bazooka
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).
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).
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).
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, our 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).
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).
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).
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).
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).
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 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).
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).
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).
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).
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 n