bazooka


DEVELOPMENTAL BIOLOGY

Embryonic

The BAZ mRNA is maternally provided and expressed in a dynamic pattern in various tissues throughout embryogenesis. At the subcellular level, the mRNA is not uniformly distributed in the cytoplasm, but is highly localized in many of the cells in which it is expressed. BAZ mRNA is concentrated beneath the apical membrane in epithelial cells of the gastrulating embryo. Later, epithelial cells of the epidermis show a similar subcellular localization of BAZ mRNA. Localized expression is also observed in neuroblasts, which are situated right below the epithelium. In the neuroblasts, the RNA is restricted to a crescent in the apical cytocortex; this is the neuroblast pole that faces the overlying epithelium. Similar to the mRNA, Baz protein is present in the apical cytocortex of epithelial cells, such as cells of the tracheal pits or the epidermis. In neuroblasts, Baz protein is detected in a submembraneous crescent in the apical cytocortex. This localization is strictly cell-cycle dependent and is only detected at metaphase; no protein has been found by immunohistochemistry during interphase (Kuchinke, 1998).

Bazooka colocalizes with Inscuteable in neuroblasts but, in contrast to Inscuteable, Bazooka is also apically localized in epithelial cells. To compare the subcellular localisation of Partner of inscuteable with Bazooka, stage 10 embryos were stained for Pins, Bazooka and DNA. Whereas Bazooka localizes to the apical cell cortex in epithelial cells, Pins is found around the cell cortex and no apical concentration is observed in wild-type embryos. In neuroblasts, however, Pins and Bazooka colocalize at the apical cell cortex. Asymmetric localisation of Pins is also observed in sensory organ precursor (SOP) cells and epithelial cells of the procephalic neurogenic region (PNR): all these cells express Inscuteable. Thus, Inscuteable, Bazooka and Pins colocalize in cells that express Inscuteable, such as neuroblasts, SOP cells and cells of the PNR, but Pins does not colocalize with Bazooka in epithelial cells, which do not express Inscuteable (Schaefer, 2000).

Slam servers as a molecular marker for polarized cell behavior revealing functions of Eve, Runt, Myosin II and Bazooka in germband extension

During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement (Zallen, 2004).

Polarized cell movement during convergent extension ultimately derives from the asymmetric localization of proteins that direct cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein (Lecuit, 2002). Slam is present in a bipolar distribution that correlates spatially and temporally with intercalary behavior. These observations indicate that Slam can serve as a molecular marker for polarized cell behavior. Pair-rule patterning genes expressed in stripes along the A-P axis are necessary for Slam localization and, conversely, altering the geometry of their expression is sufficient to reorient Slam polarity. An endogenous planar polarity in intercalating cells has been shown to be manifested by the accumulation of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 at D-V cell borders. Moreover, germband extension is defective in bazooka mutant embryos, supporting a model where molecular polarization of the cell surface is a prerequisite for polarized cell movement. Therefore, differences in gene expression along the A-P axis may direct planar polarity in intercalating cells through the creation of molecularly distinct cell-cell interfaces that differ in migratory potential (Zallen, 2004).

Cell movement during germband extension is oriented along the D-V axis, suggesting a mechanism that restricts the productive generation of motility to dorsal and ventral cell surfaces. Molecules that are asymmetrically localized during convergent extension may therefore contribute to the spatial regulation of cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein, a novel cytoplasmic factor required for cellularization in the early embryo (Lecuit, 2002). While proteins such as Armadillo/β-catenin are uniformly distributed at the cell surface, ectopic Slam is enriched in borders between neighboring cells along the A-P axis. This polarized Slam population is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. Therefore, intercalating cells have distinct apical junctional domains that differ in their capacity for Slam association (Zallen, 2004).

Interestingly, the polarized distribution of ectopic Slam protein is spatially and temporally correlated with intercalary behavior. Slam polarity is not observed in Stage 6 embryos prior to the onset of intercalation. Slam accumulation at A-P cell borders first appears in late Stage 7, when cells of the germband initiate intercalation, and reaches its full extent during the period of sustained intercalation in Stage 8. In contrast, Slam is uniformly distributed in cells of the head region and the dorsal ectoderm, tissues which do not undergo intercalary movements. These results indicate that the polarized distribution of ectopic Slam protein is specific to intercalating cells and that Slam can therefore serve as a molecular marker for the visualization of polarized cell behavior (Zallen, 2004).

The enrichment of Slam at borders between neighboring cells along the A-P axis is consistent with two modes of localization: Slam could mark one side of each cell in a unipolar distribution, or Slam could localize to both anterior and posterior surfaces in a bipolar pattern. To distinguish between these possibilities, mosaic embryos were generated where Slam-expressing cells were juxtaposed with unlabeled cells, using the Horka mutation to induce sporadic chromosome loss in early embryos. Slam protein accumulates at anterior and posterior boundaries of mosaic clone, indicating that ectopic Slam protein is targeted to both anterior and posterior surfaces of intercalating cells in a symmetric, bipolar distribution. The bipolar localization of ectopic Slam corresponds well with the bidirectionality of cell movement during germband extension, where cells are equally likely to migrate dorsally or ventrally during intercalation. Bipolar motility is also observed during convergent extension in the presumptive Xenopus and Ciona notochords and in Xenopus neural plate cells in the absence of midline structures (Zallen, 2004).

To extend the spatial and temporal correlation between Slam polarity and cell movement, it was asked if this polarized Slam localization is achieved in mutants that are defective for intercalation. Cell intercalation is dependent on the transcriptional cascade that generates cell fates along the A-P axis, in the direction of tissue elongation and perpendicular to the migrations of individual cells. A-P patterning reflects the hierarchical action of maternal, gap, and pair-rule genes. Cell fate differences along the A-P axis are abolished in embryos maternally deficient for the bicoid, nanos, and torso-like genes (referred to as bicoid nanos torso-like mutants), and these mutant embryos do not exhibit intercalary behavior. Ectopic Slam is correctly targeted to the apical cell surface in bicoid nanos torso-like mutants, but fails to adopt a polarized distribution in the plane of the epithelium (Zallen, 2004).

Downstream of the maternal patterning genes, gap genes establish overlapping subdomains along the A-P axis. A quadruple mutant for the gap genes knirps, hunchback, forkhead, and tailless lacks A-P pattern within the germband while retaining terminal structures. This quadruple mutant exhibits severely reduced cell intercalation, and mutant embryos also display a loss of Slam polarity. The absence of planar polarity in A-P patterning mutants correlates with a more hexagonal appearance of germband cells, in contrast to the irregular morphology of wild-type intercalating cells (Zallen, 2004).

In response to maternal and gap genes, pair-rule patterning genes expressed in narrow stripes act in combination to assign each cell a distinct fate along the A-P axis. In particular, the even-skipped (eve) and runt pair-rule genes are essential for germband extension. This strong requirement for eve and runt during germband extension contrasts with the more subtle effects in mutants for other pair-rule genes such as hairy and ftz. Consistent with these defects in intercalation, eve and runt mutants also display aberrant Slam localization. These results establish a correlation between intercalary behavior and the polarized localization of the ectopic Slam marker (Zallen, 2004).

The Eve and Runt transcription factors ultimately direct Slam polarity and cell intercalation through the transcriptional regulation of target genes. To identify downstream effectors involved in this process, components of the noncanonical planar cell polarity (PCP) pathway, which is required for convergent extension in vertebrates, were examined. Germband extension occurs normally in the majority of embryos lacking the Frizzled and Frizzled2 receptors. Similarly, germband extension is unaffected in the absence of Dishevelled. Moreover, dishevelled mutants exhibit a normal polarization of the Slam marker. These results demonstrate that molecular and behavioral properties of planar polarity in the Drosophila germband do not require Frizzled or Dishevelled function (Zallen, 2004).

The polarized distribution of ectopic Slam in intercalating cells provides the first clue to a molecular distinction between D-V cell interfaces that generate productive cell motility and A-P interfaces that do not. However, endogenous Slam mRNA and protein are not detected during germband extension, indicating that Slam may not play a functional role in cell intercalation. Slam colocalizes with the Zipper nonmuscle myosin II heavy chain subunit during cellularization and when Slam is ectopically expressed at germband extension (Lecuit, 2002). Therefore, the endogenous distribution of myosin II was examined during germband extension in wild-type embryos. During cell intercalation, myosin II is present in a punctate distribution at the apical cell surface, colocalizing with the adherens junction component Armadillo/β-catenin. In Stage 8 embryos, apical myosin II protein accumulates at interfaces between cells along the A-P axis. Slam can enhance this polarized localization when ectopically expressed (Lecuit, 2002), suggesting that Slam and myosin II may associate with a common localization machinery. Myosin II polarity is not apparent in Stage 6 or early Stage 7 embryos that have not begun intercalation, indicating that the enrichment of myosin II at A-P interfaces is specific to intercalating cells (Zallen, 2004).

The localized distribution of myosin II is not as pronounced as that of ectopic Slam, suggesting that additional asymmetries contribute to the polarization of intercalating cells. To identify such proteins, the localization was examined of components implicated in cell polarity in other cell types. In particular, the PDZ domain protein Bazooka/PAR-3 participates in both apical-basal and planar polarity. Bazooka/PAR-3 also exhibits a polarized distribution in intercalating cells. Bazooka, like myosin II, is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. However, in contrast to the accumulation of myosin II at A-P cell interfaces, Bazooka is enriched in the reciprocal D-V interfaces. Bazooka polarity is specific to intercalating cells, where it first appears at the onset of intercalary movements in late Stage 7. Bazooka polarity is not observed in cells of the head region, which do not undergo intercalation, nor is it observed in germband cells following the completion of germband extension at Stage 9 (Zallen, 2004).

To characterize the relationship between cell shape and the polarized localization of cortical proteins, the orientation of cell borders was measured as an angle relative to the A-P axis (with A-P interfaces closer to 90° and D-V interfaces closer to 0° and 180°). Interfaces from embryos stained for Bazooka and myosin II were ranked according to mean fluorescence intensity as a relative measure of protein distribution. These results illustrate that Bazooka and myosin II are enriched in distinct sets of cell-cell interfaces that adopt largely nonoverlapping orientations relative to the A-P axis. This quantitation confirms the visual impression from confocal images and demonstrates that the molecular composition of a cell surface domain is a reliable predictor of its orientation within the epithelial cell sheet (Zallen, 2004).

The polarized localization of Bazooka is abolished in the absence of A-P patterning information in bicoid nanos torso-like mutant embryos. A similar disruption of myosin II polarity is observed in A-P patterning mutants. The A-P patterning system may therefore mediate cell intercalation through the polarized accumulation of cell surface-associated proteins. Bazooka participates in a conserved protein complex containing the atypical PKC (DaPKC), and DaPKC is also enriched in D-V cell interfaces during germband extension (Zallen, 2004).

To determine whether the polarized Bazooka/PAR-3 protein is functionally required for germband extension, homozygous bazooka (baz) mutant embryos were examined. In zygotic baz mutants, residual Bazooka protein persists from maternal stores and is often, but not always, correctly distributed along the apical-basal and planar axes. Despite this maternal Bazooka contribution, loss of zygotic Bazooka disrupts germband extension. In wild-type embryos, the posterior end of the extended germband is located at 70% egg length from the posterior pole. Of the progeny of bazYD97/+ females and wild-type males, 72% were wild-type-like, 25% were partially defective, and 3% were strongly defective. These results demonstrate that Bazooka is required for normal germband extension (Zallen, 2004).

Bazooka/PAR-3 and the associated DmPAR-6 and DaPKC components also influence epithelial cell polarity along the apical-basal axis. To address the possibility that germband extension defects may occur indirectly as a result of disrupted apical-basal polarity, properties of apical-basal polarity were examined in zygotic baz mutants, where some functions are carried out by maternal gene products. Zygotic baz mutant embryos exhibit several signs of normal apical-basal polarity at gastrulation, including a monolayer epithelial morphology in the germband and the correct distribution of proteins to apical and lateral membrane domains. This is consistent with findings that zygotic baz mutants exhibit proper localization of the Armadillo/β-catenin adherens junction component prior to Stage 10 of embryogenesis. These results demonstrate that properties of apical-basal polarity are established correctly in baz mutant embryos during germband extension, consistent with a direct role for Bazooka in cell movements along the planar axis, independent of its later effects on apical-basal polarity (Zallen, 2004).

The local reorientation of planar polarity in response to Eve and Runt expression argues that planar polarity is generated by cell-cell interactions, rather than a distant polarizing cue. In addition to these local effects of Eve and Runt on planar polarity, Slam polarity frequently adopted a circular pattern in mosaic embryos, even when Eve and Runt were not present along the entire circumference of the circle. This unexpected configuration indicates that polarizing information can propagate from cell to cell downstream of an Eve-dependent signal. A similar relay mechanism is suggested by the swirling patterns of wing hair polarity that persist in Drosophila mutants defective for the PCP signaling pathway. Therefore, mechanisms of cell-cell communication may reinforce local polarizing events in the organization of a two-dimensional cell population (Zallen, 2004).

Planar polarity in Drosophila germband extension is locally established through the concentration of specific proteins at sites of contact between cells with different levels of Eve and Runt expression. Cells can monitor the identity of their neighbors through qualitative or quantitative differences in the activity of cell surface proteins, perhaps through ligand-receptor mediated signaling events or adhesion-based cell sorting. Transcriptional targets of Eve and Runt are therefore likely to include components that mediate intercellular signaling events involved in the transmission of polarizing information during multicellular reorganization (Zallen, 2004).

The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila

Cell polarity is critical for epithelial structure and function. Adherens junctions (AJs) often direct this polarity, but it has been found that Bazooka (Baz) acts upstream of AJs when epithelial polarity is first established in Drosophila. This prompted an investigation into how Baz is positioned and how downstream polarity is elaborated. Surprisingly, it was found that Baz localizes to an apical domain below (basally to) its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In fact, Baz positioning is independent of aPKC and PAR-6, relying instead on cytoskeletal cues, including an apical scaffold and dynein-mediated basal-to-apical transport. AJ assembly is closely coupled to Baz positioning, whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical domain with Baz and AJs localizing basally to aPKC and PAR-6, and specific mechanisms were identified that keep these proteins apart. These results reveal key steps in the assembly of the apical domain in Drosophila (Harris, 2005).

These results frame a model of apical domain assembly during epithelial polarity establishment in Drosophila. During cellularization, Baz acts as a primary polarity landmark that positions AJs and aPKC. Baz, itself, is positioned by two cues (an apical scaffold and dynein-mediated transport). Baz recruits and colocalizes with AJ proteins in a subapical region while helping direct aPKC to the extreme apical region. During gastrulation, a third cue becomes important for Baz and AJ positioning. At this stage, aPKC becomes required for maintaining Baz and AJs. PAR-6 is also recruited to the extreme apical region and maintains Baz and AJs. Although Baz can interact with aPKC and PAR-6 at this stage, Crb blocks these interactions. It is proposed that this interaction network establishes a robust, stratified apical domain from the earliest stages of epithelial development (Harris, 2005).

AJs are often key polarity landmarks. However, Baz positioning is AJ independent at the time that epithelial polarity is first established in Drosophila. Here, Baz appears to act as a primary polarity landmark, but what cues position Baz? The data indicate that Baz is initially positioned by cytoskeletal cues that support an apical Baz-binding scaffold and mediate basal-to-apical Baz transport. The apical scaffold is saturable. Its function requires actin; Baz becomes basally mislocalized after actin disruption. However, since Baz overlaps only the basal reaches of the apical actin network, it is unlikely that Baz simply binds actin. Interestingly, Baz remains largely membrane associated when actin is disrupted. One caveat is that there is some residual actin. However, the same treatment dissociates APC2 from the cortex. Actin is also required for PAR-3 cortical association in C. elegans one-cell embryos. During Drosophila cellularization, it is speculated that Baz may have other cortical anchors and that actin may control their distribution -- of course, actin is critical for many cellular processes and could play other roles in positioning Baz. It will be important to identify the apical scaffold for Baz (Harris, 2005).

Baz positioning also requires the minus-end-directed MT motor dynein. Live imaging of BazGFP revealed basal-to-apical translocation of BazGFP puncta during cellularization. Baz-GFP that diffuses to ectopic basal positions appears to engage a preexisting, dynein-based, basal-to-apical transport system. Such a system transports Golgi vesicles apically during cellularization. Baz-dynein associations appear to cease once dynein brings Baz to the apical region, where Baz presumably docks with its apical scaffold. Although BazGFP puncta move slower than in vitro dynein velocity measurements, dynein-mediated lipid droplet movements have similar speeds during Drosophila cellularization. In vivo, BazGFP puncta may be slowed because they form large cortical complexes. Indeed, DE-Cad, aPKC, and PAR-6 associate with these puncta and Baz oligomerization may promote complex assembly. Further supporting a role for dynein, endogenous Baz is positioned near MT minus ends in WT embryos, but mislocalizes basally in dhc64Cm/z mutants. dhc64C mutations also enhance the baz mutant embryonic phenotype. This is the first report of dynein positioning Baz or its homologues (Harris, 2005).

Analysis of dynein mutants also revealed a third mechanism that can reposition Baz apically during gastrulation. Perhaps the apical Baz-binding scaffold is strengthened during this stage. Alternatively, a distinct polarizing mechanism may be activated, or aPKC and PAR-6 may be involved. Having three Baz positioning mechanisms may ensure proper Baz localization for regulating downstream polarity (Harris, 2005).

Baz acts upstream of AJs as epithelial polarity is first established in Drosophila. The following model is proposed in which AJ assembly may be coupled to Baz positioning. During cellularization, AJ proteins accumulate in both apical and basal junctions. Basal junctions form transiently near the base of each invaginating furrow. Baz is not required for basal junctions, but is required for recruiting AJ proteins into apical junctions. Apical Baz may provide a landmark for apical AJ assembly (Harris, 2005).

The data also suggest that Baz may be involved in ferrying DE-Cad to the apical domain via dynein-mediated transport. Dynein is required for correct apical positioning of both Baz and DE-Cad, and their colocalization in ectopic basal complexes in dhc64Cm/z mutants suggests they may normally be transported to the apical domain together. Indeed, Baz can form complexes with DE-Cad and Arm. Although most endogenous Baz is apical during WT cellularization, its basal mislocalization in dhc64Cm/z mutants suggests that some Baz may normally move basally. In fact, excess BazGFP displaced from the apical domain preferentially accumulates at basal junctions. It is hypothesized that some Baz may normally interact transiently with basal junctions. From there, it may help ferry AJ proteins apically via dynein-mediated transport. MT motors have been implicated in AJ assembly. For example, dynein interacts with ß-catenin and may tether MTs to AJs assembling between PtK2 cells. Kinesin transports AJ proteins to nascent AJs in cell culture, and the mitotic kinesin-like protein 1 is required for apical targeting of AJs and other cues in C. elegans epithelia. It will be important to see if these targeting mechanisms have commonalities with AJ positioning in Drosophila, and if Baz homologues are involved (Harris, 2005).

Finally, it is hypothesized that the third Baz-AJ positioning mechanism revealed in dhc64Cm/z mutants might be related to the normal maturation/stabilization of AJs at gastrulation. At this stage, precursory spot AJs fuse into continuous belt junctions around the top of each cell. In mammalian cell culture, aPKC is required for such AJ maturation. Similarly, aPKC is required for proper AJ and Baz positioning during Drosophila gastrulation, as has been shown for PAR-6. Considering aPKC and PAR-6 are positioned apically as dhc64Cm/z mutants gastrulate, they might recruit Baz and AJs apically in this context as well (Harris, 2005).

Based on their shared roles in polarity in C. elegans, characterized physical interactions, and colocalization in mammalian cells, Baz, aPKC, and PAR-6 are thought to function, at least in some cases, as an obligate tripartite complex. The data suggest that the bulk of cortical Baz and aPKC/PAR-6 do not form obligate complexes during epithelial development in Drosophila. Instead, aPKC and PAR-6 localize to an apical region above Baz and AJs, and are positioned there by distinct mechanisms. Baz/PAR-3 also segregates from aPKC and PAR-6 in other cell types. In C. elegans one-cell embryos, PAR-3, aPKC, and PAR-6 each localize in clusters on the anterior cortex, but these different clusters have limited colocalization (60%-85% fail to colocalize. aPKC and PAR-6 colocalize without PAR-3 at the leading edge of migrating mammalian astrocytes. In Drosophila photoreceptors, Baz colocalizes with AJs below aPKC, PAR-6, and Crb. Even in polarized MDCK cells, aPKC and PAR-6 show some segregation above PAR-3, and although they mainly colocalize at tight junctions, mammalian PAR-3 can regulate tight junction assembly independently of aPKC and PAR-6. Thus, in many contexts interactions between Baz/PAR-3, aPKC, and PAR-6 are dynamic and/or regulated (Harris, 2005).

Baz (PAR-3), aPKC, and PAR-6 often recruit each other to the cortex, but the assembly pathways vary. In C. elegans, one-cell embryos, PAR-3, aPKC, and PAR-6 are mutually dependent for their cortical recruitment. However, in Drosophila neuroblasts, Baz can be positioned without aPKC and PAR-6. Similarly, apical Baz is positioned without aPKC and PAR-6 during Drosophila cellularization. In contrast, apical aPKC recruitment requires Baz, whereas PAR-6 is largely nonpolarized at this stage. Given the lack of extensive colocalization of Baz and aPKC in WT embryos, Baz may control aPKC positioning indirectly, perhaps regulating binding to a separate apical scaffold. Alternately, cortical recruitment might involve cytoplasmic Baz-aPKC complexes. Apical PAR-6 accumulates at gastrulation, and this appears partially Baz independent. Indeed, cdc42 recruits PAR-6 at this stage, and at the same time aPKC and PAR-6 become required for maintaining apical Baz. Thus, although Baz is first positioned independently of aPKC and PAR-6, these cues soon develop complex interdependencies (Harris, 2005).

Although Baz can directly bind both aPKC and PAR-6, at least two mechanisms keep them apart. During cellularization, Baz colocalizes with aPKC and PAR-6 when overexpressed, but normally it localizes with AJs below aPKC and PAR-6. This normal segregation may thus involve competition with other binding partners. After cellularization, Crb also becomes important for segregating Baz and AJs from aPKC and PAR-6. These segregation mechanisms help form a stratified apical domain from the earliest stages of epithelial development (Harris, 2005).

A stratified apical domain may strengthen the boundary between the apical and basolateral domains. This boundary forms via reciprocal antagonism between polarity cues. For example, aPKC phosphorylates and excludes Lethal giant larvae (Lgl) from the apical domain in Drosophila epithelia and Lgl appears to repel PAR-6 from the basolateral domain. The Crb and Dlg complexes also have mutual antagonism. It is proposed that the subapical Baz-AJ region may insulate the apical and basolateral domains. For example, it may inhibit active aPKC from moving basally. Indeed, PAR-3 binding can block mammalian aPKC kinase activity. The Baz-AJ subapical region could also block basolateral cues, since AJs are required to segregate Dlg. In this way, the Baz-AJ subapical region could help define a distinct apical-basolateral boundary (Harris, 2005).

To conclude, Baz appears to be a primary epithelial polarity landmark in Drosophila. It is positioned by multiple mechanisms, including an apical scaffold and dynein-mediated transport, and organizes a stratified apical domain, in which it colocalizes with AJs below its typical partners aPKC and PAR-6 (Harris, 2005).

Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila

Diverse types of epithelial morphogenesis drive development. Similar cytoskeletal and cell adhesion machinery orchestrate these changes, but it is unclear how distinct tissue types are produced. Thus, it is important to define and compare different types of morphogenesis. Cell flattening and elongation were investigated in the amnioserosa, a squamous epithelium formed at Drosophila gastrulation. Amnioserosa cells are initially columnar. Remarkably, they flatten and elongate autonomously by perpendicularly rotating the microtubule cytoskeleton - this is called 'rotary cell elongation'. Apical microtubule protrusion appears to initiate the rotation and microtubule inhibition perturbs the process. F-actin restrains and helps orient the microtubule protrusions. As amnioserosa cells elongate, they maintain their original cell-cell contacts and develop planar polarity. Myosin II localizes to anterior-posterior contacts, while the polarity protein Bazooka (PAR-3) localizes to dorsoventral contacts. Genetic analysis revealed that Myosin II and Bazooka cooperate to properly position adherens junctions. These results identify a specific cellular mechanism of squamous tissue morphogenesis and molecular interactions involved (Pope, 2008).

Amnioserosa tissue morphogenesis involves dramatic cell shape change. Before amnioserosa morphogenesis, cells are columnar with lateral MT bundles in a basket-like array along the apicobasal axis. With amnioserosa morphogenesis, the cells elongate and flatten. Amnioserosa cells could change shape by symmetrically re-positioning cellular contents (full cytoskeleton and/or membrane reorganization) or by perpendicularly rotating cellular components to reorient the long axis of the cell into the plane of tissue extension. To distinguish these possibilities, 3D cell organization was studied over time; it was discovered that the MT array plus the nucleus, centrosomes and ER rotate, apparently as a unit, into the plane of tissue extension. More symmetric adherens junction (AJ) and cortical reorganizations appear to accompany the rotation. MT arrays also reorient to polarize cells during chemotaxis and tissue migration, and to reposition cell contents as occurs during cortical rotation in early Xenopus embryos. These results reveal rotation of the MT cytoskeleton linked to cell shape change and amnioserosa morphogenesis. Similar mechanisms may underlie the development of other squamous epithelial monolayers (Pope, 2008).

Regulated apical MT protrusion appears to initiate amnioserosa rotary cell elongation. MT inhibition perturbs initial elongation, and the process normally begins with MTs protruding into the apical domain, bending perpendicularly and then extending in the axis of cell elongation. Pre-existing lateral MT bundles appear to protrude across the apical domain - they are mainly non-centrosomal and contain older (acetylated) MTs. However, EB1-GFP imaging also revealed bi-directional MT growth across the apical domain. This indicates that the bundles are dynamic, but argues against MT bundle protrusion through polarized individual MT polymerization. Instead, MT bundle protrusion may involve greater net renewal of bundles apically versus basally, motors sliding MTs past MTs in the bundles and/or motors moving bundles along the cell cortex. Distinguishing these models requires further study. As MTs extend apically the actin cytoskeleton appears to inhibit them, as weakening actin leads to excessively long and randomly oriented MT-based protrusions. Actin is normally found around the full apical circumference as amnioserosa cells elongate. By contrast, Myosin II becomes enriched at AP contacts and is gradually lost from the full cell cortex. Thus, different pools of actin may regulate MT protrusion. It is speculated that the gradual overall loss of cortical actin-myosin complexes permits, and may help orient, regulated MT protrusion. Actin also antagonizes cortical MTs in other systems. MT-based primary axons form where cortical actin is weakest. Actin inhibits cortical MT protrusion in neutrophils and Myosin IIA inhibits cortical MTs in mammalian cells. Actin might physically block MT protrusion, but direct or indirect molecular interactions may also be involved (Pope, 2008).

In Drosophila embryos, MT-actin interactions also affect germband cells. At stage 7-8, actin disruption enhances AJ planar polarity at DV contacts. MT disruption suppresses this, suggesting that actin inhibits MT-based AJ positioning in these cells - however, germband cells show minimal shape change with actin disruption at this stage. Remarkably, the same actin disruption causes stage 9-10 germband cells to rotate analogously to early amnioserosa cells. Their apical domains elongate and their lateral regions rotate perpendicularly, becoming exposed to the embryo surface. Implicating MTs in this change, lateral MT bundles run into the extended apical domains and simultaneous MT disruption suppresses the cell shape change. Thus, actin may inhibit apical MTs to regulate tissue structure in many parts of the embryo. This MT inhibition may also involve coordination with AJs, as disrupted germband cells in armm/z mutants also display MTs protruding into extended apical domains (Pope, 2008).

How do MTs elongate the apical domain and how is this linked to the rotation of the full MT cytoskeleton in the amnioserosa? It is proposed that rotary cell elongation occurs in two phases. In phase one, MT imaging and inhibitor studies indicate that regulated MT protrusion elongates the apical domain. This may involve a combination of physical force, membrane delivery and/or relaxation of actin-myosin contractility. The AJ clustering observed at abnormal apical MT protrusions formed with actin inhibition in both early amnioserosa cells and the later germband suggests that MTs may apply force to AJs. Consistent with this idea, MT inhibition affected both initial amnioserosa cell elongation and later amnioserosa cell-cell interactions. However, amnioserosa cell elongation in armm/z mutants suggests that MTs may not necessarily engage AJs directly. Since Baz localizes apically in early armm/z mutants, and is enriched at DV amnioserosa cell contacts to which MTs rotate in wild type, it is a strong candidate for coordinating these interactions. However, severe early defects in bazm/z mutants would confound analysis of amnioserosa development - this may require conditional mutants. Phase two of rotary cell elongation requires full perpendicular rotation of the MT array, apical and basal membrane growth, and lateral membrane removal. Although it is unclear how full rotation occurs, MT rotation and cortical remodeling may occur in concert. For example, membrane remodeling may explain how amnioserosa cells remain elongated with MT disruption during later development (Pope, 2008).

For rotary cell elongation to translate into tissue extension, cell contacts and AJs must be remodeled. Remarkably, amnioserosa cells maintain their neighbor relationships as they elongate, and two contact types develop; highly elongated AP contacts and lesser elongated DV contacts. Each appears to involve unique AJ remodeling. Intriguingly, Myosin II and Baz localize to AP and DV contacts, respectively -- the same reciprocal planar polarized relationship displayed in the germband. In the amnioserosa, Myosin II and Baz synergize to control overall AJ positioning, a regulatory interaction that has not been shown elsewhere (Pope, 2008).

Myosin II and Baz may regulate specific AJ remodeling events occurring at AP and DV contacts, respectively. Amnioserosa cells increase their apical circumference 10-fold, initially doubling the length of their AP cell contacts every 5-10 minutes. Remarkably, AJs localize around the full circumference as this occurs. This contrasts elongating Drosophila follicle cells, which lose AJ continuity, suggesting specific mechanisms for maintaining AJ continuity during amnioserosa morphogenesis. amnioserosa AJs do lose continuity with actin disruption, suggesting a role for actin. More specifically, AJ fragmentation in baz zip double mutants suggests a role for Myosin II. In the neighboring ventral furrow and germband, actin-myosin contractility is coupled to AJs during apical constriction and cell intercalation, respectively. The actin-myosin complexes enriched along amnioserosa AP contacts may also be contractile, but here they may counterbalance MT protrusion. Slowing apical elongation may indirectly allow AJ remodeling. However, Myosin II may also have direct affects on AJs (Pope, 2008).

Baz may regulate distinct AJ re-modeling at DV contacts. It is hypothesized that MT protrusion applies force to the DV contact at the cell 'front', and that cell elongation may also pull the 'rear' contact. Either force could detach AJs and necessitate AJ remodeling. Dynamic looping of DE-CadGFP and ArmCFP was observed at D-V contacts, and BazGFP partly colocalized with these loops. Although further experiments (e.g., photobleaching) are needed to understand this and other amnioserosa AJ remodeling, Baz localization at DV contacts and abnormal AJ aggregation at DV contacts in baz zip double mutants suggests a role for Baz in AJ remodeling at these sites. Baz appears to interact with MTs and Dynein to initially position AJs during Drosophila cellularization, and Baz might re-position AJs at DV amnioserosa contacts in a similar way (Pope, 2008).

In four different cases, cells were observed elongating towards potential sources of pulling forces. First, wild-type amnioserosa cells elongate along the DV axis towards the germband (potential source of DV pulling forces during convergent extension) and the ventral furrow (potential source of DV pulling forces during invagination). Second, amnioserosa cells elongate along the DV axis of bcd nos tsl mutants, in which germband extension fails, but ventral furrow formation occurs. Third, in dl mutants in which the ventral furrow does not form and the amnioserosa forms a ring around the DV axis, amnioserosa cells reoriented along the AP axis towards ectopic contractile furrows. Fourth, in the stage 9-11 wild-type germband, cells artificially induced to flatten and elongate did so in coordinated groups oriented towards contractile regions of the germband. Thus, polarized pulling forces across a tissue may orient rotary cell elongation. In wild-type embryos, these forces may come from germband extension and/or ventral furrow formation. However, Zen must first trigger the amnioserosa cell shape change, while AP patterning may specifically regulate AJ remodeling (Pope, 2008).

Increased cell bond tension governs cell sorting at the Drosophila anteroposterior compartment boundary

Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. This study quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. Mechanical tension was found to be approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632, an inhibitor of Rho-kinase whose main effector is Myosin II. Simulations using a vertex model demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. These results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries (Landsberg, 2009).

A long-standing hypothesis to explain the maintenance of compartment boundaries is based on differential cell adhesion (or cell affinity). Cell adhesion molecules required for the maintenance of compartment boundaries, however, have not been identified. More recently, it has been proposed that actin-myosin-based tension is important for keeping the dorsoventral compartment boundary of the developing Drosophila wing smooth and straight. However, whether a similar mechanism operates at the anteroposterior compartment boundary (A/P boundary) is unclear. Moreover, a physical measurement of differential mechanical tension at compartment boundaries has not been reported. Furthermore, whether and how differential mechanical tension governs cell sorting at compartment boundaries is not well understood (Landsberg, 2009).

To test whether actin-myosin-based tension is increased at the A/P boundary, the levels of Filamentous (F)-actin and nonmuscle Myosin II (Myosin II) were quantified. The A/P boundary in the wing disc epithelium was particularly well defined by the cell bonds located at the level of adherens junctions, indicating that mechanisms maintaining the boundary operate at this cellular level. F-actin and the regulatory light chain of Myosin II (encoded by spaghetti squash, sqh) were increased at these cell bonds along the A/P boundary. Cell bonds displaying elevated levels of Myosin II correlate with decreased levels of Par3 (Bazooka in Drosophila), a protein organizing cortical domains, at the dorsoventral compartment boundary and during germ-band extension in Drosophila embryos. Likewise, Bazooka was decreased at cell bonds along the A/P boundary, indicating a common mechanism of complementary protein distribution of Myosin II and Bazooka. The level of E-cadherin, a component of adherens junctions, was not altered along the A/P boundary (Landsberg, 2009).

To identify signatures of increased tension in the vicinity of the A/P boundary, the morphology of cells were quantitatively analyzed at the level of adherens junctions. Line tension and mechanical properties of cells have been proposed to contribute to cell shape and to influence angles between cell bonds. Line tension associated with adherens junctions, here termed cell bond tension, can be defined as the work, per unit length, performed as a cell bond changes its length. Cell bond tension results from actin-myosin bundles and other structural components at junctional contacts that generate tensile stresses. Wing discs from late-third-instar larvae were stained for E-cadherin and engrailed-lacZ, a marker for the posterior compartment. Cell bonds were identified, and morphological parameters were analyzed. Adjacent anterior and posterior cells (A1 and P1, respectively) displayed a significantly enlarged apical cross-section area compared to cells farther away from the compartment boundary, indicating that apposition of anterior and posterior cells alters specifically the properties of A1 and P1 cells. Angles between adjacent cell bonds along the A/P boundary were larger compared to angles between bonds of the remaining cells and were significantly smaller in mutants for Myosin II heavy chain (encoded by zipper; zip2/zipEbr). Thus, the unique morphology of A1 and P1 cells depends on Myosin II. These data are consistent with an increased Myosin II-based tension of cell bonds located along the A/P boundary (Landsberg, 2009).

Cells on opposite sides of the A/P boundary differ in gene expression. The homeodomain-containing proteins Engrailed and Invected as well as the Hedgehog ligand are only expressed on the posterior side. The Hedgehog signal is transduced exclusively on the anterior side. Hedgehog signal transduction and the presence of Engrailed and Invected are required to maintain this compartment boundary. Whether the altered cell morphology at the A/P boundary could be reproduced by ectopically juxtaposing Hedgehog signaling and non-Hedgehog signaling cells was tested. Clones of cells that expressed Hedgehog from a transgene and that were also mutant for the gene smoothened (encoding an essential transducer of the Hedgehog pathway) were generated. In the P compartment, which is refractory to Hedgehog signal transduction, clones displayed a normal morphology. In the A compartment, a response to Hedgehog that is secreted by the clones is elicited in the surrounding wild-type cells. These clones had a rounder appearance, and at the clone border, but not away from it, apical cross-section area and bond angles were increased. Similarly, juxtaposing cells expressing engrailed and invected with cells that are mutant for these genes resulted in increased apical cross-section area and increased bond angles at the clone border. It is concluded that the morphology that is characteristic of cells at the A/P boundary can be imposed on cells within a compartment by juxtapositioning cells with different activities of Hedgehog signal transduction or Engrailed and Invected (Landsberg, 2009).

Ablating cell bonds generates cell vertex displacements, providing direct evidence for tension on cell bonds. Individual cell bonds were ablated by using a UV laser beam focused in the plane of the adherens junctions. Single-cell bonds were cut, and the displacement of vertices of neighboring cells, visualized by E-cadherin-GFP, was recorded. The P compartment was visualized by expression of GFP-gpi under control of the engrailed gene via the GAL4/UAS system. The increase in distance between the two vertices of the ablated cell bond and the initial velocity of this vertex separation were analyzed. The ratio of initial velocities in response to cell bond ablation is a measure of the tension ratio on these cell bonds. Initial velocity and extent of vertex separation were indistinguishable between anterior (A/A) and posterior (P/P) cell bonds located away from the A/P boundary. This was also the case when specifically cell bonds between the first and second row of anterior cells were ablated. By contrast, ablation of bonds between adjacent anterior and posterior cells (A/P cell bonds) gave rise to a larger vertex separation. This result was not due to the fact that A/P cell bonds have a preferred orientation. Moreover, the initial velocity of ablated A/P bonds was 2.37-fold higher compared to the mean of initial velocities of A/A and P/P bonds. This value provides an estimate of the ratio λ of cell bond tension along the A/P boundary relative to the average tension of cell bonds. In the presence of the Rho-kinase inhibitor Y-27632, the ratio of initial velocity of vertex separation of A/P cell bonds relative to A/A cell bonds was reduced to 1.46. Given that Myosin II is the main effector of Rho-kinase, these results strongly suggest that Myosin II-based tension acting on cell bonds is locally increased along the A/P boundary (Landsberg, 2009).

To quantify λ by an independent method, the displacement field was calculated after laser ablation. Using a vertex model, two populations of adjacent cells were introduced and cell bond ablations were simulated, varying λ between 1 and 4. When λ = 2.5, the vertex displacement, and in particular the anisotropy of displacements, in the simulations closely matched the vertex displacements in the experiment. In the vertex model, λ = 2.5 also resulted in increased bond angles at the interface of the two cell groups, similar to the A/P boundary in the wing disc. Thus, on the basis of two different methods, the data demonstrate that cell bond tension is increased approximately 2.5-fold along the A/P boundary compared to the remaining tissue (Landsberg, 2009).

To test whether a 2.5-fold increase in cell bond tension is sufficient to maintain a compartment boundary, the vertex model was used to simulate the growth of two adjacent cell populations for λ = 1, 2.5, and 4. For λ = 1, the interface between two growing cell populations became increasingly irregular. By contrast, for λ = 2.5 and 4, a well-defined interface was maintained. Moreover, corresponding changes in cell bond tension at borders of simulated clones resulted in the morphology and sorting behavior of cell patches that resembled those of experimental cell clones compromised for Hedgehog signal transduction or Engrailed and Invected activity. The roughness of the interface in the simulations decreased with increasing λ, showing that cell bond tension is sufficient to maintain straight interfaces between growing cell populations. For λ = 2.5, the roughness of the interface was still larger than the roughness of the A/P boundary in wing discs. This suggests that additional mechanisms might contribute to further reduce the roughness of the A/P boundary. Also, because of the uncertainty of the mechanical properties of A1 and P1 cells, which differ from those of the remaining cells, the value of λ, inferred from laser ablation of cell bonds, might be underestimated. Remarkably, the roughness of the A/P boundary could be altered in mutant conditions. In zip2/zipEbr mutant wing discs, the roughness of the compartment boundary was significantly larger than in controls, demonstrating a role for Myosin II in maintaining a sharp and straight A/P boundary (Landsberg, 2009).

In summary, by applying physical approaches and quantitative imaging, this work for the first time demonstrates and quantifies an increase in tension confined to the cell bonds along the A/P boundary. Moreover, simulations show that this increase in tension suffices to maintain a stable interface between two proliferating cell populations. Genetic studies demonstrated that cells of the two compartments differ in their expression profiles and signaling activities. It has therefore been proposed that biophysical properties of cells within the P compartment differ from those within the A compartment, and that such differences could drive cell sorting. When quantifying cell morphology and vertex displacements after laser ablation, no differences were detected in the biophysical properties of cells between the two compartments. However, the two rows of abutting A and P cells show clear differences in biophysical properties from other cells. Most importantly, the cell bond tension along the A/P boundary is increased. Cell divisions in the vicinity of the A/P boundary were randomly oriented in the epithelial plane. Thus, taken together with the simulations, these results suggest a sorting mechanism by which an increased cell bond tension guides the rearrangement of cells after cell division to maintain a straight interface. Increased cell bond tension and the roughness of the A/P boundary depend on Rho kinase activity and Myosin II, indicating a role for actin-myosin-based tension in this process. Because cell bond tension also depends on cell-cell adhesion, differences in the adhesion between A1 and P1 cells as compared to the remaining cells might also contribute to sorting. The heterotypic, but not homotypic, interaction of molecules presented on the surface of A and P cells might trigger the local increase in cell bond tension. Hedgehog signal transduction and the presence of Engrailed and Invected might control the expression of these heterotypically interacting molecules. These data indicate an important role for cell bond tension directing cell sorting during animal development (Landsberg, 2009).

The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila

Apical constriction is a major mechanism underlying tissue internalization during development. This cell constriction typically requires actomyosin contractility. Thus, understanding apical constriction requires characterization of the mechanics and regulation of actomyosin assemblies. This study analyzed the relationship between myosin and the polarity regulators Par-6, aPKC and Bazooka (Par-3) (the PAR complex) during amnioserosa apical constriction at Drosophila dorsal closure. The PAR complex and myosin accumulate at the apical surface domain of amnioserosa cells at dorsal closure, the PAR complex forming a patch of puncta and myosin forming an associated network. Genetic interactions indicate that the PAR complex supports myosin activity during dorsal closure, as well as during other steps of embryogenesis. It was found that actomyosin contractility in amnioserosa cells is based on the repeated assembly and disassembly of apical actomyosin networks, with each assembly event driving constriction of the apical domain. As the networks assemble they translocate across the apical patch of PAR proteins, which persist at the apical domain. Loss- and gain-of-function studies show that different PAR complex components regulate distinct phases of the actomyosin assembly/disassembly cycle: Bazooka promotes the duration of actomyosin pulses and Par-6/aPKC promotes the lull time between pulses. These results identify the mechanics of actomyosin contractility that drive amnioserosa apical constriction and how specific steps of the contractile mechanism are regulated by the PAR complex (David, 2010).

The repeated assembly and disassembly of apical actomyosin networks is an integral part of amnioserosa tissue morphogenesis during DC. Restricting myosin to the amnioserosa alone is sufficient for amnioserosa apical constriction and overall DC. Dynamic apical myosin has been described in the amnioserosa. This study defined these dynamics as repeated assembly and disassembly cycles of actomyosin networks. Moreover, assembly and disassembly are linked to apical constriction and relaxation, respectively. This is consistent with laser ablation studies showing that the apical surfaces of amnioserosa cells maintain tension across the tissue. Moreover, AJ live imaging has revealed general pulsing of amnioserosa cells from germband retraction through DC. The pulsing actomyosin networks arise with this same developmental timing. A 230±76 second periodicity of cortical pulsing has been described at DC, similar to that of the pulsing actomyosin networks. This study found that increased network durations and decreased lull times with amnioserosa-targeted Baz overexpression coincide with faster DC, as compared with amnioserosa-targeted Par-6 plus aPKC-CAAX overexpression, which increases lull times. It is concluded that the pulsing actomyosin networks mediate the constriction of individual amnioserosa cells and that this contributes to DC (David, 2010).

Remarkably, a single amnioserosa apical constriction event is followed by an almost equal relaxation. However, over many constrictions the cells progressively reduce their apical surface area. This suggests that ratcheting mechanisms incrementally harness the constrictions for overall tissue change. Intracellular and extracellular ratchets are possible. Cells of the Drosophila ventral furrow also display pulsed contractions of apical actomyosin networks as they apically constrict. However, there is minimal relaxation after each constriction. Instead, residual myosin filaments are retained between pulses, and may act as intracellular ratchets to harness the pulsed contractions. By contrast, residual myosin filaments were rarely observed between actomyosin pulses in amnioserosa cells, possibly explaining their relaxation after each cell constriction. It has been proposed that the leading edge actomyosin cable of the surrounding epidermis acts as an extracellular ratchet to harness amnioserosa contractility. However, the ability of myosin expression in the amnioserosa alone to drive DC suggests that other mechanisms contribute. Indeed, DC is a robust process with redundant contributions from both amnioserosa and epidermis. At later stages, filopodia-based epidermal zippering at the canthi could provide another extracellular ratchet. In addition, each amnioserosa cell has a persistent circumferential actin belt that might act as an intracellular ratchet, and other uncharacterized processes, such as membrane trafficking or basal activities, could also contribute (David, 2010).

Actomyosin activity also appears to be linked between cells. The networks display preferential D-V movement, and a network in one cell appears to promote network formation in neighbors. Overall amnioserosa cell shape changes are also coordinated between neighbors. Moreover, myosin activity in isolated amnioserosa cells can elicit cortical myosin accumulation in neighboring epidermal cells. It is speculated that feedback from epidermal cells might orient the D-V movement of amnioserosa actomyosin networks. Interestingly, amnioserosa cells also preferentially contract along the D-V axis. Although the actomyosin networks move in this direction, it is unlikely that they are solely responsible for the directional cell shape changes -- the networks affect the cell circumference both along the axis of their trajectory and perpendicular to it, and, as discussed, both effects are transient. Thus, forces from the epidermis might be needed for the biased D-V amnioserosa cell contraction, and they might also direct the D-V movement of amnioserosa actomyosin networks to facilitate DC (David, 2010).

As the actomyosin networks assemble and disassemble, they translocate across a persistent PAR protein patch. These transient associations and lack of specific colocalization between the actomyosin networks and the PAR proteins argue against PAR proteins being integral parts of the actomyosin networks. However, the current results show that the PAR proteins regulate the networks. Genetic interaction tests indicate that Baz, Par-6 and aPKC support myosin activity for proper DC. Strikingly, the live imaging revealed that Baz and Par-6/aPKC regulate distinct phases of the myosin assembly/disassembly cycle. Together, the loss-of-function and gain-of-function studies show that Baz promotes network durations, whereas Par-6 and aPKC promote lull times between pulses. Baz overexpression also decreased lull times, which could result indirectly from increased network durations or from more direct inhibition of the lull phase. Importantly, overexpression experiments indicate that the effects occur specifically in amnioserosa cells, and analyses of cell polarity and AJs indicate that the PAR proteins have relatively direct effects on the actomyosin networks. However, it remains possible that the PAR proteins have additional functions in the amnioserosa (David, 2010).

A number of molecular interactions must control PAR protein activity in the apical domain of amnioserosa cells. The PAR proteins often, but not exclusively, colocalize in amnioserosa cells, suggesting a dynamic relationship consistent with separate Baz and Par-6/aPKC functions. They also show colocalization with Crb, an apical transmembrane protein at the core of the Crb polarity complex. Interestingly, Crb is known to regulate DC, and Par-6 and aPKC can bind Crb complex components. Thus, Crb might be one anchor for PAR proteins at the apical surface of amnioserosa cells (David, 2010).

Molecular mechanisms connecting PAR proteins to myosin and actin have been implicated in a number of studies. For example, aPKC phosphorylates and inhibits mammalian myosin IIB, although these sites are not present in Drosophila Myosin II (Zipper). Par-6/aPKC also inhibits Rho by activating the ubiquitin ligase Smurf1 in mammalian cells. Additionally, Baz and aPKC immunoprecipitate with Sqh from Drosophila egg chambers. Analogous to amnioserosa morphogenesis, mammalian Par-3 and Par-6/aPKC regulate distinct aspects of cell shape change through different cytoskeletal regulators during dendritic spine morphogenesis: Par-3 inhibits cell protrusions by inhibiting Rac through sequestering the RacGEF Tiam1, whereas Par-6/aPKC promotes protrusions by inhibiting Rho via p190 RhoGAP (David, 2010 and references therein).

Amnioserosa cell apical constriction has similarities to endoderm precursor cell apical constriction during C. elegans gastrulation. Here, myosin activity drives cell ingression. Similar to in amnioserosa cells, the PAR complex and myosin accumulate at the center of the apical surface of these cells and of earlier cells as well. However, these C. elegans actomyosin networks do not appear to undergo full assembly/disassembly cycles and instead progressively accumulate or display continual network flows. Interestingly, apical myosin enrichment requires PAR-3 in C. elegans endodermal precursor cells. Apical myosin enrichment also requires Baz, Par-6 and aPKC in Drosophila egg chamber follicle cells. These results suggest that the PAR complex initiates actomyosin network assembly, contrasting with the amnioserosa, in which networks can assemble without detectable Baz and are inhibited by Par-6/aPKC. Perhaps, actomyosin networks with full assembly/disassembly cycles are regulated distinctly. In the one-cell C. elegans embryo, PAR protein puncta move with a multifaceted cortical myosin network to the embryo anterior. Each facet of the network assembles and disassembles with durations similar to those of the amnioserosa actomyosin networks. The network can also form without the PAR proteins, but the overall flow of the network fails with loss of PAR-3, PAR-6 or aPKC. It would be interesting to test whether PAR-3, PAR-6 and aPKC have distinct effects on the individual facets of these networks (David, 2010).

What triggers actomyosin network assembly in amnioserosa cells? It appears to be independent of Baz, and must overcome Par-6/aPKC inhibition. The Rho pathway triggers actomyosin contractility in many contexts. However, amnioserosa-targeted expression of dominant-negative Rho does not appear to block DC. Alternatively, actin assembly might trigger the networks. Actin networks appear larger and last longer than myosin networks as both start forming during germband retraction. This suggests that actin might organize these networks during germband retraction and possibly DC. Intriguingly, Rac inhibition disrupts DC and reduces amnioserosa actin levels. The trigger might also involve intercellular forces from networks in neighboring cells (David, 2010).

How is the actomyosin assembly/disassembly periodicity regulated? Since more than one network per cell is rarely observed, network assembly might require disassembly of the existing network. Disassembly might begin a cascade that ultimately triggers formation of the next network. For cycling, assembly might likewise elicit disassembly. The data indicate that the PAR proteins are important elements of the regulatory network that is involved. Once a network is triggered, Baz prolongs it, but as the network persists, trigger and maintenance signals must be overcome for network disassembly. With disassembly, Par-6/aPKC activity appears to inhibit new assembly, promoting lull times. With time, this Par-6/aPKC activity must diminish and/or be overwhelmed by the trigger mechanism for new network assembly to occur. Identifying trigger and feedback mechanisms within this cycle will be key for understanding how pulsed actomyosin contractions are regulated in the amnioserosa (David, 2010).

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, 'budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g., cortical mark) or an extrinsic cue (e.g., cell-cell signal). Using live imaging and in vitro culture, and using the distributions of Baz and aPKC as markers, it was found that neuroblasts in contact with epithelial cells always 'bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remain anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. It is concluded that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity (Siegrist, 2006).

This study shows that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor their centrosome(s) at the apical side of the cell, induce Par cortical polarity at prophase, and position Par cortical crescents at the apical cortex. How does the extrinsic cue stabilize centrosome position throughout multiple rounds of cell division? It is likely to stabilize centrosome-cortex interactions, perhaps by regulating association of microtubule plus-ends with the apical neuroblast cortex. During mitosis, the apical cortex is enriched with several proteins with the potential to interact with microtubules directly and indirectly, such as Pins, Gαi, Dlg and Insc, but it remains unknown whether one or more of these are involved in transducing the extrinsic cue that promotes centrosome anchoring. During interphase, none of these proteins shows apical enrichment, although several have uniform cortical localization (e.g., Dlg, Galphai) and could help stabilize the neuroblast centrosome following the completion of telophase (Siegrist, 2006).

The epithelial extrinsic signal is also required for the timing and position of Par cortical polarity in embryonic neuroblasts. In the presence of the extrinsic cue, Par polarity, as evidenced by the distribution of Baz and aPKC, is established around the G2/prophase transition; without the extrinsic cue, Par polarization is delayed until prometaphase/metaphase. Because adjacent neuroblasts divide asynchronously, it is likely that the epithelial cue is always present, but the neuroblast only becomes competent to form the Par crescent at the G2/prophase transition. The best candidates would be mitotic kinases or phosphatases that change levels at the G2/prophase transition (Siegrist, 2006).

The position of the Par cortical crescent is also determined by the epithelial cue. In isolated neuroblasts, the Par cortical crescent forms at random positions during subsequent cell cycles, correlating with randomization of the cell division axis. It is not known how Par protein crescents are formed in wild-type embryonic neuroblasts exposed to the epithelial cue or in isolated neuroblasts that lack extrinsic signals. In wild-type neuroblasts, the initial events in Par protein polarization are likely to involve polarization of Baz or Insc, the two most upstream components in the Par cortical polarity pathway. In isolated neuroblasts, Par crescents form over one pole of a randomly oriented mitotic spindle, raising the possibility that astral microtubules may induce Par crescents, similar to their ability to trigger Pins/Galphai/Dlg crescents. Although Par crescents can still form in the absence of both microtubules and extrinsic cues (such as in Colcemid-treated isolated neuroblasts), astral microtubules may be necessary to direct the position of Par crescents in isolated neuroblasts (Siegrist, 2006).

In the future, it will be important to determine the relationship between centrosome position and position of cortical polarized Par proteins. Both require an extrinsic signal from the overlying epithelium, but they could be independently regulated by two different signals, independently regulated by the same signal, or they could act in a linear pathway. For example, a single extrinsic cue could anchor the G2 centrosome pair, and then the centrosome pair could induce apical cortical polarity at the G2/prophase transition, similar to centrosome-induced cortical polarity in the C. elegans zygote (Siegrist, 2006).

One of the best candidate pathways for regulating orientation of the neuroblast division axis by extrinsic cues is the non-canonical Wnt signaling pathway, because it is known to orient cell divisions in Danio rerio, C. elegans and Drosophila. This pathway uses the Frizzled (Fz) receptor and the cytoplasmic Disheveled (Dsh) and Gsk3 proteins from the Wnt pathway, but does not use a Wnt ligand. In addition, these three components are joined by the two transmembrane proteins Strabismus (Stm) and Flamingo (Fmi) during planar cell polarity signaling in Drosophila. However, no evidence was found to support a role for this pathway in orienting embryonic neuroblast divisions. RNAi of each of the four Drosophila Fz receptors, individually and in combination, had little effect on neuroblast spindle orientation or cortical polarity. Nor were spindle orientation defects observed following expression of a dominant-negative Fz1 lacking the cytoplasmic domain, expression of the Wnt pathway antagonist Axin, or in dsh maternal zygotic mutants, fmi zygotic mutants, stm maternal zygotic mutants or fz1 fz2 double mutants. The non-canonical Wnt pathway may still be involved in the ectodermal signal that regulates neuroblast orientation, but its role may be masked by genetic redundancy (Siegrist, 2006).

A second candidate pathway for regulating epithelial-to-neuroblast signaling is an extracellular matrix (ECM)-integrin pathway. ECM is deposited by the basal surface of epithelia, which is where neuroblasts contact the overlying embryonic epithelia. However, no major integrin ligand, Laminin, is detected at the basal surface of the embryonic ectoderm during stages 9-11, nor was the core ß-integrin protein detected in neuroblasts. In addition, maternal zygotic mys mutants lacking ß-integrin show normal embryonic neuroblast spindle orientation. It is unlikely that the ECM-integrin signaling regulates embryonic neuroblast spindle orientation (Siegrist, 2006).

Interestingly, neuroblasts located in the procephalic neural ectoderm are reported to undergo asymmetric cell divisions within the plane of the epithelium and reproducibly orient along the apicobasal embryonic axis to bud GMCs towards the interior of the embryo. Similarly, during adult PNS development, the pIIb cell lies within the imaginal disc epithelium yet divides along the apicobasal axis. In both cases, the reproducibly apicobasal spatial pattern of cell divisions occurs independent of an overlaying polarized epithelium. It remains unknown whether the oriented pattern of these cell divisions is regulated by intrinsic cues or extrinsic cues (e.g., more internal cells). Unlike ventral cord embryonic neuroblasts, neuroblasts in the brain and in the PNS contain several cell-cell junctions, including cadherin-containing adherence junctions and septate junctions. These signaling rich sites could provide spatial information for spindle orientation as seen in other cell types (Siegrist, 2006).

Although the nature of the cue required to orient embryonic neuroblasts is not clear, there are several approaches to identify potential genes required for this process. As extrinsic cues are required for early localization of Par proteins and because baz and insc mutants have mis-oriented spindles relative to the epithelium, identifying binding partners for either Insc or Baz could be informative. In addition, a small genetic deficiency has been identified that, when homozygous, results in embryonic neuroblast spindle orientation defects relative to the overlying ectoderm without affecting epithelial morphology; one or more genes within this genetic interval would be excellent candidates for components of the extrinsic signaling pathway (Siegrist, 2006).

Finally, does neuroblast cell behavior in culture accurately reflect neuroblast behavior in vivo? It has previously been shown that in vivo embryonic neuroblasts establish apicobasal spindle orientation through one of two behaviors. Either the mitotic spindle first forms parallel to the overlaying epithelium and then rotates 90° to align orthogonal to the overlaying epithelium or the spindle forms as it rotates into its proper orientation. Centrosome separation and rotation behavior were not described. Both behaviors were also observed in cultured neuroblasts, however, with several differences: (1) rotations of fully formed spindles were observed at a very low frequency and this behavior usually correlated with an unhealthy culture; (2) if both centrosomes moved basally or away from the epithelial contact site after separation, an initial spindle formation coinciding with rotation into a position orthogonal to epithelial cells is frequently observed, similar to some of the reported in vivo cases. One additional difference in the analysis between these two systems involves the Drosophila stocks used for live imaging. Following microtubule behavior from cells relied on expressing endogenous levels of a microtubule-associated protein fused in frame to GFP, rather than upon overexpression of a tau:GFP fusion protein. This difference alone could account for the observed differences between the two studies (Siegrist, 2006).

Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein Btsz: Bazooka is required for Btsz localization

Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. This study identifies an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. Two polarized cues have been identified that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. Btsz binds to the Ezrin-Radixin-Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, these data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia (Pilot, 2006).

Homotypic binding of the cell-adhesion molecule E-cadherin (E-cad) at the adherens junctions of epithelial cells organizes the formation of multiprotein complexes, composed in part of the ß-catenin and alpha-catenin proteins, and their dynamic interaction with actin filaments (F-actin). F-actin is required to stabilize E-cad-ß-catenin-alpha-catenin complexes. Moreover, E-cad regulates its own stability through the organization of actin filaments through alpha-catenin: alpha-catenin binds Formin (also known as Diaphanous) and suppresses branching by competing with Arp2/3 (Drees, 2005). When epithelia form through the mesenchymal-epithelial transition, the sites of initial cell contact serve as spatial landmarks for the recruitment of E-cad-ß-catenin-alpha-catenin complexes during the formation of adherens junctions. In primary embryonic epithelia, however, adherens junctions do not form through specific cell contacts, and the spatial cues positioning the adherens junctions in the AJR are less characterized and may be different. The identification of such spatial cues and the mechanisms whereby these cues organize structural, cytoskeletal components associated with the formation and/or stabilization of adherens junctions is an important challenge (Pilot, 2006).

This problem was addressed in the early Drosophila embryo. Formation, stabilization and remodelling of adherens junctions occur in a tightly and genetically controlled sequence involving e-cad (or shotgun), armadillo (or ß-catenin), par-6 , par-3 (or bazooka, baz), aPKC, crumbs and others. A microarray-based RNA interference (RNAi) screen of epithelial morphogenesis identified btsz, a gene previously known to control growth in adult flies (Serano, 2003), as a regulator of embryonic epithelial integrity. In embryos injected with double-stranded RNA (dsRNA) probes specific for btsz (hereafter called btszRNAi embryos), gastrulation is severely affected and the epithelium fails to extend properly. Defects are either strong or medium; that is, they are visible at the beginning of gastrulation or about 15 min later, respectively. The defects are penetrant (80%) and dose dependent. Four different, nonoverlapping probes produce these defects and embryos were not affected with control probes (Pilot, 2006).

Btsz is the only Drosophila member of the synaptotagmin-like protein (SLP) family characterized by the presence of tandem carboxy-terminal C2 boxes. btsz encodes several isoforms (Serano, 2003). In early embryos, btsz1 was not detected but btsz2 and btsz3 are expressed together with btsz0, another isoform not previously reported. At least one of these isoforms is maternally and zygotically provided. The most efficient dsRNA probes used recognizes all three maternally and zygotically expressed isoforms. These isoforms were strongly reduced in btszRNAi embryos, suggesting that RNAi produces a severe btsz loss-of-function phenotype (Pilot, 2006).

Two btsz alleles have been described (Serano, 2003): btszK13-4 introduces a deletion in the amino terminus of btsz2 (residues 501-1,494), btszJ5-2 corresponds to a frameshift mutation that introduces a stop codon at amino acid 390, which leads to a truncation in Btsz0 and Btsz2, and probably the absence of Btsz3. btszK13-4 homozygous female escapers can be recovered and were crossed to heterozygous btszK13-4 or btszJ5-2 males. Although many embryos were not fertilized, those that were reached cellularization and showed epithelial defects during gastrulation: 26% of btszK13-4/btszK13-4 and 46% of btszK13-4/btszJ5-2 embryos. btszJ5-2 germline clones do not produce eggs and btszJ5-2 is lethal. Trans-heterozygous embryos were examined with a deficiency removing the btsz locus (Df(3R)Exel6275, called Dfbtsz): 12% of embryos from crosses of Dfbtsz/btszK13-4 females and wild-type males showed epithelial defects. This proportion went up to 39% when males were heterozygous btszK13-4/+. It is concluded that btsz is zygotically and maternally required. Whereas RNAi targeted all three btsz isoforms, btszK13-4 left intact a large fraction of Btsz2 and Btsz0, probably explaining the weaker penetrance of phenotypes in btszK13-4 (26%) or btszK13-4/btszJ5-2 (46%) mutants, as compared with btszRNAi embryos (80%). Notably, despite its essential role in the formation of epithelia in early embryos, the recovery of adult escapers suggests that btsz may be dispensable in adult epithelia (Pilot, 2006).

Overexpression of a btsz2 isoform lacking the 3' untranslated region (UTR) rescues the phenotypes produced by an RNAi probe targeting the 3' UTR of all btsz isoforms. Overexpression of btsz2 more robustly rescues the btszRNAi phenotype than does btsz3 overexpression, suggesting that btsz2 has a prominent role. The injection of morpholino antisense oligonucleotides (morpholinos) specific to each btsz isoform confirmed this: a control morpholino showed no defect, a mix of btsz0, btsz2 and btsz3 morpholinos caused penetrant defects (92%), and a btsz2-specific morpholino alone caused defects in 73% of embryos. Experimental focus was therefore placed on Btsz2, a 286-kDa protein (2,645 residues) (Pilot, 2006).

The expression of a Glu-epitope-tagged variant of Btsz2 (Btsz2-Glu) was strongly reduced in btszRNAi embryos. The epithelium failed to maintain its regular morphology in btszRNAi embryos, btsz mutants and btsz morphants. Although cellularization proceeds similarly in btszRNAi and control embryos, at the onset of gastrulation the epithelium collapses and becomes multilayered in btszRNAi and btszK13-4/btszJ5-2 mutant embryos, as compared with controls. A similar phenotype was observed in e-cadRNAi embryos. Thus, btsz controls the stable architecture of primary embryonic epithelia (Pilot, 2006).

These data suggested that btsz might regulate the formation of adherens junctions. In contrast to the wild type, in which E-cad is uniformly present at the adherens junctions, E-cad expression is heterogeneous and the adherens junctions appears severely fragmented in btsz mutants and btszRNAi embryos. Time-lapse recordings of E-cad fused to green fluorescent protein (GFP) showed that adherens junctions forms properly in the AJR of btszRNAi embryos but that, subsequently, E-cad-GFP expression disappears, suggesting a defect in the stabilization but not targeting of E-cad. E-cad-GFP, or endogenous E-cad, disappears in small patches at cell contacts, pointing to defects in actin organization. Indeed, the actin belt in the AJR is fragmented in btszRNAi embryos. Tested were performed to see whether actin organization or the E-cad-ß-catenin-alpha-catenin complexes was the primary cause of the disassembly of adherens junctions in btszRNAi embryos. In e-cadRNAi embryos, in which E-cad was undetectable in the nascent AJR, the actin belt is not considerably affected during early gastrulation and clearly less affected than in btszRNAi embryos at the same stage. Subsequently, however, F-actin was disorganized in e-cadRNAi embryos. This suggests that Btsz is part of an E-cad-independent pathway controlling actin organization in the AJR and consequently junction stability (Pilot, 2006).

Next, Btsz2 localization was examined. Btsz2-Glu is a functional protein that rescues the btszRNAi phenotype. Btsz2-Glu was previously reported to localize apically in follicular epithelial cells (Serano, 2003). In early embryos, Btsz2-Glu is detected at the AJR together with E-cad from the end of cellularization until about 30 min into gastrulation. Subsequently, Btsz2-Glu was found in a subapical compartment. At these early stages, E-cad colocalizes with Par-3 (also known as Baz). Therefore the possible role of E-cad and Par-3/Baz in Btsz2 localization in the AJR was addressed. In e-cadRNAi embryos, the recruitment of E-cad in the AJR is blocked and Btsz2 is normal; by contrast, in par-3/bazRNAi embryos Btsz2 is largely cytoplasmic, like PatJ, another marker of AJR at this stage. Btsz2 is thus a target of the early polarity marker Par-3/Baz, which is required for E-cad localization in the AJR (Pilot, 2006).

The role of the two C2 boxes (C2AB) in the localization of Btsz2 was tested. Purified glutathione S-transferase (GST)-tagged C2AB binds to phosphatidylinositol mono- and bisphosphate species in a Ca2+-dependent fashion in vitro. The in vivo relevance of this binding was assessed. A tagged form of Btsz2 lacking the C2 boxes (Btsz2-DeltaC2-HA) expressed in gastrulating embryos was cytoplasmic and failed to localize at the AJR. Conversely, an epitope-tagged form of C2AB (C2AB-HA) localizes at the plasma membrane in gastrulating embryos. Notably, C2AB is polarized and concentrates in the apical surface and in the AJR. Of all the phosphoinositides that C2AB binds in vitro, phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) is the most abundant at the plasma membrane, suggesting that PtdIns(4,5)P2 could be required for Btsz2 localization in the AJR. Injection of cellularizing embryos with neomycin, a compound that binds and inhibits PtdIns(4,5)P2, resulted in epithelial defects similar to btsz, par-3 or e-cadRNAi, and inhibits the recruitment of Btsz2 at the plasma membrane and the AJR. A fusion between GFP and the pleckstrin homology (PH) domain of phospholipase Cdelta (PLCdelta), which specifically binds PtdIns(4,5)P2, localizes apically and in the AJR, similar to the Btsz C2 boxes. It is concluded that PtdIns(4,5)P2 is a polarized spatial cue required for localization of Btsz in the AJR, and hence for adherens junction stability, together with Par-3/Baz (Pilot, 2006).

How does localized Btsz organize F-actin in the AJR? A large-scale two-hybrid screen identified an interaction between Btsz and Moesin, the only Drosophila member of the Ezrin-Radixin-Moesin (ERM) family of F-actin binding proteins that has been implicated in various aspects of epithelial polarity. This interaction was confirmed and Btsz was shown to bind to the third F3 subdomain of Moesin. A minimal region in Btsz that binds Moesin was narrowed down. This interaction occurred in GST pull-down assays of Drosophila S2 cell lysates and embryonic extracts. The functional relevance of this interaction was assessed. Moesin and the phosphorylated active form of Moesin, which binds F-actin, localizes in early embryos in the apical surface and in the AJR, together with Btsz2 and E-cad, suggesting that the interaction between Btsz and Moesin may spatially define a domain of actin organization in the AJR required to stabilize E-cad. In agreement with this, in btszRNAi and btsz mutant embryos, Moesin localization was diminished in the AJR as compared with controls, and E-cad and Moesin segregated in distinct domains as E-cad progressively disappeared (Pilot, 2006).

Whether Moesin is required for epithelial stability was tested in early embryos. Moesin has a major maternal contribution and is a very stable protein. Moreover, females whose germline is mutant for moesin do not lay eggs. Thus, no phenotype was identified using either various moesin mutant alleles or RNAi. Therefore a dominant-negative construct of Ezrin, a mammalian Moesin orthologue that lacks the C-terminal actin-binding domain and acts as a dominant-negative in Drosophila (EzrinDN, containing residues 1-280) was expressed in early embryos . Embryos expressing EzrinDN during gastrulation showed epithelial defects (41% of embryos) similar to btsz mutants. In particular, cellularization was normal, adherens junctions formed properly, but E-cad was no longer present homogeneously around the AJR (Pilot, 2006).

These results shed light on the mechanisms underlying the spatial control of actin filament and the stability of the adherens junctions in the Drosophila primary embryonic epithelium. In Btsz, an E-cad independent pathway has been identified that participates in F-actin organization in the AJR, together with Moesin. Btsz and Moesin bind and colocalize in the AJR in a btsz-dependent fashion, and expression of a mutant form of Ezrin that does not bind F-actin disrupts adherens junctions stability similar to loss of btsz. Notably, this work identifies upstream polarity cues (Par-3/Baz and PtdIns(4,5)P2) that control the spatial order of actin organization at the AJR through the localization of Btsz. The fact that PtdIns(4,5)P2 acts as a key regulator of epithelial polarity in the early embryo raises the issue of how PtdIns(4,5)P2 metabolism is spatially regulated in epithelial cells. The observation that Par-3 binds PTEN, which converts PtdIns(3,4,5)P3 into PtdIns(4,5)P2, suggests that Par-3/Baz may be part of this process. Thus a key intermediate between polarity cues and structural elements of adherens junctions important for embryonic epithelial stability has been identified. Five SLPs and two SLP-related (Slac2) proteins are close orthologues of Btsz in mammals. It would be worth investigating their potentially conserved role in the dynamic organization of actin at adherens junctions in embryonic epithelia (Pilot, 2006).

Rho-kinase directs Bazooka/Par-3 planar polarity during Drosophila axis elongation

Cell rearrangements shape the Drosophila embryo via spatially regulated changes in cell shape and adhesion. This study of axis elongation (germband extension) shows that Bazooka/Par-3 (Baz) is required for the planar polarized distribution of myosin II and adherens junction proteins and polarized intercalary behavior is disrupted in baz mutants. The myosin II activator Rho-kinase is asymmetrically enriched at the anterior and posterior borders of intercalating cells in a pattern complementary to Baz. Loss of Rho-kinase results in expansion of the Baz domain, and activated Rho-kinase is sufficient to exclude Baz from the cortex. The planar polarized distribution of Baz requires its C-terminal domain. Rho-kinase can phosphorylate this domain and inhibit its interaction with phosphoinositide membrane lipids, suggesting a mechanism by which Rho-kinase could regulate Baz association with the cell cortex. These results demonstrate that Rho-kinase plays an instructive role in planar polarity by targeting Baz/Par-3 and myosin II to complementary cortical domains (de Matos Simões, 2010).

The spatially regulated activity of protein kinases with multiple substrates provides an efficient strategy for the control of cell polarity in different contexts. This study shows that Rho-kinase is an asymmetrically localized protein that plays an instructive role in planar polarity in the Drosophila embryo by excluding its substrate Baz/Par-3 from the cell cortex. Rho-kinase prevents expansion of the Baz domain and Baz in turn directs the localization of contractile and adherens junction proteins that are required for axis elongation, converting a localized source of kinase activity into a robust bias in polarized cell behavior. The effect of Rho-kinase on Baz planar polarity appears to be independent of its role in regulating myosin II, as Baz localization is not affected in myosin mutants and activated myosin does not reproduce the effects of Rho-kinase in culture. Instead, Rho-kinase can directly phosphorylate the Baz C-terminal coiled-coil domain that is required for Baz association with the cortex. Deletions within the Baz C-terminal domain or replacement of the Baz C-terminus with a heterologous phospholipid binding motif abolish Baz planar polarity in vivo. These results are consistent with a model in which Rho-kinase directly inhibits the association of the Baz C-terminal domain with specific regions of the cell cortex (de Matos Simões, 2010).

Rho-kinase has been shown to phosphorylate mammalian Par-3 in cultured cells, disrupting its interaction with the Par complex proteins Par-6 and aPKC (Nakayama, 2008). The Par complex is necessary for some aspects of epithelial organization but dispensable for others. Par-6 and aPKC are not required for Baz planar polarity in Drosophila, suggesting that the role of Rho-kinase in this process is unlikely to occur through a similar mechanism. This study provides evidence for a different mechanism of regulation by Rho-kinase involving the Baz C-terminal domain, which is phosphorylated by Rho-kinase in vitro and is necessary for Baz planar polarity in vivo. The Baz C-terminus has been shown to bind directly to phosphoinositide membrane lipids including PI(3,4,5)P3, PI(3,4)P2 and PIP (Krahn, 2010). This study shows that Rho-kinase inhibits the association of Baz with phosphoinositide membrane lipids in vitro, consistent with a model in which Rho-kinase directly regulates Baz association with the cortex. Alternatively, Rho-kinase could regulate Baz localization indirectly through other proteins that interact with the Baz C-terminal domain. Despite potential differences in the mechanism, these results demonstrate that the regulation of Par-3 localization or activity by Rho-kinase is a conserved feature of cell polarity in Drosophila and mammals (de Matos Simões, 2010).

The results demonstrate that Rho-kinase is an asymmetrically localized protein that initiates a cascade of events required for the planar polarized distribution of contractile and adherens junction proteins in intercalating cells. The upstream signals that generate localized Rho-kinase activity are not known. Differences between cells conferred by striped or graded patterns of gene expression orient cell movement during axis elongation, and AP patterning genes expressed in stripes are necessary for the asymmetric localization of Rho-kinase. These findings raise the possibility that planar cell polarity may be generated by the local activation of a Rho GTPase signaling pathway. The Drosophila genome contains 21 RhoGEFs and 19 RhoGAPs that are candidate upstream regulators in this process. Rho GTPase pathways are activated by a number of upstream signals including G protein-coupled receptors, receptor tyrosine kinases, cytokine receptors, and cell-cell and cell-substrate adhesion. Identification of the signals upstream of Rho-kinase will help to elucidate the spatial cues that initiate planar polarity in the Drosophila embryo (de Matos Simões, 2010).

The role of Rho-kinase in planar cell polarity is reinforced by the effect of Baz on the localization of contractile and adherens junction proteins. The relationship between Baz and myosin II is complex. In the C. elegans zygote, a contractile myosin network carries PAR-3 to the anterior cell cortex, suggesting a positive relationship between these proteins. In other cell types myosin appears to be dispensable for Baz localization. PAR-3 is required to sustain myosin contractility in C. elegans and Drosophila, and Baz promotes myosin apical localization during C. elegans gastrulation and in the Drosophila follicular epithelium. The ectopic association of myosin with DV cell boundaries in baz mutants, and the complementary distributions of Baz and myosin in several contexts, raise the possibility of inhibitory effects of Baz on myosin. This regulation could also occur indirectly through effects of Baz on apical-basal polarity (de Matos Simões, 2010).

Differential adhesion is sufficient to drive cell sorting in culture and has been proposed to influence tissue morphogenesis in vivo. This study shows that Rho-kinase and Baz regulate the planar polarized localization of the adherens junction protein β-catenin. Rho-kinase has been shown to downregulate adhesion in culture, an activity that is thought to occur through myosin II, which can play positive and negative roles in junctional stabilization. The ability of Rho-kinase to exclude the Baz/Par-3 junctional regulator from the cortex suggests an alternative mechanism for the regulation of adherens junctions by Rho GTPases. These results suggest that Rho-kinase can both promote contractility and inhibit adhesion, providing a single molecular mechanism linking cortical contraction with adherens junction disassembly during tissue morphogenesis (de Matos Simões, 2010).

Microtubule-dependent apical restriction of recycling endosomes sustains adherens junctions during morphogenesis of the Drosophila tracheal system

Epithelial remodelling is an essential mechanism for organogenesis, during which cells change shape and position while maintaining contact with each other. Adherens junctions (AJs) mediate stable intercellular cohesion but must be actively reorganised to allow morphogenesis. Vesicle trafficking and the microtubule (MT) cytoskeleton contribute to regulating AJs but their interrelationship remains elusive. This study reports a detailed analysis of the role of MTs in cell remodelling during formation of the tracheal system in the Drosophila embryo. Induction of MT depolymerisation specifically in tracheal cells showed that MTs were essential during a specific time frame of tracheal cell elongation while the branch extended. In the absence of MTs, one tracheal cell per branch overelongated, ultimately leading to branch break. Three-dimensional quantifications revealed that MTs were crucial to sustain E-Cadherin (Shotgun) and Par-3 (Bazooka) levels at AJs. Maintaining E-Cadherin/Par-3 levels at the apical domain required de novo synthesis rather than internalisation and recycling from and to the apical plasma membrane. However, apical targeting of E-Cadherin and Par-3 required functional recycling endosomes, suggesting an intermediate role for this compartment in targeting de novo synthesized E-Cadherin to the plasma membrane. The apical enrichment of recycling endosomes was dependent on the MT motor Dynein and essential for the function of this vesicular compartment. In addition, E-Cadherin dynamics and MT requirement differed in remodelling tracheal cells versus planar epithelial cells. Altogether, these results uncover an MT-Dynein-dependent apical restriction of recycling endosomes that controls adhesion by sustaining Par-3 and E-Cadherin levels at AJs during morphogenesis (Le Droguen, 2015).

This study has reveal the importance of the functional interplay between MTs, vesicular trafficking and the control of AJ dynamics in cells undergoing extensive remodelling through collective migration. MT depletion in tracheal cells induces the formation of intracellular dots containing E-Cad and Par-3. Interestingly, these intracellular accumulations are only seen in remodelling tracheal cells and not in planar epithelia. These dots are still detected when endocytosis is affected, showing that they are de novo synthesis route intermediates. Altogether, this suggests that maintaining the correct level of E-Cad/Par-3 at the apical domain requires a continuous supply of newly synthesized proteins, which could be essential for the intensive AJ reorganisation that occurs during cell intercalation and elongation of the tracheal branch (Le Droguen, 2015).

Using the photo-convertible E-Cad-EosFP in flat epithelium, a previous study showed that E-Cad that is engaged in homophilic interactions at the AJs forms very stable domains. This study has demonstratde that MT depletion does not affect the integrity of this 2D epithelium. In addition, it was shown that the pool of photo-converted E-Cad-EosFP is less stable in tracheal cells than in epidermal cells. Together with a FRAP assay suggesting that AJs are more dynamic in tracheal cells than in epithelial cells, the results highlight a specific fine-tuning of AJ components in tracheal cells undergoing cell movement and cell shape changes in 3D through cell intercalation, cell elongation and thereby organ formation. This fine-tuning is likely to cycle between internalisation, recycling, degradation and de novo synthesis, the latter being MT dependent. When the balance is altered in the absence of MTs, and thus when E-Cad or Par-3 are reduced at AJs, tracheal cells overelongate after completing intercalation. During branch elongation without MTs, the two migrating leading cells generate a pulling force on the following stalk cells, which display a critical reduction in AJ components. Either the tip cell or the base cell of the stalk becomes unable to maintain its integrity in response to this force. Consequently, this tracheal cell overelongates by a factor 1.8, preventing the remaining stalk cells from reaching their average size. As a result, DBs present a single overelongated cell and several underelongated cells (Le Droguen, 2015).

An overlap was detected between the E-Cad intracellular dots generated in the absence of MTs and recycling endosome vesicles. Interfering with Rab11 function in tracheal cells induces cell overelongation and affects E-Cad and Par-3 distribution at the AJs, as does MT depletion. These results illustrate that the E-Cad de novo synthesis pathway passes through the Rab11-positive recycling endosome compartment. The overlap of E-Cad and recycling endosome markers represents only a small proportion of the total intracellular E-Cad, suggesting a transient residence in this vesicular compartment for this newly synthesized protein. Recent studies conducted in different model systems have revealed that some newly synthesized apical plasma membrane proteins, such as E-Cad and Rhodopsin, leave the trans-Golgi network to cross Rab11-positive recycling endosome compartments before reaching the apical surface). This apical trafficking route is used specifically in tracheal cells and requires the MT network. Quantification of Rab11DN-associated defects upon tracheal branch formation reveals that impairing recycling endosome function has a similar effect to altering the distribution of E-Cad at AJs by depleting the MT network. Moreover, interfering with Rab11 function reduces Par-3 levels at the AJs of tracheal cells. Interestingly, Par-3 does not colocalise with the E-Cad cytoplasmic pool, indicating that functional recycling endosomes are required by Par-3 and E-Cad to assemble as a complex and to be targeted to the apical domain of tracheal cells. However, as E-Cad and Par-3 can be apically targeted in the absence of the other, this suggests that apical targeting of E-Cad and Par-3 can be independent in tracheal cells or that redundant pathways could sustain the localisation of each protein. For example, the Nectin protein Echinoid is required for Par-3 localisation at AJs in shg mutant cells. Moreover, the apical distribution of PI(4,5)P2 in the Drosophila follicular epithelium sustains Par-3 apical anchorage at the plasma membrane. Furthermore, Par-3-independent localisation of E-Cad has been observed (Le Droguen, 2015).

Dynamic MTs in the ectoderm locally upregulate AJ turnover through RhoA activity. RhoA stabilises cellular contacts through acto-myosin regulation. In tracheal cells, MT depletion does not alter actin distribution. Moreover, tracheal cells mutant for the Myosin light chain zipper (zip) and also expressing a dominant-negative form of Zip do not present obvious defects in E-Cad distribution at stage 14. By contrast, MT depletion induces the cytoplasmic accumulation of E-Cad and Par-3 in tracheal cells only and not in ectodermal cells at the same developmental stage. Thus far, cytoplasmic accumulation of E-Cad and Par-3 has only been observed after colchicine-induced MT depolymerisation during polarity establishment at embryo cellularisation, when AJs are extremely dynamic and vesicular trafficking is strongly active. This study demonstrated that the E-Cad distribution in tracheal cells is more sensitive to MT depolymerisation and to Rab11DN overexpression than that in the overlying ectodermal cells. The comparison of the maximum recovery of the E-Cad signal in a FRAP assay in tracheal cells and in ectodermal cells, together with the differences in stability of the photo-converted E-Cad-EosFP between these two tissues, suggest that AJs are more dynamic in tracheae. It is thus conceivable that tracheal cells, which undergo extensive cell shape changes through collective cell migration, require more dynamic AJs as sustained by an efficient targeting of E-Cad. These discrepancies between ectodermal and tracheal epithelia underline the importance of investigating MT function in different morphogenetic contexts under different constraints (Le Droguen, 2015).

This study has demonstrated that the MT minus-end motor Dynein is essential for the restricted localisation of recycling endosomes in a developing organism. The Dynein requirement for the apical enrichment of recycling endosomes is in agreement with the MT minus ends being anchored at the apical plasma membrane. The asymmetric distribution of recycling endosome vesicles has been observed in various differentiated cell types, especially during cell division. Vesicles are found enriched either in the apical domain or at the microtubule-organising centre (MTOC; i.e. the centrosome) during mitosis. Indeed, in vivo, Dynein physically interacts with Nuf. During metaphase, Dynein is required for the maintenance of Nuf at the centrosome. This study demonstrates that Dynein is also required for the apical distribution of recycling endosomes in non-dividing tracheal cells. In a context in which Dynein function is altered, Nuf-positive recycling endosome vesicles are dispersed but colocalise with E-Cad and Par-3 intracellular dots, indicating that the recycling endosome compartment remains functional for the assembly of such a complex (Le Droguen, 2015).

A previous study has characterised the relocalisation of the MTOC in tracheal cells; MTs are nucleated and anchored at the apical domain just above the AJs. It has also been demonstrated that such MT organisation is crucial for tracheal morphogenesis. Non-centrosomal MT organisation occurs in many differentiated cell types but the functional relevance of such an organisation is still poorly understood. It will be informative to investigate whether such non-centrosomal MT organisation provides a means to regulate epithelial remodelling by controlling the apical enrichment of recycling endosomes and thus AJ dynamics (Le Droguen, 2015).

Larval and Pupal

Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the orientation of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. Drosophila E-Cadherin-Catenin (Shotgun-Armadillo) complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized Shotgun-Armadillo complexes. While a complete loss of Shotgun function disrupts the apical-basal polarity of the epithelium, both a partial loss of Shotgun function and expression of a dominant-negative form of Shotgun affect the orientation of the pIIa division. Furthermore, expression of dominant-negative Shotgun also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Shotgun regulates the orientation of asymmetric cell division (Le Borgne, 2002).

Three distinct mechanisms regulate the stereotyped orientation of the first three asymmetric cell divisions in the seemingly simple lineage that generates the sense organs on the Drosophila notum. (1) In the pI cell, Fz signaling orients the mitotic spindle along the AP axis of the body, regulates the formation of the Dlg/Pins and Baz complexes at the anterior and posterior poles, respectively, and thereby directs the asymmetric localization of the Numb crescent to the anterior cortex. (2) By analogy to the neuroblasts, an apical Baz/Insc/Pins complex is thought to direct the apical-basal orientation of the pIIb division. This analogy is supported by the observation that Pins, Baz, and Insc colocalize at the apical cortex of the dividing pIIb cell. (3) The pIIa cell divides with the same orientation as its mother cell in a Fz- and Insc-independent manner. In the pIIa cell, a specific cortical domain formed at the region of cell-cell contact between the pIIb/pIIIb and pIIa cells appears to regulate the precise orientation of this division. Five lines of evidence support this last conclusion: (1) Shotgun (Shg), Arm, and alpha-Catenin-GFP localize asymmetrically in a cortical patch at the anterior pole of the dividing pIIa cell; (2) the mitotic spindle of the pIIa cell rotates to specifically line up with this cortical domain; (3) expression of a dominant-negative form of Shg perturbs both the formation of this cortical domain, the orientation of the pIIa division, and the precise positioning of Pins at the anterior lateral cortex; (4) loss of Shg activity in clones leads to defects in the orientation of the pIIa division; (5) Pins localizes opposite of Baz in the pIIa cell along a polarity axis defined by the patch of Shg, and dominant-negative Shg affects the orientation of these two domains relative to this patch. Noticeably, a strong loss of Shg function does not randomize the orientation of the mitotic spindle or of the Pins/Baz domains. Thus, one function of Shg in the pIIa cell is to ensure precision in the orientation of the polarity axis. Although loss of Fz activity randomizes the orientation of the pI cell, Shg appears to play a role formally similar to Fz in defining the polarity axis in the pIIa cell. This is the first evidence of a regulatory role of E-Cadherin in the orientation of asymmetric cell divisions (Le Borgne, 2002).

A study of APC1 and APC2 examines asymmetric protein localization in larval neuroblasts

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. The functions of the Drosophila APCs is further explored using the larval brain as a model. Both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. These experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins (Akong, 2002).

One striking feature of the asymmetric localization of APC2 is that it is present throughout the cell cycle and is particularly strong during interphase. During embryonic neuroblast divisions, most asymmetric markers are localized only during mitosis. However, less is known about their localization in larval neuroblasts. Several asymmetric markers in larval neuroblasts were examined, and their localization was compared with that of APC2. In embryonic neuroblasts, the transcription factor Prospero (Pros) and its mRNA are GMC determinants that are asymmetrically localized to the GMC daughter. Pros protein then becomes nuclear and helps direct cell fate. In larval neuroblasts, a similar localization is observed. Pros is not detectable in interphase neuroblasts, when the cortical APC2 crescent is strongest. A small amount of Pros transiently localizes to an asymmetric crescent during mitosis. Pros is present at low levels in GMC nuclei and at higher levels in the nuclei of ganglion cells (Akong, 2002).

Mira is basally localized in embryonic neuroblasts, and required there for localization of Pros protein and mRNA. In central brain neuroblasts, Mira is diffusely cytoplasmic during interphase, when the APC2 crescent is the strongest. As cells enter mitosis, Mira first becomes cortical and then begins to accumulate asymmetrically on the side of the neuroblast where the daughter will be born. By metaphase, Mira asymmetry is very pronounced. The center of the Mira crescent is always precisely aligned with one spindle pole. As a result, in cells with the spindle pointing toward the center of the APC2 crescent, the Mira and APC2 crescents substantially overlap, while in cells in which the spindle points to the edge of the APC2 crescent, the two crescents are offset. Mira is partitioned into the GMC during anaphase, while APC2 relocalizes to the cleavage furrow. Mira could still be detected in some GMCs, which are thought to be those that were recently born (Akong, 2002).

In contrast to Mira and Pros, Inscuteable (Insc) and Bazooka (Baz) localize to the apical sides of embryonic neuroblasts, where they play essential roles in asymmetric divisions. Insc is asymmetrically localized in larval neuroblasts. Insc localizes to the side of the neuroblast opposite that of APC2 through much, if not all, of the cell cycle. Interestingly, there is a weak Insc crescent during interphase, that becomes stronger through prophase and metaphase. During anaphase, Insc localizes to the neuroblast cortex but not the GMC daughter. Baz localization was similar to that of Insc, though no cortical localization during interphase was detected. During prophase and metaphase, Baz localizes to a crescent opposite APC2, and as the chromosomes begin to separate, Baz localizes to a tight cap opposite the future GMC. Together, these data confirm that larval and embryonic neuroblasts asymmetrically localize many of the same proteins, and that APC2 localizes on the GMC side (basal) of the neuroblast, overlapping Mira and opposite Baz and Insc, which localize apically (Akong, 2002).

Arm also localizes asymmetrically in neuroblasts. Extending this, an examination was made of the localization of Arm's adherens junction partners DE-cadherin and ß-catenin. When central brain neuroblasts undergo a sequential series of asymmetric divisions, the GMCs remain associated with their neuroblast mother, resulting in a cap of GMCs in association with each neuroblast. APC2 localizes strongly to the boundary between the neuroblast and each GMC, and more weakly to the borders between the GMCs. APC2 is present at lower levels in ganglion cells and differentiating neurons (Akong, 2002).

The adherens junction proteins DE-cadherin, Arm, and ß-catenin all show a striking and asymmetric localization pattern in central brain neuroblasts. All precisely colocalize both at the boundary between neuroblasts and GMCs and at the boundaries between GMCs. DE-cadherin, Arm, and ß-catenin are also all expressed in epithelial cells of the outer proliferation center. The localization of DE-cadherin and the catenins is consistent with the idea that cadherin-catenin-based adhesion could help ensure that GMCs remain associated with each other, via association with their neuroblast mother (Akong, 2002).

To further explore this, how successive GMCs are positioned relative to their older GMC sisters was examined using two different approaches. First Mira was used to mark the newborn GMCs and DE-cadherin was used to mark the neuroblast and all of her GMC daughters. Mira localizes to a crescent on the side of the neuroblast where the daughter will be born (basal side), and then is segregated into the daughter. Mira persists for some time in newborn GMCs, and it remains detectable in the other GMCs as well, thus allowing the position of newborn GMCs to be examined relative to their older sisters. In many cases, new GMCs are clearly born at the edge of the cluster of older GMCs. This is particularly striking in neuroblasts with many progeny. It is worth noting that the cluster of daughters is three-dimensional, comprising a 'cap' of daughters in three dimensions rather than a two-dimensional line of daughters. It is thus suspected that new daughters are born near the edge of this cap (Akong, 2002).

These data suggest that neuroblasts and their GMC progeny remain closely associated. The GMCs then divide to form ganglion cells and ultimately neurons. The data further suggest that these latter cells may also remain associated and send their axons together toward targets in the central brain. When sections were made more deeply into the brain, below each cluster of neuroblasts and GMCs, structures that appear to be axons were detected projecting from these groups of cells. These axons label with Arm, DE-cadherin, and APC1. Arm also localizes to the axons of the neuropil, while DE-cadherin and APC2 are present at low levels or are absent from this structure (Akong, 2002).

Bazooka and synaptic plasticity

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. At Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1B-related protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, Baz and Par-6 are also expressed at synapses and their synaptic localization depends on aPKC activity. These findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory (Ruiz-Canada, 2004).

During expansion of the NMJ, parent boutons located at the distal end of a branch give rise to new synaptic boutons by budding. New buds separate from parent boutons by the formation of a neck, and NMJ branches extend by neck elongation and bouton enlargement. Throughout this process, the postsynaptic membrane and underlying cytoskeleton impose a barrier to presynaptic extension, since synaptic boutons and their buds are completely surrounded by the muscle cell membrane and underlying cytoskeleton. During branch elongation, a presynaptic signal may induce the retraction of the postsynaptic cytoskeleton barrier. It is proposed that changes in both the pre- and postsynaptic cytoskeleton during branch elongation mediate these events and that these processes are regulated by aPKC with the collaboration of Baz and Par-6 in both locales (Ruiz-Canada, 2004).

The results show that changes in aPKC activity affect both postsynaptic MT and Actin domains. Based on the lack of aPKC within the Actin domain and the enrichment of aPKC at the MT domain, a primary action of aPKC in muscle cells might be through MTs that surround the peribouton area. Alternatively, the effect of aPKC activity on muscle MTs may arise as a consequence of changes in Baz and Par-6 in the Actin-rich peribouton area, which is spatially segregated from postsynaptic MTs (Ruiz-Canada, 2004).

An interesting finding is that both an increase and decrease in aPKC activity, either pre- or post-synaptically, result in reduction of NMJ expansion. This may reflect the possibility that the pre- and post-synaptic cytoskeleton antagonize one another during NMJ expansion and that an asymmetric perturbation of the cytoskeleton in each cell prevents normal synaptic growth. An alternative or additional possibility is that aPKC is asymmetrically regulated at the pre- and postsynaptic cell, being activated in one cell and inhibited in the other. In this regard, it was noteworthy that while increasing aPKC activity increases the stability of presynaptic microtubules, increasing aPKC postsynaptically results in microtubules that appeared to retract from the junctional area (Ruiz-Canada, 2004).

These studies indicate that Baz and Par-6 are colocalized with aPKC, although this colocalization is only partial. Further, a decrease in Baz or Par-6 gene dosage has been shown to alter NMJ growth and the genes interact genetically with aPKC. That all three proteins coimmunoprecipitate supports the notion that they exist in a tripartite complex. However, it is also likely that at different regions of the NMJ, the composition of the complex is reduced to aPKC-Par-6 or Baz-Par-6. This is suggested by the colocalization studies showing that only Par-6 and aPKC are concentrated at the MT bundle and that only Par-6 and Baz are concentrated at the peribouton area (Ruiz-Canada, 2004).

Baz and Par-6 are localized to the Actin/Spectrin peribouton area, and loss of Baz in dapkc mutants or baz4/+ mutants decreases peribouton Spectrin localization, suggesting that Baz regulates the Actin/Spectrin network. In epithelial cells, Baz is required for the maintenance of the zonula adherens, an Actin belt that encircles the cell just below its apical face. At the NMJ, Baz may similarly contribute to the maintenance of the Actin-rich domain (Ruiz-Canada, 2004).

The composition of the Baz/Par-6/aPKC complex is likely to be regulated by the kinase activity of aPKC; expressing PKM increased the amount of Baz associated to the complex. Mammalian Par-6 is known to bind to both aPKC and Baz at distinct sites, and Par-6 activates aPKC when bound to activated Cdc42 and Rac1. Mammalian Baz/Par-3 is also known to bind to both aPKC and Par-6 at distinct sites, but in contrast to Par-6, Baz inhibits aPKC activity. This inhibition can be suppressed by aPKC-dependent Baz phosphorylation at a highly conserved protein region, and this phosphorylation promotes the dissociation of Baz and aPKC. At the NMJ, it was found that increasing PKM, which lacks the Par-6 binding site, increases the binding between Par-6 and Baz, suggesting that Baz phosphorylation may promote the association between Baz and Par-6. A potential scenario is that Baz and aPKC may exist as an inactive complex at the muscle cortex. Phosphorylation of Baz dissociates the complex and phosphorylated Baz may accumulate at the peribouton region. In agreement with this model, it was found that overexpressing PKM postsynaptically results in an expansion of the peribouton area and increased accumulation of Baz at this area (Ruiz-Canada, 2004).

Electrophysiological studies show that aPKC activity also influences synaptic efficacy. This may result from cytoskeletal changes, which may alter the localization of synaptic proteins, such as GluRs. Indeed, changes in aPKC activity were found to affect both GluR levels or distribution and mEJP amplitude. Many synaptic receptors are anchored to the Actin submembrane matrix. For example, the scaffolding protein DLG, which is responsible for the clustering of Shaker K+ channels and the cell adhesion molecule FasII at the peribouton area, depends on normal Spectrin levels for proper localization at this area. Similarly, in mammals, the DLG homolog SAP97 binds to band 4.1, which is anchored at the Actin/Spectrin network, and NMDA receptors bind alpha-Actinin, an Actin binding protein. Therefore, the changes in GluR levels and distribution found in dapkc mutants may result from alterations in the postsynaptic Actin network (Ruiz-Canada, 2004).

Despite the changes in mEJP amplitude, synaptic junction efficacy (represented by quantal content) was decreased in both aPKC gain- and loss-of-function mutants. This is in contrast to other mutants that affect synaptic transmission in which quantal content is maintained despite changes in postsynaptic sensitivity. For example, reduction of GluR at the postsynapse results in an increase in the amount of neurotransmitter release at Drosophila NMJs. The results raise the possibility that aPKC may be affecting the mechanism that controls retrograde regulation of neurotransmitter release (Ruiz-Canada, 2004).

In addition to changes in quantal content and mEJP amplitude, a reduction was also observed in mEJP frequency. Changes in the frequency of mEJP may arise from a decrease in the probability of release or in the number of release sites. At the NMJ, the reduction in mEJP frequency may reflect the reduction in bouton number observed in dapkc mutants (Ruiz-Canada, 2004).

In the mammalian hippocampus, atypical PKMzeta is necessary and sufficient for LTP maintenance. In flies, overexpression of PKMzeta enhances memory in a Pavlovian olfactory learning paradigm. Moreover, aPKC inhibition using a kinase dead dominant-negative or chelerythrine treatment, which specifically inhibits the catalytic domain of aPKC, diminishes memory without affecting learning. Although these studies suggest that aPKC is involved in functional plasticity of synapses, the cellular mechanism for this effect is unknown (Ruiz-Canada, 2004).

Recent studies suggest that morphological modifications of dendritic spines accompany synapse plasticity, and therefore, changes in spine structure might be at the core of learning and adaptive mechanisms. Spines are particularly enriched in Actin, and interfering with the Actin cytoskeleton inhibits spine motility. Further, many members of the postsynaptic complex, including NMDA receptors, CaMKII, PSD-95, SPAR, and Shank associate with F-Actin through Actin binding proteins. MTs, in contrast, localize to dendritic shafts and are believed to constitute a more stable component. This partitioning between MT and microfilament domains, however, is reminiscent of these domains in growth cones, where Actin and MT dynamics are highly interdependent and ultimately responsible for growth cone dynamics. Similarly, in these studies it has been shown that interfering with normal MT dynamics though modifications in aPKC activity has important consequences for the arrangement of the Actin-rich peribouton area and the normal localization of GluRs. Therefore, although the influence of MTs in spine structure has received less attention, it may be the case that spine architecture is ultimately defined by an interplay between Actin- and MT-rich domains (Ruiz-Canada, 2004).

These studies demonstrate that changes in MT organization are an essential aspect of synapse development and that the aPKC/Baz/Par-6 complex plays an important role in their regulation. In addition, the results show that at the postsynaptic cell, changes in aPKC activity result in dramatic changes in both the MT and Actin networks. Commensurate with the behavioral and electrophysiological studies in which increasing aPKC activity enhanced LTP and memory maintenance, it was found that increases and decreases in aPKC activity inversely regulated the synaptic cytoskeleton. These observations raise the attractive possibility that aPKC regulates synapse plasticity, at least in part, by affecting the organization of the synaptic cytoskeleton (Ruiz-Canada, 2004).

The role of the actomyosin cytoskeleton in coordination of tissue growth during Drosophila oogenesis; active myosin is apically anchored by the Bazooka/Par-6/aPKC complex

The Drosophila egg chamber is an organ composed of a somatic epithelium that covers a germline cyst. After egg-chamber formation, the germline cells grow rapidly without dividing while the surface of the epithelium expands by cell proliferation. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. This study identifies a role for the actomyosin cytoskeleton in this process. Myosin activity is restricted to the epithelium's apical surface, which is facing the growing cyst. The epithelium collapses in the absence of myosin activity; the force that deforms the epithelium originates from the growing cyst. Thus, myosin activity maintains epithelial shape by balancing the force emanating from cyst growth. Further, these data indicate that cyst growth induces cell division in the epithelium. In addition, apical restriction of myosin activity is controlled. Myosin is activated at the apical cortex by localized Rho kinase and inhibited at the basolateral cortex by PP1β9C. In addition, these data indicate that active myosin is apically anchored by the Bazooka/Par-6/aPKC complex (Wang, 2007).

The apical membrane domain is established by the Crumbs (Crb)/Stardust (Sdt)/Patj complex and the Bazooka (Baz)/Par-6/aPKC complex. All these proteins localize, like pRMLC, to the apical membrane of the follicular epithelium. In epithelia lacking crb, myosin restriction is affected as revealed by the interrupted apical pRMLC pattern and by ectopic activity at the basal membrane. However, apical myosin activity is not completely disrupted as broad regions of the epithelium still concentrate higher levels of pRMLC at the apical compared to the basal cortex. In contrast, par-6, aPKC and baz mutants abolish the formation of the apical myosin activity. In these mutants, apical pRMLC restriction is lost, and ectopic myosin activity is detectable in the cytoplasm and at the basal cortex. To test whether the two apical complexes cooperate in apical myosin restriction, baz sdt double mutants were examined. The phenotype of the double mutants is, however, very similar to that of the baz single mutants, suggesting that apical myosin activity is controlled by the Baz/Par-6/aPKC complex (Wang, 2007).

To further analyze this interaction, the Baz/Par-6/aPKC complex was immunoprecipitated from ovaries using an antibody against Baz. Western-blot analysis of the precipitated protein complex reveals a strong enrichment of Baz and aPKC. Notably, pRMLC is also present in the precipitated protein complex, indicating an association of Baz and active myosin. Taken together, these genetic data show that baz, par-6, and aPKC are required for apical myosin restriction, and biochemical data show that Baz associates with pRMLC. This suggests the Baz/Par-6/aPKC complex anchors active myosin at the apical cortex. To further analyze the role of the complex in the apical restriction of myosin activity, its localization was examined in mutants that affect pRMLC localization. Consistent with a function in the anchoring of active myosin, it was found that apical aPKC localization is not affected in arm mutants, in which pRMLC is apically restricted. Further, apical aPKC localization is interrupted in crb mutants, in which pRMLC localization is also interrupted. In summary, the data suggest that the Baz/Par-6/aPKC complex anchors active myosin at the apical cortex independently of the adherence junctions (Wang, 2007).

To examine how myosin activity is inhibited during early oogenesis at the basal and lateral cortex, the localization and function of PP1β9C, the phosphatase that deactivates phosphorylated RMLC, was examined. In follicle cells, PP1β9C is ubiquitously distributed as revealed by a hemagglutinin (HA) fusion protein. PP1β9C is encoded by flap wing (flw). Western-blot analysis of the viable flw1 allele showed that the total pRMLC levels in ovaries are increased 2.8-fold compared to those of the wild-type. The total increase is the result of ectopic myosin activity in the follicular epithelium. This is revealed by flw6 follicle cell clones and homozygous flw1 mutant egg chambers, which show pRMLC staining at the basal and lateral cortex. Interestingly, the ectopic Myosin activity is accompanied by an irregular and wavy appearance of the apical surface of the epithelium. In addition, flw mutant egg chambers are not round or ellipsoid like the wild-type but are either stretched or develop bulges. The coincidence of the altered shape with the ectopic pRMLC staining in the follicular epithelium suggests that the abnormal shape is the result of ectopic myosin activity. This is confirmed by the finding that the expression of constitutively active RMLC results in a very similar phenotype. The defects in flw mutants are not secondary effects of mislocalization of the Baz/Par-6/aPKC complex as the localization of aPKC is indistinguishable from that of the wild-type. In summary, these results show that PP1β9C activity is required to prevent myosin activity at the basal and lateral cortex. They further suggest that during early oogenesis, myosin activity has to be restricted to the apical cortex to ensure the development of normally shaped egg chambers (Wang, 2007).

Diversity of epithelial morphogenesis during eggshell formation in drosophilids

The eggshells of drosophilid species provide a powerful model for studying the origins of morphological diversity. The dorsal appendages, or respiratory filaments, of these eggshells display a remarkable interspecies variation in number and shape, and the epithelial patterning underlying the formation of these structures is an area of active research. To extend the analysis of dorsal appendage formation to include morphogenesis, this study developed an improved 3D image reconstruction approach. This approach revealed considerable interspecies variation in the cell shape changes and neighbor exchanges underlying appendage formation. Specifically, although the appendage floor in Drosophila melanogaster is formed through spatially ordered neighbor exchanges, the same structure in Scaptodrosophila pattersoni is formed through extreme changes in cell shape, whereas Drosophila funebris appears to display a combination of both cellular mechanisms. Furthermore, localization patterns of Par3/Bazooka suggest a self-organized, cell polarity-based origin for the variability of appendage number in S. pattersoni. these results suggest that species deploy different combinations of apically and basally driven mechanisms to convert a two-dimensional primordium into a three-dimensional structure, and provide new directions for exploring the molecular origins of interspecies morphological variation (Osterfield, 2015).

Polarity proteins and Rho GTPases cooperate to spatially organise epithelial actin-based protrusions

Different actin-filament-based structures co-exist in many cells. This study characterises dynamic actin-based protrusions that form at distinct positions within columnar epithelial cells in the Drosophila pupal notum, focusing on basal filopodia and sheet-like intermediate-level protrusions that extend between surrounding epithelial cells. Using a genetic analysis, it was found that the form and distribution of these actin-filament-based structures depends on the activities of apical polarity determinants, not on basal integrin signalling. Bazooka/Par3 acts upstream of the RacGEF Sif/TIAM1 to limit filopodia to the basal domain, whereas Cdc42, aPKC and Par6 are required for normal protrusion morphology and dynamics. Downstream of these polarity regulators, Sif/TIAM1, Rac, SCAR and Arp2/3 complexes catalyse actin nucleation to generate lamellipodia and filopodia, whose form depends on the level of Rac activation. Taken together, these data reveal a role for Baz/Par3 in the establishment of an intercellular gradient of Rac inhibition, from apical to basal, and an intimate association between different apically concentrated Par proteins and Rho-family GTPases in the regulation of the distribution and structure of the polarised epithelial actin cytoskeleton (Georgiou, 2010).

Although many studies have used the segregation of apical, junctional and basolateral markers as a model of epithelial polarity, and a number of studies have reported the existence of cell protrusions in the notum and other epithelia, these structures and the genes regulating their formation have not been characterised in detail. This study used Neuralized-Gal4 to express GFP-fusion proteins in isolated epithelial cells to reveal the dynamic shape of cells within the dorsal thorax of the fly during pupal development. Using this method, distinct populations of protrusions were characterised based on their form, dynamics and location within the basolateral domain of columnar epithelial cells. The analysis reveals dynamic protrusions at three distinct locations within the epithelial cell: apical microvillus-like structures, intermediate-level sheet-like protrusions and basal-level lamellipodia and filopodia. Importantly, although these are all dependent on continued actin filament dynamics, these populations of protrusions rely on different gene activities for their formation (Georgiou, 2010).

Cdc42, Rac, SCAR/WAVE and the Arp2/3 complex are required for the formation of basal lamellipodia and filopodia, but not for the formation of the apical microvillus-like structures. This analysis also confirms that HSPC300 should be considered to be a functional component of the SCAR complex. Moreover, the SCAR and Arp2/3 complexes are required to induce the formation of both lamellipodia and filopodia in this system. Although many studies have suggested that Rac activates the SCAR complex to induce branched Arp2/3-dependent actin nucleation that underlies lamellipodial formation, whereas Cdc42 is required to induce filopodial formation, this analysis suggests that the macroscopic form of the protrusion in a tissue context is not dictated by the nucleator used. In this, the current results are in line with several recent studies in cell culture. Instead, the macroscopic structure generated depends on the local level of Rac activity, with high levels of Rac driving filopodial formation and low levels leading to lamellipodial formation. Since the forces required to distort the membrane to generate finger-like protrusions are likely to be greater than those required to generate the equivalent section of a sheet-like protrusion, protrusion morphology might be a product of a force balance between membrane tension, extracellular confinement and local actin-filament formation. Since wild-type cells have a graded distribution of protrusions, with lamellipodia predominating apically and filopodia basally, wild-type cell morphology might reflect a gradient in the level of Rac activation, from high basal levels to low apical levels (Georgiou, 2010).

Within this system, Cdc42-Par6-aPKC and Baz/Par3 appear to have antagonistic roles in the formation of basolateral protrusions. Cdc42-Par6-aPKC is required for actin filament formation and protrusion dynamics, whereas Baz/Par3 ensures the separation of basal and intermediate protrusions by limiting the extent of basal filopodia along the apical-basal axis. In this, the current analysis adds to the growing body of evidence that Baz/Par3 and Par6-aPKC have distinct molecular targets. Moreover, the data confirm that Par6-aPKC act together with the Rho-family GTPase Cdc42. Significantly, the loss of Baz/Par3 phenocopies gain-of-function mutations in Rac and the overexpression of the Rac-GEF Sif/TIAM1, a Par3-interacting protein. Baz/Par3 might therefore serve as a cell-intrinsic cue to polarise the dynamic actin cytoskeleton along the epithelial apical-basal axis, giving epithelial cells their characteristic polarised morphology (Georgiou, 2010).

Baz/Par3 has previously been implicated in the restriction of actin polymerisation to specific subcompartments within a cell, allowing for the formation of distinct populations of protrusions. This has been studied most extensively in hippocampal neurons, in which Par3 was shown to interact with TIAM1 to regulate the activation of Rac within distinct domains of the cell during axon specification and dendritic spine morphogenesis. Indeed, it has been suggested that the formation of a Cdc42-Par6-Par3-TIAM1-Rac1 complex is required to establish neuronal polarity. The current study suggests that Baz/Par3 acts in a similar fashion in the morphogenesis and positioning of dynamic protrusions in epithelia. However, this analysis reveals an antagonistic relationship between Sif/TIAM1 and Baz/Par3 in protrusion formation. Baz/Par3 might sequester Sif/TIAM1 to prevent its association with Rac. Furthermore, because the loss of Cdc42, Par6 or aPKC results in the loss of basolateral protrusions and a marked reduction in the GFP:Moe reporter (a phenotype that can be rescued by the coexpression of RacV12 or Sif) Cdc42, Par6 and aPKC are probably required for the basal activation of Rac in epithelial cells in the Drosophila notum. Thus, signals from apically concentrated polarity determinants appear to be communicated and translated into local protrusion formation within the basolateral domain. Whether this occurs through the diffusion of an apically localised regulator or via long-range transmission of polarity information e.g. via microtubules, will be an important area of future research. An intriguing correlation is the largely apical localisation of Baz and its proximity to intermediate-level sheet-like protrusions. This would suggest a possible gradient of Baz/Par3-mediated Rac inhibition, allowing sheet-like protrusions at an intermediate level and restricting filopodial protrusions to the very base of the cell. Since Baz/Par3 has been shown to localise PTEN to apical junctions, it is possible that Baz recruits PTEN, which acts on PtdIns(3,4,5)P3 to generate a PtdIns(3,4,5)P3 gradient from high levels basally to low levels apically. PIP3 could then act to aid in the recruitment and activation of Rac at the membrane (Georgiou, 2010).

Taken together, these data demonstrate that different components of the apical determinants of cell polarity act in conjunction with the Rho-family GTPases Cdc42 and Rac to regulate the positioning of lamellipodial and filopodial protrusions over the entire span of the apical-basal cell axis. Significantly, in this tissue context, Rac, SCAR and Arp2/3 complexes promote the formation of both lamellipodia and filopodia, whose structure appears to depend on the level of Rac activation (Georgiou, 2010).

Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo

The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. This study found that the small GTPase Roughened/Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. The current linear model for polarity establishment was tested. Both Bazooka and aPKC regulate Canoe localization despite being 'downstream' of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data suggest that polarity establishment is regulated by a protein network rather than a linear pathway (Choi, 2013).

Polarity is a fundamental property of all cells, from polarized cell divisions in bacteria or fungi to the elaborate polarity of neurons. Among the most intensely studied forms of polarity in animal cells is epithelial apical-basal polarity. Polarity of epithelial sheets is key to their function as barriers between body compartments, and is also critical in collective cell migration and cell shape change during morphogenesis, as cytoskeletal and apical-basal polarity often go hand in hand. Loss of apical-basal polarity is a hallmark of metastasis. Significant advances have been made in defining the machinery required for cell polarity in many settings, but fundamental questions remain unanswered (Choi, 2013).

Cadherin-catenin complexes, which assemble into adherens junctions (AJs) near the apical end of the lateral cell interface, are critical polarity landmarks that define the boundary between apical and basolateral domains. Studies in C.elegans and Drosophila identified other key regulators of apical-basal polarity. In the textbook view, the apical domain is defined by the Par3/Par6/aPKC and Crumbs/Stardust(Pals1)/ PATJ complexes, while Scribble, Dlg, Lgl, and Par1 define the basolateral membrane (Choi, 2013).

Complex cross-regulatory interactions between apical and basolateral proteins maintain these mutually exclusive membrane territories. These proteins also regulate other types of polarity during morphogenesis; e.g., fly Par3 (Bazooka; Baz), aPKC, and AJ proteins are planar-polarized during fly convergent extension, thus regulating polarized cell movements (Choi, 2013).

Polarized cytoskeletal networks also play key roles in establishing and maintaining apical-basal and planar polarity. These networks are thought to be physically linked to apical junctional complexes. The earlier model suggesting that cadherin-catenin complexes link directly to actin via α-catenin is now viewed as over-simplified. Instead, different proteins are thought to mediate this connection in different tissues and at different times (Choi, 2013).

Among the linkers is Canoe (Cno)/Afadin, an actin-binding protein that binds transmembrane nectins via its PDZ domain. While originally hypothesized to be essential for cell adhesion, subsequent work supports a model in which afadin modulates adhesive and cytoskeletal machinery during cell migration in vitro and the complex events of mouse gastrulation. Afadin has two N-terminal Ras association domains for which the small GTPase Rap1 is the major binding partner, and Afadin and Rap1 are functionally linked in both flies and mice. Rap1, Cno, and the Rap1 GEF Dizzy/PDZGEF are all essential for maintaining effective linkage between AJs and the apical actomyosin cytoskeleton during apical constriction of Drosophila mesodermal cells during fly gastrulation. Rap1 regulates Cno localization to the membrane. Cno plays a related role during convergent extension, though its role is planar polarized during this process. Cno also regulates collective cell migration, signaling, and oriented asymmetric divisions. The Rap1/Cno regulatory module is also important in disease, as Afadin and Rap1 are implicated in congenital disorders of the cardiovascular system and cancer metastasis. It remains unclear whether these diverse roles all involve junction-cytoskeletal linkage or whether some are independent functions (Choi, 2013 and references therein).

The small GTPase Rap1 plays diverse cellular roles. Mammalian Rap1 isoforms are perhaps best known for regulating integrin-based cell matrix adhesion, but Rap1 also regulates cell-cell AJs in both Drosophila and mice. In murine endothelial cells, for example, Rap1, its effector Krit1, and VE-cadherin form a complex that regulates endothelial cell junctions and stabilizes apical-basal polarity (Choi, 2013 and references therein).

In Drosophila imaginal disc cells, Rap1 regulates the symmetric distribution of DE-cadherin (DEcad) around the apical circumference of each cell. Rap1 carries out these functions via a diverse set of effector proteins, including Krit1, TIAM, RIAM, and Cno/Afadin. Thus, Rap1 and its effectors are candidate proteins for regulating interactions between AJs, polarity proteins and the cytoskeleton during polarity establishment and maintenance (Choi, 2013).

The early Drosophila embryo provides among the best models of establishing and maintaining apical-basal polarity. Flies start embryogenesis as a syncytium, with 13 rounds of nuclear division without cytokinesis. Membranes then simultaneously invaginate around each nucleus, forming ~6000 cells in a process known as cellularization. Prior to cellularization, the egg membrane is already polarized and serves as a polarity cue for underlying nuclei. This ultimately becomes the apical end of the new cells. Epithelial apical-basal polarity is initiated during cellularization. In the absence of cadherin-catenin complexes, cells form normally but then lose adhesion and polarity as gastrulation begins. These data and earlier work from cell culture suggested AJs are the initial apical cue. However, it was found that Bazooka (Baz)/Par3 acts upstream of AJs in this process. Strikingly, Baz and DEcad apically co-localize in spot AJs from cellularization onset. In the absence of Baz, DEcad loses its apical enrichment and redistributes all along the lateral membrane, while in the absence of AJ proteins, Baz remains apically localized, and a subset of cells retain residual apical-basal polarity, although cell shapes are highly abnormal. Cadherin-catenin and Baz complexes form independently before cellularization, and Baz then helps position DEcad in the apicolateral position where spot AJs will form. This placed Baz atop of the polarization network, raising the question of how it is positioned apically. Two cytoskeletal networks play important roles in initial Baz positioning (Choi, 2013).

Disrupting dynein led to Baz spreading along the lateral membrane, suggesting polarized transport along microtubules (MTs) plays a role. Depolymerizing actin also destabilized apical Baz, as did significantly overexpressing Baz, suggesting an actin-based scaffold with a saturable number of binding sites anchors Baz apically. While both actin and MTs are required for initial Baz polarization, they are not the only cues. Mislocalized Baz is re-recruited or re-stabilized apically at gastrulation onset if either initial cue is disrupted, suggesting a third cue perhaps involving aPKC/Par6 or Par1. Thus, the current model for initial establishment of apical-basal polarity involves a relatively simple pathway in which Baz is positioned apically, and then positions other apical polarity players. However, once initial polarity is established, events become more complex, with a network of mutually reinforcing and inhibitory interactions between apical and basolateral polarity complexes leading to polarity elaboration and maintenance. These were significant advances, but the proteins directing apical accumulation of Baz remained unknown. Work on apical constriction in the fly mesoderm, convergent extension during gastrulation, establishment of anteriorposterior polarity in one cell C. elegans embryos, and on apically constricting Drosophila amnioserosal cells, suggested that a complex network of interactions link AJs, the apical polarity proteins Baz and aPKC, and the actomyosin cytoskeleton. Recent work on Canoe and Rap1's roles in mesoderm apical constriction and convergent elongation (Sawyer, 2011) suggested they also fit into this network. These data led to an exploration of whether Rap1 and Cno play roles in initial apical positioning of AJs and Baz and thus in the establishment and early maintenance of polarity (Choi, 2013).

In regulating polarity establishment, Rap1 and Cno could act by several possible mechanisms. Their role in AJ positioning may be solely due to their effects on Baz localization, or alternatively Rap1 and Cno may independently affect the localization of both Baz and AJs. In the latter case, Cno may directly link AJs to the apical actin scaffold, as it was suggested to act in apical constriction. Rap1 and Cno also clearly regulate Baz positioning. Since Baz apical positioning requires an apical actin scaffold and dynein based MT transport, whether Rap1 and Cno act indirectly by regulating cytoskeletal organization was examined. However, the data suggest this is not the case: both the MT and actomyosin cytoskeletons appear normal in mutants. Thus the most likely model is that Rap1 and Cno are required for anchoring Baz apically. Consistent with this, when Cno was ectopically localized to artificial cell-cell contacts in cultured fly cells, it was able to recruit Baz to that site. This could occur directly, for example, by Cno binding Baz, or indirectly, via unknown intermediaries. Strikingly, however, when Baz was over-expressed in cellularizing embryos, presumably saturating its apical binding sites, it accumulated basolaterally and recruited DEcad but not Cno to these ectopic sites. Thus Cno and Baz do not co-localize obligatorily. It likely that each has multiple binding partners and that when pools are limiting, as Cno may be in this latter experiment, ectopic Baz cannot recruit Cno away from a preferred binding site. Of course, it remains possible that Cno and Rap1 also regulate Baz positioning through effects on MT transport or, given Cno's apical localization, unloading at an apical docking site. It will be important to test these possibilities. As is discussed in more detail below, it will also be important to define the Cno- and Rap1-independent mechanisms that partially restore apical Baz localization after gastrulation onset (Choi, 2013).

Since Rap1 is uniformly distributed along the apical-basal axis during cellularization, the most likely hypothesis is that it is locally activated apically by a GEF. A number of Rap1GEFs exist, many of which are conserved between mammals and flies. Recent work from the Reuter lab demonstrated that, like Cno and Rap1, the Rap1 GEF Dizzy (Dzy/PDZ-GEF) plays an important role in coordinated mesodermal apical constriction, suggesting it is the GEF acting upstream of Cno and Rap1 in that process. They also suggest that Rap1 and Dzy help regulate establishment of AJs. While similar in outline, their analysis of AJs differs from this one in detail, as they see strong effects on DEcad localization without similar effects on Arm. This is surprising, since these two proteins of the cadherin-catenin complex generally localize very similarly at the cortex. However, these differences aside, their data are consistent with Dzy acting with Cno and Rap1 in AJ establishment-it will be important to examine the effects of Dzy on Baz localization. It will also be important to determine how pre-existing egg membrane polarity is translated into localized Rap1 activity (Choi, 2013).

In addition to the parallel roles of Rap1 and Cno in regulating initial apical-basal polarization, this study identified a second role for Rap1 in establishing and maintaining columnar cell shape. The data suggest that this is partially or completely Cno-independent, and thus one of the many other Rap1 effectors may play a role in this process. It will be exciting to examine embryos mutant for other Rap1 effectors, such as Krit1/Bili, TIAM/Still life, RIAM/Pico, or RhoL to see if they are required for establishing columnar cell shape. baz and aPKC mutants also had defects in establishing columnar cell architecture. It is possible that each protein provides an independent mechanistic input into this process. This is consistent with the observed differences in the details of how columnar cell shape is disrupted, with Baz and aPKC primarily regulating apical cell area, while Rap1 affects cell shape at multiple apical-basal positions. A more speculative but perhaps less likely possibility is that Rap1 uses Baz and aPKC as effectors in establishing columnar cell shape. Fly Rap1 can form a complex with aPKC and Par6, and Rap1 acts upstream of cdc42/Par3/aPKC in regulating polarity of cultured neurons (Choi, 2013 and references therein).

Having identified Rap1's direct effector(s) in regulating cell shape, it is necessary to move downstream. Based on analogies with other epithelial tissues in fly development, it is hypothesized establishing columnar cell shape involves regulating apical tension. Other small GTPases play key roles in this; e.g., Rho and cdc42 have striking and opposing roles in apical tension regulation during fly eye development. In that context, Rho acts via separate effectors to maintain AJs and apical tension-it regulates tension via Rok, Diaphanous, and ultimately myosin contractility. It will be interesting to determine whether the defects in apical cell shape in the absence of Rap1, Baz, or aPKC also reflect unbalanced contractility in different nascent cells, and which contractility regulators are involved. However, for now, this is speculative (Choi, 2013).

Previous work has suggested a linear hierarchy regulating polarity establishment, with Baz at the top, positioning AJs and aPKC. The current work extends this hierarchy, positioning Rap1 and Cno upstream of Baz in this process. However, the data further suggest that viewing polarity establishment as a linear process is significantly over-simplified. It is now known that all of the relevant players -- including the AJ proteins, Baz, Cno and aPKC -- are at the cortex in syncytial embryos, prior to cellularization and the initiation of apical-basal polarity. This places them in position to cross-regulate one another. Consistent with this, the data suggest that viewing relationships with an 'upstream-downstream' point of view misses important reciprocal interactions that occur as polarity is established. Two examples point this out most clearly. First, earlier work suggested that localization of aPKC occurs 'downstream' of Baz, as apical positioning of aPKC at gastrulation onset requires Baz function. The new data reveal that Rap1 and Cno are, in turn, 'upstream' of Baz, and thus, if things work in a strictly linear fashion, Rap1 and Cno should be 'upstream' of aPKC. However, in contrast to this simple view, this study found that precise positioning of Cno during cellularization requires aPKC - in its absence, Cno is not cleared from the apical region, and the apical-basal cables of Cno at tricellular junctions are not properly assembled. In a similar fashion, Baz, which in a linear model is 'downstream' of Cno, also regulates precise positioning of Cno during cellularization. aPKC and Baz also play important roles in Cno localization during the early polarity maintenance phase beginning at gastrulation onset. Together, these data suggest that initial positioning of proteins along the apical-basal axis involves a network of protein interactions, similar to that previously suggested to regulate polarity elaboration during the extended germband phase and beyond, as cells develop the full suite of epithelial junctions. It will now be important to define mechanisms by which aPKC and Baz act to precisely position Cno: two broad possibilities are that they act on Cno directly, or that they modulate the fine scale architecture of the actin cytoskeleton, with indirect effects on Cno. It will also be exciting to determine if other polarity determinants, like the basolateral proteins Discs Large, Scribble or Lgl, or the basolateral kinase Par1 also play roles in polarity establishment, as they do in polarity maintenance. Consistent with this possibility, recent work from the Harris lab suggests Par1 is important for the gastrulation onset rescue of Baz localization in embryos in which early cues are disrupted. Finally, it will be interesting to identify the cues that come into play at gastrulation onset, which partially restore apical Baz localization, as part of the increasingly complex network of partially redundant regulatory cues that give polarity its robustness (Choi, 2013).

Effects of Mutation or Deletion

The zonula adherens (ZA) belongs to a family of actin-associated cell junctions called adherens junctions. Antibodies specific to cellular junctions and nascent plasma membranes have been used to study the formation of the zonula adherens in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. ZA formation begins during cellularization; the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, genes have been identified that are required for the formation of the ZA and the generation of basolateral membrane polarity. Embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting by mid-gastrulation in the formation of a multilayered sheet of cells. Electron microscope analysis of mutant embryos reveals a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influences ZA formation, embryos with reduced maternal and zygotic supply of wild-type Armadillo protein were studied. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium (Müller, 1996).

The mutations baz and sdt belong to a group in which mutant embryos show severe abnormalities in the differentiation of the larval cuticles, including the genes crumbs (crb) and shotgun (shg). Although the similarity in the late phenotypes of these mutants shows that the respective genes are all required for the same process, i.e., epithelial differentiation, it is difficult to determine whether all these genes act in a common pathway. Nevertheless, the genes crb and sdt show an interesting genetic interaction. Using chromosomal duplications, it has been shown that the phenotype of crb (null) embryos can be rescued by an additional copy of sdt but not vice versa (Tepass, 1993). Based on these findings, a model has been proposed that positions sdt downstream of crb in a regulatory hierarchy (Tepass, 1993). This model is complicated by the fact that sdt regulates Crb protein distribution (Tepass, 1993). A more attractive model might be that sdt functions in a parallel pathway, and, in sufficient dosage, bypasses the requirement for crb (Muller, 1996).

It is equally complicated to arrange sdt and baz in a linear pathway, given that the double mutant of zygotic null alleles shows a stronger phenotype than the single mutants. The product of the baz gene is provided maternally and zygotically. Although maternal baz may rescue the hemizygous baz phenotype to a certain extent, it is difficult to explain the enhancement of the presumed null phenotype of sdt that occurs when baz is removed, if the two genes would function in a strict pathway. In summary, it is suggested that although baz, crb and sdt are important for the same process, it is most likely that they act in different, but related pathways (Muller, 1996)

Embryos that are mutant for bazooka frequently fail to coordinate the axis of cell polarity with that of the embryo. This is manifested as defective spindle orientation and mispositioning of the GMC daughter cell after division. Mislocalization of GMCs do not alter the pattern of neural lineage markers Even-skipped and Engrailed. Delocalization of neurons is only occasionally observed. The only conspicous patterning defect in the CNS for all the baz alleles analyzed is the failure to develop one of the longitudinal axon pathways, which are formed by multiple axons including the axons of the MP2 neurons. MP2s differ from most neuroblasts in that they divide only once. Embryos that are mutant for baz show less than the normal four MP2 descendents per segment, suggesting that MP2 neurons are more sensitive to the loss of baz than are other neurons. A more detailed analysis will give further insight into the function of baz in these particular neurons (Kuchinke, 1998).

Asymmetric localization is a prerequisite for inscuteable to function in coordinating and mediating asymmetric cell divisions in Drosophila. Partner of Inscuteable (Pins), a new component of asymmetric divisions, is required for Inscuteable to asymmetrically localize. In the absence of pins, Inscuteable becomes cytoplasmic and asymmetric divisions of neuroblasts and mitotic domain 9 cells show defects reminiscent of insc mutants. Pins colocalizes with Insc and interacts with the region of the Insc protein necessary and sufficient for directing its asymmetric localization. Analyses of pins function in neuroblasts reveal two distinct steps for Insc apical cortical localization: a pins-independent, bazooka-dependent initiation step during delamination (interphase) and a later maintenance step during which Baz, Pins, and Insc localization are interdependent (Yu, 2000).

In the absence of baz function, Insc does not localize apically even in delaminating NBs and is cytoplasmic later in the cell cycle. Not surprisingly, in embryos lacking both maternal and zygotic baz, Pins distribution in mitotic NBs is mostly cortical, similar to its distribution in insc mutant NBs. Interestingly, Baz localization to the apical cortex of NBs is itself affected by pins and insc loss of function. In Pins- NBs, the apical cortical Baz crescents normally present in WT mitotic NBs cannot be detected from metaphase onward. However, occasional weak crescents can be found in mutant interphase/prophase NBs and these are always localized to the apical cortex. The Baz distribution in insc mutant NBs is similar to that seen in Pins- embryos. These observations suggest that the maintenance and/or stability of apical Baz in NBs requires both insc and pins. Taken together these results indicate that the initial localization of Insc (e.g., to the apical stalk) requires baz but not pins; however, the maintenance of apical Baz/Pins/Insc later in the cell cycle (e.g., at metaphase) are mutually dependent, requiring all three components (Yu, 2000).

Par-3/Baz, Par-6, and aPKC are evolutionarily conserved regulators of cell polarity, and overexpression experiments implicate them as axon determinants in vertebrate hippocampal neurons. Their mutant and overexpression phenotypes were examined in Drosophila melanogaster. Mutants neurons have normal axon and dendrite morphology and remodel axons correctly in metamorphosis, and overexpression does not affect axon or dendrite specification. Baz/Par-6/aPKC are therefore not essential for axon specification in Drosophila (Rolls, 2004).

Therefore, Drosophila Baz, Par-6 and aPKC are not required for axon specification in vivo, and their overexpression has no effect on axon specification or outgrowth. In contrast, overexpression of Par-3 or Par-6 in cultured mammalian hippocampal neurons results in multiple axon-like processes, leading to the hypothesis that these proteins are axon determinants. How can these apparently paradoxical results be reconciled? One possibility is that vertebrate neurons require Par-complex proteins for axon specification, whereas Drosophila neurons do not. If this is the case, it would be interesting to learn how different molecular pathways in mammals and flies generate the same functional subcellular domain (the axon). Another possibility is that neither fly nor vertebrate neurons use Par proteins to specify axon identity in vivo; cultured hippocampal neurons are separated from normal external polarity cues and may use a different mechanism for axon specification. Polarity cues from surrounding cells may also inhibit neurons in vivo from changing polarity in response to extra Par-3 or Par-6, explaining the different effects of overexpressing these proteins in Drosophila and in hippocampal neurons. A third possibility is that the overexpression experiments, where proteins are present at higher-than-normal levels, do not reflect the in vivo functions of the proteins. Loss-of-function and overexpression experiments that examine vertebrate neurons in vivo or in slice preparations will be crucial for fully understanding the role of Par complex proteins in vertebrate axon specification (Rolls, 2004).

Adherens junctions inhibit asymmetric division in the Drosophila epithelium

The asymmetry of neuroblast cell divisions might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. By disrupting adherens junctions the symmetric epithelial division of epidermal cells can be changed into asymmetric division. The adenomatous polyposis coli (APC) tumor suppressor protein is recruited to adherens junctions, and both APC and microtubule-associated Partner of numb (Pon) and Miranda (Lu, 2001 and references therein).

The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?

To characterize epithelial division by monitoring it in live embryos, transgenic embryos expressing Pon and tau proteins fused with green fluorescent protein (GFP) were used. During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains oriented along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).

Double-stranded (ds) CRB RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% (n = 200) of crb(RNAi) embryos, the organization of the ectodermal epithelium is disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos reveals that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).

After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicates that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).

Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. The effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra show coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).

In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. The function of Baz in epithelial division was examined. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orients in random directions. After cytokinesis, two equally sized daughter cells are produced and Pon-GFP is equally distributed between them (Lu, 2001).

Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. In crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos appeared similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).

To investigate the molecular mechanism underlying the planar positioning of spindles by the adherens junction, the function of proteins associated with the adherens junction was examined. A ubiquitously expressed, epithelial-cell-enriched APC (E-APC) is localized to the adherens junction, and, in shotgun and crb mutants, this adherens junction localization of E-APC is disrupted. The human APC protein interacts with a microtubule-associated EB1 protein, and the yeast homolog of EB1 (Bim1), together with the cortical marker Kar9, has been implicated in a search-and-capture mechanism of spindle positioning. Therefore, the function of E-APC in epithelial cell division was tested (Lu, 2001).

In about 60% of E-APC(RNAi) embryos, the positioning of Pon-GFP crescent and orientation of mitotic spindle became tightly coupled during epithelial division. At cytokinesis, epithelial cells divided asymmetrically to produce two unequally sized daughter cells, and Pon-GFP was always segregated to the smaller daughter cell. The asymmetric segregation of Pon-GFP and the ability to undergo unequal cytokinesis all depend on Baz, because in baz and E-APC double RNAi embryos, Pon-GFP is equally segregated to two similarly sized daughter cells. Therefore, in the absence of E-APC, epithelial cells divide asymmetrically in a Baz-dependent fashion. This suggests that adherens-junction-associated E-APC promotes spindle positioning along the planar axis and prevents the coupling of spindle positioning with asymmetric basal protein localization (Lu, 2001).

To test whether E-APC functions with EB1 to orient the mitotic spindle, RNAi was performed on a closely related fly homolog of EB1 (dEB1). In dEB1(RNAi) embryos, the epithelial divisions are also asymmetric, producing two unequally sized daughter cells, with Pon-GFP segregated to the smaller cell. The penetrance of dEB1(RNAi) phenotype (20%) is lower than that of E-APC(RNAi). Since there is strong maternal contribution of dEB1, the low penetrance might be due to a perdurance of maternal dEB1 protein. Alternatively, it might be due to functional compensation by two other distantly related EB1 homologs in the fly genome. It has been noted that E-APC lacks the carboxy-terminal domain, which is required for interaction with EB1, and no direct interaction between E-APC and EB1 could be detected in in vitro binding assays. It therefore remains to be determined whether the two are functionally linked together in vivo through some cofactor(s), or whether E-APC functions mainly to maintain adherens junction integrity and EB1 interacts with other unidentified molecules to orient spindles (Lu, 2001).

These results indicate that two sets of polarity cues exist for spindle positioning in epithelial cells: a planar polarity cue mediated by the adherens junction and an apical-basal polarity cue regulated by Baz. The division pattern of wild-type epithelial cells suggests that the planar polarity cue is normally dominant over the apical-basal polarity cue. Epithelial cells within the procephalic neurogenic region (PNR) that express endogenous Insc or epithelial cells outside of the PNR that express ectopic Insc are known to orient their mitotic spindle along the apical-basal axis during division. This suggests that the dominance of planar polarity over apical-basal polarity can be overcome by the expression of Insc. The normal appearance of the adherens junction in epithelial cells in the PNR, together with the observation that these cells divide along the planar axis and maintain their normal monolayer organization in an insc mutant, suggests that Insc functions by strengthening the apical-basal polarity instead of weakening the planar polarity through changing the behavior of the adherens junction (Lu, 2001).

When neuroblasts delaminate from the epithelium layer, they undergo morphological changes from columnar to round shape, lose their contacts with the surrounding cells and thus the adherens junction structures. This situation may be reminiscent of epithelial cells in adherens-junction mutants in which the planar polarity cue is lost. In both cases, the Baz-mediated polarity pathway takes over. That one polarity cue can dominate over another cue in orienting axis division may have its precedent in other organisms. Budding yeast can divide in either an axial or a bipolar pattern. Mutations in genes such as AXL1, BUD3, BUD4 and BUD10/AXL2 result in loss of polarity cue for axial bud formation and the cells divide in a bipolar fashion. This suggests that axial and bipolar cues coexist and that the axial cue is normally dominant over the bipolar cue. During mammalian cortical neurogenesis, neural progenitors switch from early symmetric divisions to later asymmetric divisions. It will be interesting to determine whether similar mechanisms and molecules are used to control this division symmetry switch in mammals. These results on E-APC highlight the importance of tumor suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).

Par-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila

Drosophila neuroblasts arise from polarized epithelial cells. Par-6 localization was followed during neuroblast delamination and through neuroblast cell division. Par-6 is localized in an apical stalk that extends into the epithelium during neuroblast delamination, and in an apical cortical crescent in delaminated interphase and metaphase neuroblasts. During telophase, the crescent becomes wider and weaker, indicating that the protein becomes delocalized and finally disappears. This subcellular localization is reminiscent of Bazooka and, indeed, double staining for Par-6 and Bazooka shows colocalization of the two proteins in epithelial cells and neuroblasts. Thus, Par-6 and Bazooka colocalize at the apical cell cortex of epithelial cells and neuroblasts. In neuroblasts, colocalization of Par-6 and Inscuteable is also observed. Par-6 has also been shown to physically associate with Bazooka in vitro (Petronczki, 2001).

Par-6 colocalizes with Bazooka in epithelial cells and neuroblasts. Whether there is a functional connection between the two proteins was tested by analysing Par-6 localization in RNA interference (RNAi) mutants of bazooka (bazookaRNAi) in which both maternal and zygotic bazooka function are disrupted. Whereas Par-6 is apically localized in epithelial cells and forms an apical cortical crescent in 93% of metaphase neuroblasts in control-injected embryos, Par-6 was homogeneously distributed in the cytoplasm of bazookaRNAi mutant embryos. In these embryos, 100% of metaphase neuroblasts that had lost Bazooka protein showed cytoplasmic localization of DmPAR-6. Thus, both apical and cortical localization of Par-6 require bazooka. Whether mislocalization of Bazooka can redirect Par-6 localization was tested by overexpressing Bazooka in Drosophila embryos using the UAS-GAL4 system. Overexpression of bazooka perturbs epithelial polarity and results in accumulation of Bazooka protein at ectopic sites of the cell cortex. Co-staining of Bazooka overexpressing embryos for Bazooka and Par-6 has revealed that the two proteins colocalize at these ectopic positions, indicating that Bazooka is not only required but also sufficient for localization of Par-6 (Petronczki, 2001).

The function of Bazooka in neuroblasts, at least in part, is to localize Inscuteable to the apical cortex. Bazooka is strictly required for Inscuteable localization, but Inscuteable is dispensable for Bazooka localization even though Bazooka crescents become weaker in Inscuteable mutants. Therefore Par-6 localization was analyzed in inscuteable mutants. Whereas 88% of metaphase control neuroblasts showed a strong apical crescent, normal localization of Par-6 was only observed in 14% (n = 42) of inscuteableP72 mutant neuroblasts. In 52% of these neuroblasts, Par-6 was localized into an apical crescent that was weaker and extended further to the lateral cortex than in control embryos and in 33% of the metaphase neuroblasts, Par-6 was not asymmetrically localized. Thus, although Bazooka is strictly required for Par-6 localization, absence of Inscuteable only causes a partially penetrant defect in Par-6 localization (Petronczki, 2001).

Therefore Drosophila Par-6 has an important function in both maintaining apical-basal polarity of epithelial cells and directing asymmetric cell division of neuroblasts in Drosophila. Physical interaction, colocalization and functional similarity of Par-6 with Bazooka, the Drosophila PAR-3 homolog, all indicate that these two proteins may cooperate closely in these functions. In neuroblasts Inscuteable may be a functional part of this complex and is recruited into this complex through direct interaction with Bazooka (Petronczki, 2001 and references therein).

Bazooka is required for localization of determinants and controlling proliferation in the sensory organ precursor cell lineage in Drosophila.

Asymmetric divisions with two different division orientations follow different polarity cues for the asymmetric segregation of determinants in the sensory organ precursor (SOP) lineage. The first asymmetric division depends on frizzled function and has the mitotic spindle of the pI cell in the epithelium oriented along the anterior-posterior axis, giving rise to pIIa and pIIb, which divide in different orientations. Only the pIIb division resembles neuroblast division in daughter-size asymmetry, spindle orientation along the apical-basal axis, basal Numb localization, and requirement for inscuteable function. Because the PDZ domain protein Bazooka is required for spindle orientation and basal localization of Numb in neuroblasts, it was of interest to enquire whether Bazooka plays a similar role in the pIIb in the SOP lineage. Surprisingly, in pI and all subsequent divisions, Bazooka controls asymmetric localization of the Numb-anchoring protein Partner of numb, but not spindle orientation. Bazooka also regulates cell proliferation in the SOP lineage; loss of bazooka function results in supernumerary cell divisions and apoptotic cell death (Roegiers, 2001).

For Bazooka to be involved in every asymmetric division of the adult SOP lineage one might expect Bazooka to be expressed in every precursor cell. Indeed, Bazooka is asymmetrically localized in every dividing cell of the SOP lineage. Starting with a strong accumulation at the apical surface of interphase pI cells, specifically at junctions with neighboring epithelial cells, Bazooka becomes enriched at the posterior cortex during mitosis and shows no overlap with the anterior Numb crescent at metaphase. By early anaphase, Bazooka forms a smooth posterior crescent. At anaphase B, Bazooka is localized to the posterior cortex, although a significant amount remains anterior to the cleavage furrow. The pI division is followed by division of the pIIb cell, which exhibits an apical-posterior crescent of Bazooka at mitosis. Subsequently, in the mitotic pIIa, Bazooka accumulates in the cell cortex and a strong patch of Bazooka is detected at the anterior cortex, a region that coincides with the position of the anterior-most centrosome of the mitotic spindle. Finally, in the mitotic pIIIb cell, Bazooka forms an apical-posterior crescent similar to the one observed in the pIIb cell. In the pI, pIIb, and pIIIb divisions, Bazooka is localized opposite the Numb crescent in mitosis; whereas in the mitotic pIIa cell, the anterior accumulation of Bazooka may colocalize with Numb. Following completion of these asymmetric divisions, Bazooka expression is enriched at the apical borders in cells of the developing es organ, specifically the hair and socket cells. Based on these observations, it is concluded that Bazooka is expressed in all precursor cells within the SOP lineage; it is asymmetrically enriched during each cell division of the SOP lineage, and its expression is maintained in the postmitotic cells that will give rise to the external structures of the es organ, the hair and socket cells (Roegiers, 2001).

During embryonic neuroblast divisions, Bazooka is required not only to localize Inscuteable to the apical cortex and Numb, Miranda, Prospero, and Pon to the basal cortex, but also to orient the mitotic spindle along the apical-basal axis. To determine the requirement of bazooka in the asymmetric divisions of the adult SOP lineage, the MARCM system was used to generate baz mutant clones expressing both Pon-GFP (as a reporter for Numb localization) and Tau-GFP (as a reporter for spindle orientation) under the control of scabrous-GAL4, which is strongly expressed in the SOP cell and in the SOP lineage. The movements of Pon-GFP and Tau-GFP were monitored in live tissue throughout all asymmetric divisions of the SOP lineage. In bazxi106 or bazEH171 null mutant clones, pI cells underwent mitosis at ~15 h APF as in wild type. However, in all mutant pI cells observed, Pon-GFP remained uniformly distributed and never formed an anterior crescent as seen in dividing wild-type pI cells. Nor did Pon-GFP crescents form in the subsequent divisions in the lineage. Thus, although only the pIIb resembles the embryonic neuroblast in its orientation of division and requirement for Inscuteable, Bazooka is required for the asymmetric Pon/Numb localization in the pI division, as well as all subsequent divisions (Roegiers, 2001).

Because Numb functions as an asymmetrically localized cell-fate determinant in the SOP lineage, the absence of Numb crescents in baz mutant clones could lead to cell-fate transformations in the daughters of the pI cell. Thus the anterior daughter cell of the pI in bazooka mutant clones (the pIIb cell in the wild type) is referred to as pIIbb, and the posterior daughter cell as pIIab. It is worth noting, however, that either loss-of-function or misexpression of numb causes cell-fate transformation only in a subset of sensory organs, presumably because the Notch-mediated mutual inhibition may still allow the two daughter cells to adopt different cell fates, albeit without a bias set by the Numb crescent. Transformation of pIIa to pIIb cell fate is known to alter the timing of mitosis of the transformed pIIa cell. Timing of the pIIbb, pIIab, and pIIIbb divisions is indistinguishable from wild-type pIIb, pIIa, and pIIIb cells. In addition, the pI and pIIab spindles align along the A-P axis in all mutant clones. And in eight of the nine clones examined the pIIbb spindles were oriented along the apical-basal axis as in wild type (the remaining pIIbb cell divided before the pIIab, but had its spindle oriented along the anterior-posterior axis). Because an apically localized Inscuteable is required for mitotic spindle positioning in the pIIb cell, Inscuteable localization was also examined in the pIIbb cell in bazooka mutant clones. Inscuteable is localized to an apical stalk in pIIbb, similar to the wild-type pIIb (n = 12). Thus the great majority of pIIbb and pIIab cells resemble wild-type pIIb and pIIa cells in their timing and orientation of division, as well as the expression of Inscuteable in the pIIbb. It therefore appears that these bazooka mutations do not cause detectable cell-fate transformation in most of the pIIbb and pIIab cells, although it remains possible that there are partial transformations and cell-fate changes in a subset of these cells. In light of these observations, the complete loss of Pon-GFP crescents in every mitotic pIIbb and pIIab cell examined strongly supports a model wherein Bazooka controls Pon/Numb asymmetric localization in not only pI but also pIIb and pIIa cells (Roegiers, 2001).

Some cell-fate changes apparently take place in the progeny of pIIa and pIIb. On adult nota, mutant clones contain patches of bald cuticle and regions with small bumps that may represent lost ES organs, and sockets without hairs. Ectopic Prospero-expressing cells were found in baz mutant clones, indicating that additional sheath and/or glial cells are present because of cell-fate transformations in the pIIbb lineage. Interestingly, in six of nine mutant SOP lineages examined, ectopic divisions in the pIIab cell lineage were found. Whereas the wild-type pIIa cell divides only once to give rise to two external cells of the ES organ (the hair and socket cells), in baz mutant clones each daughter of the pIIab cell undergoes another round of division, causing the SOP lineage to produce a cluster of seven cells, as opposed to the normal five cells (Roegiers, 2001).

In the wild-type SOP lineage, shortly after the last division of pIIIb (~24 h after pupa formation) one of the pIIIb daughters forms a neuron and extends an axon. Within an hour after completion of the pIIIb mitosis, the small glial cell migrates away from the cluster along the axon. By following the development of the ES organs in vivo in baz mutant clones to observe their morphogenesis, no axon extension or glial cell migration was found in 16 of 20 ES organs examined. Moreover, within 3-6 h after the last mitosis, clusters of cells underwent apoptosis in ten of twenty clones examined. Apoptotic bodies formed and dispersed rapidly. Cell death of ES organ cells is specific to the bazooka mutants, because apoptosis was never observed in wild-type ES cells (Roegiers, 2001).

Bazooka and its homolog Par-3 in C. elegans are known to be required for asymmetric divisions, in embryonic neuroblasts in Drosophila and in the zygote and early blastomeres of the worm embryo, respectively. In Drosophila neuroblasts, Bazooka forms a complex with Pins and localizes Inscuteable, which coordinates the asymmetric localization of Numb and spindle orientation. In the mitotic pI cell, an anterior Pins/Dlg complex has been shown to be required for Bazooka localization, and Bazooka is required for Numb localization. This study of the adult SOP lineage reveals several functions for Bazooka. (1) Bazooka is the first molecule to be required for the asymmetric localization of Pon, the adapter protein for Numb, in every division of the SOP lineage, even though only the pIIb division resembles embryonic neuroblast division both in its orientation along the apical-basal axis and its dependence on Inscuteable. It thus appears that Bazooka may localize Pon and Numb in a pathway independent of Inscuteable. (2) Bazooka is not required for proper spindle orientation in the asymmetric divisions of the SOP lineage. The function of Bazooka in the SOP lineage, therefore, concerns only determinant localization but not spindle orientation. Unlike the neuroblast, in the pIIb cell Inscuteable orients spindles along the apical-basal axis in the absence of Bazooka, indicating that pIIb cells may have a Bazooka-independent mechanism for Inscuteable localization. (3) Although bazooka mutations did not cause detectable cell-fate transformation in most pIIb and pIIa cells, there is apparent cell-fate transformation occurring in the pIIbb lineage, and possibly partial cell-fate transformations of the pIIab lineage leading to formation of cells of indeterminate cell fates, such as sockets without hairs or the bumps in bazooka mutant clones. (4) Loss of Bazooka function leads to apoptosis. The possibility that inadequate cell-fate specification results in apoptosis cannot be ruled out. However, the cell-fate transformations in the SOP lineage in various mutants reported thus far have not been associated with apoptosis, indicating that cell-fate transformation per se does not necessarily lead to apoptosis. The terminal fates of mutant es cell clusters are difficult to determine with certainty, because most undergo apoptosis rather than differentiation. (5) Bazooka appears to limit the number of cell cycles of the pIIa to one; the pIIab daughters in bazooka mutant clones often proceed with mitosis instead of differentiating into hair and socket cells. Similarly, antiproliferative activity has been found in follicle cells of the ovary. (6) Finally, loss of Bazooka function leads to failure of ES neuron axonal outgrowth and glial cell migration. These defects could reflect cell-fate changes in the pIIbb lineage or a requirement for bazooka in differentiation of ES organ cells (Roegiers, 2001).

In summary, Bazooka has a much broader spectrum of function in the SOP lineage than previously suspected. In bazooka mutant clones the Numb-anchoring protein Pon fails to form a crescent in every division of the SOP lineage, regardless of the requirement for Inscuteable. The function of Bazooka in the SOP lineage also differs from that in embryonic neuroblasts because Bazooka controls spindle orientation in neuroblasts but not in the SOP lineage. The pI, pIIb, and pIIa cells show little evidence of cell-fate transformation in bazooka mutant clones, and yet exhibit a total loss of Pon-GFP crescent formation. It thus appears that Bazooka controls Pon/Numb crescent formation in these precursors with different planes of division. Although in bazooka mutant clones there appears to be partial cell-fate transformation, in later divisions in the lineage it is striking that asymmetric localization of determinants is abolished in all divisions. The loss of Pon-GFP crescent is fully penetrant, in contrast to the variable and partially penetrant cell-fate transformation phenotype. These observations suggest that Bazooka is the general link between polarity cue and the localization of cell-fate determinants in all asymmetric cell divisions. Other previously uncharacterized functions uncovered in this study include the ability of Bazooka to restrict the number of divisions in the SOP lineage and to promote differentiation instead of apoptosis. It is worth noting that the function of Bazooka in the central nervous system has been previously studied only for the neuroblast division. Based on these findings in the SOP lineage, it will be interesting to learn whether in the CNS Bazooka also has an Inscuteable-independent role in controlling asymmetry of subsequent divisions, as well as in regulating proliferation and apoptosis. Given that Bazooka/Par-3 is part of an evolutionarily conserved gene cassette, these findings of a myriad of previously uncharacterized functions of Bazooka in the sensory organ lineage raise the possibility that Bazooka/Par-3 may have a similarly wide range of functions in vertebrates (Roegiers, 2001).

Functions of Bazooka in Inscuteable-independent apicobasally oriented asymmetric divisions in the Drosophila embryonic CNS

Inscuteable is the founding member of a protein complex localized to the apical cortex of Drosophila neural progenitors that controls progenitor asymmetric division. Aspects of asymmetric divisions of all identified apicobasally oriented neural progenitors characterized to date, in both the central and peripheral nervous systems, require inscuteable. The generality of this requirement has been examined. Many identified neuroblast lineages, in fact, do not require inscuteable for normal morphological development. To elucidate the requirements for apicobasal asymmetric divisions in a context where inscuteable is not essential, focus was placed on the MP2 ---> dMP2 + vMP2 division. For MP2 divisions, asymmetric localization and segregation of Numb and the specification of distinct dMP2 and vMP2 identities require bazooka but not inscuteable. It is concluded that inscuteable is not required for all apicobasally oriented asymmetric divisions and that, in some cellular contexts, bazooka can mediate apicobasal asymmetric divisions without inscuteable (Rath, 2002).

Two obvious candidate molecules that might be responsible for mediating the MP2 asymmetric division are Baz and Partner of Inscuteable (Pins). pins appears not to be a major player since, in embryos lacking both maternal and zygotic pins, only 5.7% of the hemisegments show dMP2 duplication, as demonstrated by three odd-positive cells. Assessing the role of baz is problematic since loss of zygotic function has no effect, and removing both the maternal and zygotic baz results in embryos with severe morphological defects that prevent scoring of dMP2 and vMP2 fates in older embryos. These problems were circumvented by performing RNAi with baz double-stranded RNA on AJ96 embryos, which yielded ~25% embryos with reduced Baz protein but without the severe morphological defects that prevented scoring of vMP2 and dMP2 identities. In such embryos, vMP2>dMP2 transformations are observed in the great majority of hemisegments (57/60), as demonstrated by the presence of two cells double positive for ß-Gal and Odd. Moreover, localization of Pon and Numb becomes cortical in dividing MP2 (38/40). However, there does not appear to be a dramatic defect on the orientation of the cell division, with almost all of the MP2 divisions oriented within 45° of the A/B axis (32/34). These defects associated with baz RNAi cannot merely be due to secondary effects associated with a disruption of the epithelium since, in crumbs loss of function, Baz (12/12) and Pon (10/10) remain correctly localized to the MP2 apical and basal cortex, respectively. These results indicate that Baz is required for the asymmetric localization of Pon and Numb in the MP2 asymmetric division (Rath, 2002).

Although apical complex members, like Baz, Insc and Pins, are expressed and apically localized in both MP2 and NBs, their behavior appears to differ somewhat in the two cellular contexts. For example, when baz function is attenuated, Insc is in the cytosol of NBs while Insc remains localized as an apical crescent in most dividing MP2 cells; similar results are seen in dividing MP2s of embryos derived from bazXi106 germline clones, which lack both maternal and zygotic function. Moreover, it is interesting to note that in the absence of insc function, Baz and Pins can be localized to the apical cortex of metaphase MP2s, although the intensity of staining is always reduced compared with WT MP2s, and in some cases the weak apical crescent of Baz can be difficult to detect. These observations suggest that even a small amount of apically localized Baz is sufficient to mediate basal localization of Pon and direct the MP2 asymmetric division. Strikingly, Baz, but not Insc and Pins, seems to play a dominant role in mediating Pon/Numb basal localization in MP2. When baz function is attenuated, Pon/Numb become cortically localized even though Insc and Pins can remain apically localized. These observations indicate that the precise requirements for asymmetric protein localization differ between MP2s and NBs (Rath, 2002).

MP2 appears to be the only known A/B-oriented asymmetric division that does not require insc. Although MP2 delaminates from the neuroectoderm and divides in an apico-basal fashion like NBs, there are unique features that set MP2 apart from other neuroblasts. Unlike NBs that divide in a stem-cell-like mode, MP2 undergoes one differentiative division, making it more like a GMC or a pIIb division. Insc is present in both GMC/SOP lineages. A/B-oriented asymmetric GMC divisons, like those of GMC4-2a, require insc. While the first SOP division (the anterior-posterior pI > pIIa + pIIb) does not require insc, recent work has shown that the spindle orientation of the strikingly GMC-like A/B division of the pIIb cell is dependent on Insc. Finally, unlike NBs, Pros shows nuclear localization in MP2. There is evidence supporting the view that Pros acts to terminate cell proliferation during Drosophila neurogenesis. It is plausible that both GMCs and the MP2 precursor use nuclear Pros as a cue to reduce their mitotic potential and undergo a single differentiative division. Two recent reports have shown that planar asymmetric divisions undertaken by pI in the peripheral nervous system and epithelial cells with disrupted adherens junctions both require baz and not insc. It has been demonstrated in this study that insc is not required for all A/B-oriented asymmetric divisions. These findings further support the view that baz is a more general mediator of asymmetric divisions than insc, and can act to promote both A/B and planar asymmetric divisions in the absence of insc (Rath, 2002).

Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions - Effect of bazooka mutation

Cell division often generates unequally sized daughter cells by off-center cleavages, which are due to either displacement of mitotic spindles or their asymmetry. Drosophila neuroblasts predominantly use the latter mechanism to divide into a large apical neuroblast and a small basal ganglion mother cell (GMC), where the neural fate determinants segregate. Apically localized components regulate both the spindle asymmetry and the localization of the determinants. Asymmetric spindle formation depends on signaling mediated by the Gβ subunit of heterotrimeric G proteins. Gβ13F distributes throughout the neuroblast cortex. Its lack induces a large symmetric spindle and causes division into nearly equal-sized cells with normal segregation of the determinants. In contrast, elevated Gβ13F activity generates a small spindle, suggesting that this factor suppresses spindle development. Depletion of the apical components also results in the formation of a small symmetric spindle at metaphase. Therefore, the apical components and Gβ13F affect the mitotic spindle shape oppositely. It is proposed that differential activation of Gβ signaling biases spindle development within neuroblasts and thereby causes asymmetric spindles. Furthermore, the multiple equal cleavages of Gβ mutant neuroblasts accompany neural defects: this finding suggests indispensable roles of eccentric division in assuring the stem cell properties of neuroblasts (Fuse, 2003).

During mitosis, neuroblasts localize the cell fate determinants Prospero and Numb to the basal cortex and orient the mitotic spindle along the apical-basal axis to segregate the determinants into GMCs. These processes are regulated by the apical protein complex that includes Inscuteable, Bazooka, atypical protein kinase C (DaPKC), the G protein subunit Gαi, and Partner of Inscuteable (Pins). The depletion of any single apical component does not severely affect the cell size difference between the neuroblast daughters. However, a recent study shows that the two signaling pathways, Bazooka/DaPKC and Pins/Gαi, within the apical complex control in parallel the production of unequal-sized daughters (Fuse, 2003).

During a mutational screen with Miranda, the adaptor protein of Prospero, the f261 mutant, which is defective in unequal-sized neuroblast divisions, was obtained. In germline clone embryos that are both maternally and zygotically mutant for f261 (f261 mutant), the neuroblasts produce nearly equal-sized daughters, although the GMC is still slightly smaller than the sibling neuroblast after the initial divisions. Nevertheless, after a slight delay in crescent formation, Miranda localizes normally in f261 neuroblasts and segregates to the GMC. Consequently, Prospero is inherited by the GMCs. The abnormal division in f261 causes neuroblasts to be smaller and smaller after each succeeding division. The f261 mutant turned out to be a protein null mutant of the Gβ13F gene that encodes a β subunit of heterotrimeric G proteins. In wild-type neuroblasts, this protein distributes uniformly at the cell cortex. A deletion mutant of Gβ13F has been reported (Schaefer, 2001) to show delayed localization of Miranda and randomized orientation of neuroblast division, as well as gastrulation defects, all of which occur in f261 embryos, but cell size defects have not been described. Deletion mutants lacking the entire Gβ13F coding sequence have been created. Such a mutant, Gβ13FΔ15, as well as the deletion mutant reported previously (Schaefer, 2001) indeed show the same neuroblast phenotypes as those of f261 embryos. Therefore, the loss of Gβ13F activity affects cell size asymmetry but essentially does not affect the segregation of the cell fate determinants. The neuroblast phenotypes observed in the Gβ13F mutants are not consequences of morphological defects before neuroblast formations because the neuroblast-specific expression of Gβ13F rescues the phenotype of cell size asymmetry (Fuse, 2003).

In canonical heterotrimeric G protein signaling, the Gβ and Gγ complex (Gβγ) associates with the GDP form of Gα, but the conversion of GDP to GTP releases Gβγ from Gα; both Gβγ and Gα can then signal downstream. In Drosophila neuroblasts, it is unlikely that GTP-Gαi acts as a signal. Instead, it has been suggested that the GDP form of Gαi binds to Pins to release the Gβγ subunit (Schaefer, 2001). According to this model, Pins-dependent activation of Gβ signaling occurs at the apical cortex, where Pins and Gαi are colocalized. Unlike the Gβ13F mutants, defects in unequal-sized divisions are observed only in a small fraction of pins mutant neuroblasts, probably because of the bazooka/DaPKC activity that functions in parallel to form asymmetric spindles. Therefore the effects of pins and Gβ13F on microtubule development were compared under conditions in which bazooka activity is simultaneously depleted. In the absence of both and bazooka, metaphase neuroblasts form a large symmetric spindle resembling that seen in f261. In contrast, the simultaneous loss of pins and bazooka activities results in the formation of a small symmetric spindle at metaphase, which is rather similar to the basal half of the wild-type spindle. Therefore, Gβ and Pins exert opposite effects on spindle formation during metaphase in the absence of bazooka. This reciprocal effect of Pins and Gβ on spindle development is not straightforwardly deduced from the model that shows that Pins induces the free and active Gβγ. These states of the mitotic spindle in the double mutants appear to persist throughout mitosis because the midbody, the bundled central spindle at telophase, is notably narrower in the pins-bazooka double mutant than in the Gβ-bazooka mutant. In comparison, astral microtubules develop to a similar extent from anaphase onward under those two mutant conditions. The asters in these double mutants develop more than the basal half of wild-type but less than that seen in f261 and appear at an intermediate level. The differential influence of the mutations on the mitotic spindle (or central spindle) and asters may originate from different mechanisms that regulate these microtubule structures. This possibility has been suggested by the existence of asterless mutants, in which asters are apparently absent, whereas the mitotic spindle appears to develop normally. The role of astral microtubules in cell size asymmetry is controversial because asterless mutant neuroblasts still bud off small GMCs by forming an asymmetric central spindle (Fuse, 2003).

Effects of mutation: Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions

Drosophila neuroblast asymmetric divisions generate two daughters of unequal size and fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apicobasal orientation of the mitotic spindle, but how daughter cell size is controlled has remained unclear. Mitotic spindle geometry and unequal daughter cell size were shown to be controlled by two parallel pathways (Bazooka/DaPKC and Pins/Galphai) within the apical complex. While the localized activity of either pathway alone is sufficient to mediate the generation of an asymmetric mitotic spindle and unequally sized neuroblast daughters, loss of both pathways results in symmetric divisions. In sensory organ precursors, Bazooka/DaPKC and Pins/Galphai localize to opposite sides of the cortex and function in opposition to generate a symmetric spindle (Cai, 2003).

Thus members of the NB apical protein complex control the generation of daughter cells of unequal size. There are two redundant pathways: (1) Baz/DaPKC/ (and presumably DmPar6) as well as (2) Pins/Gαi, either of which, when asymmetrically localized to the NB cortex, can lead to the formation of an asymmetric mitotic spindle through the preferential elongation of the proximal spindle arm and the displacement of the spindle toward the distal cell cortex, resulting in the production of unequal-sized daughter cells. In addition, in NBs, Insc is required for the function of the Baz/DaPKC/(DmPar6) pathway. When both pathways are inactivated/attenuated, spindle asymmetry and displacement fail to occur and equal-sized daughter cells are produced at high frequency. In the PNS progenitor, pI, where Baz/DaPKC are localized to the posterior cortex and Pins/Gαi are localized to the anterior cortex, the mitotic spindle is symmetric. Consistent with this hypothesis that both pathways can act to cause the preferential elongation of the proximal spindle arm relative to the distal spindle arm, removing posterior baz function without abolishing the localization and function of the anterior components results in the production of an asymmetric spindle with an anterior bias; removing anterior pins function without affecting the function of the posterior components results in a posteriorly biased asymmetric spindle; if components of both pathways are localized to the anterior cortex through the ectopic expression of Insc, an anteriorly biased asymmetric spindle results. These findings suggest that DaPKC and hetrotrimeric G protein signaling work in conjunction in the NB to produce an asymmetric spindle and in opposition in pI to produce a symmetric spindle (Cai, 2003).

Several lines of evidence suggest that localized signaling is essential to generate an asymmetric spindle and daughter cells of unequal size. (1) When both signaling pathways are abolished/attenuated (e.g., in insc/pins double mutant) or when signaling is uniform, which is assumed to be the case when Baz/DaPKC/Pins/Gαi are all uniformly localized throughout the cell cortex (e.g., in the case of Gαi overexpression in wt NBs), equal-sized daughters are generated. (2) When pins function is removed and DaPKC/Baz is asymmetrically localized (e.g., in pins mutant NB) or when Pins/Gαi are uniformly cortical but DaPKC/Baz are asymmetrically localized (e.g., in 69% [n = 51] of wt NBs overexpressing C-Pins), the site of the DaPKC/Baz localization coincides with the position where the larger daughter forms. (3) When Pins/Gαi is asymmetrically localized but baz/DaPKC function has been compromised (e.g., in insc mutant) or when Pins/Gαi is asymmetrically localized but Baz/DaPKC is uniformly cortical (in the case of NBs with basal Pins-C-Pon crescents), the site of localization coincides with the larger daughter and the extended spindle arm. These observations indicate that just one localized signal source, mediated presumably by either heterotrimeric G protein or DaPKC, is sufficient to cause proximal spindle arm elongation and the generation of unequal-sized daughters (Cai, 2003).

The situation is different in pI where Baz/DaPKC/(DmPar6) and Pins/Gαi act in opposition and where Insc is not required for the function of the Baz/DaPKC/(DmPar6) with respect to spindle elongation. Here, a distinction can be made between two possible models for explaining how spindle asymmetry/geometry is mediated. The first model is that the presence of either asymmetrically localized Baz/DaPKC/(DmPar6) or Pins/Gαi on one side of the cell is sufficient to cause elongation of the proximal spindle arm, regardless of what occurs on the other side of the cell. A second model would be that the signals from the opposite sides of the cortex are integrated and the bias in the spindle geometry depends on the relative magnitude of the two signals. The simplest prediction of the first model would be that the distance from the cleavage furrow to the spindle pole of wt telophase pI should be equivalent to the longer of the two spindle arms in telophase pI mutant for either baz or pins. This appears not to be the case. The average length of the longer spindle arm in telophase pI mutant for pins or baz is greater than that of a wt spindle arm and the length of the shorter of the spindle arms in mutant pI is less than that of a wt spindle arm . An equivalent analysis is difficult to do with NBs, since the size of the 30 or so NBs found in each hemisegment is more variable. Nevertheless, based on these observations the second type of model is favored (Cai, 2003).

Previous work has shown that Pins binds to the GDP bound form of Gαi and can cause Gαi to dissociate from Gβ13F; moreover, some phenotypes seen when Gαi is overexpressed in wt NBs (e.g., equal size divisions) are not seen when GαiQ205L, an activated form of Gαi lacking GTPase activity that should be in the GTP bound form, is overexpressed, or when Gβ13F function is abolished. These phenotypes therefore are unlikely to be induced by GTP bound Gαi or by depletion of Gβγ, suggesting that the GDP bound form of Gαi may be responsible for the equal size NB divisions seen when wt Gαi is overexpressed. These findings clearly support the view that the Pins/GDP-Gαi complex has a role for generating the signal associated with spindle asymmetry. (1) Equal size divisions seen when Gαi is overexpressed in wt NBs is drastically reduced when overexpression is performed in the absence of Pins. (2) Whenever unequal size division occurs when Baz/DaPKC function is compromised, Pins and Gαi are always colocalized to the side of the cell where the future larger daughter is formed (Cai, 2003).

Although in the nematode embryo generation of unequal-sized daughters involves only the posterior displacement of a symmetric spindle, there appears to be some parallels between the two model systems. In the wt nematode P0 division, the magnitude of the forces acting on the two spindle poles apparently depend on the character of the anterior and posterior cortex. In wt P0, PAR-3 and PAR-2 localize to the anterior and posterior cortex, respectively, and the mitotic spindle is displaced toward the posterior pole, correlating with a greater net posterior force acting on the posterior spindle pole relative to the net anterior force acting on the anterior spindle pole. In par-2 mutants, PAR-3 expands to occupy the whole of the cortex, imparting anterior character throughout, and the net force acting on both spindle poles has a magnitude equivalent to that of the wt force acting on the anterior spindle pole. Conversely in par-3 P0, PAR-2 becomes cortical, imparting posterior character to the entire cortex, and the magnitude of both forces acting on the spindle poles is equivalent to that of the wt posterior acting force. In both par-2 and par-3 mutants, the forces acting on the spindle poles are equalized, mitotic spindle is no longer displaced, and equal-sized daughters result (Cai, 2003).

In Drosophila NBs, although spindle displacement occurs, the generation of an apically biased mitotic spindle mediated by either asymmetrically localized Baz/DaPKC or Pins/Gαi makes the major contribution to the difference in daughter cell size. It is proposed that the asymmetric localization of components of either of these pathways can make the region of the cell cortex they occupy different from the cortical regions that they don't occupy through localized DaPKC or heterotrimeric G protein signaling mediated through Pins/Gαi. In wt NBs, the components of either pathway would impart apical character to the cell cortex where they are localized. One effect of the asymmetric signaling is to generate the preferential elongation of the spindle arm closest to the site of the localized signal. If signaling is symmetric, for example either when Baz/DaPKC and Pins/Gαi are all uniformly cortical, or when Baz/DaPKC and Pins/Gαi are localized to opposite sides of a dividing progenitor, as in pI, a symmetric spindle results. Hence, in both the nematode P0 and in Drosophila NBs the generation of unequal-sized daughters is regulated by asymmetrically localized cortical components. In the nematode there is compelling evidence that differential forces acting on the two spindle poles mediate spindle displacement and the generation of unequal daughters. However, NBs of Drosophila asterless mutants are apparently devoid of functional centrosomes and astral microtubules, yet they form functional asymmetric anastral mitotic spindles and undergo unequal cytokinesis to generate unequal size daughters. It remains to be seen how the localized properties of the NB cell cortex influences its spindle geometry (Cai, 2003).

Effects of Mutation: Bazooka function in oogenesis

The anterior-posterior axis of C. elegans is defined by the asymmetric division of the one-cell zygote, and this is controlled by the PAR proteins, including PAR-3 and PAR-6, which form a complex at the anterior of the cell, and PAR-1, which localizes at the posterior. PAR-1 plays a similar role in axis formation in Drosophila: the protein localizes to the posterior of the oocyte and is necessary for the localization of the posterior and germline determinants. PAR-1 has recently been shown to have an earlier function in oogenesis, where it is required for the maintenance of oocyte fate and the posterior localization of oocyte-specific markers. The homologs of PAR-3 (Bazooka) and PAR-6 are also required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that develop 16 nurse cells and no oocyte. Furthermore, oocyte-specific factors, such as Orb protein and the centrosomes, still localize to one cell but fail to move from the anterior to the posterior cortex. Thus, PAR-1, Bazooka, and PAR-6 are required for the earliest polarity in the oocyte, providing the first example in Drosophila where the three homologs function in the same process. Although these PAR proteins therefore seem to play a conserved role in early anterior-posterior polarity in C. elegans and Drosophila, the relationships between them are different, since the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm (Huynh, 2001).

Drosophila oogenesis begins in region 1 of the germarium when a germline stem cell divides asymmetrically to give rise to a new stem cell and a cystoblast, which then undergoes four rounds of division with incomplete cytokinesis to produce a cyst of 16 cells interconnected by ring canals. A vesicle-rich organelle called the fusome ensures that the pattern of divisions is invariant by anchoring one pole of each spindle at every division to give rise to a cyst that contains two cells with four ring canals, two with three, four with two, and eight with one. The two cells with four ring canals both start to develop as oocytes and are therefore referred to as pro-oocytes. One of them then becomes a nurse cell along with the other 14 cells in the cyst, while the other differentiates as the oocyte. The determination of the oocyte requires the activity of BicD and Egalitarian (Egl) proteins, which both localize to this cell. How this cell is chosen remains unclear, but several lines of evidence suggest that this depends on the asymmetric segregation of the fusome during the cyst divisions, since one of the pro-oocytes always inherits more fusome than the other cells (Huynh, 2001).

PAR-1 localizes to the fusome in regions 1 to 2 of the germarium and is required for the determination of the oocyte, since all 16 cells become nurse cells in par-1 null mutant germline clones. Although this has provided the first example of a fusome component that plays a specific role in oocyte determination, a detailed analysis of the par-1 phenotype reveals that it is not required for the initial selection of the oocyte but for the maintenance of its fate. The determination of the oocyte can be followed with three kinds of markers: (1) oocyte-specific cytoplasmic proteins, such as Orb, BicD, and Egl, accumulate first in the pro-oocytes and then in the oocyte in a microtubule-dependent manner; (2) the centrosomes migrate along the fusome from the other cells of the cyst into the oocyte in a process that is not disrupted by microtubule-depolymerizing drugs; and (3) the oocyte is the only cell of the cyst to remain in meiosis, and this can be followed by the formation of the synaptonemal complex as the chromosomes pair during pachytene. The paired chromosomes then condense to form a hollow sphere called the karyosome, whereas the 15 nurse cells endoreplicate their DNA to become polyploid. Oocyte-specific proteins and the centrosomes still accumulate in one cell in par-1 null mutant germline clones, and this cell remains in meoisis longer than the other 15 cells. However, the centrosomes and oocyte-specific cytoplasmic proteins fail to translocate from the anterior to the posterior of the oocyte, and this cell soon exits meiosis and becomes a polyploid nurse cell. Thus, the par-1 phenotype identifies a new step in oocyte determination, which involves an anterior-posterior movement within the oocyte (Huynh, 2001 and references therein).

To investigate whether this early anterior-posterior polarity in the Drosophila oocyte shows further similarities with the anterior-posterior polarization of the first cell division in C. elegans, whether the Drosophila homologs of other PAR proteins play a role in oogenesis was examined. The best characterized of these is Bazooka, the homolog of PAR-3, which localizes to the apical side of ectodermal cells and neuroblasts in the embryo and is required for epithelial polarity and the localization of cell fate determinants during asymmetric neuroblast divisions. To test for a requirement for bazooka during oogenesis, germline clones of the strongest available allele, baz815-8, were induced; these clones were marked by the loss of green fluorescent protein (GFP). The majority of baz mutant egg chambers stop growing at stage 5 and adopt an oval shape. Furthermore, none of the cells in the cyst accumulate the higher levels of cortical actin that are normally found in the oocyte, although the two cells with four ring canals lie at the posterior of mutant egg chambers, as they do in wild-type. This phenotype suggests that baz mutants fail to form an oocyte, and therefore another oocyte marker, Staufen protein, was stained examined. Staufen accumulates in wild-type oocytes at around stage 5, but the majority of baz mutant cysts show no asymmetric accumulation of Staufen, even when they are older than wild-type cysts that have already localized the protein. Wild-type egg chambers always develop 15 polyploid nurse cells and an oocyte with its DNA compacted into a karyosome. In contrast, staining of baz mutant egg chambers with the DNA stain Hoechst reveals that they usually contain 16 polyploid cells. Thus, the loss of Bazooka activity from the germline gives rise to egg chambers in which all 16 cells develop as nurse cells (Huynh, 2001).

When germline clones of baz815-8 are generated using the ovoD technique to arrest the development of nonmutant cysts, the females produce a few fertilized eggs, indicating that baz mutant egg chambers sometimes develop a normal oocyte. The penetrance of the baz oogenesis phenotype was examined by scoring the frequency of egg chambers with an identifiable oocyte at two different times after the clones were induced. When females are dissected 2 days after eclosion, 14% of the mutant cysts contain a normal oocyte, whereas 86% arrest at stage 5 with the 16 nurse cell phenotype. The frequency of normal egg chambers falls to about 3%, however, when the females are dissected after 6 days. This suggests that Bazooka is essential for oocyte determination but that some egg chambers escape because the wild-type protein perdures for a long time after the clones are induced (Huynh, 2001).

Egg chambers mutant for egl, BicD, or par-1 also contain 16 nurse cells and no oocyte, but, in egl and BicD cysts, oocyte-specific proteins, such as Orb, never become restricted to one cell, whereas these proteins transiently localize to the anterior of the oocyte in par-1 mutant cysts. To test when Bazooka is required in oocyte determination, the localization of Orb protein was examined in baz815-8 germline clones. In wild-type cysts, Orb localizes to the oocyte in late region 2a of the germarium, where it accumulates at the anterior of the cell before translocating to the posterior in region 3. Orb protein still localizes to the anterior of the oocyte in baz mutant cysts, although this is slightly delayed compared to wild-type. The protein never relocalizes to the posterior, however, and is no longer enriched in the oocyte by stage 3 (Huynh, 2001).

The determination of the oocyte can also be followed by the migration of the centrosomes, which move along the fusome to cluster at the anterior of the oocyte in region 2b, and then translocate to the posterior of the oocyte in region 3. In baz mutant cysts, the centrosomes accumulate at the anterior of the oocyte but never move to the posterior cortex. Furthermore, alpha-tubulin stainings of mutant cysts indicate that microtubules remain focused on the anterior of the oocyte and fail to rearrange. These phenotypes are identical to those produced by par-1 mutants, indicating that Bazooka and PAR-1 act in the same step in oocyte determination. Neither is necessary for the initial selection of the oocyte, because Orb and the centrosomes still become restricted to one cell, but they are required for the maintenance of oocyte fate, since the oocyte soon dedifferentiates and becomes a nurse cell. This failure to maintain oocyte identity correlates with a block in the movement of oocyte-specific factors and the centrosomes from the anterior to the posterior of the cell, suggesting that this early polarization is important for the further development of the oocyte (Huynh, 2001).

PAR-6 has been shown to localize to the same protein complex as PAR-3 in C. elegans, Drosophila, and mammalian cells and is essential both for the localization and the function of this complex. In Drosophila, Bazooka and PAR-6 colocalize to the apical side of the embryonic ectoderm, where they are necessary for the maintenance of epithelial polarity, and both proteins are also inherited by the neuroblasts when they delaminate and are required for the basal localization of cell fate determinants during their asymmetric divisions. To test if Drosophila PAR-6 also functions with Bazooka during oogenesis, germline clones were generated of the par-6Delta226 allele, which is a deletion of the promoter, the start codon, and the first 121 amino acids of the protein and is therefore a strong loss of function mutation if not a null. The majority of mutant egg chambers appear small, oval-shaped, and contain 16 polyploid nurse cells and no oocyte, indicating that PAR-6 is also required for oocyte determination. Furthermore, Orb and the centrosomes accumulate in one cell at the posterior of the cyst, although with a slight delay compared to wild-type. Both remain at the anterior of the oocyte, however, and fail to translocate to the posterior pole. Thus, the loss of PAR-6 from the germline gives an identical phenotype to Bazooka and PAR-1. As is the case for bazooka germline clones, some of the par-6 mutant egg chambers escape the early arrest and go on to produce normal eggs. When the females are scored 2 days after eclosion, half of the egg chambers form a normal oocyte, and about a quarter still do so after 10 days. This increase in the penetrance of the phenotype with age shows that PAR-6 protein perdures for many days after the clones are produced. Consistent with this, PAR-6 appears to be unusually stable in the embryo; the protein can be detected throughout embryogenesis in zygotic par-6 null embryos, at levels that are only slightly lower than in wild-type. However, the continued presence of escapers after 10 days suggests that PAR-6 may not be essential for oocyte determination in all cases and that there may be redundant pathways that can partially compensate for its absence (Huynh, 2001).

During the asymmetric divisions of the neuroblasts, the Bazooka/PAR-6 complex recruits Inscuteable to the apical side of the cell, where it plays a role in directing the basal localization of Miranda protein. Germline clones of null mutants in inscuteable or miranda cause no visible defects in oocyte determination or the posterior localization of Orb, however, and give rise to normal eggs that can be fertilized. Furthermore, neither protein shows any asymmetric localization in early egg chambers. Thus, some of the downstream effectors of early oocyte and neuroblast polarity are different, despite the similar roles of Baz and PAR-6 in the two processes (Huynh, 2001).

To investigate the relationships between Bazooka, PAR-6, and PAR-1 during oocyte determination, their localizations were analyzed in both wild-type and mutant germaria. In region 2a to region 3 of the germarium, Bazooka localizes around the ring canals, in a ring that is about twice the diameter of that formed by actin. This localization is very similar to that of the adherens junction components Shotgun (E-cadherin) and Armadillo. A double staining was therefore performed for Arm and Baz. Although Arm localizes to these rings before Bazooka in early region 2a, the two proteins colocalize from the middle of region 2a until region 3, when they both disappear. Bazooka also colocalizes with Shotgun and Armadillo in the zonula adherens of the embryonic epithelium, which provides a boundary between the apical and basolateral membrane domains. This raises the possibility that the Shotgun, Armadillo, and Bazooka rings in the germarium perform a similar function by marking the separation between an anterior and a posterior domain within the oocyte. It is unclear whether PAR-6 also localizes to these rings, since none of the available antibodies give any significant staining that disappears in par-6 null germline clones (Huynh, 2001).

In C. elegans, the PAR-3/PAR-6 complex is required for the posterior localization of PAR-1. This is not the case during Drosophila oogenesis, however, since PAR-1 shows a wild-type localization to the fusome in baz and par-6 germline clones. Furthermore, the localization of Bazooka around the ring canals does not require PAR-6, since it is unaffected in mutant germline clones. This is in marked contrast to both the C. elegans zygote and Drosophila neuroblasts and epithelia, where the localizations of PAR-3/Baz and PAR-6 depend on each other. Bazooka and PAR-6 also localize to the apical sides of the somatic follicle cells of the egg chamber, and mutants in either gene disrupt the localization of both proteins and cause the cells to overproliferate and lose their apical-basal polarity. Thus, the relationship between Bazooka and PAR-6 is different in the germline and the somatic follicle cells, where they appear to have a similar role to that described in other epithelia (Huynh, 2001).

These results show that PAR-1, Bazooka, and PAR-6 act in the same step in oocyte determination, providing the first example in Drosophila where these three homologs of C. elegans PAR proteins participate in the same process. Furthermore, mutants in all three genes disrupt the movement of oocyte-specific proteins and the centrosomes from the anterior to the posterior of the oocyte, which is the earliest visible sign of polarity within the oocyte. Given the role of these PAR proteins in other systems, it seems very likely that their primary function in the germarium is in the anterior-posterior polarization of the oocyte, and that the failure to maintain oocyte fate is a consequence of this defect (Huynh, 2001).

It is intriguing that this very early anterior-posterior polarity of the Drosophila oocyte requires three of the PAR proteins that mediate the anterior-posterior polarization of the first cell division in C. elegans. Although this suggests that these proteins act in a conserved pathway for generating cell polarity in these two systems, the relationships between the localizations of these proteins are quite different in the Drosophila oocyte and C. elegans zygote. Thus, at least some aspects of their function are not conserved, and it will therefore be interesting to determine whether the downstream pathways that generate other cellular asymmetries in response to this polarity are related (Huynh, 2001).

Bazooka and atypical protein kinase C are required to regulate oocyte differentiation in the Drosophila ovary

The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote, subsequently have been shown to regulate cellular polarity in diverse cell types by means of an evolutionarily conserved protein complex including PAR-3, PAR-6, and atypical protein kinase C (aPKC). The Drosophila homologs (in parentheses) of C. elegans par-1, par-3 (bazooka), par-6 (DmPar-6), and pkc-3 (aPKC; DaPKC) each are known to play conserved roles in the generation of cell polarity in the germ line as well as in epithelial and neural precursor cells within the embryo. In light of this functional conservation, the potential role of baz and DaPKC in the regulation of oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of initial anterior-posterior polarity within germ-line cysts and maintenance of oocyte cell fate. Germ-line clonal analyses indicate both proteins are essential for two key aspects of oocyte determination: the posterior translocation of oocyte specification factors and the posterior establishment of the microtubule organizing center within the presumptive oocyte. Baz and DaPKC colocalize to belt-like structures between germarial cyst cells. However, in contrast to their regulatory relationship in the Drosophila and C. elegans embryos, these proteins are not mutually dependent for their germ-line localization, nor is either protein specifically required for PAR-1 localization to the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).

To examine the potential oogenic function of baz and DaPKC, protein null germ-line mutant clones were generated for both baz and DaPKC. Germ-line clones, identified by the absence of nuclear GFP expression, were counterstained with the chromatin marker propidium iodide to examine the number and ploidy of the germ-line nuclei. In contrast to wild-type egg chambers, which invariably contain 15 nurse cells and a single oocyte, baz mutant germ-line clones, while containing the normal complement of germ-line nuclei, fail to differentiate an oocyte, resulting in a 16-nurse cell phenotype as revealed by the polyploid state of all 16 germ-line nuclei. Similarly, DaPKCk06403 germ-line clones also fail to differentiate an oocyte as indicated by the presence of 16 polyploid nurse cell nuclei in germ-line mutant egg chambers. These results reveal a germ-line autonomous requirement for DaPKC and confirm the role of Baz in oocyte differentiation and/or maintenance (Cox, 2001).

These analyses further reveal that germ-line depletion of either DaPKC or baz function from the follicle cells leads to their multilayering, which disrupts the normal partitioning of germ-line nuclei to successively mature egg chambers caused by mispositioning of mutant follicle cells. These mispartitioned baz+ nurse cell nuclei, into an otherwise baz null germ-line clone, may provide the threshold of germ-line Baz required to rescue the oocyte differentiation defect and allow production of a mature egg. Alternatively, the Baz protein may exhibit a long perdurance after mitotic clone induction, which depletes over a period of days, resulting in the cessation of egg production in these mosaic females. These results, however, in no way invalidate the previous conclusions that maternally provided Baz masks the severity of the embryonic polarity phenotype in both epithelial cells and neuroblasts. Rather, the results indicate that these embryos are not likely to represent a complete maternal depletion for Baz (Cox, 2001).

Oocyte differentiation requires the polarized accumulation of oocyte specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in the localization of these factors, mutant germ-line clones for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst cells in region 2a, and then both molecules are targeted first to the two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In contrast, ORB fails to translocate from the anterior to a posterior crescent in both baz and DaPKC null germ-line cysts in germarial region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in baz and DaPKC null germ-line clones in germarial region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).

Furthermore, in contrast to wild-type germ-line cysts in which DHC64C localizes to a single posterior cell, in DaPKC null germ-line clones DHC64C fails to localize to a single cell posteriorly, but rather accumulates in the two posterior-most presumptive pro-oocytes of the mutant germ-line cyst. Therefore, baz and DaPKC display essentially identical phenotypes in germ-line mutant clones with regards to oocyte differentiation and the establishment of initial A-P polarity within the oocyte. The failure to maintain oocyte identity in either baz or DaPKC mutant cysts can therefore be directly correlated with defects in the A-P translocation of oocyte specification factors within a single posterior cell of a germ-line cyst, suggesting oocyte differentiation depends on this early polarization event (Cox, 2001).

The posterior assembly of a functional MTOC has been directly implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the failure to translocate these factors to a posterior crescent in region 3 baz or DaPKC mutant cysts may result from a defect in microtubule reorganization within these mutant cysts. In contrast to wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of oocyte specification factors in these mutants are likely caused, at least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).

In addition to the microtubule network, both ring canals and the fusome play critical roles in cyst polarization and oocyte differentiation. The formation and spatial distribution of ring canals as well as fusome morphogenesis was examined in both baz and DaPKC mutant germ-line cysts. In baz mutant cysts, ring canal formation and spatial distribution are indistinguishable from wild-type germ-line cysts. Furthermore, the wild-type spatial arrangement of ring canals in baz null germ-line cysts suggests there is no apparent disruption in germ cell adhesion within the cyst. Similarly, no defects were observed in ring canal formation or spatial distribution in DaPKC mutant cysts (Cox, 2001).

These analyses indicate baz mutant cysts display relatively normal fusome morphology, although mutant fusome branches appear slightly thinner when compared with wild-type fusomes within the same germarium. Similarly, no apparent defect is observed in fusome morphology in DaPKC null germ-line cysts, indicating DaPKC is dispensable in the germ line for proper fusome morphogenesis (Cox, 2001).

To investigate the mechanism by which Baz and DaPKC exert their effects on oocyte differentiation, the localization of these proteins was analyzed in the germ line and soma during oogenesis. The specificity of the Baz and DaPKC antibodies was verified by using mosaic ovaries containing baz or DaPKC null mutant clones. Baz is first detected in the germarium as a belt-like specialization on germ cell membranes at sites of germ cell interconnection. These belt structures are reminiscent of ring canals with respect to their position between germ-line cyst cells; however, in contrast to ring canals these "Baz belts" are approximately 2-fold greater in diameter. Germaria double-labeled for Baz and rhodamine phalloidin reveal that the Baz belts localize adjacent to ring canals and further reveal an approximate 1:1 ratio between the two structures within germ-line cysts. The microtubule cytoskeleton as well as the fusome were observed projecting through individual cystocytes coincident with the site of Baz belt expression on the germ cell membrane. Later in oogenesis, Baz is transiently enriched in the oocyte cytoplasm at stages 5-6 before the onset of vitellogenesis and is subsequently undetectable in the germ line of vitellogenic egg chambers. As with Baz localization to the apical junctional zone in the embryonic epithelium, tight apical localization of Baz is also observed in follicular epithelia (Cox, 2001).

DaPKC localizes to the Baz belts in the germarium, whereas in follicle cells DaPKC is apically constricted consistent with Baz localization in these cells (Cox, 2001).

In embryonic epithelia, Baz, DmPAR-6, and DaPKC apically colocalize and partially overlap with the apico-laterally enriched Arm and DE-cadherin proteins in the region of the apical zonula adherens. Furthermore, these proteins are required to maintain epithelial cell polarity and are mutually dependent for their proper localization. DE-cadherin is localized to belt-like structures adjacent to ring canals in region 2 germarial cysts reminiscent of Baz belt localization, suggesting that these adherens junction components may similarly colocalize in the germ line. To further investigate the molecular and functional nature of Baz belt expression in the germarium, germaria were labeled with anti-Baz, anti-Arm, and anti-DE-cadherin antibodies and their potential colocalization within the germarium was examined. These analyses reveal Baz colocalizes with both DE-cadherin and Arm to the Baz belts within the germarium. Furthermore, consistent with the colocalization of Baz and DaPKC to Baz belts within the germarium, partial colocalization of DaPKC with DE-cadherin is observed in wild-type germarial cysts. To assay whether these proteins are mutually dependent for their germ-line localization, the localization of DE-cadherin and Arm was examined in baz null germ-line clones. In contrast to their mutual dependence in embryonic epithelia, these analyses indicate germ-line Baz function is dispensable for the localization of either DE-cadherin or Arm to the Baz belts in the germarium. DaPKC is dispensable for the localization of either DE-cadherin or Arm to these structures. These results indicate Baz and DaPKC function are not required for the formation of these structures because both Arm and DE-cadherin localization to these belts in baz or DaPKC mutant cysts is indistinguishable from that observed in wild-type cysts. Previous studies have revealed that germ-line clones of a strong allele of shotgun (shgIG29), the gene encoding DE-cadherin, disrupts the arrangement of germ cells in region 2b germarial cysts, suggesting DE-cadherin may mediate germ cell adhesion. In contrast, these analyses of ring canal spatial distribution in baz and DaPKC mosaic cysts strongly suggests Baz and DaPKC function are dispensable for normal germ cell adhesion. Furthermore, germ-line clonal analyses of either shg or arm reveal neither gene is required for oocyte differentiation, whereas both baz and DaPKC are essential in oocyte determination. These results suggest the components of the Baz belts likely mediate diverse cellular functions essential for germ-line cyst development, which may include cell adhesion, cell signaling, and cyst polarization (Cox, 2001).

In the C. elegans zygote, the PAR-3/PAR-6/PKC-3 complex is localized to the anterior where it is required for the posterior localization of PAR-1. To investigate the potential regulatory relationship between baz and par-1 in the Drosophila germ line, baz null germ-line clones were generated and PAR-1 expression and localization was analyzed. In wild-type germ-line cysts, PAR-1 is localized to the spectrosome and fusome in germarial regions 1 and 2a and subsequently is down-regulated on fusomes in regions 2b and 3. In baz mutant germ-line cysts, PAR-1 localization to the spectrosome is unaffected and is likewise present on fusomes although somewhat weaker localization is observed on fusome branches of baz mutant germ-line cysts when compared with wild-type germ-line cysts. Taken together, these results suggest germ-line baz is dispensable in the germarium for normal PAR-1 expression and localization (Cox, 2001).

To determine whether par-1 may regulate Baz expression, par-1 null germ-line clones were generated and Baz localization was examined. No defect was observed in Baz belt expression in par-1 mutant germ-line cysts. Furthermore, in contrast to wild-type stage 5-6 egg chambers in which Baz is transiently enriched in the oocyte cytoplasm, in par-1 mutant egg chambers Baz localization is abolished from the posterior presumably due to the defect in oocyte differentiation observed in par-1 mutant egg chambers. These results indicate germ-line par-1 is dispensable for normal Baz belt expression and localization (Cox, 2001).

To assay the regulatory relationship between baz and DaPKC the localization of DaPKC was analyzed in baz mutant germ-line cysts, as well as the localization of Baz in DaPKC mutant germ-line cysts. In contrast to their mutual dependence for localization in the embryo, both Baz and DaPKC localization within the germ line is mutually independent. These results indicate that despite the apparent functional conservation of Baz, DaPKC, and PAR-1 in generating oocyte polarity, the regulatory relationships among these genes are not conserved in the germ line (Cox, 2001).

The colocalization of Baz, DaPKC, Arm, and DE-cadherin to the Baz belt structures in the germarium strongly suggests the components of the Baz belts are capable of mediating a multiplicity of functions in germ-line cysts. In embryonic epithelia, these proteins function in the formation of the apical zonula adherens junction and are mutually dependent for their apical localization. The germ-line colocalization of these molecules to the Baz belts suggests these structures may represent a potential polarity cue on the germ cell plasma membrane. The restricted localization of Baz belt components to germ cell membranes at points of cell-cell contact represents an asymmetry on the plasma membrane of germ-line cyst cells with regard to the A-P axis of the cyst and stage 1 oocyte. The asymmetric localization of these molecules to one side of the germ cell plasma membrane may act as a polarity cue in defining anterior versus posterior within individual cystocytes of the 16-cell germ-line cyst and thus contribute to the establishment of an initial A-P axis and to subsequent germ-line cyst polarization. These results further suggest that Baz and DaPKC likely function in a signaling capacity, rather than a structural one, to mediate oocyte differentiation, whereas DE-cadherin and Arm are more likely to function in maintaining germ cell adhesion and cyst integrity (Cox, 2001).

Consistent with par gene function in other systems, these results indicate that Baz, DaPKC, and PAR-1 are required for the establishment and maintenance of cellular polarity in the Drosophila germ line; however, the regulatory relationships observed between these genes in the germ line versus that observed in embryonic blastomeres, epithelial cells, and neural precursor cells indicates that, while these molecules are functionally conserved, the mechanisms by which these genes act appear to be less well conserved. In contrast to their mutual dependence for localization in the embryo, Baz, DaPKC, and PAR-1 each are mutually independent for their localization in the germ line. Taken together, these results underscore the functional utility of the par genes and their effectors as a molecular module for generating cellular polarity in diverse cell types. Furthermore, the independence of Baz, DaPKC, and PAR-1 in their germ-line localization provides a unique opportunity to probe new mechanisms by which these highly conserved proteins function in regulating diverse processes such as cellular polarity, asymmetric cell division, or growth control (Cox, 2001).

The fusome and microtubules enrich, Par-1 in the oocyte, where it effects polarization in conjunction with Par-3, BicD, Egl, and Dynein

After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Par-1 is shown to become restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. baz is required for Par-1 relocalization within the oocyte and the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization (Vaccari, 2002).

It has been reported that, like Par-1, the D. melanogaster homolog of Par-3 Bazooka (Baz) is necessary for oocyte maintenance. Germline clones of a baz null allele are defective in oocyte polarization, establishing a functional parallel with C. elegans, in which the PAR genes polarize the early embryo. In the worm, restriction of Par-1 activity to the posterior cortex of the embryo crucially depends on Par-3, but the reverse is not the case. In the Drosophila germarium, Par-1 localization to the fusome appears to be independent of Baz. To assess whether the subsequent localization of Par-1 in the oocyte requires Baz, Par-1 distribution was further evaluated in baz germline clones. Par-1 is initially detected at the anterior of the oocyte in region 2b, but the protein disappears in region 3, revealing that Par-1 relocalization is baz dependent (Vaccari, 2002).

The relative distributions of Par-1 and Baz were investigated during later stages of oogenesis in wild-type ovaries. Par-1 is detected at the posterior of the oocyte as of germarial region 3 and becomes tightly associated with the cortex between oogenesis stages 3 and 5. Concomitantly, Baz becomes transiently localized to the anterior of the oocyte. The anterior localization of Baz is specific; it is not observed in germline clones of a baz null allele. Interestingly, maximal expression of Baz at the anterior coincides with the apparent tightening of Par-1 signal at the posterior cortex, suggesting a role for Baz in sharpening Par-1 localization. At this stage, the localization of the two proteins appears to be mutually exclusive. The anterior enrichment of Baz between stages 2 and 5 is absent in germline clones of the par-1 null allele (Vaccari, 2002).

The mutually exclusive distribution of Par-1 and Baz in the Drosophila oocyte is strikingly reminiscent of that observed in the C. elegans embryo. The ability to visualize the two proteins has allowed their respective roles in achieving this distribution to be genetically evaluated. The seeming dependence of Baz localization on par-1 is in contrast to results in C. elegans, in which localization of Par-3 is independent of Par-1. However, the absence of Baz in par-1 germline clones may well reflect the loss of oocyte fate and the onset of its degeneration that occurs at stage 1. Nonetheless, the anterior localization of Baz during stages 2–5 suggests that, after acting in oocyte polarization in region 2b/3, baz may be required again during oogenesis. The existence and nature of such a second requirement for baz after oocyte polarization is not yet clear (Vaccari, 2002).

Research has shown that the fusomal localization of Par-1 is unaffected in baz mutants, suggesting a difference between the Drosophila oocyte and the C. elegans embryo, in which Par-1 localization depends on par-3. Remarkably, at the time when oocyte polarization takes place, baz is in fact required for Par-1 localization within the oocyte. Hence, it appears that a similar relationship exists between Par-1 and Baz at the time when their activities are critical in the two organisms (Vaccari, 2002).

Effects of Mutation: 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; rather it 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 interpretation is consistent with the observation that, during border cell migration, baz function is clearly distinct from that of DE-cadherin, which, though essential for cell motility, does not affect border cell adhesion. The requirement of DE-cadherin for border cell motility appears to be independent of its role as a structural component of the ZA since another essential ZA component, the cytoskeletal linker protein Armadillo, is not required for border cell migration. In mosaic border cell clones with DE-cadherin mutant cells, the mutant cells consistently lag behind wild-type cells, indicating that the mutant cells have a compromised migratory ability and that they are being dragged along by their wild-type neighbor cells. Conversely, in mosaic border cell clones, bazEH171 mutant cells were found in every position within the migrating border cell clusters, including the leading position, indicating normal motility. Indeed, cohesion between the migrating cells appears to be defective since mosaic cell clones were frequently dispersed and misarranged. Therefore, baz and DE-cadherin appear to have different functions during border cell migration (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).

Requirement for Par-6 and Bazooka in Drosophila border cell migration

Polarized epithelial cells convert into migratory invasive cells during a number of developmental processes, as well as when tumors metastasize. Much has been learned recently concerning the molecules and mechanisms that are responsible for generating and maintaining epithelial cell polarity. However, less is known about what becomes of epithelial polarity proteins when various cell types become migratory and invasive. This study reports the localization of several apical epithelial proteins, Par-6, Par-3/Bazooka and aPKC, during border cell migration in the Drosophila ovary. All of these proteins remain asymmetrically distributed throughout migration. Moreover, depletion of either Par-6 or Par-3/Bazooka by RNAi results in disorganization of the border cell cluster and impaired migration. The distributions of several transmembrane proteins required for migration were abnormal following Par-6 or Par-3/Bazooka downregulation, possibly accounting for the migration defects. Taken together, these results indicate that cells need not lose apical/basal polarity in order to invade neighboring tissues and in some cases even require such polarity for proper motility (Pinheiro, 2004).

Therefore, border cells retain an asymmetric distribution of the apical epithelial proteins Baz, Par-6 and aPKC throughout their migration, raising the question as to why. One possibility could be that these proteins contribute to the cells' direction-sensing mechanism. However, neither Par-6 nor Baz localized asymmetrically with respect to the direction of migration, making this possibility seem less likely. In premigratory border cells, the apical domain is oriented towards the nurse cells and the direction of migration. However, once the cells separate from the epithelium, the side of the cluster with the highest levels of Baz, Par-6 and aPKC was found to be roughly orthogonal to the direction of migration. These findings are consistent with observations regarding the distribution of Crumbs, another apical marker, and suggest that early in migration the entire cluster rotates so that the leading edge is roughly perpendicular to the apical domain (Pinheiro, 2004).

A second possibility is that maintaining some aspects of epithelial polarity during migration eliminates the need to re-establish polarity de novo when the border cells reach the oocyte. While possible, this hypothesis is difficult to test and cannot be the only function for Par-6 and Baz in border cells, since these proteins are also required during migration (Pinheiro, 2004).

A third possibility is that cellular asymmetry is retained during border cell migration in order to achieve the proper asymmetries in the distributions of other proteins. Consistent with this proposal, the normally asymmetric accumulations of E-cadherin and ßps-integrin within border cells are dramatically altered in cells depleted of Baz or Par-6. Loss of E-cadherin from border cells has been shown to inhibit migration, and misdistribution of E-cadherin at border cell/nurse cell boundaries correlates with a migration defect. The defects in the distributions of E-cadherin and other membrane-associated proteins in border cells either depleted of, or overexpressing Par-6 and Baz, may collectively lead to the observed migration defect (Pinheiro, 2004).

A large number of mosaic egg chambers containing clones mutant for par-6 or baz were examined and delays were observed in border cell migration as well as defects in cohesion within the cluster. It has been reported that mosaic clones of baz show a lack of adhesion within the border cell cluster but no migratory defects. It is likely that this difference is due to clone size and/or protein perdurance, since only large clones in which the majority of the border cells were mutant, showed defects in border cell migration. Consistent with this, RNAi-mediated reduction of Par-6 and Baz in the border cells results in delayed migration, suggesting that the strongest migration defects are observed only when all the border cells lack Par-6 or Baz. This is not unusual. Mutations in slbo, jing, stat92E and shotgun, which encode E-cadherin, exhibit similar behavior such that clusters containing some wild-type cells can migrate. These findings seem to indicate that wild-type cells can 'drag' a few mutant cells, but when the number of migration-defective cells exceeds the number of migration-competent cells, migration slows or stops (Pinheiro, 2004).

RNAi-mediated reduction of Par-6 and Baz in the polar cells, in addition to the outer border cells, exacerbates the defects caused by expression in the migratory cells alone. This suggests that polar cells contribute to organizing the cluster. Cohesion of the cluster may be necessary in order for the migratory cells to receive continuous activation of the JAK/STAT pathway during migration. Consistent with this, in those clusters that split, those cells that remain attached to the polar cells migrate further than the cells that become detached. Polar cells require the migratory cells to reach the oocyte because they are not motile themselves, but the migratory cells also appear to need the polar cells in order to sustain their motility. This mutual requirement may serve to ensure that the migratory cells do not run off without the polar cells, since the polar cells are required at the oocyte surface to form the pore in the micropyle through which a sperm enters at fertilization (Pinheiro, 2004).

The observations presented in this study demonstrate that Par-6 and Par-3/Baz are distributed asymmetrically in migrating border cells, suggesting that not all epithelial polarity is lost when these epithelial cells become motile. In spite of this, the morphology of the border cells, particularly at the border cell/nurse cell interface, can appear fibroblast-like. This interface must support protrusive behavior and dynamic adhesion, so that the cells can move along the nurse cells, while they simultaneously remain firmly attached to each other and to the polar cells. Therefore, migrating border cells possess both epithelial and mesenchymal characteristics (Pinheiro, 2004).

It is proposed that the Par-3/Par-6/aPKC complex functions in these cells, as it does in an epithelium or in asymmetrically dividing neuroblasts, to maintain distinct protein distributions and functional domains in different parts of the cell. In the case of the border cells, three important domains are the interfaces between border cells and nurse cells, between border cells and polar cells and between adjacent border cells. Such distinct domains may be present in other types of cells that maintain contacts with an intact epithelium while they migrate, such as motile keratinocytes at a wound edge or leading endothelial cells during angiogenesis. Tumor cells that metastasize in groups or 'nests' may also possess both epithelial and mesenchymal characteristics. Thus the Par-3/Par-6/aPKC complex may contribute to the invasiveness of other cell populations as well (Pinheiro, 2004).

Bazooka is required for polarisation of the Drosophila anterior-posterior axis

The Drosophila anterior-posterior (AP) axis is determined by the polarisation of the stage 9 oocyte and the subsequent localisation of bicoid and oskar mRNAs to opposite poles of the cell. Oocyte polarity has been proposed to depend on the same PAR proteins that generate AP polarity in C. elegans, with a complex of Bazooka (Baz; Par-3), Par-6 and aPKC marking the anterior and lateral cortex, and Par-1 defining the posterior. The function of the Baz complex in oocyte polarity has remained unclear, however, because although baz-null mutants block oocyte determination, egg chambers that escape this early arrest usually develop normal polarity at stage 9. This study characterised a baz allele that produces a penetrant polarity phenotype at stage 9 without affecting oocyte determination, demonstrating that Baz is essential for axis formation. The dynamics of Baz, Par-6 and Par-1 localisation in the oocyte indicate that the axis is not polarised by a cortical contraction as in C. elegans, and instead suggest that repolarisation of the oocyte is triggered by posterior inactivation of aPKC or activation of Par-1. This initial asymmetry is then reinforced by mutual inhibition between the anterior Baz complex and posterior Par-1 and Lgl. Finally, it was shown that mutation of the aPKC phosphorylation site in Par-1 results in the uniform cortical localisation of Par-1 and the loss of cortical microtubules. Since non-phosphorylatable Par-1 is epistatic to uninhibitable Baz, Par-1 seems to function downstream of the other PAR proteins to polarize the oocyte microtubule cytoskeleton (Doerflinger, 2010).

The baz358-12 allele causes a fully penetrant defect in the localisation of bcd and osk mRNAs and in the positioning of the oocyte nucleus and gurken mRNA, providing the first demonstration that Baz is required for the polarisation of the Drosophila AP and dorsal-ventral axes. This raises the question of why baz-null mutant germline clones that escape the block in early oogenesis sometimes develop into eggs with normal polarity. Although it is formally possible that Baz is not absolutely essential for oocyte polarity and that the baz358-12 allele has a dominant-negative effect, this seems very unlikely. First, baz358-12 behaves like a typical hypomorphic mutation as it is recessive and fails to complement the lethality of baz-null alleles. Second, nearly half of the escapers from baz-null germline clones show similar polarity defects to baz358-12 at stage 9, indicating that this is a loss-of-function phenotype. Thus, it seems more likely that whatever allows a few of the null germline clones to escape the early-arrest phenotype also allows some of them to escape the polarity defect at stage 9. For example, other polarity pathways might be activated in baz-null mutant germaria that can partially compensate for the loss of Baz in both oocyte determination and axis formation (Doerflinger, 2010).

The observation that baz358-12 does not cause any defects in the initial polarisation of the oocyte, although it is essential for the AP polarisation at stage 9, indicates that there must be some differences in the functions of Baz at each stage. During early oogenesis, Baz localises in a ring around each ring canal at the anterior of the oocyte and shows perfect colocalisation with DE-cadherin (Shotgun - FlyBase) and Armadillo. Since the PDZ domains of Baz have been shown to interact with Armadillo, it might be recruited to the anterior rings through this interaction, which should still occur normally in the baz358-12 mutant. By contrast, the truncated Baz protein does not localise to the cortex of the oocyte at stages 7-9, indicating that the C-terminal region is necessary for its cortical recruitment at this stage. The only identified domain in this region is CR3, which binds to the kinase domain of aPKC. However, a point mutation in CR3 that disrupts its interaction with aPKC has no effect on the cortical localisation of Baz at stage 9. There must therefore be another domain in the C-terminal region of Baz that is required for its recruitment to the oocyte cortex (Doerflinger, 2010).

Another important difference between the initial polarisation of the oocyte and the repolarisation at mid-oogenesis is the relationship between the PAR proteins. During early oogenesis, the localisation of Baz is unchanged by loss of Par-1 and vice versa. By contrast, Baz and Par-1 show mutually exclusive localisations at stage 9, with Par-1 spreading around the lateral cortex in baz mutants, and Baz and Par-6 localising to the posterior in par-1 mutants. Baz is required to recruit Par-6 to the cortex in mid-oogenesis, as Par-6 disappears from the anterior cortex in baz358-12 clones and localises to the posterior with BazS151A S1085A-GFP. Thus, Baz, Par-6 and presumably also aPKC form a complex in the stage 9 oocyte, making the arrangement of PAR proteins much more similar to that in the C. elegans zygote, with Baz (PAR-3), Par-6 and aPKC defining the anterior and lateral cortex and Par-1 the posterior. As in C. elegans, these complementary localisations are also maintained by mutual antagonism between the anterior and posterior PAR proteins. It has been shown that Par-1 phosphorylates Baz to exclude it from the posterior. This study shows that mutation of the conserved aPKC site in the Par-1 linker region leads to the mislocalisation of Par-1 around the anterior and lateral cortex, strongly suggesting that aPKC phosphorylates this site to restrict Par-1 to the posterior (Doerflinger, 2010).

Although the final pattern of PAR proteins in the stage 9 Drosophila oocyte is similar to that in the C. elegans zygote, this pattern develops over a much longer period of time and in a different way. Baz-GFP is enriched at the posterior of the oocyte at the beginning of stage 7 and gradually spreads anteriorly during the succeeding 12 hours, before finally disappearing from the posterior at stage 9. Since Par-1 appears at the posterior early in stage 7, Baz and Par-1 overlap at the posterior for some considerable time. By contrast, Par-6-Cherry starts to disappear from the posterior during stage 7, and already shows a complementary pattern to Par-1 at stage 8. This raises the question of why Par-6, which is recruited to the cortex by Baz, disappears more rapidly from the posterior. Although this might mean that they are excluded by different mechanisms, both Par-6 and Baz localise to the posterior in par-1 mutants and in BazS151A S1085A-GFP-expressing oocytes, indicating that their exclusion depends on the phosphorylation of Baz by Par-1. Thus, Par-1 phosphorylation might first release Par-6 from Baz, and then more gradually displace Baz from the cortex. The phosphorylation of serine 1085 of Baz by Par-1 disrupts the interaction of Baz with aPKC and this might be sufficient to release the Par-6-aPKC complex. However, Par-6 also binds directly to the PDZ domains of Baz, and the phosphorylation of serine 1085 alone would not be expected to interfere with this interaction. Thus, Par-1 might also act in some other way to release Par-6, perhaps by promoting the posterior recruitment of Lgl, as the latter is known to inhibit the interaction of Par-6-aPKC with Baz in neuroblasts (Doerflinger, 2010).

The gradual evolution of PAR protein localisation during stages 7-9 argues against the idea that the oocyte is polarised by a cortical contraction, as in C. elegans, and no any evidence has been observed for cortical movements of the actin cytoskeleton. This raises the question of how this asymmetry arises. Two possible scenarios for how the polarising signal from the posterior follicle cells triggers PAR protein asymmetry can be invisioned. First, the initial cue could remove or inactivate aPKC and Par-6 at the posterior, which would then allow Par-1 to localise there because aPKC is no longer present or able to exclude it. Although aPKC can be inhibited at the posterior by Lgl, this seems unlikely to provide the cue because Lgl localises to the posterior after Par-1 and is not essential for oocyte polarity. Alternatively, the initial asymmetry could be generated by the posterior recruitment and activation of Par-1. Work in mammals has shown that LKB1 (STK11) phosphorylates the activation loop of PAR-1 (MARK2) to turn on its kinase activity, and this is likely to be case in Drosophila as well, as lkb1 mutants exhibit a very similar phenotype to par-1 mutants. LKB1 activity is regulated by protein kinase A (PKA), which is required for the transduction of the polarising follicle cell signal in the oocyte. Thus, it is possible that the initial asymmetry is generated by a kinase cascade at the posterior of the oocyte, consisting of PKA, which activates LKB1, which activates Par-1 (Doerflinger, 2010).

Once the PAR polarity has been established, it must somehow polarise the oocyte microtubule cytoskeleton to direct the localisation of bcd and osk mRNAs. The epistasis experiment suggests that Par-1 provides the primary output from the PAR system, as uniformly distributed Par-1 makes the whole cortex behave like the posterior cortex regardless of whether Baz is also uniformly distributed or not. Based on the par-1 loss- and gain-of-function phenotypes in the oocyte and follicle cells, Par-1 might act to stabilise microtubule plus ends at the cortex and to inhibit the nucleation or anchoring of microtubule minus ends (Doerflinger, 2010).

One key remaining question is the identity of the Par-1 substrates that mediate its effect on microtubule organisation. In addition to Baz, Par-1 has also been shown to phosphorylate Exuperantia and Ensconsin in the oocyte to regulate bcd mRNA localisation and the activity of Kinesin. However, neither of these targets can account for the dramatic effects of Par-1 on microtubule organisation. It has recently been claimed that Par-1 regulates the oocyte microtubule cytoskeleton by phosphorylating the microtubule-stabilising protein Tau, thereby destabilising the microtubules at the posterior of the oocyte. This conclusion was based on the observation that germline clones of tauDf(3R)MR22 produce a partially penetrant defect in the anchoring of the oocyte nucleus. However, the tauDf(3R)MR22 mutation is a 65 kb deletion that removes eight other genes as well as tau, and the phenotype could therefore be due to the loss of one of these other loci. More importantly, tau can be specifically removed without deleting any other genes by generating heterozygotes for two overlapping deficiencies, and these tau-null flies are homozygous viable and fertile and develop normally polarised oocytes. Thus, it seems highly unlikely that Tau is a relevant substrate for Par-1 in the polarisation of the oocyte. A full understanding of oocyte polarity will therefore depend on the identification of the Par-1 targets that control microtubule nucleation, anchoring and stability (Doerflinger, 2010).

A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. This study explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. Surprising parallels were found between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change--cell extension along the anterior-posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. A model is proposed in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension (Sawyer, 2011).

The data suggest that coupling AJs to a contractile apical actomyosin cytoskeleton plays an important role in a very different cell movement: convergent extension during Drosophila GBE. A novel cell shape change, AP cell elongation, was identified that contributes to WT GBE. Furthermore, it was found that Cno is required for maintaining attachment of the apical actomyosin network AJs in a planar-polarized way. Disrupting this connection results in failure of GBE and prevents coordination of apical myosin contractility and cell shape change. These data are consistent with a model in which Cno tightly couples apical actomyosin to AP AJs and coordinates apical polarity proteins with the network, helping to integrate individual cell shape changes across the tissue (Sawyer, 2011).

Previous studies illustrated how an apical contractile actomyosin network powers apical constriction. In contrast, convergent extension during Drosophila GBE was thought to involve planar-polarized enhancement of contractile actomyosin cables, driving cell intercalation and body elongation. It was surprising to find that, in addition to junctional cables, germband cells also have an apical actomyosin network that undergoes cyclical constriction and relaxation. This coincides with and may help to drive cell shape change. The asymmetric cue of planar-polarized myosin is likely to impose asymmetry. Together, asymmetric cortical myosin and cyclical contractions may help to extend cells in one dimension instead of shrinking them in all dimensions, thus contributing to tissue elongation. While this manuscript was being revised, two other papers independently discovered and described the apical network: the Lecuit lab data further suggest that myosin condensations preferentially move toward AP borders, helping to drive cell rearrangement (Rauzi, 2010; Fernandez-Gonzalez, 2011). Both the current data on Cno and the Lecuit lab's data on β-catenin further suggest that different proteins linking this apical network to AJs are critical for the fidelity and coupling of apical myosin contraction to cell shape change (Sawyer, 2011).

Also, a novel cell shape change was identified that may help to drive AP body axis extension - AP cell elongation. Cno and presumably linkage of the apical actomyosin network to AJs are important for this cell shape change. One speculative possibility is that an asymmetric ratchet acts in germband cells, selectively preventing elongation along the DV body axis while allowing cell elongation along the AP body axis. It is also possible that outside forces, such as shape changes of the first cells to divide, help reshape ectodermal cells, but it is thought that this is less likely, as cell shapes were examined during early GBE before germband mitotic domains divide. Ratchets have also been proposed during mesoderm invagination and during dorsal closure, where amnioserosal cells apically constrict. Before dorsal closure onset, amnioserosal cells have periodic apical actomyosin contractions, but cells only retain changes in shape after a junctional actomyosin purse string appears. Disrupting the purse string disrupts dorsal closure, suggesting that a junctional actomyosin cable can act as a ratchet (Sawyer, 2011).

Studies in Xenopus suggest that the role of a dynamic, planar-polarized apical actomyosin network in convergent extension is conserved. Myosin organizes actin into dynamic foci that move within intercalating cells along their mediolateral axis. In myosin's absence, actin foci are lost and convergent extension is disrupted. Thus dynamic actomyosin foci may play a conserved role in convergent extension (Sawyer, 2011).

It will be interesting to identify regulators shaping contractile activity in different tissues. Jak/Stat signaling restricts apical constriction to the mesoderm; in its absence apical myosin accumulates in the ectoderm, and those cells inappropriately apically constrict. Thus, although both mesoderm and ectoderm share an apical contractile network, its regulation is tuned differently. Furthermore, different actin regulators regulate apical and junctional myosin, with Wasp regulating the apical pool (Sawyer, 2011).

Linking AJs to actin is key in diverse processes from adhesion itself to morphogenetic movements as different as apical constriction and collective cell migration. Cno regulates linkage during mesoderm apical constriction, but isn'n required for cell adhesion (Sawyer, 2009). Other AJ-actin linkers act in other contexts, suggesting that cells use distinct linkers in circumstances with different force regimes. The current data suggest that during GBE, Cno regulates AJ:actomyosin network connections, acting specifically along AP borders (Sawyer, 2011).

Core AJ proteins are more reduced on AP borders in cnoMZ mutants than in WT. In WT, slightly reducing AJ proteins on AP borders may facilitate shrinkage of these borders during GBE. It is tempting to speculate that Cno enhancement along AP borders provides extra support when DEcad/Arm is reduced, strengthening AJ:actomyosin linkages along AP borders yet still allowing cell shape change. In this model, when Cno is absent, AJ:actomyosin linkage is weakened at AP borders, leading to inefficient cell shape change, impairing GBE, and accentuating reduction of AJ proteins (Sawyer, 2011).

The data further suggest that Cno is not the only AJ:actomyosin linker during GBE. Although the actomyosin network detaches from AJs in cnoMZ, it does not collapse into a ball; instead, cables remain 0.2-0.5 microm distant from AJs. A second connection is also supported by the appearance of apical strands of DEcadherin stretching from the cortex to detached myosin in cnoMZ. It will be interesting to determine what proteins compose these other AJ:actomyosin links. β-Catenin regulates actin:AJ linkage just prior to this stage and also plays a role in GBE, although how β-catenin mediates linkage remains mysterious (Sawyer, 2011).

Both myosin and Baz/Par3 are important GBE regulators. One of the most surprising consequences of Cno loss was dramatic change in Baz and aPKC localization. Their strong reduction along AP borders and restricted localization along DV borders correlates well with altered localization of apical actomyosin, which detached from AP AJs and retracted along DV borders from vertices. These data suggest that coordination of the actomyosin network and Baz/aPKC facilitates efficient cell shape change. Consistent with this, an interesting recent paper demonstrated that Baz is required for reciprocal planar-polarized distribution of myosin and AJs. Baz localization, in turn, is restricted by the cytoskeletal regulator Rho-kinase (Rok), leaving Baz enriched at DV borders (Simoes Sde, 2010). This suggests a complex network of interactions (Sawyer, 2011).

In C. elegans a contractile actomyosin cytoskeleton positions apical-polarity proteins (PAR3/PAR6/aPKC) anteriorly in one-cell embryos, and this complex then alters the actomyosin network, promoting asymmetric cortical flow to maintain anterior and posterior domains. It is tempting to speculate that the germband contractile actomyosin network plays a similar role. In this model, planar polarization of the network would create a symmetry break, helping to trigger Baz/aPKC planar polarization. They, in turn, may feed back to modulate actomyosin contractility, driving GBE. Strengthening AJ:actomyosin linkages via Cno could help to ensure efficient cell shape changes that are integrated across the tissue (Sawyer, 2011).

Several mechanistic hypotheses are consistent with these data that are not mutually exclusive. First, Cno may directly affect Baz/aPKC localization during assembly or maintenance, working in parallel or in series with Rok (Simoes Sde, 2010), with actomyosin positioning and contractility then modulated by Baz/aPKC. Consistent with this, previous work revealed that Baz remains apical in the absence of AJs; residual epithelial cells retain polarized actin but have hyperconstricted apical ends. Furthermore, PAR proteins regulate actomyosin contractility during DC. Second, Cno could alter the actomyosin network, which in turn may affect proper Baz/aPKC localization. Baz apical positioning requires the actin cytoskeleton. Actin disruption and Cno loss alter Baz localization similarly, consistent with this hypothesis. Finally, Baz/aPKC may mediate Cno apical positioning, as Baz does for AJs. Of course, more complex interplay with feedback between actomyosin and Baz/aPKC seems likely, creating a network of interactions rather than a linear pathway. Teasing out the complex coordination of AJs, apical polarity protein, and the actomyosin network during morphogenesis is an exciting challenge (Sawyer, 2011).

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC 'buds' off. This compensatory mechanism has been referred to as 'telophase rescue'. The Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (TRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. TRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires TRAF1. These data suggest that TRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway (Wang, 2006).

In telophase NBs, segregation of cell fate determinants, such as Pros, into future GMCs, is critical for their proper development. Telophase rescue appears to be one of the safeguard mechanisms that acts to ensure that GMCs inherit the cell fate determinants and adopt the correct cell identity when the mechanisms, which normally operate during NB divisions, fail (e.g., in insc mutant). Telophase rescue is a phenomenon for which the underlying mechanism involved remains largely unknown. The current data demonstrate that TRAF1 and Egr are two members of the Insc-independent telophase rescue pathway specific for Mira/Pros (Wang, 2006).

Although it is apically enriched in mitotic NBs and can directly interact with Baz in vitro, TRAF1 does not seem to be involved with the functions normally associated with the apical complex proteins. One distinct feature of TRAF1 differs from the other known apical proteins is its localization pattern; it is cytoplasmic in interphase and the apical crescent is prominent only at metaphase. In contrast, proteins of the apical complex are largely undetectable during interphase and form distinct apical crescents, starting from late interphase or early prophase. The protein localization difference between TRAF1 and other apical proteins suggests that TRAF1 and apical proteins are not always colocalized during mitosis. If TRAF1 is a bona fide member of the apical complex, the localization defects of other apical proteins are expected to be observed in TRAF1 mutant, as well as mislocalization of basal proteins, which was not detect. In addition, no spindle orientation or geometry defects were observed in the absence of TRAF1. Based on these observations, it is concluded that TRAF1 is not involved with the functions normally associated with the apical complex proteins (Wang, 2006).

The in vitro GST fusion protein pull-down assay suggests that TRAF1 may physically bind to Baz. This result is consistent with genetic data, indicating that TRAF1 acts downstream of baz and that its apical localization requires baz. These observations are consistent with the view that TRAF1 is recruited to the apical cortex by apical Baz in mitotic NBs. Baz, even at very low levels, can recruit TRAF1 to the apical cortex of the mitotic NBs. For example, in insc mutant NBs, TRAF1 remains apical probably owing to the low levels of Baz that remain localized to the apical cortex. This speculation is supported by Mira/Pros telophase rescue data, which clearly demonstrate that the telophase rescue seen in insc mutant NBs is severely damaged in baz mutant, suggesting that the Baz function required for Mira/Pros (and Pon/Numb) telophase rescue is intact in insc mutant NBs (Wang, 2006).

It has been shown that Pins/Gαi asymmetric cortical localization can be induced at metaphase by the combination of astral microtubules, kinesin Khc-73 and Dlg in the absence of Insc; this coincides with the observation that TRAF1 also forms tight crescent only at metaphase in both WT and insc mutant NBs. Does TRAF1 apical crescent formation also require the functions of astral microtubules, kinesin Khc-73 and Dlg? The data do not favor this hypothesis based on the following observations. (1) In TE35BC-3, a small deficiency uncovering sna family genes insc is not expressed but Pins and Gαi are asymmetrically localized, indicating that the astral microtubules, kinesin Khc-73 and Dlg pathway remain functional. TRAF1 is delocalized and is uniformly cortical in this deficiency line. (2) Similarly, in egr insc NBs, TRAF1 is cytoplasmic whereas the functions of astral microtubules, kinesin Khc-73 and Dlg are intact. (3) In egr NBs TRAF1 is cytoplasmic, whereas the apical complex is normal and astral microtubules, kinesin Khc-73 and Dlg are present. (4) TRAF1 apical localization remains unchanged in dlg mutant NBs. Based on these observations, it is concluded that TRAF1 apical localization is unlikely to share similar mechanism with Pins and Gαi and is likely to be independent of astral microtubules, kinesin Khc-73 and Dlg. TRAF1 apical localization appears to specifically require Egr and Baz (Wang, 2006).

In TRAF1 insc double-mutant embryos, the complete segregation of Mir/Pros into future GMCs occurs only in about 12% of the total population, and in the remaining NBs, only a fraction of Mira/Pros segregate into future GMCs as indicated by the Mira 'tail' extending into the future NBs at telophase. As it is difficult to address the global effect of this partial segregation of Mira/Pros on GMC specification in TRAF1 insc double mutant, focus was place on a well-defined GMC, GMC4-2a in NB4-2 lineage, to evaluate this issue. It is assumed that as long as the RP2 neuron (progeny of GMC4-2a, Even-skipped (Eve)-positive) was identified in a particular hemisegment, the GMC cell fate of GMC4-2a in that hemisegment should have been correctly specified. In insc mutants, almost all hemisegments contain RP2s, indicating that GMC4-2a has adopted the correct GMC cell fate in 99% of the total hemisegments. When TRAF1 insc double-mutant embryos were stained with anti-Eve, it was found the frequency of loss of Eve-positive RP2 neuron increased (to 8%) in late embryos, suggesting that about 8% of the GMCs in TRAF1 insc double mutant did not inherit sufficient Pros to specify the GMC fate in these embryos. The relatively low frequency (8%) of mis-specification of GMCs suggests that the threshold amount of Pros protein needed is sufficiently low such that just a partial inheritance of Pros, even when telophase rescue is compromised, is sufficient for most GMCs to be correctly specified (Wang, 2006).

Although Mira/Pros and Pon/Numb share similar basal localization patterns in insc NBs, further removal of either TRAF1 or Egr compromised telophase rescue only for Mira/Pros, but not for Pon/Numb. This difference between Mira/Pros and Pon/Numb indicates that the detailed mechanisms of basal localization and segregation of Mira/Pros differ from those of Pon/Numb, which is consistent with the observations that the dynamics of Mira/Pros and Pon/Numb localization early in mitosis are different and the basal localization for Mira/Pros and Pon/Numb requires different regions of the Insc coding sequence (Wang, 2006).

Dlg/Lgl/Scrib are required for correct basal localization of Mira/Pros and Pon/Numb in mitotic NBs. Dlg has been shown to be involved in the Mira telophase rescue. In dlg insc double-mutant NBs, not only was spindle geometry symmetric but Mira telophase rescue was also affected. It would be interesting to know if Dlg belongs to the same pathway as TRAF1 and Egr and if Dlg is also involved in Pon/Numb telophase rescue (Wang, 2006).

Two other members of the TRAF family have also been identified in Drosophila: DTRAF2 (DTRAF6) and DTRAF3. In contrast to the specific and strong expression of TRAF1 in the embryonic NBs, only low levels of ubiquitous signals similar to the control background were seen in the NBs with DTRAF2 and DTRAF3 probes. It is likely that DTRAF2 and DTRAF3 are not expressed in NBs and do not play an important role in Mira/Pros telophase rescue pathway as the Mira/Pros telophase rescue is dramatically compromised in TRAF1 insc and egr insc NBs (Wang, 2006).

In mammals, the TNF pathway works as a typical receptor-mediated signal transduction pathway. TNFR is a key player in transducing external signal to the cytoplasm. In the Drosophila compound eyes, ectopic Egr, Wgn and TRAF1 seem to work in a similar receptor-mediated signal pathway to induce apoptosis through the activation of the JNK pathway. Does the same Egr, Wgn and TRAF1 receptor-mediated signal pathway play a role in Mira/Pros telophase rescue? If it does, the coexpression of Egr, Wgn and TRAF1 might be expected to be seen in dividing NBs, along with the potential interaction between TRAF1 and the cytoplasmic domain of Wgn. Three observations argue against this hypothesis: (1) wgn is not expressed in embryonic NBs but in the mesoderm. (2) The domain analysis suggests that the Drosophila Wgn cytoplasmic domain is unique with no sequence homology to any mammalian TNFR family members and has neither a TRAF-binding domain nor a death domain, which is required for the interaction between TNFR and TRAF in mammals. (3) More informatively, Wgn knockdown by a UAS head-to-head inverted repeat construct of wgn (UAS-wgn-IR) driven by a strong maternal driver, mata-gal4 V32A, in WT embryos did not affect TRAF1 apical localization. These observations are consistent with the view that the receptor Wgn may not be involved in Mira/Pros telophase rescue or is redundant in this pathway. If this is the case, then how do TRAF1 and Egr function in Mira/Pros telophase rescue? It has been reported that TRAFs associate with numerous receptors other than the TNFR superfamily in mammals. It is speculated that Egr and TRAF1 may adopt an alternative receptor in NBs for Mira/Pros telophase rescue. However, until an anti-Wgn antibody and wgn mutant alleles are available, the possibility that Wgn is involved in Mira/Pros telophase rescue cannot be ruled out (Wang, 2006).

JNK signaling controls border cell cluster integrity and collective cell migration

Collective cell movement is a mechanism for invasion identified in many developmental events. Examples include the movement of lateral-line neurons in Zebrafish, cells in the inner blastocyst, and metastasis of epithelial tumors. One key model to study collective migration is the movement of border cell clusters in Drosophila. Drosophila egg chambers contain 15 nurse cells and a single oocyte surrounded by somatic follicle cells. At their anterior end, polar cells recruit several neighboring follicle cells to form the border cell cluster. By stage 9, and over 6 hr, border cells migrate as a cohort between nurse cells toward the oocyte. The specification and directionality of border cell movement are regulated by hormonal and signaling mechanisms. However, how border cells are held together while they migrate is not known. This study shows that negative-feedback loop controlling JNK activity regulates border cell cluster integrity. JNK signaling modulates contacts between border cells and between border cells and substratum to sustain collective migration by regulating several effectors including the polarity factor Bazooka and the cytoskeletal adaptor D-Paxillin. A role for the JNK pathway is anticipated in controlling collective cell movements in other morphogenetic and clinical models (Llense, 2008).

In an analysis of the mechanisms regulating the expression of puckered (puc), the gene encoding the Drosophila Jun N-terminal kinase (JNK) dual-specificity phosphatase (DSP) (Martin-Blanco, 1998) regulatory sequences (PG2) were uncovered directing its expression to border cells. PG2 expands across the first and second introns of puc, in which the pucB48 insertion is located. This expression is also observed in puc enhancer (pucB48) and protein trap lines (Llense, 2008).

JNKs represent a signaling hub with pivotal functions in cell proliferation, differentiation, and death. JNKs are inactivated by DSPs, and transcriptional induction of DSP expression is well documented as a negative-feedback mechanism. In Drosophila, this loop modulates JNK activity in processes such as epithelial expansion and overexpression of dominant-negative constructs relies on JNK signaling. Further, Puc overexpression leads to inhibition of JNK activity. Thus, Puc implements a negative-feedback loop in border cells (Llense, 2008).

Defects caused by the loss of JNK function in border cells included cluster dissociation and impaired motility. Instead of collectively following a leader cell, JNK-minus border cells autonomously disperse at the late step of migration, with most exhibiting long cellular extensions (LCEs) and actin-rich protrusions. JNK signaling does not affect polar cell specification or border cell recruitment (Llense, 2008).

Dissociation phenotypes are also observed in JNK-specific but not ERK-specific loss-of-function conditions for D-Fos, a major MAPK target, thereby ruling out potential interference via ERK. Indeed, reduced D-Fos suppresses border cell migration defects induced by elevated JNK activity (Llense, 2008).

Does JNK act in a linear pathway or does it target multiple independent effectors simultaneously to produce a multifaceted phenotype? Cells that migrate as part of a group cling firmly to each other while adhering transiently to the substrate. So, during migration, border cells show apicobasal polarity and remain attached to one another and to polar cells. Cell contacts are enriched in the adherens junctions (AJs) components, DE-Cadherin and Armadillo (β-Catenin). In electron microscopy (EM) preparations, border cells are tightly bound, whereas interfaces between border and nurse cells exhibit multiple interdigitations (Llense, 2008).

In JNK-minus conditions, namely after Puc overexpression or in bsk (JNK) clones, cell polarity is disrupted and only remnants of apical markers, such as Bazooka (Baz), are present. Adhesion is impaired, and DE-Cadherin and Armadillo are downregulated. Reduction of JNK activity also resulted in β-Integrin accumulation at ectopic actin-rich protrusions. These also accumulate MyoVI, consistent with its role in force generation. In summary, upon depletion of JNK activity, border cells lose apicobasal polarity and progress into a mesenchymal phenotype. Indeed, EM preparations show that border-border cell contacts are less tight than wild-type cell contacts and cell membranes detach from each other at multiple sites. The end result is a cluster with multiple leading edges and residual cell-cell contacts (Llense, 2008).

How does the JNK pathway become activated in border cells? Rho, Rac, and Cdc42 GTPases are potential candidates. Loss of Rac completely abolishes border cell migration. However, phenotypes for RhoA and Cdc42 expression of dominant-negative forms -- RhoADN and Cdc42DN) closely resemble JNK-minus induced dissociation. Furthermore, in Cdc42DN, polarity, cell contacts, and redistribution of substrate adhesion and motor markers are similarly affected. Most importantly, reporters of JNK activity such as Jun phosphorylation and the expression of the pucB48 transgene are also downregulated. Null cdc42 MARCM clones display the same phenotype, although frequency and penetrancy were very low. Therefore, a role for other GTPases, such as RhoA, in JNK activation cannot be ruled out (Llense, 2008).

Border cell clusters deficient for Baz (BazRNAi) resemble JNK loss of function (which leads to Baz downregulation) and exhibit dissociation and downregulation of DE-Cadherin. Thus, Baz, a critical landmark of epithelial polarity, could serve as an effector for the control of border-border cell contacts. To test this, Baz was overexpressed in cells lacking JNK activity (or expressing Cdc42DN); Baz was strongly rescued cluster integrity and DE-Cadherin expression (Llense, 2008).

Epithelial cells use a specialized repertoire of integrin receptors to mediate contacts and migration. However, border cells lacking β-Integrin were still able to adopt a leading migratory position, although the effect of complete removal of integrins from the cluster has not been reported (Llense, 2008).

Interestingly, β-Integrin antibodies reveal a rosette staining in border cell clusters that colocalize with AJ markers. Thus, β-Integrin could participate in the stabilization or strengthening of cell contacts, as shown for amnioserosa and larval epithelial cells in Drosophila, mammalian keratinocytes, and carcinoma cell clusters. Furthermore, β-Integrin, after JNK inactivation, strikingly accumulates at the front of LCEs suggesting a second function in cell invasiveness, as observed in leukocytes (Llense, 2008).

Direct evidence for β-Integrin involvement in border cell migration was obtained by RNAi in a sensitized JNK-minus condition. The expression of β-Integrin dsRNAs in border cells reduced β-Integrin levels but did not cause migration or integrity defects. However, in the presence of Puc, β-Integrin RNAi led to a strong enhancement of cluster dissociation and prevented the full extension of LCEs, which become mostly blunted. Moreover, an adhesion dominant negative (diβ) integrin chimera showed weak, but reproducible, dissociation phenotypes. Thus, β-Integrin turns out to participate in, first, the stabilization of border-border cell contacts and, second, the promotion of LCEs extension. The integrin countereceptors that facilitate border cell attachment and invasiveness are not yet known (Llense, 2008).

D-Paxillin was present in border cell contacts but was downregulated in JNK-minus conditions. Genomic-profiling analyses of JNK mutants suggests a transcriptional control of D-Paxillin expression. However, other options, such as subcellular relocation after phosphorylation, could also explain why D-Paxillin may be absent from JNK-minus border cells. Expression in border cells of two different D-Paxillin dsRNA lines was found to result in JNK loss-of-function-like dissociation, DE-Cadherin downregulation and β-Integrin accumulation at LCEs. Expression of a Talin RNAi line does not produce any migration phenotype, although it impairs follicle epithelia integrity (Llense, 2008).

In migratory leukocytes, PKA-mediated integrin phosphorylation prevents Paxillin accumulation at the leading front. Paxillin-integrin interactions in lateral positions lead to the inhibition of Rac, whose activation is thus spatially limited to the leading edge where it induces lamellipodia. Consequently, D-Paxillin might stabilize β-Integrin in border-border cell contacts. Its absence, in JNK-minus conditions, would lead in lateral and trailing cells to Rac activation, dissociation of border-border cell contacts, and extension of β-Integrin-rich ectopic lamellipodia. Indeed, the PKA-RII subunit is expressed in border cells, and border cells mutant for PKA show migration defects (Llense, 2008).

Interestingly, D-Paxillin overexpression rescued the border cell defect resulting from loss of JNK activity (or expression of Cdc42DN). DE-Cadherin relocated to border-border cell contacts, and β-Integrin expression was partially eliminated from residual LCEs. D-Paxillin overexpression alone had no effects (Llense, 2008).

It was further asked whether the control of cell polarity and cytoskeletal adaptor proteins by JNK were related. Paxillin expression was strongly reduced in baz mutant conditions, whereas Baz expression was only slightly affected by interference in Paxillin expression (Llense, 2008).

JNK signaling regulates border cells clustering by controlling at least two key elements, cell polarity (Baz) and cytoskeletal adaptor proteins (D-Paxillin), and as a consequence cell-cell contacts and cell-substrate attachments. Interestingly, the overexpression of Hindsight (Hnt), a target and negative regulator of JNK, results in similar defects to those caused by inhibition of JNK. Because re-expression of a variety of proteins (Baz, D-Paxillin, DE-Cadherin, and Armadillo) can rescue the dissociation phenotype and given that each time rescue is achieved, DE-Cadherin and Armadillo expression are restored, a plausible explanation for the effects observed with JNK-minus and Hnt overexpression is that there is an overall loss of multiple cell-cell adhesion complexes. The restoration of any of them would provide sufficient cell-cell adhesion to enable the cluster to move as a collective (Llense, 2008).

The individual migratory abilities of JNK-minus border cells could be partially explained by the observed β-Integrin relocalization to LCEs (border-nurse cell contacts). Alternatively, border cells could have lost their capacity to respond to positional gradients leading to random outward movements. Border cells use PVF and EGF to guide their migration. Blocking PVR and EGFR does not reduce the ability of border cells to extend protrusions but abolishes their directionality, with protrusions now extending in all directions. However, in these conditions, border cell clusters do not dissociate, thereby ruling out the possibility that dissociation in JNK mutants is due only to loss of directional guidance. A directionality index (DI) can be calculated. A DI of 0 indicates equal numbers of protrusions extending forward and backward. A DI of 1 indicates that cells only extend protrusions in the direction of migration. This study found a DI of 0.59 for wild-type clusters. In the absence of JNK, however, clusters show a DI ranging from -0.2 to 0, suggesting that JNK-minus border cells are blind to positional cues. This fact accounts for recently described synergistic effects of JNK and PVR signaling on border cells (Llense, 2008).

This model makes a significant prediction: JNK hyperactivation should increase adhesiveness and eventually block migration. Accordingly, ut was observed that the overexpresssion of a constitutively active form of Hep, the overexpresssion of a constitutively active form of Misshapen, or loss of function clones of puc resulted in nonmigratory and strongly compacted clusters. Occasionally, the death of a number of border cells was observed (Llense, 2008).

So far, the molecular and cellular study of collective versus individual migration both in developmental and cancer models has mainly focused on the analysis of structural elements. The identification of the JNK cascade as a key determinant of migratory responses in border cells could have an important impact in the understanding of collective movements. Border cell migration could serve as a good model for studying migratory transitions, thus impacting on the understanding of cancer metastasis and invasiveness, during which so little is known about the signaling mechanisms controlling migratory behavior (Llense, 2008).

A modifier screen for Bazooka/PAR-3 interacting genes in the Drosophila embryo epithelium

The development and homeostasis of multicellular organisms depends on sheets of epithelial cells. Bazooka (Baz; PAR-3) localizes to the apical circumference of epithelial cells and is a key hub in the protein interaction network regulating epithelial structure. This study sought to identify additional proteins that function with Baz to regulate epithelial structure in the Drosophila embryo. The baz zygotic mutant cuticle phenotype could be dominantly enhanced by loss of known interaction partners. To identify additional enhancers, molecularly defined chromosome 2 and 3 deficiencies were screened. 37 deficiencies acted as strong dominant enhancers. Using deficiency mapping, bioinformatics, and available single gene mutations, 17 interacting genes encoding known and predicted polarity, cytoskeletal, transmembrane, trafficking and signaling proteins, where identifed. For each gene, their loss of function enhanced adherens junction defects in zygotic baz mutants during early embryogenesis. To further evaluate involvement in epithelial polarity, GFP fusion proteins were generated for 15 of the genes which had not been found to localize to the apical domain previously. It was found that GFP fusion proteins for Drosophila ASAP, Arf79F, CG11210, Septin 5 and Sds22 could be recruited to the apical circumference of epithelial cells. Nine of the other proteins showed various intracellular distributions, and one was not detected. This enhancer screen identified 17 genes that function with Baz to regulate epithelial structure in the Drosophila embryo. A secondary localization screen indicated that some of the proteins may affect epithelial cell polarity by acting at the apical cell cortex while others may act through intracellular processes. For 13 of the 17 genes, this is the first report of a link to baz or the regulation of epithelial structure (Shao, 2010).

Microarray data indicate that all 17 genes are expressed in early Drosophila embryos. mRNA localization data from the Berkeley Drosophila Genome Project and Flyfish show that some of them are expressed in distinctive patterns. During cellularization and early gastrulation, CG30372 is expressed as a wide central band ending at the anterior and posterior termini of the embryo, while fj is expressed in two stripes that appear to overlap the two ends of the CG30372 band -- these are interesting patterns given the role of the anterior-posterior patterning system in controlling Baz planar polarization and cell intercalation at gastrulation. asp mRNA is apical in both epithelial cells and in neuroblasts, similar to Baz mRNA and protein localization. CG5823, rho1, par-1, sds22 and hk mRNAs have ubiquitous expression at cellularization. CG1951, CG11210 and roc2 mRNAs are also in all cells at cellularization but are excluded from the apical domain. mRNA localization data was not available for the six other interacting genes (cul-5, arf79f, sep5, muskelin, alt and CG10702) (Shao, 2010).

The genetic interactions identified in the screen appear to be especially important for regulating dynamic epithelia. AJ disruption was observed in both the amnioserosa and the neurectoderm. These tissues have specific demands for AJ remodeling. During gastrulation, the amnioserosa undergoes a transition from a columnar epithelium into a flattened squamous epithelium. The flattening of these cells greatly enlarges their circumferences and Baz has been shown to regulate AJ remodeling as this occurs. In the neurectoderm, a reduction of DE-cad leads to loss of AJs because of dynamic AJ remodeling associated with neuroblast delamination. Recently, Cdc42, PAR-6, aPKC and Baz have been shown to indirectly stabilize neurectoderm AJs by controlling the trafficking of Crb. The proteins this study has implicated appear to directly or indirectly affect AJs during these processes as well. The partial tissue specificity observed may reflect separable regulatory networks important for AJ positioning in each tissue. Based on localization studies, many of the proteins could act directly in the apical domain while the others may impact apical polarity indirectly from various intracellular sites (Shao, 2010).

In the pilot screen it was found that reduction of apkc or crb substantially enhances the baz mutant cuticle phenotype. The deficiency screen also found genetic interactions with par-1 and fj. Baz/PAR-3 is known to interact with aPKC in a complex with PAR-6 to regulate cell polarity in many contexts. In the follicular epithelium, PAR-1 has been shown to localize the basolateral membranes where it phosphorylates and inhibits Baz to maintain apical Baz polarity. Similarly, knock-down of PAR-1 in the early embryo leads to abnormal spreading of AJs in the apicolateral region, but in embryonic epithelia PAR-1 is enriched in the apicolateral region versus the basolateral domain (Shao, 2010).

Although direct links between Baz and the planar polarity regulator fj have not been made, Baz localizes in a planar polarized pattern during germband extension. Germband extension occurs independently of the canonical planar polarity genes Frizzled and Dishevelled, but other planar cell polarity genes, such as fj, have not been tested. fj has an intriguing striped mRNA expression pattern at this stage, suggesting a link to the A-P patterning system, which regulates planar polarity in the tissue. Fj is a golgi-associated protein, consistent with localization data, which can phosphorylate transmembrane proteins en route to the plasma membrane. Thus, Fj may affect the apical domain via transport from the Golgi (Shao, 2010).

Intracellular membrane trafficking plays a central role in controlling epithelial cell polarity. More specifically, Baz/PAR-3 and its interaction partners PAR-6 and aPKC have been implicated in regulating the endocytosis of apical proteins and AJs in Drosophila epithelia and to impact general endocytic traffic in C. elegans. Thus, genes implicated in trafficking were assorted by gene ontogeny. A number of additional proteins were found that appear to localize to intracellular compartments. Three of these localized to the apical cortex as well (Shao, 2010).

We found that Arf79F and CG30372 can localize to the apical domain. Generally, Arfs function in the formation and targeting of vesicles in the cell. Arf79F is the Drosophila version of Arf1 and has been implicated in lipid droplet transport and the regulation of the apical domain during Drosophila rhabdomere formation. Intriguingly, CG30372, encodes a putative ArfGAP. Although not characterized in Drosophila, CG30372 has a similar domain structure to the ASAP proteins (Arf GAPs with Src homology 3, ankyrin repeat, and pleckstrin homology domains), which have been implicated in the regulation of actin and endocytosis. It will be interesting to test whether Arf79F and Drosophila ASAP interact to regulate epithelial structure. Of note, CG11210::GFP has a similar distribution as Arf79F, localizing to the apical cortex and intracellular compartments. CG11210 is an uncharacterized protein predicted to have 10–11 transmembrane helices. It is hypothesized that these proteins may co-ordinate membrane trafficking with the apical cortex (Shao, 2010).

Five other proteins localized to intracellular compartments without apparent cortical localization. As discussed, Fj appears to localize to the Golgi. CG1951, an uncharacterized kinase, appears to localize to scattered small vesicles. Alt, CG5823 and CG10702 appear to localize to ER membranes. Alt is functionally uncharacterized, but displays some sequence similarity with Myosins and MT associated proteins CLP190 and NUMA (BLAST search) and has been co-fractionated with lipid droplets from early embryos. CG5823 has been implicated in ubiquitination, and CG10702 is a predicted receptor tyrosine kinase. hk localizes to intermediate sized vesicles consistent with past localization studies in other Drosophila cell types and hk's role in trafficking to the multivesicular body. These five proteins might affect cell polarity through intracellular trafficking (Shao, 2010).

The cytoskeleton also plays a major role in regulating epithelial structure. The screen found that rho1, sep5 and asp genetically interact with baz. Rho1 localizes to the apical domain and other parts of Drosophila embryonic epithelia, and has been shown to have a general role in regulating epithelial structure. It was also found that Sep5 can be recruited to the apical domain. In mammalian cells, Septin 2 has been shown to regulate AJs. Asp functions at the centrosomes to control the structure of the mitotic spindle. At stage 15, Asp::GFP was detected in linear parallel arrays consistent with the organization of MTs in these cells. Thus, it is speculated that Asp affects cell polarity via MTs. Muskelin is functionally uncharacterized, but contains kelch motifs found in cytoskeletal and other proteins. However, Muskelin::GFP localized diffusely through the cytoplasm (Shao, 2010).

It was found that Sds22 localizes to the apical domain of embryonic epithelial cells. Sds22, a regulatory subunit of protein phosphatase 1 (PP1), has recently been linked to regulation of cell shape and apical-basal polarity in Drosophila imaginal disc and follicular epithelia, where a GFP-tagged form of Sds22 localized to the cytoplasm and nucleus. Sds22 binds to all four Drosophila PP1 isoforms, and sds22 phenotypes correlated with elevated phosphorylation of Myosin regulatory light chain and Moesin. Intriguingly, PP1alpha has been shown to de-phosphorylate PAR-3 and affect tight junction formation in mammalian cell culture (Shao, 2010).

Cul-5, Roc2 and CG5823 are involved in protein ubiquitination. PAR-3 has been shown to interact with a ubiquitin ligase in the generation of neuronal polarity and ubiquitination has also been linked to polarized cell protrusion. Cul-5 regulates the neuromuscular junction in Drosophila, but roles for Cul-5, Roc2 and CG5823 in epithelial structure have not been described. Intriguingly, Roc2 and Cul-5 form a complex in Drosophila. Perhaps this complex supports epithelial structure by down-regulating inhibitors of the apical domain (Shao, 2010).

In this screen, attempts were made to identify additional proteins that function with Baz to regulate epithelial structure in the Drosophila embryo. From the 655 possible interacting genes identified through deletion screening and mapping, gene ontogeny terms were used to select genes with possible functions in polarity, the cytoskeleton, membrane trafficking or signaling, as well as transmembrane proteins. This was done based on the known roles for Baz/PAR-3 in controlling cell structure at the cortex, but the approach would miss novel Baz functions and interactions with genes with unknown function or that are unique to Drosophila. Nonetheless, 13 of the 17 genes implicated by the screen have not been previously shown to interact with Baz or to affect epithelial structure, and thus should be of interest for future studies (Shao, 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).

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, 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. 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, it was 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 under the heatshock promoter was tested, 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).

Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors

Photoreceptor morphogenesis in Drosophila requires remodelling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. This study shows that overexpression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodelling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant (Muschalik, 2011).

Strikingly, CrbFLAGintra can only affect photoreceptor cell (PRC) shape and adhesion when expressed during late larval and early pupal development. During this period, PRCs undergo substantial morphogenetic changes to adopt their final shape. It is noteworthy that the epithelial cells of the imaginal disc are already well polarised, with an elaborated ZA encircling the apices of the cells. Therefore, the transition from a larval epithelial cell into the highly modified PRC does not require establishment of polarity, but rather mechanisms that control remodelling of polarity and AJs. This study shows that early expression of CrbFLAGintra interferes with this process. Similar conclusions were drawn from studies in the Malpighian tubules, where proper Crb levels are essential for maintenance of polarity and epithelial integrity only during the process of tube elongation, which depends on major cell rearrangements. Once most of the morphogenetic changes and remodelling of the ZA have been completed, PRCs are less susceptible to elevated CrbFLAGintra levels. This is reflected by the observation that cells in which the intracellular domain of Crb is expressed during late pupal development and in the adult, exhibit a normal polarised shape, although junctional and polarity proteins are severely mislocalised in these cells. Two explanations might account for this difference. First, the apical and basolateral membrane domain, as well as the ZA, might be more stable at later stages, so that ectopic apical and junctional components recruited by CrbFLAGintra are unable to affect apico-basal polarity and AJs. Second, some of the downstream factors required for ectopic apical pole formation might no longer be available at later stages. In fact, Baz is removed from the ZA at ~60% of pupal development and becomes enriched in the rhabdomere, similar to aPKC. Furthermore, Par-6 can be found at the basolateral membrane in adult PRCs. Although the polarised shape is unaffected, PRCs overexpressing CrbFLAGintra during later stages display defects in ZA positioning and show an increase in stalk membrane length, the development of which is regulated by crb (Muschalik, 2011).

Loss-of-function studies show that crb is not required for the development of an apical pole, yet, as shown in this study, overexpression of its cytoplasmic tail is sufficient to induce formation of ectopic apical membranes. This raises the question of how ectopic apical poles develop under these conditions. The results, from localisation studies and genetic interactions, indicate that, once initiated, development of an ectopic apical membrane domain relies on the same events and requires identical components to those required for formation of the original apical domain. It is suggested that CrbFLAGintra assembles a new Crb-dependent membrane-associated protein platform at the basolateral membrane domain, enabling the recruitment of effector proteins essential to develop apical features. One of these is βH-spec, which might stabilise the CrbFLAGintra complex by linking it to the underlying spectrin-based membrane skeleton. In fact, removal of one copy of kst strongly suppresses the overexpression phenotype and F-actin accumulates at CrbFLAGintra-positive membranes. In addition, the actin-based cytoskeleton is likely to be directly involved in the formation of ectopic rhabdomeres, as rhabdomeres are composed of microvilli and the terminal web, both of which are actin-rich structures (Muschalik, 2011).

In addition to βH-spec, Par-6 and aPKC are also recruited into the CrbFLAGintra complex and both are required to mediate the CrbFLAGintra-induced overexpression phenotype, as demonstrated by genetic interactions. Furthermore, by using different hypomorphic alleles of aPKC, the function of aPKC in this process could be shown to depend on its ability to bind Par-6 and the presence of an intact kinase domain. In the embryonic epidermis, aPKC ensures apical identity by phosphorylation of the tumour suppressor Lgl, thereby excluding it from the apical domain and restricting its activity to the basolateral side of the cells. Lgl, in contrast, prevents Baz from promoting apical membrane characteristics basolaterally. It is proposed that, upon overexpression of CrbFLAGintra, Lgl is removed from CrbFLAGintra-positive sites through phosphorylation by aPKC, which weakens basolateral membrane identity. The observation that other basolateral markers are absent from ectopic rhabdomeres and diminished at membranes surrounding ectopic rhabdomeres supports this assumption. Furthermore, removal of Lgl from the basolateral membrane upon overexpression of CrbFLAGintra would be consistent with the finding that the lgl loss-of-function phenotype of PRCs mimics the CrbFLAGintra overexpression phenotype. This is similar to the situation in Drosophila embryonic epithelia, and suggests that there is a conserved mechanism for both cell types. Moreover, it might explain why lowering the dose of lgl does not cause an enhancement of the overexpression phenotype. By contrast, an enhancement was found with yrt, which negatively regulates Crb activity, demonstrating that the experimental approach is suitable for the identification of enhancers. Besides Lgl, aPKC also phosphorylates Baz, as shown in the Drosophila follicle epithelium, the embryonic epidermis and PRCs. Phosphorylation of Baz is required to exclude it from the apical membrane, thereby restricting AJs to more basal positions. Apical exclusion of Baz also requires Crb, which prevents binding of Baz to Par-6. It is suggested that the following scenario occurs upon CrbFLAGintra overexpression. First, removal of Lgl from the basolateral membrane enables Baz to spread basolaterally. However, under these conditions, Baz becomes immediately excluded from CrbFLAGintra-positives sites by the same mechanisms occurring at the original apical domain. Delocalisation of Baz, in turn, affects AJs and alters the adhesive properties of the cells, as Baz localisation defines the position of the ZA. The model is consistent with observations from genetic interactions, which have shown that simultaneous expression of CrbFLAGintra and a non-phosphorylatable version of Baz (GFP-Baz-S980A) strongly suppressed the CrbFLAGintra overexpression phenotype. This suppression could be the result of Baz S980A either binding to aPKC-Par-6, or to Sdt, therefore preventing aPKC-Par-6 or Sdt from binding to CrbFLAGintra. Alterations in PRC adhesion might also explain the disruption of the basal lamina and the elimination of PRCs. As no obvious decrease in cell number was noticed at 45-55% of pupal development, elimination is likely to occur during late pupal development (Muschalik, 2011).

Formation of distinct membrane domains also requires polarised protein trafficking. The ectopic localisation of Rh1 and Spam (Eyes shut) upon overexpression of CrbFLAGintra during late larval and pupal development suggests that the apical secretory machinery becomes reorganised under these conditions. In Drosophila PRCs, delivery of various apical proteins, including Rh1, depends on the small GTPase Rab11 and the exocyst component Sec6. A redistribution of these proteins upon overexpression of CrbFLAGintra in developing PRCs might account for the delivery of apical transport vesicles to CrbFLAGintra-positive membranes, which facilitates the formation of a second apical pole. In case of cells with reversed apico-basal polarity the majority of apical vesicles might be targeted to the ectopic apical pole so that the original apical membrane domain receives only minor amounts of apical proteins, with it eventually adopting basolateral membrane identity (Muschalik, 2011).

Another crucial component in polarised vesicle delivery and targeting are phosphoinositides. In developing Drosophila PRCs, PtdIns(3,4,5)P3 is enriched at the apical membrane, whereas PtdIns(4,5)P2 predominantly localises at the ZA. Studies in MDCK (Madin-Darby canine kidney) cells have shown that ectopic localisation of either of the above two phosphoinositides is sufficient to cause a switch from one membrane identity to the other. Strikingly, Baz recruits the lipid phosphatase PTEN (phosphatase and tensin homolog) to the AJs of PRCs and embryonic epidermal cells, and Baz is delocalised upon CrbFLAGintra expression in pupal PRCs. Mutations in, or overexpression of, PTEN cause severe morphogenetic defects, including loss of PRCs and absence or splitting of rhabdomeres, phenotypes that are also observed upon overexpression of CrbFLAGintra. Given these data, it is tempting to speculate that ectopic CrbFLAGintra and its associated proteins cause a modification in the lipid composition of the basolateral membrane domain, thereby remodelling the polarity of PRCs (Muschalik, 2011).

A Cdc42-regulated actin cytoskeleton mediates Drosophila oocyte polarization

Polarity of the Drosophila oocyte is essential for correct development of the egg and future embryo. The Par proteins Par-6, aPKC and Bazooka are needed to maintain oocyte polarity and localize to specific domains early in oocyte development. To date, no upstream regulator or mechanism for localization of the Par proteins in the oocyte has been identified. This study analyzed the role of the small GTPase Cdc42 in oocyte polarity. Cdc42 was shown to be required to maintain oocyte fate, which it achieves by mediating localization of Par proteins at distinct sites within this cell. Cdc42 localization itself is polarized to the anterolateral cortex of the oocyte, and Cdc42 is needed for maintenance of oocyte polarity throughout oogenesis. The data show that Cdc42 ensures the integrity of the oocyte actin network and that disruption of this network with Latrunculin A phenocopies loss of Cdc42 or Par protein function in early stages of oogenesis. Finally, it was showm that Cdc42 and Par proteins, as well as Cdc42/Par and Arp3, interact in the context of oocyte polarity, and that loss of Par proteins reciprocally affects Cdc42 localization and the actin network. These results reveal a mutual dependence between Par proteins and Cdc42 for their localization, regulation of the actin cytoskeleton and, consequently, for the establishment of oocyte polarity. This most likely allows for the robustness in symmetry breaking in the cell (Leibfried, 2013).

The findings show that Cdc42 is required for oocyte polarity throughout oogenesis. The following findings were made: (1) Cdc42 localizes to the anterolateral cortex of the young oocyte; (2) Cdc42 interacts with Par proteins in the germline in vivo; (3) mutants for Cdc42, aPKC or Baz display a disrupted actin cytoskeleton at the anterolateral cortex; and (4) disrupting the actin cytoskeleton with Latrunculin A results in loss of anterior-to-posterior movement of the oocyte-specific protein Orb, phenocopying loss of Cdc42 or the Par proteins. Thus, the cortical actin cytoskeleton is crucial for the establishment of oocyte polarity (Leibfried, 2013).

This is in line with previous observations linking the actin cytoskeleton and Par proteins in the generation of cell polarity. Loss of Baz results in an increase in actin protrusions in Drosophila epithelia and a decrease in actin at synapses. In C. elegans, active CDC-42 localizes to the anterior during the polarity maintenance phase, when it is important for PAR-6 localization, and the anterior actin cap is depleted in par-3 mutants during polarity establishment. Similar to the current observations, actin depolymerization does not affect Par protein localization in C. elegans. By contrast, drug-induced actin depolymerization has been shown to disrupt Baz apical localization during cellularization and to interfere with its cortical association during gastrulation in Drosophila (Leibfried, 2013).

Although the molecular relationship between Par proteins and actin has not been clearly delineated, in mammals Par-3 (Pard3) associates with actin regulators, including the RacGEF Tiam1 and LIM kinase 2. In the current study, it was shown that Cdc42 localization depends on the Par complex and that Cdc42, aPKC, Baz and Par-6 interact in vivo in biochemical or genetic assays. This interaction is required for oocyte polarity. Par-6 interacts biochemically with Cdc42 and Baz via its semi-CRIB and PDZ domains and via its PB1 domain with the PB1 domain of aPKC. Baz interacts biochemically with the kinase domain of aPKC. Indeed, a quaternary complex of Myc-Cdc42, HA-Par-6b, PKCτ/λ and Par-3 can be isolated from transfected COS-7 cells. In Drosophila, a Baz mutant lacking its aPKC-interaction domain supports early oogenesis and an aPKC mutant that cannot bind Par-6 also develops late egg chambers. Together, these results and the current data indicate that interaction of Cdc42, Par-6, aPKC and Baz is required for their correct function in the germline, and that the binding of aPKC to either Par-6 or to Baz is sufficient to ensure this interaction, highlighting the role of all three Par proteins in actin regulation via their interaction with Cdc42. This quaternary relationship seems important for the regulation of polarity establishment, whereas studies in mature epithelial cells have delineated separate functions of Par-6/aPKC/Cdc42 and Baz for polarity maintenance (Leibfried, 2013).

Early oocyte polarity and its maintenance were previously linked to the microtubule network. Microtubules play an important role in early oogenesis, as their disruption with Colchicine leads to a 16-nurse-cell phenotype. Indeed, oocyte specification depends on the accumulation of the oocyte-specific protein BicD, which is a component of the microtubule-related dynactin complex (Leibfried, 2013).

The results point to a sequential involvement of actin and microtubules in polarizing the oocyte: in the early stages, after oocyte specification, the Par proteins together with Cdc42 establish cortical domains and a pronounced cortical actin cytoskeleton. The interdependence of these proteins for their localization persists during oogenesis, allowing for robustness of symmetry breaking. At later stages, knockdown of Cdc42 results in reduced amounts of Baz and Par-1 at the anterior and posterior of the oocyte, respectively. As Par-1 is required for microtubule organization, this most likely leads to the observed mislocalization of axis determinants. Similarly, disrupting the actin cytoskeleton with drugs or by knockdown of actin-binding proteins has been shown to result in bundling of microtubules and premature ooplasmic streaming, leading to loss of oocyte polarity. Hence, microtubules act in oocyte specification and late polarity events, whereas Cdc42 and actin dominate in the establishment and maintenance of polarity in the developing oocyte (Leibfried, 2013).

Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells

Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells

Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. This study reassesses this issue by comparing the phenotypes of the commonly used baz4 and baz815-8 alleles with those of the so far uncharacterized bazXR11 and iEH747 null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, strong discrepancies were observed in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in bazEH747 and bazXR11 while baz4 and baz815-8 show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz4 and baz815-8 alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type (Shahab, 2015).


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bazooka: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 August 2018

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