MTG is crucial for synaptomatrix glycan patterning and functional synaptic development. MTG regulates VVA synaptomatrix labeling, suggesting a mechanistic link between the VVA and MTG changes in Mgat1 mutants. The MTG elevation observed in Mgat1 nulls provides a plausible causative mechanism for strengthened functional differentiation (Parkinson, 2013).

Consistent with recent glycosylation gene screen findings (Dani, 2012), Mgat1 nulls exhibit increased synaptic growth and structural overelaboration. Therefore, complex and hybrid N-glycans overall provide a brake on synaptic morphogenesis, although individual N-glycans may provide positive regulation. Likely players include MGAT1-dependent HRP-epitope proteins (e.g., fasciclins, Neurotactin, Neuroglian), and position-specific (PS) integrin receptors and their ligands, all of which are heavily glycosylated and have well-characterized roles regulating synaptic architecture. An alternative hypothesis is that Mgat1 phenotypes may result from the presence of high-mannose glycans on sites normally carrying complex/hybrid structures, suggesting possible gain of function rather than loss of function of specific N-glycan classes. NMJ branch and bouton number play roles in determining functional strength, although active zones and GluRs are also regulated independently. Thus, the increased functional strength could be caused by increased structure at Mgat1 null NMJs. However, muscle-targeted UAS-Mgat1 rescues otherwise Mgat1 null function, but has no effect on structural defects, demonstrating that these two roles are separable. Presynaptic Mgat1 RNAi also causes strong functional defects, showing there is additionally a presynaptic requirement in functional differentiation. Neuron-targeted Mgat1 causes lethality, indicating that MGAT1 levels must be tightly regulated, but preventing independent assessment of Mgat1 presynaptic rescue of synaptogenesis defects (Parkinson, 2013).

Presynaptic glutamate release and postsynaptic glutamate receptor responses drive synapse function. Using lipophilic dye to visualize SV cycling, this study found Mgat1 null mutants endogenously cycle less than controls, but have greater cycling capacity upon depolarizing stimulation. The endogenous cycling defect is consistent with the sluggish locomotion of Mgat1 mutants, whereas the elevated stimulation-evoked cycling is consistent with electrophysiological measures of neurotransmission. Similarly, mutation of dPOMT1, which glycosylates VVA-labeled Dystroglycan, decreases SV release probability (Wairkar, 2008), although dPOMT1 adds mannose not GalNAc. Null Mgat1 mutants display no change in SV cycle components (e.g. Synaptobrevin, Synaptotagmin, Synaptogyrin, etc.), but exhibit reduced expression of the key active zone component Bruchpilot. Other examples of presynaptic glycosylation requirements include the Drosophila Fuseless (FUSL) glycan transporter, which is critical for Cacophony (CAC) voltage-gated calcium channel recruitment to active zones, and the mammalian GalNAc transferase (GALGT2), whose overexpression causes decreased active zone assembly. Postsynaptically, Mgat1 nulls show specific loss of GLURIIB-containing receptors. Similarly, dPOMT1 mutants exhibit specific GLURIIB loss (Wairkar, 2008), although dystroglycan nulls display GLURIIA loss. Selective GLURIIB loss in Mgat1 nulls may drive increased neurotransmission owing to channel kinetics differences in GLURIIA versus GLURIIB receptors (Parkinson, 2013).

Bidirectional trans-synaptic signaling regulates NMJ structure, function and pre/postsynaptic composition. This intercellular signaling requires ligand passage through, and containment within, the heavily glycosylated synaptomatrix, which is strongly compromised in Mgat1 mutants. In testing three well-characterized signaling pathways, this study found that Wingless (Wg) accumulates, whereas both GBB and JEB are reduced in the Mgat1 null synaptomatrix. WG has two N-glycosylation sites, but these do not regulate ligand expression, suggesting WG build-up occurs owing to lost synaptomatrix N-glycosylation. Importantly, WG overexpression increases NMJ bouton formation similarly to the phenotype of Mgat1 nulls, suggesting a possible causal mechanism. GBB is predicted to be N-glycosylated at four sites, but putative glycosylation roles have not yet been tested. Importantly, GBB loss impairs presynaptic active zone development similarly to Mgat1 nulls, suggesting a separable causal mechanism. JEB is not predicted to be N-glycosylated, indicating that JEB loss is caused by lost synaptomatrix N-glycosylation. Importantly, it has been shown that loss of JEB signaling increases functional synaptic differentiation similarly to Mgat1 nulls (Rohrbough, 2013). In addition, jeb mutants exhibit strongly suppressed NMJ endogenous activity, similarly to the reduced endogenous SV cycling in Mgat1 nulls. Moreover, the MTG lectin negatively regulates JEB accumulation in NMJ synaptomatrix, consistent with elevated MTG causing JEB downregulation in Mgat1 nulls (Parkinson, 2013).

Trans-synaptic signaling drives recruitment of scaffolds that, in turn, recruit pre- and postsynaptic molecular components. Specifically, DLG1 and L(2)GL scaffolds regulate the distribution and density of both active zone components (e.g. BRP) and postsynaptic GluRs, and both of these scaffolds are reduced at Mgat1 null NMJs. Importantly, dlg1 mutants display selective loss of GLURIIB, with GLURIIA unchanged, similar to Mgat1 nulls, suggesting a causal mechanism. Moreover, l(2)gl mutants display both a selective GLURIIB impairment as well as reduction of BRP aggregation in active zones, similarly to Mgat1 nulls, suggesting a separable involvement for this synaptic scaffold. DLG1 and L(2)GL are known to interact in other developmental contexts, indicating a likely interaction at the developing synapse. Although synaptic ultrastructure has not been examined in l(2)gl mutants, dlg1 mutants exhibit impaired NMJ development, including a deformed SSR. These synaptogenesis requirements predict similar ultrastructural defects in Mgat1 mutants, albeit presumably due to the combined loss of both DLG1 and L(2)GL scaffolds. Future work will focus on electron microscopy analyses to probe N-glycosylation mechanisms of synaptic development (Parkinson, 2013).

Oogenesis

A Fasciclin 2 morphogenetic switch organizes epithelial cell cluster polarity and motility: Fas2 polarization is then directed by PC Dlg and Lgl

Little is known about how intercellular communication is regulated in epithelial cell clusters to control delamination and migration. This problem has been investigated using Drosophila border cells as a model. Just preceding cell cluster delamination, expression of transmembrane immunoglobulin superfamily member, Fasciclin 2, is lost in outer border cells, but not in inner polar cells (PCs) of the cluster. Loss of Fasciclin 2 expression in outer border cells permits a switch in Fasciclin 2 polarity in the inner polar cells. This polarity switch, which is organized in collaboration with neoplastic tumor suppressors Discs large and Lethal-giant-larvae, directs cluster asymmetry essential for timing delamination from the epithelium. Fas2-mediated communication between polar and border cells maintains localization of Discs large and Lethal-giant-larvae in border cells to inhibit the rate of cluster migration. These findings are the first to show how a switch in cell adhesion molecule polarity regulates asymmetry and delamination of an epithelial cell cluster. The finding that Discs large and Lethal-giant-larvae inhibit the rate of normal cell cluster movement suggests that their loss in metastatic tumors may directly contribute to tumor motility. Furthermore, these results provide novel insight into the intimate link between epithelial polarity and acquisition of motile polarity that has important implications for development of invasive carcinomas (Szafranski, 2004).

How does developmentally programmed loss of Fas2 expression in BCs permit Fas2 polarization in PCs? The data indicate that this is a multistep process. Initially, Fas2 homophilic interactions between BCs and PCs are lost, and several experiments indicate that they are replaced by Fas2 heterophilic interactions with a putative BC receptor. These interactions are essential for maintaining Fas2 in PC membranes contacting BCs. Next, loss of Fas2 from BCs causes relocation of the majority of PC Fas2 to the interface between PCs, where it is maintained because of homophilic interactions with Fas2 from the adjacent PC. In support of this interpretation, misexpression of Fas2 in PCs appears to oversaturate Fas2 between PCs, causing its circumferential accumulation at the contact sites with BCs. It is concluded that the accumulation of Fas2 between PCs ensures that Fas2 is kept at sufficiently low levels at the sites of contact with BCs to allow its polarization to the leading half of PCs. Fas2 polarization is then directed by PC Dlg and Lgl, as evidenced by the observation that loss of function of either protein causes loss of Fas2 polarity. However, Fas2 can also polarize Dlg and Lgl; loss of Fas2 causes loss of Dlg and Lgl polarity, while ectopic Fas2 redirects Dlg and Lgl localization. Thus, Fas2 is in a positive feedback loop with Dlg and Lgl that ensures the build up of a PC signaling and adhesion complex at the leading half of the PCs. These data indicate that Fas2 is involved in intercellular interactions crucial for organizing polarity, an important criterion for a function specifically involved in regulation of motility in multicellular clusters (Szafranski, 2004).

Significantly, the results indicate that molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. The timing of the reorganization of epithelial polarity is crucial for timing delamination. Fas2 therefore plays a direct role in mediating intercellular interactions that modulate movement, a second property proposed for a function specifically involved in regulating cluster motility as opposed to single cells. It is concluded that the Fas2 morphogentic switch facilitates development of motile polarity essential for timely BC delamination. A similar switch mechanism may be important in other processes that crucially depend on timing of Fas2 activity, such as axon pathfinding, and learning and memory (Szafranski, 2004).

Fas2 polarity appears to compartmentalize PCs into distinct functional domains in order to control functionally distinct intercellular communication with leading versus trailing BCs. Leading BCs play a functionally distinct role by pioneering invasion between germ cells while simultaneously detaching from the epithelium. Trailing BCs are likely to play a less active role in invasion, but must mediate precisely timed detachment from the epithelium. Fas2 polarization is thus likely to be crucial for facilitating coordination of the distinct functional requirements of leading versus trailing BCs, by establishing distinct sets of intercellular contact and communication between the PCs and leading versus trailing BCs. In support of this hypothesis, previous studies have suggested that leading and trailing BCs are functionally distinct. In BC clusters comprising a mixture of wild type and slbo, jing, taiman or DE-cadherin mutant cells, wild-type BCs always lead invasion. Furthermore, additional structural evidence has been documented for cluster asymmetry. Amphiphysin, a vesicle trafficking protein that regulates Dlg and Lgl localization, is expressed at higher level in trailing BCs compared to leading BCs. Amph, Dlg and Lgl, are thus good candidates for proteins that differentially regulate cortical and cell surface activities needed to mediate distinct interactions of leading and trailing BCs with adjacent epithelial cells and germ cells during the delamination process (Szafranski, 2004).

Since only Dlg and Lgl are mislocalized in Fas2 clusters, but not Fas3, alpha-Spec or Crb, the data suggest that Fas2 directs localization of specific molecules within distinct regions of different cells of the cluster to control motility. A putative Fas2-binding BC receptor may be another molecule whose polarity is controlled by Fas2. Interaction with this putative receptor appears to facilitate organization of the global polarity of the cluster, since the orientation of delamination, mediated by the BCs, directly correlates with Fas2 polarity in PCs. These data thus suggest that Fas2 coordinates directional mass motion between cells that are potentially capable of motion in any direction, and that it helps to determine the locomotive-active regions of these cells, additional criteria for a function specifically involved in regulating cluster motility. Thus, because Fas2 is required for regulation of several activities that distinguish how single cells versus clusters move, the data provide the first molecular model for understanding the organization of epithelial cluster polarity during delamination and movement. One argument against this proposal might be that the PCs appear to be highly specialized. However, it is thought that this is likely to be of less significance, since PCs express epithelial polarity proteins in a pattern similar to adjacent follicle epithelial cells (Szafranski, 2004).

As has been shown for BC clusters, several vertebrate studies have shown that transmembrane proteins are differently expressed within different cell subpopulations in migrating clusters. Furthermore, the structure and functions of Fas2, Dlg and Lgl homologs are conserved across phylogeny. Thus, the involvement of Fas2, Dlg and Lgl in organizing cell cluster motility also may be conserved. It is concluded that although the precise mechanism of cluster movement may not be conserved in vertebrates, the information gleaned about how BCs regulate epithelial polarity to dynamically organize cluster polarity and movement will be generally useful for understanding how cell cluster motility is organized across phylogeny (Szafranski, 2004).

The role of Fas2 in regulating migration is discussed. Loss- and gain-of-function experiments demonstrate that PC Fas2 acts as a signal to inhibit the rate of BC migration. This work builds on previous studies demonstrating the importance of PCs in determining BC fate. However, this work is the first example of an intercellular signal that specifically organizes cluster movement, rather than determining cell fate. Fas2 clearly has a signaling function, since PCs do not contact the migration substrate. Thus, these data demonstrate for the first time the existence of intercellular communication between cells of a migratory cluster, that is specifically required to modulate migration (Szafranski, 2004).

PC Fas2 signaling inhibits the rate of cluster movement by maintaining Dlg and Lgl localization in BCs. The putative BC receptor with which Fas2 interacts may control Dlg and Lgl localization in BCs. Since Dlg is localized to the cortex of BCs, Dlg must inhibit the rate of migration through cortical activities in BCs. One cortical activity controlled by Dlg is the recruitment of Lgl to the membrane. Since lgl clusters have very similar migration phenotypes to dlg clusters, the data indicate that Lgl and Dlg cooperate to inhibit BC movement. The importance of Dlg and Lgl in regulating cell movement probably derives from the same scaffolding activities they use to organize and control membrane, cytoskeletal and signaling specialization during the polarization of epithelial and neuronal cells. It is proposed that Dlg and Lgl scaffolding organizes and integrates transmembrane signaling and adhesion proteins with signaling, trafficking and cytoskeletal effectors in the cortex of BCs to mediate contact-inhibition of cluster movement (Szafranski, 2004).

BCs resemble mutant dlg invasive tumor cells in that they lose epithelial polarity by accumulating Dlg and Lgl around their circumference, but in contrast to BCs, mutant dlg tumor cells migrate between germ cells without temporal or spatial control. The data demonstrate that Dlg and Lgl not only control polarity and delamination of epithelial clusters, but also actively inhibit movement. Thus, mutant dlg tumor invasion is likely to be caused by a combination of loss of epithelial polarity and over-activation of motility pathways. In this context the results appear to be paradoxical in that loss of epithelial polarity is generally considered to be crucial for facilitating acquisition of motility, but it is seen that loss of polarity in normal migrating clusters delays initiation of movement. The data resolve this paradox in that during normal development, molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. It therefore seems likely that in carcinomas, inappropriate loss of epithelial polarity simultaneously disrupts acquisition of motile polarity, but this phenomenon is not appreciated because ultimately the tumor cells migrate. Thus, it is postulated that overactivation of motility pathways, as is seen with loss of Dlg and Lgl in BCs, may be especially crucial for achieving carcinoma invasion. Consistent with this hypothesis, some dlg mutations that cause loss of epithelial polarity do not lead to tumor invasion, suggesting that acquisition of motility is a separate Dlg function (Szafranski, 2004).

Gene expression data for human cancers suggests that mutations that promote tumor formation, through loss of epithelial polarity and increased proliferation, may be the same mutations that subsequently cause tumor cell invasion. Based on the observation that Dlg is required to maintain polarity, inhibit proliferation and inhibit movement, it is proposed that tumor suppressors such as Dlg that regulate signaling and adhesion at epithelial junctions may unify human gene expression data by providing an ultrastructural target that controls contact inhibition of both proliferation and movement. Progressive deterioration of epithelial junctions may thus provide a common mechanism through which multiple tumor suppressor pathways impact the cascade from cell proliferation to tumor invasion, either through mutation or mislocalization of critical junctional proteins (Szafranski, 2004).

Effects of Mutation or Deletion

Mutations of the discs large-1 tumor suppressor gene causes a non-epithelial overgrowth or neoplastic transformation, resulting in tumor-like imaginal discs and enlarged larvae that never pupariate. dlg mutant wing discs develop convoluted monolayers of epithelial cells characterized by well-defined apical-basal polarity. These layered cells secrete large amounts of basement membrane material. Drosophila laminin and collagen are components of this matrix. Late in development clusters or 'rosettes' of separated cells form, lacking both cell-cell junctions and apical-basal polarity. In in vitro culture experiments, dlg wing discs do not respond to a pulse of exogenous ecdysone by secreting cuticle or losing basement membrane as do normal discs. These results are consistent with the hypothesis that cell-cell interaction and communication is required for termination of disc cell proliferation, which must occur prior to a cellular response to ecdysone (Abbott, 1991).

A weak mutation of dlg results in minor bristle defects, including missing and duplicate bristles. A stronger allele fails to hatch and dies during late embryogenesis with a failure of dorsal closure [Images] and terminal defects. Mutants in an intermediate allele die as pharate adults with severe bristle and eye defects. The antennae and legs of these animals have small overgrowth regions and the eyes show defects in the planar polarity of the ommatidial bristles: they are found at random around the ommatidial cluster (Woods, 1996).

DLG is expressed at one type of glutamatergic synapse of the neuromuscular junction and is associated with both presynaptic and postsynaptic membranes. Mutations in dlg alter the expression of dlg and cause striking changes in the structure of the subsynaptic reticulum, a postsynaptic specialization at these synapses. These results indicate that dlg is required for normal synaptic structure and offers insights regarding the role of dlg homologs at vertebrate synapses (Lahey, 1994).

Inhibition of patterned cell shape change and cell invasion by Discs large during Drosophila oogenesis.

During oogenesis Drosophila Discs large (Dlg) protein is involved in maintaining the structure of one population of follicular cells (the follicular epithelium or FE), and in the migratory activity of another population of follicular cells (border cells or BC). These two follicle cell populations migrate at stage 9 of oogenesis. Most follicle cells (the FE cells) surrounding nurse cells move to the oocyte as an epithelial sheet along the outside of the egg chamber, whereas the BCs move through the center of the egg chamber, in concert with the epithelium. Six to seven BCs break from the follicular epithelium and adopt a mesenchymal-like morphology, then migrate to the oocyte, as they contact anterior nurse cells, then posterior nurse cells, before reaching their destination. The interaction of BCs with posterior nurse cells is particularly dramatic. Nurse cells maintain an invariant quadrihedral-like architecture throughout most stages of oogenesis, but following initiation of BC movement, one nurse cell adjacent to the oocyte extends a cytoplasmic process that contacts the migrating cells. Nurse cell processes are never observed preceding BC migration, although BC fate has already been established. Nurse cell processes show a clear directionality. They are never observed to extend from anterior nurse cells to BCs that have moved close to the oocyte at the posterior of the egg chamber. These observations suggest that nurse cells adjacent to the oocyte play an active role in guiding the movement of BCs to the oocyte (Goode, 1997).

Drosophila Dlg is required to block cell invasion. Loss of dlg activity during oogenesis causes FE cells to change shape and invade in a pattern similar to BCs, yet dlg mutant cells have not adopted a border cell fate. Specifically these defectively migrating FEs do not express slow border cells or FasIII, both diagnostic indicators of the BC fate. dlg-invasive FE cells share an apolar morphology with BCs but other aspects of BC morphology, such as absence of lamellipodia-like structures, are not shared with BCs. Nevertheless, both oocyte and nurse cells attached to the oocyte extend processes that contact invasive FEs (Goode, 1997).

Both functional and morphological evidence indicates that cooperation between germ cell and follicle cell Dlg, probably mediated by Dlg PDZ domains, is crucial for regulating cell mixing, suggesting a novel developmental mechanism and mode of action for the Dlg family of molecules. Dlg is expressed in both germ and follicle cells from the time that the germ cell cyst becomes surrounded by follicle cells in the germarium. Dlg appears to be expressed at equivalent levels in both tissues throughout the growth phases, as the germ cell cyst expands in size and the number of follicular epithelial cells increases greater than 10-fold. Following cessation of follicle cell proliferation, levels of Dlg protein appear to dramatically decrease in germ cells, corresponding to the time just preceding and including BC migration to the oocyte. At the cellular level, Dlg is localized to sites of contact between follicle cells and to sites of contact between germ cells, but appears to be excluded at sites of contact between germ cells and follicle cells. Dlg is required in both germ cells and follicle cells. When dlg function is eliminated in both follicle cells and germ cells, follicle cells always invade along the BC pathway. Dlg also appears to be required to prohibit cell proliferation of follicular cells at the poles of the egg chambers. Mutations in SH3 or GuK domains of Dlg fail to confer premature cell mixing. Dlg is also required to prevent premature BC migration These findings suggest that Dlg does not simply inhibit individual cell behaviors during oogenesis, but rather acts in a developmental pathway essential for blocking cell proliferation and migration in a spatio-temporally defined manner. It is suggested that the PDZ domains of Dlg are required for prohibiting follicle cell invasion (Goode, 1997).

Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. Cells in epithelial sheets are characterized by columnar or cuboidal shape, strong cell-cell adhesion, and pronounced apicobasal polarity. However, tumors of epithelial origin lose these characteristics as they progress from benign growth to malignant carcinoma, and this loss is associated with poor clinical prognosis. Evidence is provided that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribbled (Scrib) is a cell junction-localized protein required for polarization of embryonic and imaginal disc and follicular epithelia. The tumor suppressor scrib was isolated in a screen for maternal effect mutations that disrupt aspects of epithelial morphogenesis such as cell adhesion, shape and polarity. scrib encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein. The structure of the embryonic cuticle was used to reflect the organization of the underlying epithelial epidermis that secretes it. The wild-type cuticle forms a smooth, continuous sheet, but embryos that are maternally and zygotically mutant for scrib produce a corrugated cuticular surface that is riddled with holes, hence the name scribbled (Bilder, 2000a).

Two tumor suppressors, lethal giant larvae (lgl) and discs-large (dlg), have the identical effects as scrib mutation on epithelial structure. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Thus, these three tumor suppressors act together in a common pathway to regulate cell polarity and growth control (Bilder, 2000b).

Because follicle cell epithelia require scrib, lgl, and dlg, the functions of lgl and dlg were examined in the embryonic epidermis, where scrib acts to restrict apical proteins and adherens junctions to their appropriate positions within the cell membrane (Bilder, 2000a). Embryos lacking both maternal and zygotic contributions of lgl and dlg, (hereafter referred to as lgl and dlg embryos) were stained with antibodies to polarized proteins and cellular junction components. During mid-embryogenesis, lgl and dlg embryos show defects in apicobasal polarity, revealed by aberrant distribution of the apical protein Crumbs (Crb) and disruption of adherens junctions. These defects are similar to those of scrib embryos; the terminal phenotypes of scrib, lgl, and dlg embryos, as indicated by cuticle deposition, are also nearly identical. Thus, lgl and dlg, like scrib, act to properly localize apical proteins and adherens junctions to organize epithelial architecture in embryos (Bilder, 2000b).

The similarity of mutant phenotypes in different epithelia suggests that the three proteins are components of the fundamental machinery that creates the distinctive architecture of epithelial cells and tissues. To test this assertion, the scrib phenotype was compared to that of lgl and dlg in a third major epithelium, the larval imaginal disc. Discs isolated from late third instar larvae zygotically mutant for scrib are profoundly disorganized and also massively overgrown. scrib discs contain 4.7 times as many cells as wild-type (WT) discs and consist of spherical masses of tightly packed cells, as opposed to the folded monolayer epithelium seen in WT larvae. The apical polarization of actin evident in WT discs is absent in scrib discs. This loss of epithelial organization accompanied by overproliferation corresponds to the phenotype described for lgl and dlg zygotic mutant discs. Additional features of lgl and dlg larval phenotypes, such as overgrowth of brain tissue, are also present in scrib larvae. Together, these data indicate that scrib and the two previously characterized Drosophila malignant neoplastic tumor suppressors, lgl and dlg, share a role in growth control as well as epithelial polarity. Epistatic relations between scrib, lgl, and dlg were investigated by determining the localization of each protein in embryos mutant for the other two genes. These experiments have shown that dlg is required for the stable association of Scrib with the cell membrane and scrib is required for the cortical association of Lgl; all three genes act to localize Scrib and Dlg to the apical margin of the lateral membrane (ALM) of the embryonic epidermal epithelium (Bilder, 2000b).

These results provide strong evidence that Scrib, Dlg, and Lgl act in a common pathway to regulate cell architecture and cell proliferation control. Of the ~50 Drosophila genes in which mutation gives rise to overproliferation, only scrib shares with dlg and lgl the concomitant loss of tissue organization that groups the three together as malignant neoplastic tumor suppressors. Previous analyses have described a role for dlg and lgl in imaginal disc polarity; the demonstration in this work of genetic interactions with scrib and codependence for protein localization indicates a functional link between the three tumor suppressors. Furthermore, involvement of the tumor suppressors in embryonic epithelial polarity provides a well-studied context in which to understand their activities. These findings suggest that, in the WT gastrula, intrinsic, perhaps adhesion-based cues localize Dlg at the ALM; Dlg stabilizes Scrib at this position, and finally Scrib acts on the cortical cytoskeleton to bring Lgl to the membrane. The three proteins may then collaborate to maintain the proper distribution of polarized factors, including themselves (Bilder, 2000b).

The correlation between loss of membrane-associated Lgl in scrib and dlg mutants and defective cell polarity suggests models of action for this group of proteins. Whereas the PDZ domains of Scrib and Dlg are likely to bind to transmembrane proteins that organize the epithelial cell surface, the role of Lgl in polarity determination may derive from its function in targeted secretion of membrane proteins. Lgl homologs from humans and yeast can bind to plasma membrane t-SNARE proteins and promote the fusion of cargo-carrying vesicles with target membranes. In yeast undergoing polarized growth, the broadly distributed Lgl homologs function primarily at the bud tip, the site of the 'exocyst' complex required for vesicle trafficking and addition. In vertebrate epithelia, exocyst components are found at the tight junction, a structure analogous to the septate junction where Dlg and Scrib localize. In Drosophila epithelia, recruitment of Lgl into the proximity of membrane t-SNAREs requires proper localization of Scrib and Dlg, thus potentially linking the transmembrane proteins that establish polarity to the protein-targeting system that preserves it (Bilder, 2000b and references therein).

In many epithelial-derived cancers, cytoarchitectural changes are hallmarks of oncogenic transformation. The disruption of epithelial architecture seen in scrib, dlg, and lgl animals could affect growth control by several mechanisms. Many growth factor receptors are polarized to a specific membrane domain, and mislocalization of such proteins may affect signaling pathways that maintain cells in a differentiated, nonproliferative state. Additionally, the aberrant cell-cell junctions formed in scrib, dlg, and lgl mutants could compromise contact inhibition. Finally, disruption of cell-cell contacts may release junction-localized signaling components, such as Arm or APC, that have been implicated in regulating cell proliferation; indeed, a human Dlg homolog has been shown to bind APC and associate with beta-catenin, the human homolog of Arm. Because the modes of action of Scrib, Dlg, and Lgl are likely to be conserved between vertebrates and invertebrates, investigation into a tumorigenic role for the multiple human homologs of these genes is warranted. Further analysis of the mechanisms by which Scrib, Dlg, and Lgl keep Drosophila cell growth in check will likely enhance an understanding of mammalian oncogenesis as well (Bilder, 2000b).

Regulation of synaptic plasticity and synaptic vesicle dynamics by the PDZ protein Scribble

The Drosophila tumor suppressor Scribbled (Scrib) is required for maintaining epithelial cell polarity. At the larval neuromuscular junction, Scrib colocalizes and indirectly interacts with another tumor suppressor and PDZ protein, Discs-Large (Dlg). Dlg is critical for development of normal synapse structure and function, as well as for normal synaptic Scrib localization. Scrib is also an important regulator of synaptic architecture and physiology. The most notable ultrastructural defect in scrib mutants is an increase in the number of synaptic vesicles in an area of the synaptic bouton thought to contain the reserve vesicle pool. Additionally, the number of active zones is reduced in scrib mutants. Functionally, the scrib synapse behaves relatively normally at low-frequency stimulation. However, several forms of plasticity at this synapse are drastically altered in the mutants. Specifically, scrib mutants exhibit loss of facilitation and post-tetanic potentiation, and faster synaptic depression. In addition, FM1-43 imaging of recycling synaptic vesicles shows that vesicle dynamics are impaired in scrib mutants. These results identify Scrib as an essential regulator of short-term synaptic plasticity. Taken together, these results are consistent with a model in which Scrib is required to sustain synaptic vesicle concentrations at their sites of release (Roche, 2002).

Mutations in dlg lead to prominent defects in both synapse structure and function. At the ultrastructural level these defects include an increase in bouton size and number of active zones, as well as a poorly developed subsynaptic reticulum (SSR), an elaborate folding of the postsynaptic membrane at the NMJ. Dlg is colocalized with Scrib, and mutations in dlg also result in severe mislocalization of synaptic Scrib. In contrast, although localization of Scrib to the NMJ is completely disrupted in scrib mutants, the localization of Dlg is not affected. Analysis of the NMJs in scrib mutants has shown that the general morphology is not affected. It is hypothesized that some of the defects in dlg mutants might be the consequence of Scrib mislocalization. This hypothesis was tested by serially sectioning type I synaptic boutons in several scrib mutant allelic combinations and examining their ultrastructure using electron microscopy. It was found that the synaptic structure in these mutants was drastically altered; however, these defects were quite distinct from those in dlg mutants. One of the most prominent defects was an abnormally high density of synaptic vesicles. In wild type, synaptic vesicles are organized into at least two pools: a pool in direct proximity to the T-shaped active zones [thought to represent the readily releasable pool (RRP)], and a pool localized in a broad area at the periphery of the entire synaptic bouton [representing the reserve vesicle pool (RP)]. Typically, the central region of the bouton is devoid of synaptic vesicles and contains endosomes and mitochondria, as well as other nonvesicular material. In contrast, boutons in the null allele scrib2 and scrib2/Df, but not those from the less severe allele scrib1, are filled with synaptic vesicles and lack an empty core. The number and area of mitochrondrial profiles, however, is unchanged in scrib mutant boutons. Overall, in these mutants there is a significant increase in synaptic vesicle density, as measured by determining the total number of vesicles at the central cross section of the boutons divided by the area of this cross section. In addition to this striking defect in vesicle distribution and density, many boutons contained morphologically abnormal vesicular material at the core (Roche, 2002).

To determine whether both the RRP and the RP are affected in scrib mutants, the number of vesicles was counted in an area 100, 150, and 200 nm around the active zone, which likely encompasses the RRP. The number of vesicles in these areas of scrib2/Df mutant boutons was not significantly different from wild type. Thus, it is the distribution and density of the RP that appear to be specifically affected in scrib mutants (Roche, 2002).

In addition to the defect in the RP, the average number of active zones in both scrib2 and scrib2/Df is slightly lower than wild type, although this difference was statistically different only at scrib2 homozygous boutons. Unlike dlg mutants, the SSR appeared normal, and neither the number of SSR layers nor the SSR density is significantly different from wild-type controls. This is in contrast to the observations in severe dlg mutants, in which the number of active zones is increased several fold and the SSR length is reduced (Roche, 2002).

One might hypothesize, on the basis of the mislocalization of Scrib in dlg mutants, that by mutating dlg one would see, in addition to a dlg-specific phenotype, the scrib phenotype as well. Indeed this seems to be the case in other cell types in which mutation of either dlg or scrib causes similar phenotypes: formation of tumors and loss of cell polarity. However, at the Drosophila NMJ the effects of scrib mutation are quite different from those in several dlg mutants. These differences may stem from the fact that residual synaptic Scrib is still present in dlgXI-2 mutants, although at a lower level, and hence the remaining Scrib may be sufficient to override the synaptic scrib phenotype. Interestingly, levels of synaptic Scrib have an opposite influence on the regulation of the number of active zones than do levels of synaptic Dlg. Although a decrease in Dlg levels in severe dlg mutants causes an increase in active zone number, the same phenotype is observed by increasing Scrib levels. This observation is consistent with the notion that at synapses Scrib may negatively regulate Dlg function. This is in contrast to the observation in epithelial cells, where Dlg and Scrib appear to function in a similar manner during the determination of cell polarity and tumor suppression. This may reflect the ability of Dlg and Scrib to bind different protein partners with different functions in the two cell types. Indeed, partners such as Fasciclin II bind to Dlg at synapses but are absent in epithelial cells. Thus, the specific influence of scaffolding proteins in different cell types may be highly dependent on the availability of specific binding partners (Roche, 2002).

Synaptic development is controlled in the periactive zones of Drosophila synapses

A cell-adhesion molecule Fasciclin 2, which is required for synaptic growth, and Still life (Sif), an activator of Rac, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. betaPS integrin and Discs large, both involved in synaptic development, also decorate the zone. However, Shibire (Shi), the Drosophila dynamin that regulates endocytosis, is found in the distinct region. Mutant analyses show that sif genetically interacts with Fas2 in synaptic growth and that the proper localization of Sif requires Fas2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).

To characterize the periactive zone, especially in identifying its functional significance, the distribution patterns of other molecules were examined with the aid of Pak staining. Monoclonal antibody, MAb1D4, against Fas2 labels the boutons in a complementary pattern with Pak staining. Fas2 staining surrounds the Pak-positive regions and forms concentric patterns as observed for Sif staining. The cross-section profile also shows similar patterns as Sif and Pak double staining. Sif and Fas2 are indeed co-localized in overlapping network-like patterns. Fas2 is involved in synaptic growth, stabilization and structural plasticity, possibly through its homophilic adhesion. These data suggest that Fas2 controls these synaptic events locally in the periactive zones. Thus, the periactive zone is characterized by the specific localization of two distinct types of molecules: a cell adhesion molecule (Fas2) that controls synaptic development and an intracellular molecule (Sif) that is a GEF to Rac (Sone, 2000).

In an attempt to understand the function of the periactive zone further, other molecular markers that stain the zone were sought. MAb6G11, the monoclonal antibody against betaPs integrin (Myospheroid) that is structurally similar to the vertebrate integrin beta1 subunit also shows staining that is complementary to Pak staining. Unlike Sif and Fas2, however, MAb6G11 staining is observed much more diffusely on the muscle surfaces surrounding the outside of the bouton, suggesting the staining in the postsynaptic specialization: the subsynaptic reticulum. In mutants of the mys gene the extent of the cell contact between nerve terminals and muscles is altered by either a primary or secondary effect of the mutation, and the growth of larval neuromuscular synapses is affected. These synaptic defects observed in the betaPs integrin mutants may represent its function in the periactive zones (Sone, 2000).

The polyclonal antibody against Dlg protein stains synaptic boutons in a way similar to MAb6G11. The Dlg staining also appears to be moderately diffused on the muscle surfaces surrounding the bouton. This pattern is complementary with the anti-Pak staining when the bouton is scanned at the surface level. In dlg mutants, the structural properties of synapses, including the formation of subsynaptic reticulum at the postsynapses and the number of active zones at the presynapses, are altered. Furthermore Dlg regulates the synaptic localization of Fas2 by binding directly to the cytoplasmic tail of Fas2. Therefore, one of the roles for Dlg in synaptic development is probably the localization of Fas2 to the periactive zone. These observations indicate that two additional molecules, betaPs integrin and Dlg, are present in synaptic areas including the periactive zones. They are both involved in the structural development of the neuromuscular synapses, and therefore appear to participate in the control of synaptic development in the periactive zones (Sone, 2000).

The mutant larvae could be distinguished from the wild type by the blind test for the Sif and Pak co-staining patterns. Similar results were obtained in the heterozygotes with Fas2e76 and a Fas2 null allele, suggesting that the alteration of Sif localization is not due to a second-site mutation on the Fas2e76 chromosome. To investigate the altered distribution of Sif further, the mutant boutons were examined under the electron microscope. A large number of Sif signals are occasionally present in the medial portions of the electron-dense regions in the Fas2e76 boutons and these signals are still associated with the plasma membrane, as are the signals observed in the wild type. It is therefore concluded that the reduction of Fas2 in the periactive zones results in the improper localization of Sif along the plasma membrane. Previous study has shown that the synaptic localization of Fas2 requires Dlg. Therefore, the localization of Sif was examined in the dlg mutant background, but no apparent alteration was found in the network pattern. A considerable amount of Fas2 is still present in the periactive zones of dlg mutant boutons, while faint or no staining is detected in the Fas2e76 boutons. This residual Fas2 seems to be sufficient to sustain the proper localization of Sif in the dlg mutants (Sone, 2000).

The periactive zone has been indicated as a region for the control of synaptic development. The periactive zone surrounds the active zone, which is the site for vesicle exocytosis or neurotransmission. This concentric organization suggests that the two zones specialize for the different cellular functions and constitute an elemental unit for the presynaptic structure. Investigation of how these zones are incorporated into the synaptic bouton during development will be of interest. The segregated distribution of the two zones suggests that the mechanisms controlling synaptic development and neurotransmission may be separable. This view is supported by the mutant analyses for Fas2 and Sif; both mutations affect structural properties of synapses without changing basic electrophysiological functions. In the NMJs of Fas2 mutants, the bouton number is decreased or increased depending on the alleles but the total synaptic strength is maintained at the normal level. Functional strength of the synapse is regulated only through the activity of a transcription factor, cAMP-response-element-binding protein (CREB), which functions independently of Fas2. Also in sif mutants, the basic electrophysiological properties of NMJs are normal. These observations clearly contrast with the mutant phenotypes for the proteins controlling vesicle exocytosis: Synaptotagmin, Cysteine string protein, n-Synaptobrevin and Syntaxin 1A. Mutants in genes coding for all these proteins show impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically separable phenomena and are regulated by independent pathways. It is proposed that these genetically separable phenomena are spatially segregated into the two zones on the presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).

Role of cortical tumor-suppressor proteins in asymmetric division of Drosophila neuroblast

In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Lethal (2) giant larvae (Lgl) is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumor-suppressor protein, Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells (Ohshiro, 2000).

In mitotic neuroblasts, the Prospero transcription factor and Numb, an antagonist of Notch signaling, associate with their respective adapter proteins, Miranda and Partner of Numb (Pon), and thereby localize to the basal cortex. In contrast, Inscuteable (Insc), Bazooka (Baz) and Partner of Inscuteable (Pins) form a ternary complex at the apical cortex independently of the basal determinants. However, the mechanisms that underlie the asymmetric protein sorting in neuroblasts are not known. To address this issue, chromosomal deficiencies have been sought that affect the subcellular distribution of Miranda. Such screening identified the lgl tumor-suppressor gene that encodes a protein containing WD40 repeats. In wild-type neuroblasts, Miranda, which localizes apically during interphase, accumulates at the basal cortex upon mitosis after a transient spread into the cytoplasm. In germline clone embryos lacking both maternal and zygotic lgl activity (lglGLC embryos), Miranda does not localize asymmetrically in mitotic neuroblasts, but rather is distributed uniformly throughout the cortex as well as in the cytoplasm, where it is concentrated along microtubule structures. Consequently, Miranda segregates into both the daughter neuroblast and the ganglion mother cell (GMC). Numb and Pon are also distributed uniformly at the cortex and in the cytoplasm (Ohshiro, 2000).

Whether other tumor-suppressor genes contribute to protein localization in neuroblasts was investigated. The tumor-suppressor gene dlg encodes a membrane-associated guanylate kinase homolog. Germline clone embryos lacking both maternal and zygotic dlg activity (dlgGLC embryos) exhibit defective localization of Miranda and Numb essentially identical to that of lglGLC embryos, suggesting that both tumor-suppressor proteins function in the same process in neuroblasts. To investigate the relationship between the roles of Dlg and Lgl, their subcellular localization in neuroblasts was compared. Both Lgl and Dlg are distributed mainly throughout the cortex, whereas the amount of Lgl in the cytoplasm appears to be greater in mitosis than in interphase. The cortical localization of Lgl appears to be important for its function -- whereas the mutant protein encoded by the temperature-sensitive allele lglts3 is distributed normally at the permissive temperature (18°C), it fails to localize cortically at the restrictive temperature (29°C). The wild-type Lgl protein exhibits a similar, abnormal cytoplasmic distribution in dlgGLC embryos, whereas Dlg localization is not affected in lglGLC embryos. Thus, the cortical localization of Lgl requires dlg activity, suggesting that Dlg may function in localization of cell-fate determinants in neuroblasts by positioning Lgl at the cortex (Ohshiro, 2000).

There are two important processes associated with the asymmetric division: (1) the asymmetric localization of cell-fate determinants, which is achieved by specific adapter proteins that themselves localize asymmetrically to the cortex in neuroblasts; and (2) the orientation of the mitotic spindle and its coordination with the polarized localization of the determinants, which requires the apical Baz-Insc-Pins complex. This study has revealed another important process mediated by Dlg, Lgl and Myosins, which is responsible for the cortical anchoring of the determinant-adapter complexes. This process occurs upstream of the first and independently or parallel to the second of those two aspects of asymmetric division, as the localization of Lgl and Dlg is independent of apical or basal components. Both Lgl and Dlg contribute to the generation or maintenance of epithelial polarity, and zygotic mutants of the corresponding genes develop epithelial cell tumors as well as brain tumors at late larval stages. These previous observations with epithelial cells, together with the data on the roles of Lgl and Dlg in protein targeting in neuroblasts, suggest that aberrant sorting of intracellular proteins may be responsible for the tumor formation apparent in larval stages of lgl and dlg mutants (Ohshiro, 2000).

The tumour-suppressor genes lgl and dlg regulate basal protein targeting in Drosophila neuroblasts

Drosophila neuroblasts are a model system for studying asymmetric cell division: they divide unequally to produce an apical neuroblast and a basal ganglion mother cell that differ in size, mitotic activity and developmental potential. During neuroblast mitosis, an apical protein complex orients the mitotic spindle and targets determinants of cell fate to the basal cortex, but the mechanisms of these two processes are unknown. The tumor-suppressor genes lethal (2) giant larvae (lgl) and discs large (dlg) regulate basal protein targeting, but not apical complex formation or spindle orientation, in both embryonic and larval neuroblasts. Dlg protein is apically enriched and is required for maintaining cortical localization of Lgl protein. Basal protein targeting requires microfilament and myosin function, yet the lgl phenotype is strongly suppressed by reducing levels of myosin II. It is concluded that Dlg and Lgl promote, and myosin II inhibits, actomyosin-dependent basal protein targeting in neuroblasts (Peng, 2000).

Embryonic Drosophila neuroblasts develop from an apical/basal polarized epithelium. Individual cells delaminate into the embryo, enlarge to form neuroblasts, and begin a series of asymmetric cell divisions; these divisions result in the production of a large mitotically active apical cell (neuroblast), and a smaller basal cell (ganglion mother cell, GMC) that differentiates into two neurons or glia. A growing number of proteins are known to be asymmetrically localized in mitotic neuroblasts: apically localized proteins include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins); basally targeted proteins include Miranda, Prospero, Partner of Numb (Pon) and Numb, which are important for GMC development. Miranda and Prospero are apically localized at late interphase before their mitosis-dependent transport to the basal cortex. The Baz/Insc/Pins apical complex is required for both apical/basal spindle orientation and basal protein targeting, but little is known about how this complex regulates either process (Peng, 2000).

To identify genes required for apical/basal protein targeting in neuroblasts, deficiency stocks were screened looking for defects in Prospero basal localization in neuroblasts. This screen identified the lgl gene, which encodes a WD-40 repeat protein with homologues in many species, including the closely related 'Lgl family' genes Lgl1/Lgl2 (human), Lgl1 (mouse), U51993 (Caenorhabditis elegans); the slightly more divergent 'Tomosyn family' genes Tomosyn (rat), KIAA1006 (human), C617762 (Drosophila ), and M01A10 (C. elegans); and recently duplicated genes similar to both families: sro7/sro77 (budding yeast). In Drosophila, lgl mutations affect protein targeting to epithelial apical junctions, epidermal cell-shape changes, and produce tumors of the brain and the imaginal disc. This spectrum of phenotypes has been noted for another tumor-suppressor gene, discs large. This study explores the role of Lgl and Dlg in regulating neuroblast cell polarity (Peng, 2000).

Apical and basal protein targeting are compared in neuroblasts from wild-type embryos and embryos that lack all maternal and zygotic Lgl or Dlg function (called lglGLC or dlgGLC embryos). Wild-type metaphase neuroblasts show apical Insc/Pins localization, and basal Miranda/Prospero/Pon crescents. In addition, Miranda and Prospero proteins can be observed around the apical centrosome and weakly on the mitotic spindle in wild-type neuroblasts. In contrast, all lglGLC and dlgGLC metaphase neuroblasts show cytoplasmic Pon and uniformly cortical and strongly spindle-associated Miranda/Prospero; the apical proteins Insc/Pins are normal or slightly expanded. Although lglGLC and dlgGLC embryos show striking defects in neuroblast basal protein localization, they also show an early loss of embryonic epithelial apical/basal polarity, which could indirectly cause the observed neuroblast defects (Peng, 2000).

To determine the neuroblast-specific function of Lgl and Dlg, Lgl- or Dlg-depleted neuroblasts were studied in embryos or larvae where epithelial development occurs normally. Initially, homozygous null lgl4 embryos were studied, in which maternal Lgl protein allows normal embryonic epithelial development (including Armadillo, Crumbs and Dlg localization). In stage 16-17 lgl4 embryos, mitotic neuroblasts show normal Baz/Insc/Pins apical crescents, and normal spindle orientation, but Miranda/Prospero are delocalized onto the spindle and around the cortex and Pon is cytoplasmic. This phenotype is less severe in early embryos but fully penetrant in older embryos, presumably due to progressive loss of maternal Lgl protein. Next, neuroblasts were assayed in lgl3344 or dlgv55 homozygous larvae -- these larval neuroblasts are persistent embryonic neuroblasts that develop from a normal embryonic epithelium due to maternal Lgl and Dlg protein function. Wild-type larval metaphase neuroblasts have Insc/Pins crescents the opposite of Miranda/Prospero/Pon/Numb crescents, whereas homozygous lgl3344 or dlgv55 larval metaphase neuroblasts show normal Insc/Pins crescents but Miranda/Prospero/Pon proteins are cytoplasmic, uniformly cortical, and weakly spindle-associated. It is concluded that Lgl and Dlg are required specifically in neuroblasts for basal protein targeting, without affecting apical protein localization or spindle orientation (Peng, 2000).

The lgl and dlg neuroblast basal localization phenotype is cell-cycle dependent. lgl and dlg mutant embryonic and larval neuroblasts show a fully penetrant loss of basal protein targeting at metaphase, but by late anaphase or telophase most neuroblasts show normal basal protein localization; 'telophase rescue' of basal protein localization also occurs in baz, insc and pon mutants. These results indicate that there are probably multiple mechanisms for basal protein localization (Peng, 2000).

Delocalization of the Prospero and Numb proteins produces defects in the nervous system and other tissues, so lgl mutant embryos were scored for cell fate defects. lglGLC embryos have severe morphological defects that preclude analysis, and lgl4 embryos can only be scored for late embryonic phenotypes, due to persistence of maternal Lgl protein. lgl4 embryos show a decrease in Even-skipped lateral (EL) neuron number at stage 17. A similar but stronger phenotype is seen in numb mutants, suggesting that the lgl phenotype may be due to delocalization of Numb during the GMC divisions that produce the EL neurons. The relatively mild lgl phenotype could be due to 'telophase rescue' of Numb protein in these GMCs, or to maternal Lgl protein (Peng, 2000).

In wild-type embryonic neuroblasts, Dlg protein is cortical with an apical crescent from late interphase to the end of mitosis that co-localizes with Baz/Insc/Pins, whereas Lgl protein is uniformly cortical and weakly cytoplasmic. Similarly, larval neuroblasts show apical Dlg and uniform cortical/cytoplasmic Lgl localization. lgl mutants show normal Dlg localization, but dlg mutants show loss of cortical Lgl protein. Thus, Dlg acts upstream of Lgl for its localization, but not necessarily its function (Peng, 2000).

Thus, in neuroblasts Lgl and Dlg regulate targeting of all known basal proteins without affecting apical protein localization or spindle orientation. In epithelia, Lgl and Dlg are necessary to restrict proteins to the apical membrane domain. Lgl could promote protein targeting to specific membrane domains in both neuroblasts (basal) and epithelia (apical), similar to the role of Lgl-related proteins in facilitating secretory vesicle fusion at specific membrane domains in yeast and mammals. If so, Lgl must act in neuroblasts via a secretory pathway that is independent of brefeldin A, because it has been shown that treatment with brefeldin A disrupts Golgi, inhibits Wingless secretion, but does not block basal protein targeting. Alternatively, Lgl may actively promote actomyosin-dependent localization of basal proteins and/or function to keep myosin II levels low so that they do not interfere with myosin-dependent basal localization. A general function of the Lgl protein family may be to increase the fidelity of protein targeting to specific domains of the plasma membrane (Peng, 2000).

Synaptic targeting and localization of Discs-large is a stepwise process controlled by different domains of the protein

Membrane-associated guanylate kinases (MAGUKs) assemble ion channels, cell-adhesion molecules and components of second messenger cascades into synapses, and are therefore potentially important for co-ordinating synaptic strength and structure. The targeting of the Drosophila MAGUK Discs-large (Dlg) to larval neuromuscular junctions has been examined. During development, Dlg is first found associated with the muscle subcortical compartment and plasma membrane, and later is recruited to the postsynaptic membrane. Using a transgenic approach, a study of how mutations in various domains of the Dlg protein affect Dlg targeting was undertaken. Deletion of the HOOK region -- the region between the Src homology 3 (SH3) domain and the guanylate-kinase-like (GUK) domain -- prevents association of Dlg with the subcortical network and renders the protein largely diffuse. Loss of the first two PDZ domains leads to the formation of large clusters throughout the plasma membrane, with scant targeting to the neuromuscular junction. Proper trafficking of Dlg missing the GUK domain depends on the presence of endogenous Dlg. It is concluded that postsynaptic targeting of Dlg requires a HOOK-dependent association with extrasynaptic compartments, and interactions mediated by the first two PDZ domains. The GUK domain routes Dlg between compartments, possibly by interacting with recently identified cytoskeletal-binding partners (Thomas, 2000).

The sequence preceding PDZ1 exhibits only weak or no homology between various MAGUKs. Several studies have implicated the amino terminus of both PSD-95 and SAP97 in junctional targeting. To determine whether the amino terminus of Dlg is of similar importance, transgenic Dlg missing the amino terminus (DeltaN), was expressed. This construct exhibits a synaptic localization indistinguishable from the control (Dlg-FLAG). This result is found both when DeltaN is expressed in the presence or absence (in dlgX1-2 mutants) of endogenous Dlg (Thomas, 2000).

In both the wild type and dlg mutant background, deletion of any single PDZ domain does not affect the synaptic localization of Dlg. Deletion of PDZ3 in combination with either PDZ1 or PDZ2 also does not affect localization. In contrast, deletion of both PDZ1 and PDZ2 (DeltaPDZ1+2) has dramatic effects on localization. This variant becomes localized at the surface of the muscle, with little synaptic localization. The immunoreactivity at the plasma membrane appears as large spots or clusters distributed throughout the muscle membrane. The DeltaPDZ1+2 variant can also be detected at the subcortical network, although the intensity of FLAG immunoreactivity is significantly weaker than in Dlg-FLAG-expressing muscles. Thus, in the absence of PDZ1 and 2, Dlg becomes transported to the muscle membrane, but fails to be directed to synaptic sites, and instead accumulates in ectopic clusters. This conclusion was reached by expressing DeltaPDZ1+2 in both the wild type and in dlgX1-2 mutants. To determine whether the distribution of endogenous Dlg is altered by the presence of these ectopic clusters, double labeling experiments were performed, in which DeltaPDZ1+2 and endogenous Dlg proteins were discriminated using anti-DlgGUK. The anti-DlgGUK antibody recognizes both endogenous and transgenic Dlg, but the anti-DlgPDZ antibody does not recognize DeltaPDZ1+2. Notably, endogenous Dlg does not become trapped in clusters containing the DeltaPDZ1+2 variant, but remains synaptically localized (Thomas, 2000).

Interestingly a transgenic protein comprising the amino terminus and all three PDZ domains (DeltaC1/2) fails to localize to the plasma membrane or synapses, and is instead found highly enriched in nuclei and cytoplasm. This result indicates that the PDZ1 and 2 domains are necessary but not sufficient for synaptic targeting (Thomas, 2000).

The involvement of the SH3 domain in synaptic targeting of MAGUKs has been controversial. To determine the role of the SH3 domain in Dlg targeting, tests were performed on both the allele dlgm30, in which the SH3 domain is affected by a point mutation, and flies expressing a Dlg variant in which the SH3 domain was deleted (DeltaSH3). In both dlgm30 and DeltaSH3 flies, Dlg is normally targeted to synaptic sites. In the case of the DeltaSH3 line, similar results were obtained in the presence and absence of endogenous Dlg (Thomas, 2000).

The HOOK region has been implicated in the association of Dlg with septate junctions in epithelial cells. This domain is among the least conserved regions of MAGUKs, but two sub-regions are moderately conserved among specific subsets of MAGUKs. A band 4.1-binding motif known as I3 is also found in SAP97, PSD-93 and p55 and may link these MAGUKs to the actin/spectrin cytoskeleton. Another short stretch (E-F region), presumed to form an alpha-helix at the amino terminus of the HOOK region, is found in all Dlg-like MAGUKs, and has been implicated in calmodulin-dependent dimerization of PSD-95 and SAP102 (Thomas, 2000 and references therein).

Deletion of the entire HOOK region (DeltaHOOK) dramatically affects the synaptic localization of Dlg both in the presence and absence of endogenous Dlg. In general, some synaptic localization is observed, although weaker than in Dlg-FLAG controls. In addition, the FLAG signal appears in the muscle nuclei and throughout the cytoplasm. Extrasynaptic localization at the plasma membrane is weak compared with Dlg-FLAG controls, and virtually no specific immunoreactivity is detected at the subcortical network. The relative intensity of the signals at synapses versus nuclei varies, even at the muscles within the same sample. Strikingly, very similar results are obtained when the amino-terminal helix within the HOOK region is disrupted by deleting only 13 amino acids (DeltaE-F). Deletion of the I3 region (DeltaI3) also results in weak synaptic localization of the transgenic protein when expressed in wild type. Unlike DeltaHOOK and DeltaE-F, however, deletion of I3 does not result in nuclear localization. Surprisingly, DeltaI3 exhibits strong synaptic localization when expressed in dlgX1-2 mutants (Thomas, 2000).

The importance of the HOOK region for targeting to both septate and synaptic junctions appears surprising, because it is not well conserved among MAGUKs. Nevertheless, a short stretch of amino acids at the amino terminus of the HOOK region of all Dlg-like MAGUKs is predicted to form an alpha-helix with a cluster of basic residues on one side. This helix has been reported to mediate a calmodulin-dependent dimerization of PSD-95 and SAP102. The finding that disruption of this helix mimics the effect of the entire DeltaHOOK deletion, confirms that it is of particular importance in vivo. It remains to be determined whether it functions in synaptic targeting by promoting dimerization of Dlg, by contributing to an intramolecular interaction, and/or by mediating heterophilic interactions (Thomas, 2000).

The role of the GUK domain in MAGUKs has remained obscure. In the case of fly epithelial cells, deletion of the GUK domain does not alter localization of Dlg to septate junctions. Various cytoskeletal and synapse-associated proteins have been reported to bind to the GUK domain of mammalian MAGUKs, suggesting that this domain may be important for synaptic localization. To ascertain the role of GUK in synaptic targeting, transgenic flies expressing a Dlg variant lacking the GUK domain (DeltaGUK) were examined. In the presence of endogenous Dlg, DeltaGUK becomes localized to synaptic sites, although this synaptic expression appeared consistently weaker than in Dlg-FLAG-expressing controls. Notably, however, synaptic localization of DeltaGUK is barely or not detectable upon expression in dlgX1-2 mutants. It is therefore concluded that endogenous Dlg mediates synaptic targeting of the DeltaGUK variant (Thomas, 2000).

None of these deletions resulted in an obvious accumulation solely at the subcortical network. This suggests that no additional domains are required to leave this compartment. A role for the GUK domain to direct subsequent transport is supported. Recent evidence suggests that targeting of PSD-95 and PSD-93 involves a microtubule-dependent transport of vesiculo-tubular structures. These MAGUKs can be linked to microtubules by binding of the PDZ3 and GUK domains to the microtubule-associated proteins CRIPT or MAP1A, respectively. Although these binding partners are suggestive for a role of these domains in MAGUK trafficking, no such function has previously been unraveled. Removal of PDZ3 has no obvious effect on synaptic localization of Dlg. Synaptic localization of the DeltaGUK protein is, however, diminished in wild-type flies and virtually abolished in dlgX1-2 mutants. This dependency on endogenous Dlg might be explained by dimerization. Alternatively, endogenous Dlg could promote vesiculo-tubular trafficking and thus allow the truncated version to hitchhike on the same vesicles. This explanation would also apply to the finding that the GUK domain is dispensable for targeting of PSD-95 in cultured neurons or slices, in which the endogenous MAGUK is expressed (Thomas, 2000).

Consistent with other studies of MAGUK targeting, it was found that the PDZ domains are neither sufficient to target Dlg to specific extrasynaptic sites nor to dock the protein at the synapse. Nonetheless, the PDZ1 and 2 domains are found to contribute to synaptic targeting. It is suggested that either the PDZ1 or PDZ2 domain is required for the final step in Dlg targeting, from plasma membrane to synapses, but is not necessary to direct Dlg to the plasma membrane. Thus, it may be assumed that the interaction with at least one PDZ binding protein is required to transport the protein to the synapse. A double mutant, in which the only known binding partners for the PDZ1 and 2 domains of Dlg, Shaker and FasII were abolished or dramatically reduced, has no obvious effect on synaptic targeting of Dlg. Interestingly, a non-synaptic PDZ-binding protein, Cypin, may regulate synaptic targeting of PSD-95 and SAP102 at extrasynaptic sites (Thomas, 2000 and references therein).

It remains to be determined whether the association of Dlg with intracellular membrane compartments serves solely to target Dlg itself. As discussed for GRIP1 and PSD-95, this step could also contribute to the sorting and co-transport of other synaptic molecules such as ion channels. The pre-formation of a junctional protein complex at intracellular compartments has been exemplified by the interaction of E-cadherin and beta-catenin at the endoplasmic reticulum. The clustering of glycine receptors by gephyrin provides a contrary example, however, since gephyrin traps receptor molecules only at developing synapses. Unfortunately, the limited sensitivity of available Shaker-specific antibodies has impaired attempts to distinguish between intracellular and synaptic assembly of the Dlg-Shaker complex. It appears obvious, however, that each targeting step represents an additional site at which the molecular composition of synaptic junctions could be regulated (Thomas, 2000).

Bazooka is a permissive factor for the invasive behavior of discs large tumor cells in Drosophila ovarian follicular epithelia

Drosophila Bazooka and atypical protein kinase C are essential for epithelial polarity and adhesion. Wild-type bazooka function is required during cell invasion of epithelial follicle cells mutant for the tumor suppressor discs large. Clonal studies indicate that follicle cell Bazooka acts as a permissive factor during cell invasion, possibly by stabilizing adhesion between the invading somatic cells and their substratum, the germline cells. Genetic epistasis experiments demonstrate that bazooka acts downstream of discs large in tumor cell invasion. In contrast, during the migration of border cells, Bazooka function is dispensable for cell invasion and motility, but rather is required cell-autonomously in mediating cell adhesion within the migrating border cell cluster. Taken together, these studies reveal Bazooka functions distinctly in different types of invasive behaviors of epithelial follicle cells, potentially by regulating adhesion between follicle cells or between follicle cells and their germline substratum (Abdelilah-Seyfried, 2003).

Border cell migration during Drosophila oogenesis is one well-studied example of invasive and directed migration. Border cells are specified within the anterior follicular epithelium that surrounds the germ cells in each egg chamber. At late egg chamber stage 8, approximately eight border cells delaminate from the monolayer epithelium and, in a highly stereotyped fashion, invade the germ cell cluster within the developing egg chamber. First, they undergo directed cell migration between nurse cells towards the anterior margin of the oocyte and then turn dorsally, coming to rest at the dorsal anterior corner of the egg chamber next to the underlying oocyte nucleus. Border cell migration displays several features that are reminiscent of metastasis by cancer cells. Initially, border cells are polarized epithelial cells that lose some homophilic cell adhesion, undergo an epithelial-to-mesenchymal transition, acquire adhesion with the substratum, and undergo cell migration. However, not all epithelial characteristics are lost during migration. The migrating cells remain attached to each other and intercellular polarized junctions containing DE-cadherin (Shotgun), Armadillo (Arm) and Crumbs are present. DE-cadherin has been demonstrated to play an essential role in both migrating border cells, and their substratum, the germ cells. These studies suggest homophilic interactions between transmembrane receptors, such as DE-cadherin, may provide the necessary adhesion between invasive cells and their substratum (Abdelilah-Seyfried, 2003 and references therein).

The results suggest that baz is an essential component of dlg mutant follicle cell invasion into the germline. During border cell migration baz is dispensable for invasion and motility but appears to be required for correct cell adhesion within the migrating cluster. baz acts downstream of dlg in controlling follicle cell invasion. Taken together, these results suggest that loss of dlg initiates epithelial-to-mesenchymal transition and results in increased follicle cell motility. One role of wild-type baz may be to ensure the proper adherence between invading cells and their substratum (Abdelilah-Seyfried, 2003).

Two lines of evidence suggest mechanistic differences between tumor cell and border cell invasion. (1) While both dlg tumor cell and border cell invasion undergo a series of similar morphogenetic behaviors, the molecular mechanisms regulating each cellular repertoire appear, at least in part, to be distinct. Whereas tumor cell invasion is dependent on baz, border cell invasion and motility are not. Therefore, baz genetically discriminates between these processes. Conversely, border cell migration requires slbo function, whereas dlg mutant follicle cell invasion can occur with much lower levels of Slbo and FasIII proteins and therefore dlg mutant cells appear not to adopt a border cell fate. (2) A second line of evidence for mechanistic differences is that the patterns of cell invasion are distinct. The timing, direction and cohesion of border cells during their migration is highly stereotyped. In contrast, dlg tumor cells can invade at any stage in egg chamber development and in any orientation relative to the oocyte, possibly due to the position where follicle cell over-accumulation and multi-layering occur. Moreover, invasion also occurs in the absence of an oocyte, for example when the germline is dlgm52;bazEH171 double mutant (Abdelilah-Seyfried, 2003).

The data suggest that wild-type Baz is a permissive factor required for follicle cell invasion but that baz gene function is dispensable for border cell specification and invasion. Therefore, in the absence of baz, the specification of Slbo-positive cells and activation of the appropriate downstream targets that are required for the orchestration of border cell migration is normal. The activation of slbo and its target genes may largely mask the permissive role of baz in follicle cell migration, a requirement that is uncovered in the context of the slbo-independent type of follicle cell invasion caused by the loss of dlg. Moreover, in contrast to DE-cadherin, another gene with an essential function during border cell migration, Baz and DaPKC levels are not increased in border cells prior to and during border cell migration. The defects observed in baz mutant border cell migration are best explained by the lack of adhesion within the border cell cluster rather than by migratory defects (Abdelilah-Seyfried, 2003).

This study provides an example of a genetic interaction between the apical PAR complex and basolateral tumor suppressor genes. This interaction was assessed based on tumor cell invasion. baz is epistatic over (functions downstream of) dlg in regulating this process. One possible explanation for the mechanism by which Dlg, a basolateral protein absent from the sites of contact between follicle and germ cells, regulates motility is that it acts via another protein complex. Evidence is presented that the apical PAR complex may serve such a function. A model is suggested in which follicle cell invasion is a two-step process: first, the loss of dlg releases a repression of motility and, second, the apical PAR complex protein Baz serves as a permissive factor for invasion. Based on mosaic analysis, a model is proposed in which invasion might be mediated by two separate baz-dependent interactions between follicle and germline cells. During invasion of dlg mutant follicle cells, Baz functions as a permissive factor to promote follicle cell invasive behavior. This invasive behavior is blocked in the absence of follicle cell Baz, since dlgm52 bazEH171 or bazEH171 mutant follicle cells lack invasive properties. Within the germline, Baz functions as both a permissive factor during invasion of dlgm52 mutant follicle cells that express Baz, possibly by stabilizing adhesion between the invading somatic cells and the germline cells and, in the absence of follicle cell Baz, as a repressor of follicle cell invasion, possibly by regulating germ cell adhesion and preventing invasion of Baz-deficient follicle cells. The repression of Baz-deficient follicle cell invasion is neutralized in dlgm52 bazEH171 mutant germ cell clones possibly by a reduction of germ cell adhesion that may increase the ease with which dlgm52 bazEH171 mutant follicle cells can invade. These observations raise the question as to the molecular machinery and the adhesion molecules that mediate baz-dependent invasion and to the mechanisms that are in place in dlgm52 bazEH171 mutant follicle and germ cells in which invasion occurs. An alternative explanation to the loss of motility is that the removal of a second cell polarity system from follicle cells may cause such severe disturbances as to prevent cell invasion. However, dlgm52 bazEH171 double mutant follicle cells retain their capability to invade into dlgm52 bazEH171 double mutant germline proper, contradicting this explanation (Abdelilah-Seyfried, 2003).

The data presented in this study raise the possibility that DaPKC serves similar, essential functions during dlg tumor cell invasion. However, this hypothesis was not tested since it was genetically not possible to generate dlg DaPKC double mutant follicle and germline clones. During border cell migration, there is a different requirement for Baz and DaPKC. Whereas Baz appears to affect adhesion within the migratory border cell cluster, DaPKC function is dispensable for normal border cell invasion, migration, and adherence (Abdelilah-Seyfried, 2003).

In contrast to previous findings, the results indicate dlg predominantly functions cell-autonomously to prevent invasion of follicle cells. This finding is consistent with the data on lgl, which also functions cell-autonomously within the follicle cell layer to prevent heterogeneous cell mixing and invasion. Indeed, cases of cell-autonomous invasions of follicle cells into the germline have been documented; these support the notion that, despite quantitative differences between the studies, dlg functions cell-autonomously within the follicle cell layer. The FLP/FRT technique combined with GFP imaging used in the study allows for the unambiguous identification of mosaic tissues, clarifying issues of cell-autonomous gene function (Abdelilah-Seyfried, 2003).

The multiple PDZ domain protein Baz and its vertebrate homolog ASIP is a membrane scaffolding factor required for assembly and sub-membrane attachment of the apical PAR complex. The effects of the PAR complex on dlg mutant follicle cell invasion may be exerted via a separate but baz-dependent transmembrane adhesion complex, the nature of which is currently unknown. In contrast to its function during border cell migration, in humans, loss of E-cadherin correlates with and appears to promote the occurrence of invasive tumor formation. It has been suggested, therefore, that E-cadherins serve distinct functions in different cell types, either by promoting or inhibiting cell motility. Further studies are required to test whether the homologous proteins of Baz (ASIP) and DaPKC (atypical PKCs iota and zeta) serve conserved functions in mammalian cells and, in contrast to E-cadherin function, whether their loss prevents tumor cell invasion. Moreover, it is unclear whether baz function is restricted to the behavior of dlg mutant follicle cells or is essential in other forms of tumor cell invasions (Abdelilah-Seyfried, 2003).

Basolateral junctions utilize warts signaling to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells

Fasciclin2 (Fas2) and Discs large (Dlg) localize to the basolateral junction (BLJ) of Drosophila follicle epithelial cells and inhibit their proliferation and invasion. To identify a BLJ signaling pathway a genome-wide screen was performed for mutants that enhance dlg tumorigenesis. Two genes were identified 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/rotund), 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 (Zhao, 2008).

The purpose of this work was to gain greater insight into how the BLJ suppresses epithelial tumorigenesis and invasion by identifying and understanding the function of new genes important for BLJ function. To do so, a genomewide screen was completed for enhancers of dlg, which encodes a scaffolding protein that is a crucial organizer of the BLJ and is a potent repressor of follicle epithelial cell tumorigenesis and invasion. Deficiencies that cumulatively span ∼80% of the autosomes, or 64% of the Drosophila genome were systematically screened. A relatively small number of enhancers, ∼1 per 1000 genes screened, were detected indicating that the screen selected for loci specifically required for dlg function. Thus, the novel dlg enhancer genes that were identified, wts, roe, ebi, as well as at least two genes yet to be identified, are likely to be key collaborators with dlg in suppressing epithelial invasion. The specificity of the interactions between dlg and these enhancers is further indicated in that more than one allele of each gene showed an interaction, in several dlg backgrounds, and the strengths of enhancement were similar to deficiencies defining each locus. wts, roe, and ebi also enhanced Fas2 and lgl, indicating that they are not just important for dlg function, but for the function of the BLJ as a whole. In addition, overexpression of all enhancers except ebi suppressed dlg and Fas2 tumorigenesis, further confirming that the identified genes function in a BLJ network (Zhao, 2008).

BLJ pathway components in the nucleus and their putative relationship to Notch: ebi encodes an F-box protein with WD repeats that promotes protein degradation of specific targets. The failure of ebi overexpression to suppress Fas2 or dlg, and the relatively mild ebi phenotypes (midoogenesis small-nucleus and epithelial-organization defects, but no defects in germinal vesicle localization), suggest that ebi may function in only one of the three branches of BLJ signaling or in a parallel pathway to the BLJ. In the eye, ebi is important for promoting differentiation and inhibiting proliferation, which appear to be separable functions. Thus ebi could enhance Fas2 and dlg tumorigenesis by functioning within the proliferation-repressing branch of the BLJ, or the importance of ebi for differentiation suggests that it could function in the EMT branch of the BLJ or both. In contrast, ebi promotes protein degradation in response to Notch (N) and Drosophila EGF receptor (EgfR) signals, suggesting that it may act in a parallel pathway. Both Ebi and its mammalian homolog, TBL1, function in a corepressor complex through association with nuclear hormone transcriptional corepressor SMRTER/SMRT (Zhao, 2008).

Interestingly, although most N appears to be localized on the apical surface of follicle cells, some N is also localized in BLJs. Thus, it is possible that N localized to the BLJ may signal directly to Ebi. Consistent with this possibility, it was found that all of the genes in the BLJ network share some midoogenesis defects with N, including the small nucleus phenotype, epithelial stratification defects, and mislocalization of the germinal vesicle. The epithelial defects are also reminiscent of N-pathway mutants brainiac and egghead, which are required in the germ line for regulating N that is localized on the apical surface of the follicle cells abutting the germ line. Thus one possibility is that N signaling activity is regulated by its localization to apical vs. basolateral junctions in response to several signaling pathways acting during midoogenesis (Zhao, 2008).

The other modest dlg enhancer that was identified, roe, encodes a Krüppel-family zinc-finger protein that appears to be a transcription factor. Roe is also implicated in Notch signaling and thus may function with Ebi in N-dependent processes as proposed above. However, in contrast to ebi, roe loss caused follicle cell tumors, suggesting that roe may function more directly in a BLJ pathway than ebi. Consistent with a direct role for Roe in BLJ signaling, it was found that roe overexpression suppressed Fas2 and dlg tumorigenesis. Further, as for Fas2, dlg, and wts, roe represses CycE and DIAP1 expression (Zhao, 2008).

Warts was of special interested because of the many similarities observed in the quality and strength of wts and scrib phenotypes, suggesting that they are components in a BLJ signaling pathway, rather than a parallel pathway that cross talks with BLJ signaling. wts encodes a serine/threonine kinase that is an ortholog of human tumor suppressors Lats1 and Lats2, both of which have been linked to highly aggressive breast cancers. The prevailing model for Wts signaling in Drosophila is based on signaling in eye and wing tissue. Wts appears to relay signals from apicolateral junction proteins Ft, Ex, and Mer in wing and eye tissues. However, the results from almost every assay, including early tumor formation, border cell migration, BrdU, PH3, CycE, and DIAP1 expression, indicated little functional overlap between Ft, ex, mer, or mer; ex and wts, thus diminishing the importance of apicolateral Ft-Ex-Mer for Wts activation in follicle cells. The exceptions were that during midoogenesis, Mer is required for border cell migration and Ex is required for the endocycle switch, while both are required for maintenance of epithelial integrity and positioning of the germinal vesicle. However, the involvement of Ex and Mer in these processes are fundamentally distinct from how they act in Wts-dependent processes in other tissues. (1) Ft is not involved; (2) no indication was observed of Ex-Mer synergism; (3) ex, mer, and mer; ex phenotypes are relatively mild when compared to wts. It is concluded that the model for Wts activation in which apicolateral junction proteins Ft, Ex, and Mer play the predominant role cannot be universally applicable in all cell types. Rather, the relative importance of Ex and Mer for Wts regulation appears to depend on developmental context (Zhao, 2008).

Consistent with this proposal, strong functional interdependence and phenotypic similarities were found between Fas2, dlg, lgl, scrib, and wts, thus indicating that the BLJ, not the apicolateral junction, plays the predominant role in Wts regulation during oogenesis. Although genetic evidence alone cannot completely rule out that Wts may act in a parallel pathway to the BLJ and impinge on a set of downstream targets that overlap with those targeted by the BLJ, the following observations favor a model in which the BLJ is more directly involved in Wts regulation (it is noted that these are not mutually exclusive alternatives): (1) over 50 tumor suppressor genes have been identified in Drosophila, but lgl, scrib, and wts were the only strong dlg enhancers identified in this genomewide screen; (2) wts showed strong genetic interactions with Fas2, dlg, and lgl, similar to or stronger than scrib, which encodes a known BLJ protein; (3) wts has early tumor phenotypes similar to dlg partial loss of function and to scrib; (4) wts has the same border cell migration phenotype as scrib; (5) wts has similar small nucleus, epithelial stratification, and germinal vesicle defects as Fas2, dlg, lgl, and scrib; (6) like lgl and scrib, wts overexpression suppressed Fas2 and dlg tumorigenesis; (7) Fas2, dlg, and wts have similar proliferation defects, and (8) Fas2, dlg, and wts similarly repress CycE and DIAP1 expression, which is especially crucial, because CycE and DIAP1 are downstream targets of Wts signaling, and ex and mer had no impact on their expression, contrary to results in other tissues. Thus, the data strongly indicate that the BLJ signals through Wts, and may impinge on Roe in the nucleus, thus suggesting the first BLJ signaling pathway in animal cells. This implies that the BLJ not only acts as a localized scaffold, but also signals to the nucleus to control gene expression, both of which cooperate to regulate epithelial polarity and dynamics (Zhao, 2008).

How can these results in follicle cells, which suggest that Wts acts predominantly downstream of the BLJ, be reconciled with findings in eye tissue, which indicate that Wts acts downstream of the apicolateral junction? Interestingly, the genetic data in the eye suggest that Ft, Ex, and Mer cannot account for all of the signals that activate Wts, because wts overgrowth and tissue disorganization phenotypes are more severe than ft or mer; ex. On the basis of these findings in follicle cells, it is possible that Wts activation in the eye requires additional input from the BLJ. This possibility may have been overlooked thus far because dlg does not appear to have an overgrowth phenotype in the eye. dlg may be essential for additional functions in the eye that are epistatic to its tumor suppressor function, thus preventing loss of cells from the epithelium that could mask an overgrowth phenotype. Consistent with this, when activated Rasv12 is combined with dlg loss, dramatic tumors develop that are larger and more invasive than those produced by Rasv12 alone (Zhao, 2008).

In contrast, Dlg may have a diminished role in Wts signaling in the eye, much as the evidence indicates a diminished role for Ex and Mer in Wts signaling in the ovary. According to this model, Wts receives predominant input from distinct lateral junctions depending on tissue context. One distinction is that ovarian follicle cells are derived from a mesodermal lineage, while the eye and wing tissues are from ectodermal lineages. Further, many genes that disrupt apical-basal polarity and epithelial morphology have only subtle phenotypes in the eye by comparison to the ovary or embryo. Finally, the follicular epithelium requires input from junctions on all three follicle cell surfaces, lateral, apical, and basal, whereas most epithelia require only two, lateral and apical or basal. Thus, ovarian and imaginal tissues are likely to organize signaling pathways acting downstream of epithelial junctions in similar, yet fundamentally different ways to meet the unique organizational requirements of their cell-tissue morphologies. Some or all of these differences may contribute to the suggested specificity observed in Wts signaling downstream of BLJs in follicle cells. In general, these findings raise the possibility for future investigation that depending on the cell-tissue morphologies of a given organ, one lateral junction may play a predominant organizational role, and Wts signaling may act as a universal signaling adapter for mediating contact inhibition from that junction (Zhao, 2008).

An especially interesting aspect of Mer and Ex function that was uncovered in follicle cells is that it appears to be restricted to predominantly postmitotic, differentiated cells, in contrast to the role of Mer and Ex in other tissues. Further, given the absence of an involvement of Ft and lack of Mer-Ex synergism it is concluded that if Mer and Ex would be involved in Wts activation in follicle cells, they would have to function via a fundamentally distinct mechanism than in other tissues. It is proposed that during early oogenesis, the BLJ alone may provide the predominant input to Wts. Then, during midoogenesis, Ex and Mer may become involved in novel interactions with Dlg or other components of the BLJ to activate Wts in spatiotemporally distinct populations of differentiating cells to help achieve their unique developmental functions (Zhao, 2008).

How do wts, scrib, and roe promote motility? It is proposed that Scrib, Wts, and Roe are all crucially involved in EMT. In EMT, cells (1) loose apical-basal polarity and become mesenchymal-like, and (2) adopt a polarity conducive to movement. scrib, wts, and roe cells clearly lose epithelial polarity and become mesenchymal-like as indicated by their rounded morphology and lateralized phenotype. However, scrib, wts, and roe tumors do not invade, and scrib, wts, and roe border cells do not move, suggesting that the second aspect of EMT, adoption of a polarity conducive to movement, is defective. Consistent with this, mammalian Scrib is required for migration and epithelial wound healing of cultured human breast epithelial cells, and is also required in vivo for wound healing in mice. Human Scrib directs migration by organizing several polarities crucial for migration, including the orientation of the microtubule and Golgi networks and the localization of Cdc42 and Rac1 to the cell's leading edge. Thus Scrib has a conserved function in directed cell migration by organizing a polarity conducive to movement. In mammalian PC12 cells Scrib is in complex with Rac1. Fly Rac1 is essential for border cell migration and invasion of Fas2 and dlg tumors, suggesting that an essential role of Scrib in Rac1 function may be of crucial importance for movement. The apparent conserved role of BLJ proteins in organizing EMT, and both promoting and repressing movement, reemphasizes the suggestion that BLJ proteins do more than merely maintain apical-basal polarity, but rather repress a cellular transformation from epithelial polarity to a mesenchymal, lateralized signature conducive to movement (Zhao, 2008).

How is the function of scrib, wts, and roe in promoting border cell movement consistent with the requirement of Fas2, dlg, and lgl in repressing border cell movement? Further, how do scrib and wts act as enhancers of dlg tumor invasion even though scrib and wts tumors are noninvasive? For border cell movement, Fas2 and dlg mutations not only accelerate movement, but also delay border cell delamination. The delay in border cell delamination suggests that the BLJ normally promotes motility, but this promoting function can be bypassed when the repression of motility branch of the BLJ pathway is simultaneously lost. Cumulative data indicate that scrib, wts, and roe act predominantly within the EMT and proliferation branches of the BLJ pathway, and not the repression of motility branch. It is suggested that without simultaneous loss of the repression of motility branch of the BLJ pathway, scrib and wts border cells cannot bypass the essential requirement for the second step of EMT, thus border cell motility is blocked (Zhao, 2008).

This interpretation is also consistent with the seemingly paradoxical function of scrib and wts as enhancers of dlg tumor invasion, even though Scrib and Wts promote rather than repress border cell movement. The noninvasive scrib and wts tumor phenotypes indicate that they are crucial for repressing the first step of EMT, loss of epithelial polarity and adoption of a lateralized, mesenchymal-like phenotype. It has been suggested that scrib and wts enhance dlg invasive tumorigenesis by increasing the rate at which dlg mutant follicle cells undergo EMT and further facilitate invasion by depressing proliferation control and increasing the number of follicle cells available for movement. Thus, even though scrib and wts are required to promote movement, it is suggested that in dlg; scrib/+ or dlg; wts/+ tumors this requirement can be bypassed because the branch of the BLJ pathway that represses motility is simultaneously disrupted (Zhao, 2008).

The noninvasive tumor phenotypes of scrib and wts are very similar to the phenotypes of dlg mutants that specifically disrupt Dlg SH3 and GuK domains. Thus Scrib and Wts may act specifically downstream of the Dlg SH3 and GuK domains. Consistent with this, Scrib appears to associate with the Dlg GuK domain in neuronal synapses via the linker protein GuK-holder. Further, whereas Fas2, dlg, and lgl cause faster border cell migration, border cell migration is very similar to wild type in the dlg SH3/GuK-specific mutants, suggesting that Dlg SH3/GuK predominantly represses the first step of EMT and proliferation but not motility. On the basis of this specificity, it is suggest that one reason that lgl may be a stronger dlg enhancer than scrib and wts is that lgl represses motility in addition to EMT and proliferation. For example, the de novo tumor formation observed when one copy of lgl, scrib, or wts is removed in dlghf/dlgsw ovaries suggests that a threshold level of BLJ activity essential for maintenance of polarity has been lost. However, the lgl interaction may be much stronger than scrib and wts because lgl additionally represses motility (Zhao, 2008).

Increased expression of CycE and DIAP1, known Wts targets, was observed in Fas2, dlg, lgl, scrib, wts, and roe cells. Thus the importance of CycE for proliferation control, and DIAP1 for control of EMT and motility, suggests that part of the mechanism by which Fas2-Dlg represses tumorigenesis is through activating Wts signaling. DIAP1 is in a complex with Rac1 and Profilin and enables border cell motility apparently by promoting actin turnover. Further, in the embryo, DIAP1 loss leads to Dlg cleavage and cellular rounding and dispersal. Too much DIAP1 also appears to be deleterious to movement, because targeted overexpression of DIAP1 specifically in border cells slows their migration (data not shown). Thus maintaining the proper balance of DIAP1 is critical for directed movement, and it may be part of the mechanism by which Scrib and Wts influence border cell movement, suggesting that interaction with Dlg and Rac1 may be another level at which Scrib regulates EMT and movement, consistent with the possibility that it functions downstream of Scrib and Wts in follicle cells to repress both EMT and proliferation (Zhao, 2008).

In contrast to the strong enhancement of dlg by scrib, Fas2 was only weakly enhanced by scrib. Given the complexity of coordinating EMT, proliferation, and motility within an epithelial field, perhaps the simplest model is that multiple Dlg complexes reside within the BLJ, each with a distinct set of ligands that control one or more morphogenetic activities (Zhao, 2008).

Another interesting difference in the enhancement of dlg and Fas2 by lgl, scrib, wts, and roe was that they all enhanced both dlg tumorigenesis and invasion, but only enhanced Fas2 tumorigenesis, without invasion. An important difference between these experiments may be that in Fas2null follicle cells, Dlg is missing Fas2 as a ligand, whereas in dlghf/dlgsw, dlghf/dlgip20, and dlghf/dlglv55 follicle cells, Fas2 is localized at sites of contact between follicle cells in both the native epithelium and in streams of invading cells, suggesting that Fas2 continues to act as a Dlg ligand in these cells. This is probably an important difference because Fas2-Dlg binding is expected to control the conformation of Dlg. Dlg conformations in turn may specify Dlg intra- and intermolecular interactions that determine the relative balance of EMT, proliferation, and invasion factors that associate with the BLJ scaffold. For example, in neuronal cells intramolecular interactions between Dlg SH3 and GuK domains regulate the strength of intermolecular binding of GuK-holder, which binds Scrib. The SH3-GuK intramolecular interaction is further modulated by intramolecular interactions with PDZ3, which are regulated by intermolecular interactions with neurolignin, a transmembrane ligand for PDZ3 (Zhao, 2008).

On the basis of this molecular model, it is proposed that in the absence of Fas2, Dlg has a distinct conformation that tilts the balance toward EMT and proliferation over invasion, when Lgl, Scrib, Wts, or Roe are reduced. This study has shown that lgl, scrib, wts, and roe are expected to act predominantly downstream of Dlg SH3 and GuK domains to repress EMT and proliferation. Thus, removal of one copy of lgl, scrib, wts, or roe in Fas2 cells may tip the ratio of factors controlling EMT, motility, and proliferation toward derepression of EMT and proliferation, masking the Fas2 requirement for invasion. One possibility is that lgl, scrib, wts, or roe are especially important for expression of a protein in the apicolateral junction, such as Par-3/Bazooka, which is essential for dlg invasion. Consistent with this, Ex upregulation is seen in both dlg and wts clones. Further, lgl enhancement at the lglts permissive temperature showed essentially the opposite trend from Fas2. Rather than enhance tumorigenesis over invasion, removal of one copy of Fas2, dlg, scrib, wts, or roe in lgl egg chambers favored invasion. Thus, it is suggested that tumor invasiveness associated with particular combinations of mutated BLJ proteins may be masked or unmasked on the basis of the balance of activities that are disrupted, rather than disruption of particular activities per se (Zhao, 2008).

In summary, this study has identified the first signaling pathway that acts downstream of the BLJ that specifically controls EMT and proliferation, and important clues have been gained as to how this signaling may be organized. Like the Drosophila follicular epithelium, the human ovarian surface epithelium, which is thought to be the site of origin of most ovarian cancers, is derived from a mesodermal lineage. The data suggest that the BLJ plays an especially crucial role in the follicle cells compared to ectodermal lineages in repressing epithelial invasion and that the follicular epithelium appears to organize signaling from epithelial junctions in distinct ways compared to other epithelia. Given the conservation in the lineage of the fly and human epithelia, and the sensitivity of this screen for detecting molecules important for invasive carcinogenesis, it is proposed that the fly egg chamber may serve as a prototype for identifying early molecular events that are crucial for invasion of human ovarian cancer and possibly other malignancies that remain undetected before they start to invade (Zhao, 2008).

Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila

Oncogenic alterations in epithelial tissues often trigger apoptosis, suggesting an evolutionary mechanism by which organisms eliminate aberrant cells from epithelia. In Drosophila imaginal epithelia, clones of cells mutant for tumor suppressors, such as scrib or dlg, lose their polarity and are eliminated by cell death. This study shows that Eiger, the Drosophila tumor necrosis factor (TNF), behaves like a tumor suppressor that eliminates oncogenic cells from epithelia through a local endocytic JNK-activation mechanism. In the absence of Eiger, these polarity-deficient clones are no longer eliminated; instead, they grow aggressively into tumors. In scrib clones endocytosis is elevated, which translocates Eiger to endocytic vesicles and leads to activation of apoptotic JNK signaling. Furthermore, blocking endocytosis prevents both JNK activation and cell elimination. These data indicate that TNF signaling and the endocytic machinery could be components of an evolutionarily conserved fail-safe mechanism by which animals protect against neoplastic development (Igaki, 2009).

Clones of cells mutant for Drosophila tumor suppressor genes, such as scrib or dlg, are eliminated from imaginal discs, suggesting an evolutionarily conserved fail-safe mechanism that eliminates oncogenic cells from epithelia. This study reports that this elimination of mutant cells is accomplished by endocytic activation of Eiger/TNF signaling. Eiger is a conserved member of the TNF superfamily in Drosophila, but its physiological function has been elusive. Although ectopic overexpression of Eiger can trigger apoptosis, flies deficient for eiger develop normally and exhibit no morphological or cell death defect. This study shows that Eiger is required for the elimination of oncogenic mutant cells from imaginal epithelia. This not only provides an explanation for previous unexplained observations, but also argues that Eiger is a putative intrinsic tumor suppressor in a fashion similar to mammalian p53 or ATM, which causes no phenotype when mutated, but protects animals as tumor suppressors when their somatic cells are damaged (Igaki, 2009).

The intrinsic tumor suppression found in scrib mutant clones was also observed in dlg mutant clones, suggesting that this is a mechanism triggered by loss of epithelial basolateral determinants. Intriguingly, it was found that mutant clones of salvador, the hippo pathway tumor suppressor, are not susceptible to similar effect of Eiger. These data suggest that the Eiger-JNK pathway behaves as an intrinsic tumor suppressor that eliminates cells with disrupted cell polarity (Igaki, 2009).

It is intriguing that Eiger's tumor suppressor-like function is dependent on endocytosis. The data show that Eiger is translocated to endosomes through endocytosis and activates JNK signaling in these vesicles. Moreover, blocking endocytosis abolishes both JNK activation and Eiger-dependent cell elimination. Endocytic activation of signal transduction has been observed for EGF and β2-adrenergic receptor signaling in mammalian cells. After endocytosis, these ligand/receptor complexes localize to endosomes, where they meet adaptor or scaffold proteins that recruit downstream signaling components. Therefore, the endocytic activation of Eiger/TNF-JNK signaling might also be achieved by the recruitment of its downstream signaling complex to the endosomes, possibly through a scaffold protein that resides in endosomes. Recent studies in Drosophila have shown that components of the endocytic pathway -- vps25, erupted, and avalanche -- function as tumor suppressors (Lu, 2005; Moberg, 2005; Thompson, 2005; Vaccari, 2005). Furthermore, mutations in endocytosis proteins have been reported in human cancers. Thus, deregulation of endocytosis may contribute to tumorigenesis. This study provides new mechanistic insights into the role of endocytosis in tumorigenesis (Igaki, 2009).

Mammalian TNF superfamily consists of at least 19 members. While many have been shown to play important roles in immune responses, hematopoiesis, and morphogenesis, the physiological functions for other members have yet to be determined. Mechanisms that eliminate damaged or oncogenic cells from epithelial tissues are essential for multicellular organisms, especially for long-lived mammals like humans. The tumor suppressor role of Eiger might have evolved for host defense or elimination of dying/damaged cells, such as cancerous cells, very early in animal evolution. Given that components of the Eiger tumor suppressor-like machinery (such as Eiger, endocytic pathway components, and JNK pathway components) are conserved from flies to humans, it is also possible that Eiger and its mammalian counterparts are components of an evolutionarily conserved fail-safe by which animals maintain their epithelial integrity to protect against neoplastic development (Igaki, 2009).

Actomyosin contractility and Discs large contribute to junctional conversion in guiding cell alignment within the Drosophila embryonic epithelium

Proper control of epithelial morphogenesis is vital to development and is often disrupted in disease. After germ band extension, the cells of the Drosophila ventral embryonic epidermis are packed in a two-dimensional polygonal array. Although epithelial cell rearrangements are being studied productively in several tissues, the ventral epidermis is of particular interest as the final cell arrangement is, uniquely, far from equilibrium. Over the course of several hours, a subset of cells within each parasegment adopts a rectilinear configuration and aligns into parallel columns. Live imaging shows that this is accomplished by the shrinkage of select cell interfaces, as three-cell junctions are converted to four-cell junctions. Additionally, it was shown that non-muscle Myosin II and the polarity proteins Discs large (Dlg) and Bazooka are enriched along cell interfaces in a complex but reproducible pattern that suggests their involvement in junctional conversion and cell alignment. Indeed, depletion of Myosin II or dlg disrupts these processes. These results show that tight spatial regulation of actomyosin contractility is required to produce this high-energy arrangement of cells (Simone, 2010).

The phenomenon of conversion of three-cell junctions to four-cell junctions has been observed in various epithelial tissues, such as during convergent extension (CE), in involuting tracheal placodes and developing wing cells. Thus, junctional conversion is a common element in tissue remodeling. However, inspection of these cases also demonstrates that there are aspects unique to each (Simone, 2010).

During CE and tracheal placode involution, two three-cell junctions are converted to a four-cell junction by the elimination of a specific contact. However, this is not what drives the rearrangements that pattern these tissues. Rather, the resulting four-cell junction resolves further into two three-cell junctions through the growth of a new cell contact orthogonal to the original contact. This orthogonal regrowth is what drives the final cell rearrangements (Simone, 2010).

Three- to four-cell junction conversion also occurs among wing hair cells. This state also resolves, but in a manner distinct from that during CE. The four-cell junction resolves back to two three-cell junctions, but now in a random manner that maximizes cell contacts to assemble a hexagonally packed cell sheet. As in CE and tracheal placode involution, it is the last step, the re-establishment of three-cell junctions, that forms the final pattern (Simone, 2010).

The alignment among the denticle field cells contrasts with these cases. Here, it is the initial shift from three- to four-cell junctions that patterns the field. These cells make denticles, and a columnar alignment would be likely to optimize locomotion. To accomplish this, the underlying epithelial cells adopt a state far from equilibrium, as a polygonal array morphs into a rectilinear array of aligned cells. Junctional conversion is one step to accomplish this. Eventually, cells along the columns tend to re-establish three-cell junctions with cells of neighboring columns, but in a manner that maintains columnar cell alignment. Presumably, once four-cell junctions are established, a secondary interaction, perhaps adhesion, becomes engaged along each column boundary to maintain alignment (Simone, 2010).

A general principle emerging from all these tissues is that the conversion from three- to four-cell junctions is central to patterning epithelia. However, there are key tissue-specific outcomes that often reflect whether and how a final re-establishment of junctions occurs. Thus, although there will be similarities between the mechanisms involved, there will also be significant differences. Currently, the analyses conducted differ in each of these cases, and so the comparisons remain incomplete (Simone, 2010).

There are some commonalities between polarity protein localizations among the above cases. During CE, Myosin II is enriched along shrinking contacts, whereas Baz is enriched along stable contacts. During tracheal placode invagination, Myosin II is enriched along both contacts that shrink and some that do not, but there was no report of Baz localization. This study shows that Baz and Dlg and the molecular motor Myosin II are enriched along specific boundaries during alignment. Crucially, both Dlg and Myosin II functionally assist in alignment. However, as is the case for the tissues described above, two questions remain to be addressed: how are these proteins targeted to specific cellular interfaces, and how do they then mediate junctional conversion (Simone, 2010)?

Considering the selection of interfaces within denticle field cells, perhaps a different polarity complex, potentially engaging in competitive exclusion to set up the enrichments, defines each contact. So far, no candidates have been identifed with compelling patterns. For instance, Crb was enriched generally among denticle field cells, but not at a specific interface, whereas aPKC was subtly enriched but only late in alignment, suggesting a subordinate role, if any. Since Baz defines the short, stable contact, whereas Dlg defines the long, stable contact, a polarity protein might be present that is enriched on the shrinking contact and both Baz and Dlg can be excluded. One candidate is Par1, which phosphorylates and excludes Baz and Dlg from membranes in other contexts. Unfortunately, for many proteins, such as these, it has been difficult to deplete embryos of function without totally disrupting the epithelium (Simone, 2010).

Although it is a compelling idea, competitive exclusion is perhaps not a prime candidate for setting up or maintaining enrichments during alignment, as Baz enrichment was not altered in dlg mutants. Similarly, during CE aPKC and Par6 are not required for Myosin II localization. In fact, during C. elegans gastrulation, PAR-3 and Myosin II actually co-localize to ingressing edges. It has been proposed that Myosin II can be templated by prior F-actin enrichment, but this too is not the case during alignment, where F-actin and Myosin become enriched at the same time (Simone, 2010).

Since junctional conversion is a recurring theme, the mechanism by which it occurs is of particular interest. During alignment and tracheal placode invagination, but not during CE, Myosin II is enriched both along contacts that are eliminated and stable contacts. Although Myosin II is required for alignment, just how actomyosin contractility contributes to contact elimination, and how this process is counteracted along Myosin II-enriched stable contacts, are unknown. As during CE, a subtle enrichment on stable junctions is occasionally observed for DE-Cadherin and Armadillo (β-Catenin). With regard to the shrinking contact, one possibility is that Myosin II pulls actin filaments past each other, collapsing the length of the contact, although how this might destabilize Cadherin-based adhesion is unknown. Another possibility is that actomyosin contractility might differentially affect endocytosis and exocytosis. When rates of endocytosis and exocytosis are balanced, interfaces are stably maintained. However, if the equilibrium is shifted towards endocytosis along a certain contact, for instance where contractility is increased, that contact will shrink (Simone, 2010).

It was found that Dlg is enriched along stable Myosin II-enriched contacts and it is hypothesized that Dlg is involved in functionally distinguishing the two types of Myosin II-enriched contact. There are two ways in which Dlg could inhibit Myosin II function where they are co-enriched. First, Dlg might counteract Myosin II activity indirectly by facilitating exocytosis and membrane deposition as it has been suggested to do during cellularization. A bias towards deposition could lead to the apparent stabilization of contacts. Secondly, Dlg might recruit a Myosin II-inhibiting protein, such as Lgl, through one of its PDZ domains (Simone, 2010).

Regardless of how Dlg counteracts Myosin II function, it was surprising that Dlg, a member of the basolateral group of epithelial polarity genes, influences cell-cell relationships apically in the epithelium at this stage. However, recent work has revealed that at about the stage when alignment begins, there is a transfer of basolateral responsibility from the Dlg group to the Yurt/Cora group (Laprise, 2009). That would free Dlg group genes to participate in other polarity events. With this in mind, attempts were made to test whether the basolateral protein Lgl, a negative regulator of Myosin II, would similarly be freed to assist Dlg in alignment. However, lgl germline clones have radically disrupted epithelia and zygotic mutants have persistent maternal contribution (Simone, 2010).

Convergent extension has some contribution from oriented cell divisions and some from junctional conversions. Although oriented cell divisions contribute to CE more in the posterior portion of the embryo, junctional conversions occur for many cells with no spatially reproducible focus for initiation. By contrast, this study found that cell alignment differs between the prospective smooth and denticle fields, and, among denticle field cells, alignment begins along specific boundaries within each parasegment (Simone, 2010).

Among smooth field cells, stretching along the DV axis appears to be the major contributor to alignment. Even though Dlg makes some contribution to alignment here, it does not appear to be enriched on particular interfaces. However, stretching makes a minimal contribution to alignment among denticle field cells. This dramatic distinction between smooth and denticle field cells, coupled to the observation that specific cell columns initiate alignment events, strongly suggests the involvement of positional signals that are limited to, or emanate from, denticle field cells. In addition to starting at the 1/2 and 4/5 boundaries, alignment also initiates from the ventral midline and progresses laterally, eventually covering a third of the ectoderm . These observations raise the possibility that the 1/2 and 4/5 boundaries, that is boundary between cell columns 1 and 2 and the boundary between cell columns 4 and 5, template the other boundaries by propagating a polarity signal from column to column. Candidate signals include Hedgehog signaling across the 1/2 boundary, Notch signaling across the 4/5 boundary and Egfr signaling across either boundary. Differential Egfr activation has been implicated in the conversion of three- to four-cell junctions in the tracheal placode, but how differential Egfr signaling translates into junctional conversion remains unknown (Simone, 2010).

Four-cell junctions have been reported in several tissues. Their appearance during development might be a hallmark of cellular realignments integral to the patterning of that tissue. During development, signaling often occurs across smooth, defined boundaries, such as the DV compartment boundary in wing and segment boundaries in leg. In fact, the tarsus/pretarsus boundary is composed of four-cell junctions that are strikingly reminiscent of those reported in this study (Simone, 2010).

A more closely studied example is the actomyosin enrichment along the boundary between the dorsal and ventral cells of the wing imaginal disk. Cells along this boundary form a smooth, lineage-restricted interface and do not intermix. The actomyosin 'fence' was implicated in maintaining this lineage-restricted signaling boundary. Notch signaling across the DV boundary enriches it for F-actin and Myosin II and depletes it for Baz. Although a role for Baz was not tested, when Myosin II contractility is compromised, the integrity of the lineage-restricted boundary is disrupted. Under these conditions, the boundary is jagged and some cells can now mix. This work would now suggest that the normal function of the actomyosin fence is to convert three- to four-cell junctions along the boundary. The failure of this would generate interdigitating cells, resulting in a non-aligned, jagged boundary, which would be the first step to cell mixing. By keeping cells aligned at the boundary, Myosin II activity might prevent them from interdigitating and support compartment integrity (Simone, 2010).

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. Such neuroblasts were genetically created, and it was found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. These results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus (Kitajima, 2010).

In this study investigated how the asymmetric mode of neuroblast division contributes to the specification and diversification of neuronal cell fate by generating neuroblasts that divide symmetrically. Combinations of dlg and Gβ13F mutants and of baz and Gβ13F mutants successfully generated neuroblasts that divide symmetrically with respect to both partition of determinants and daughter cell size during embryonic stages, allowing all progeny to differentiate into neurons that are often clonally homogeneous in temporal identity. At larval stages, dlg-Gβ13F neuroblasts generated overgrowing cell populations. Based on these results, the roles of asymmetric features of neuroblast division in the choice of self-renewal vs. differentiation and in cellular diversification are discussed (Kitajima, 2010).

Based on the observations in this study, at embryonic stages, dlg-Gβ13F neuroblast divisions occur essentially without asymmetry in either daughter cell size or in the partition of the determinants from the first division. It is possible, however, that two daughter cells occasionally inherit different amounts of the determinants, leading to the generation of cell clusters expressing differential temporal identity genes in a single neuroblast progeny such as NB7-3, as discussed below. Such fluctuations in the partition of the determinants may occur stochastically because the apical/basal components are not tightly associated with cortex in dlg-Gβ13F neuroblasts (Kitajima, 2010).

All progenies of dlg-Gβ13F neuroblasts eventually differentiate in the embryonic stages. Which feature is then critical for the differentiation of all progeny; the asymmetric partition of determinants or of cell volume? On the one hand, the basal determinants are known to function in daughter cells’ commitment to differentiation. On the other hand, all available results suggest that reduction in neuroblast cell size contributes to attenuation of cell cycle progression but not to the induction of differentiation. In the wild type, neuroblasts gradually reduce their size by budding off GMCs, and eventually enter the dormant state (Miranda+, Pros−, Elav−). In the Gβ13F single mutant, neuroblasts more rapidly lose volume by generating equal-sized daughters with the basal determinants normally segregating to one daughter, and remain in the same Miranda+, Pros−, Elav− state with the characteristic cell morphology of quiescent neuroblasts. This suggests that in the Gβ13F single mutant, neuroblasts also eventually enter the dormant state after the generation of a fewer number of GMCs. Thus, cell size reduction alone is not likely to cause neuronal differentiation of progenitors, but instead appears to cause them to remain in the undifferentiated state unless the basal determinants are present. This was confirmed in Gβ13F mutant neuroblasts at the larval stage (Kitajima, 2010).

What amount of the basal determinants is necessary to induce GMC fate? In Gβ13F mutants where neuroblast divisions give rise to daughters of equal size, a large daughter at the first division inherits most of the basal determinants and becomes differentiated into a GMC, indicating that a full amount of basal determinants can cause a daughter cell half the size of a newly born neuroblast to commit to the GMC fate. By contrast, neuroblasts undergoing symmetric divisions (dlg-Gβ13F mutants) appear to subsequently undergo at least two cell cycles and do not immediately commit to a GMC-like fate. This difference between embryonic Gβ13F mutant and dlg-Gβ13F mutant first daughters may mean that a half amount of the basal determinants is not sufficient to commit a daughter cell to the GMC fate. Alternatively, it has been argued that neuroblasts may express self-renewal factors that promote self-renewal and thereby proliferation and that asymmetrically segregate into the neuroblast. When dlg-Gβ13F neuroblasts undergo their first division symmetrically, those postulated factors and the basal determinants will be partitioned into both daughters and will counteract each other. This may cause a delayed commitment to the GMC fate, compared with the first GMC of Gβ13F mutant neuroblasts, which do not receive the self-renewal factors (Kitajima, 2010).

In the Drosophila CNS, the expression of the temporal identity genes changes sequentially in mother neuroblasts but is persistent in the sibling GMC progeny. Hence, the expression of such genes should also depend on the asymmetric mode of division. A significant difference was found in the expression of the temporal identity genes between normal neuroblast lineages and symmetrically dividing dlg-Gβ13F neuroblasts. In the latter, the neuroblast progeny frequently forms a clone of cells homogeneous for the expression of temporal identity genes, providing evidence for the importance of asymmetric division for the generation of neuronal diversity (Kitajima, 2010).

It has been shown that the first transition of temporal identity genes in embryonic neurogenesis, from Hb to Kr, requires cytokinesis, whereas the transition from Kr to Cas occurs without cell cycle progression. Symmetrically dividing neuroblasts pass through the initial transition from Hb to Kr in all lineages examined in this study (NB1-1, NB4-2, NB3-3 and NB7-3). A large proportion of neuroblast lineages appears to continue expressing the temporal genes in succession, but terminates earlier than normal, as revealed by their lack of Grh expression. Two observations suggest that the transition of temporal identity genes occurs sequentially (in the order of Hb to Kr to Pdm to Cas) in the majority of dlg-Gβ13F neuroblasts undergoing clonal expansion during early stages of neurogenesis; first, the size of Cas clusters is mainly 4 or 8 cells when Cas expression appears at 6-8 h AEL, suggesting that Cas expression starts in the clones that have already divided two or three times (some neuroblasts like NB3-3 start with Kr). Second, the cluster size of the clones become larger in the order of Hb, Kr, and Cas clusters at 8-10 h AEL (Kitajima, 2010).

The terminal temporal identity and the size of a particular neuroblast lineage are, however, not constant in dlg-Gβ13F mutants. Furthermore, a neuroblast progeny occasionally splits into two clusters with different temporal identities, as observed in the lineages of NB7-3. These observations suggest that stochastic processes are involved in the expression of temporal identity genes and cell cycle progression in the dlg-Gβ13F mutant neuroblast lineages (Kitajima, 2010).

Analysis of the relationship between clone size and clone homogeneity of NB7-3 reveals two characteristic features regarding dlg-Gβ13F neuroblast progenies. First, larger clones tend to be heterogeneous, containing both Kr-positive and Kr-negative cells (presumably Pdm-positive in their next identity), when compared to small-sized clones. Second, in heterogeneous clones, neurons with the same temporal identity form a cluster (Kr-positive and Kr-negative) and do not intermingle with each other, suggesting that cells with a different identity are also clonal instead being formed randomly during the expansion into a large heterogeneous clone. These observations regarding a single neuroblast lineage raise the possibility that a slight heterogeneity or difference created between sibling cells in early divisions become more pronounced in temporal identity in later stages as cells go through cell cycles. This and the remaining presence of a few Hb/Kr-double positive clones at late stages indicate that, in dlg-Gβ13F neuroblasts, cell cycle progression is not always linked to temporal identity progression as expected from looking at corresponding wild type lineages, although the progression of temporal identity is seen in this mutant (Kitajima, 2010).

Termination of temporal identity progression may depend on the amount of the basal determinants, including Prospero, given that the transition of temporal identity genes do not occur in wild type GMCs. Indeed, in dlg-Gβ13F neuroblasts, as cells divide, the size of the cell size is rapidly reduced to approach a GMC-like state. It is thus speculated that the progeny of symmetrically dividing neuroblasts eventually assume a GMC-like state, thereby terminating temporal identity gene progression prematurely (Kitajima, 2010).

A remarkable finding in this study is the opposite nature of the progeny of dlg-Gβ13F mutant neuroblasts in embryos and in larvae. When created at larval stages, dlg-Gβ13F mutant neuroblasts generate continuously proliferating progeny after reducing their cell size, in contrast to the embryonic situation. This difference would appear to reflect differences in the proliferation control of neuroblasts in the embryonic and larval stages. The function of the pros gene, which negatively regulates genes promoting cell cycle progression, appears to be pivotal because Pros functions as a tumor suppressor in larval brains but not in embryos. This difference in the effect of the loss of Pros has been attributed to the redundancy of Pros with Brat in embryos, while they are both necessary for normal larval lineages. pros mutant larval clones are thought to form tumors by the transformation of GMCs into proliferative cells, although proliferative dlg-Gβ13F mutant cells are likely derived from the transformation of neuroblasts into proliferative cells that undergo symmetric divisions (Kitajima, 2010).

Given that embryonic dlg-Gβ13F mutant neuroblasts appear to become GMC-like cells that inherit sufficient amounts of the basal determinants to differentiate, a simple explanation for the continuous proliferation of larval dlg-Gβ13F mutant cells is that the effective dosage of Pros (or other basal determinants) in those cells is insufficient to induce differentiation, unlike in their embryonic counterparts. Indeed, this study has shown that elevation of Pros expression can induce proliferating cells in the dlg-Gβ13F mutant clones to exit the cell cycle and differentiate. It is of interest that, in interphase dlg-Gβ13F mutant cells of both embryonic and larval stages, Miranda is mainly cytoplasmic and Pros is largely nuclear, while during mitosis these proteins appear to form a cortical complex. There may be a larval mechanism by which neuroblasts reduce the nuclear entry of Pros in both wild-type and dlg-Gβ13F mutant cells. The ability of neuroblasts to prevent the nuclear import of Pros when it is overexpressed was tested under the heatshock promoter, and it was found that larval neuroblasts do not accumulate Pros protein in the nucleus at all under conditions in which embryonic neuroblasts show Pros nuclear accumulation. These results suggest that, compared with embryonic neuroblasts, larval neuroblasts have a strong ability to prevent nuclear accumulation of Pros (Kitajima, 2010).

A recent study has shown that cell cycle exit at the end of larval thoracic neurogenesis is programmed to reduce cell volume by symmetric divisions and nuclear localization of Pros; this is regarded as the mechanism terminating neuroblast division and allowing differentiation. As shown in larval Gβ13F mutant neuroblasts, the reduction of cell volume only limits the proliferative state or rate by idling or slowing the cell cycle progression, but does not induce differentiation. Furthermore, symmetric neuroblast divisions in the dlg-Gβ13F mutant resulted in reduction of cell volume and nuclear accumulation of Pros (although at a low level), but caused continuous proliferation of daughter cells. It may be that unlike the dlg-Gβ13F mutant, the level of nuclear Pros becomes high enough to terminate the cell cycle when wild-type neuroblasts stop division in the larval thorax. Alternatively, the progression of temporal identity in neuroblasts may induce additional mechanisms that cause neuroblasts to exit from the cell cycle into the differentiated state, as in the case for embryonic neuroblasts (Kitajima, 2010).

Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model

Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. This study used a Drosophila tumor model to identify genes, which are specifically required for tumor growth. Reduction of phosphoinositide 3-kinase (PI3K) activity was found to result in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. It is concluded that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results of this study broaden insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression (Willecke, 2011).

This study employed a genetic approach to identify genes required for the neoplastic growth phenotype of RasV12, DlgRNAi tumors. It was found that RasV12, DlgRNAi tumors are highly sensitive to reductions of the PI3K pathway and that changes in PI3K activity block cell-cycle progression (Willecke, 2011).

In agreement with previous reports, this study found that RasV12 induces the PI3K pathway in Drosophila. Yet curiously, RasV12, DlgRNAi tumors exhibit low levels of PI3K signaling. A possible reason for the paradoxical result may be derived from the cell polarity defects of RasV12, DlgRNAi tumors, which are not induced when RasV12 is expressed in otherwise wild-type cells or in combination with Upd. This interpretation implies that the activation of PI3K through RasV12 depends on proper cell polarity. In support of this explanation, it has been reported that RasV12 activates the PIP3 reporter only at the apical side of cells. Additionally, studies have shown that PTEN directly binds to the polarity gene Bazooka and that the activity of the PI3K pathway is polarized in Drosophila oocyte cells. The activation of the PI3K pathway might, therefore, require the preservation of cell polarity, which could explain why RasV12 does not induce the PI3K pathway if Dlg is lost (Willecke, 2011).

Why are RasV12, DlgRNAi tumors sensitive to changes in PI3K signaling? RasV12, DlgRNAi tumor cells receive mitogenic signals from the JAK-STAT and MAPK pathways, which promote extensive tumor growth. RasV12, DlgRNAi tumors require a higher metabolic rate compared with wild-type cells, without gaining extra activation of PI3K through RasV12. As a result, PI3K signaling might be absolutely limiting for the growth of RasV12, DlgRNAi tumors. Expression of PI3KRNAi then causes PI3K activity to drop below the threshold for such cells, triggering a block in cell-cycle progression. Tumors which express RasV12 together with Upd are not sensitive to changes in PI3K levels even though they overgrow as much as RasV12, DlgRNAi tumors. An pAkt western blot shows, however, that these tumors have high levels of PI3K signaling. Expression of PI3KRNAi in this background might not cause PI3K activity to drop below a threshold for cell-cycle progression (Willecke, 2011).

Compounds that block PI3K pathway activity are known to be potent inhibitors of mammalian tumor growth and inhibitors that target PI3K, and other members of the pathway are currently being tested in clinical trials. Preclinical and clinical trials focus mainly on tumors that carry mutations in PI3K pathway components or that display abnormal levels of the biomarkers pAKT and PS6k1. The current results are, however, an example for a case where tumors with no genetic alterations in PI3K signaling components are also highly susceptible to reduction of PI3K levels. Understanding the molecular networks that create such PI3K dependency is a central topic in cancer research as it is highly relevant to identify PI3K-dependent tumors to predict the potential effectiveness of PI3K inhibitors. Genetic studies in Drosophila may therefore complement mammalian studies to more precisely determine which tumor-initiating pathways create PI3K sensitivity (Willecke, 2011).

In conclusion, this study has uncovered a synthetic interaction between the Drosophila PI3K signaling and RasV12, DlgRNAi tumor-initiating pathways. The results provide insights into the complex mechanisms underlying tumorigenesis and illustrate the power of Drosophila genetics in revealing the vulnerabilities of tumors. Identification of additional synthetic interactions through genetic screening in Drosophila may serve as a valuable resource for identifying potential drug targets in cancer therapy (Willecke, 2011).

DlgS97/SAP97, a neuronal isoform of discs large, regulates ethanol tolerance

From a genetic screen for Drosophila melanogaster mutants with altered ethanol tolerance, intolerant (intol), a novel allele of discs large 1 (dlg1) was identified. Dlg1 encodes Discs Large 1, a MAGUK (Membrane Associated Guanylate Kinase) family member that is the highly conserved homolog of mammalian PSD-95 and SAP97. The intol mutation disrupted specifically the expression of DlgS97, a SAP97 homolog, and one of two major protein isoforms encoded by dlg1 via alternative splicing. Expression of the major isoform, DlgA, a PSD-95 homolog, appeared unaffected. Ethanol tolerance in the intol mutant could be partially restored by transgenic expression of DlgS97, but not DlgA, in specific neurons of the fly's brain. Based on co-immunoprecipitation, DlgS97 forms a complex with N-methyl-D-aspartate (NMDA) receptors, a known target of ethanol. Consistent with these observations, flies expressing reduced levels of the essential NMDA receptor subunit dNR1 also showed reduced ethanol tolerance, as did mutants in the gene calcium/calmodulin-dependent protein kinase (caki), encoding the fly homolog of mammalian CASK, a known binding partner of DlgS97. Lastly, mice in which SAP97, the mammalian homolog of DlgS97, was conditionally deleted in adults failed to develop rapid tolerance to ethanol's sedative/hypnotic effects. It is proposed that DlgS97/SAP97 plays an important and conserved role in the development of tolerance to ethanol via NMDA receptor-mediated synaptic plasticity (Maiya, 2012).

Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, a chemical mutagenesis screen was carried out in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen a mutation was identified in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, actE84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous actE84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in actE84K mutants. Genetic interactions between actE84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization (Blunk, 2014).


discs large 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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