atypical protein kinase C
To see where aPKC is expressed during embryonic development, the mRNA distribution was analyzed by RNA in situ hybridization. aPKC mRNA is already detectable in freshly laid eggs before the onset of zygotic transcription and thus must be deposited maternally during oogenesis. At the cellular blastoderm stage, aPKC mRNA is present in all cells except for the pole cells. During gastrulation, strong expression of aPKC is detectable in tissues that undergo morphogenetic movements; e.g., the invaginating mesoderm, the proctodeum, and the cephalic furrow. In embryos at the extended germ band stage, prominent aPKC expression is detectable in neuroblasts. In several epithelial tissues, in particular in the fore- and hind-gut and in the Malpighian tubules, aPKC mRNA is highly enriched in the apical cytocortex, reminiscent of the polarized localization of baz and crumbs (crb) mRNAs (Wodarz, 2000).
The distribution of aPKC protein was analyzed using anti-PKCzeta antibody C20. During cellularization of the embryo, aPKC becomes localized to the apical cytocortex of all cells except for the pole cells, which do not contain detectable amounts of aPKC. Already at this stage aPKC is enriched at apico-lateral cell borders, giving rise to a honeycomb pattern in en face views of the blastoderm. After completion of cellularization, DaPKC is highly concentrated in the apico-lateral cortex and shows little overlap with the basolateral marker Nrt. Apical localization is maintained throughout embryonic development in most epithelia that are derived from the ectoderm (e. g. epidermis, fore-, and hind-gut), Malpighian tubules, and the tracheal system. The only ectodermal epithelium devoid of DaPKC expression is the amnioserosa (Wodarz, 2000).
Strong expression of DaPKC is also detected in neuroblasts, the stem cells of the embryonic CNS. During delamination of neuroblasts, aPKC is localized in the apical stalk that is wedged between adjacent cells of the neuroectodermal epithelium. In pro- and meta-phase, aPKC forms apical cortical crescents. In anaphase, aPKC staining is strongly diminished and expands over a broader region of the neuroblast cortex, but is clearly excluded from the budding ganglion mother cell. Thus, from delamination through pro- and meta-phase, localization of DaPKC in neuroblasts is very similar to that of Baz and Insc. Simultaneously with aPKC, cell outlines of epithelial cells and neuroblasts were visualized with the Nrt antibody. Similar to epithelial cells, Nrt expression is clearly polarized in neuroblasts. Strong staining for Nrt is detectable in the basal and lateral membrane of neuroblasts, whereas staining is strongly reduced in apical regions where DaPKC is expressed (Wodarz, 2000).
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 as 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).
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).
An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. The Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. It is concluded that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation (Lee, 2006a).
Some brat mutant neuroblasts show expanded aPKC cortical crescents, in some cases reaching the basal cortex. This phenotype appears specific for aPKC, because other apical cortical proteins (e.g., Baz, Pins) are unaffected. Brat might repress aPKC translation, leading to increased aPKC protein levels in brat mutants. Alternatively, the absence of Prospero or other basal cortical proteins may indirectly affect aPKC localization (Lee, 2006a).
What is the cellular origin of the brat mutant phenotype? brat mutant GMCs are compromised in three ways: they lack Brat translational repression activity, lack Prospero, and some may have ectopic aPKC. Loss of Brat translational repression activity could well play a role in the ectopic neuroblast self-renewal phenotype, because all brat mutants disrupting the NHL translational repression domain exhibit a brain tumor phenotype, and Brat has been previously shown to negatively regulate cell growth. Loss of Prospero also plays a role in the brat phenotype: prospero mutant GMCs have a failure to downregulate neuroblast gene expression and a failure in neuronal differentiation, similar to brat mutants. prospero null mutant embryos also show a slight delay in neuronal differentiation, although they appear to undergo normal neuroblast self-renewal. Finally, ectopic aPKC can also mimic aspects of the brat phenotype, including formation of supernumerary large Dpn+ neuroblasts. Interestingly, the mammalian paralogs of Drosophila aPKC (aPKCλ/ζ) are expressed in neural progenitors of the ventricular zone, and the mammalian Prospero ortholog Prox1 is expressed in differentiating neurons of the subventricular zone. Thus, identifying Prospero transcriptional targets and aPKC phosphorylation targets may provide further insight into the molecular mechanism of neural stem cell self-renewal in both Drosophila and mammals (Lee, 2006a).
Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. Atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, a balance of microtubule and actin interactions was found to control AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost (Harris, 2007).
This study reveals the roles of aPKC during polarity establishment and elaboration in Drosophila embryos. In contrast to C. elegans, aPKC is not critical during initial polarity establishment; Baz and AJs are initially localized correctly and the embryonic epithelium can undergo initial morphogenesis. However, aPKC plays an early and striking role in maintaining the symmetrical organization of AJs, via effects on MT organization, and also plays an important later role in the elaboration of polarity (Harris, 2007).
aPKC's later role in polarity elaboration may reflect effects on multiple targets. aPKC is critical for the cortical localization of its normal binding partner PAR-6 and the apical determinant Crb. This latter effect is consistent with the fact that aPKC can phosphorylate Crb, and disruption of aPKC phosphorylation sites in Crb destabilizes Crb in the apical domain. Since Crb stabilizes AJs after gastrulation, this likely contributes to the eventual AJ breakdown in apkcm/z mutants. Crb may act in concert with PAR-6 or in parallel. aPKC can also phosphorylate and exclude the basolateral cue Lgl from the apical domain, and consequenct failure to exclude Dlg from the apical domain in apkcm/z mutants. Thus, apical invasion of basolateral cues may also contribute to the eventual loss of epithelial polarity in apkcm/z mutants (Harris, 2007).
However, it is unlikely that these global changes in apical-basal cell polarity are responsible for the early clustering of AJs into planar-polarized puncta in apkcm/z mutants. Indeed, most of these other polarity players affect polarity after gastrulation. crb mutants have normal spot junctions during gastrulation and early germband extension. Lgl and Dlg are not normally excluded from the apical domain until after gastrulation. Similarly, while mammalian aPKC can restrict PAR-1 to the basolateral domain of epithelial cells, Drosophila PAR-1 is not normally excluded from the apical domain at gastrulation. Thus, effects on Crb, Lgl/Dlg, and PAR-1 cannot easily account for the focusing of AJs and Baz into discrete planar-polarized puncta as apkcm/z mutants gastrulate (Harris, 2007).
Instead, the data suggest that aPKC regulates AJ symmetry by regulating MTs. MT regulation may be a common aPKC function. For example, Drosophila aPKC promotes MT stability at synaptic boutons of neuromuscular junctions, where aPKC forms a complex with Futsch (a MAP1B-like protein) and tubulin, recruiting Futsch to boutons to stabilize MTs. aPKC also regulates MT orientation as mammalian astrocytes and fibroblasts undergo directed migration during wound healing, while in MDCK cells, aPKC helps organize the MT cytoskeleton during ciliogenesis (Harris, 2007).
MT organization and reorganization play important roles in epithelial morphogenesis, and the data demonstrate that loss of aPKC disrupts these events. During Drosophila cellularization, strong MT nucleation from apical centrosomes is likely necessary for assembling lateral MTs that support the apical transport of lipids and proteins to form cell membranes. These MTs also help direct the initial apical positioning of AJs and Baz. During later development, the analysis of apkcm/z mutants indicates that centrosomal MTs can affect the symmetric positioning of AJs around the apical domain. Without aPKC activity, the centrosomes become abnormally dominant, bipolar cues, directing AJ clustering and thus disrupting AJ symmetry. Although this abnormal MT organization differs from changes to MT organization observed in AJ mutants, the possibility cannot be ruled out that there is feedback between MTs and AJs during epithelial morphogenesis and that aPKC may regulate these interactions. Indeed, such feedback is very likely and it will be critical to define MT-AJ cross talk mechanisms in future studies (Harris, 2007).
In apkcm/z mutants, MT-associated AJ/Baz puncta assemble at the dorsal and ventral sides of the cells. This suggests that MTs may normally function in AJ assembly at these newly formed cell-cell contacts. However, these polarized AJ assembly events must also be counterbalanced to maintain AJ symmetry and proper epithelial structure. Cytoskeletal inhibitor studies suggest that AJ symmetry may normally be regulated by a balance of MT-AJ and actin-AJ interactions at this stage -- actin appears to counteract MT-based AJ assembly at dorsal and ventral cell contacts. Actin as been shown to be enriched at anterior and posterior cell contacts, suggesting that it may be an early planar polarity cue at this stage. Perhaps this planar-polarized actin stabilizes a pool of AJs at anterior and posterior cell contacts, thereby counterbalancing MT-based AJ assembly at dorsal and ventral contacts. Alternatively, lower levels of actin at dorsal and ventral cell contacts could directly counteract MT-based AJ assembly at these sites. Distinguishing these possibilities requires further study. Nonetheless, the apkcm/z mutant phenotype appears to arise from a gain-of-function effect in which MTs become overactive and the proper balance between MT-AJ and actin-AJ interactions is lost. As a result, there is a break in AJ symmetry in apkcm/z mutants, MT-associated AJ puncta eventually become randomly positioned, and the epithelium dissociates (Harris, 2007).
The data suggest a speculative mechanistic model by which aPKC could normally regulate MT-AJ interactions. This study shows that MT association is responsible for the abnormal AJ asymmetry seen in apkcm/z mutants, and that Dynein accumulates at these abnormal AJ/Baz puncta. Since Dynein plays a role in apical transport of AJ/Baz proteins during cellularization, it is proposed that aPKC may normally regulate release of AJ/Baz complexes from Dynein, allowing a complete transport cycle. In the absence of this release, AJ/Baz complexes could maintain an abnormal association with MTs, and localized Dynein activity may pull the centrosomes and spot junctions together into the abnormal puncta seen in apkcm/z mutants. This abnormal cortical Dynein activity might also stabilize MTs emanating from the centrosomes, resulting in the persistent centrosomal MTs in the mutants. Alternatively, aPKC may function at the centrosomes to decrease MT nucleation or increase MT severing. Future experiments will illuminate these mechanisms and the generality of aPKC's role in controlling MT organization and AJ positioning (Harris, 2007).
Apical constriction is a major mechanism underlying tissue internalization during development. This cell constriction typically requires actomyosin contractility. Thus, understanding apical constriction requires characterization of the mechanics and regulation of actomyosin assemblies. This study analyzed the relationship between myosin and the polarity regulators Par-6, aPKC and Bazooka (Par-3) (the PAR complex) during amnioserosa apical constriction at Drosophila dorsal closure. The PAR complex and myosin accumulate at the apical surface domain of amnioserosa cells at dorsal closure, the PAR complex forming a patch of puncta and myosin forming an associated network. Genetic interactions indicate that the PAR complex supports myosin activity during dorsal closure, as well as during other steps of embryogenesis. It was found that actomyosin contractility in amnioserosa cells is based on the repeated assembly and disassembly of apical actomyosin networks, with each assembly event driving constriction of the apical domain. As the networks assemble they translocate across the apical patch of PAR proteins, which persist at the apical domain. Loss- and gain-of-function studies show that different PAR complex components regulate distinct phases of the actomyosin assembly/disassembly cycle: Bazooka promotes the duration of actomyosin pulses and Par-6/aPKC promotes the lull time between pulses. These results identify the mechanics of actomyosin contractility that drive amnioserosa apical constriction and how specific steps of the contractile mechanism are regulated by the PAR complex (David, 2010).
The repeated assembly and disassembly of apical actomyosin networks is an integral part of amnioserosa tissue morphogenesis during DC. Restricting myosin to the amnioserosa alone is sufficient for amnioserosa apical constriction and overall DC. Dynamic apical myosin has been described in the amnioserosa. This study defined these dynamics as repeated assembly and disassembly cycles of actomyosin networks. Moreover, assembly and disassembly are linked to apical constriction and relaxation, respectively. This is consistent with laser ablation studies showing that the apical surfaces of amnioserosa cells maintain tension across the tissue. Moreover, AJ live imaging has revealed general pulsing of amnioserosa cells from germband retraction through DC. The pulsing actomyosin networks arise with this same developmental timing. A 230±76 second periodicity of cortical pulsing has been described at DC, similar to that of the pulsing actomyosin networks. This study found that increased network durations and decreased lull times with amnioserosa-targeted Baz overexpression coincide with faster DC, as compared with amnioserosa-targeted Par-6 plus aPKC-CAAX overexpression, which increases lull times. It is concluded that the pulsing actomyosin networks mediate the constriction of individual amnioserosa cells and that this contributes to DC (David, 2010).
Remarkably, a single amnioserosa apical constriction event is followed by an almost equal relaxation. However, over many constrictions the cells progressively reduce their apical surface area. This suggests that ratcheting mechanisms incrementally harness the constrictions for overall tissue change. Intracellular and extracellular ratchets are possible. Cells of the Drosophila ventral furrow also display pulsed contractions of apical actomyosin networks as they apically constrict. However, there is minimal relaxation after each constriction. Instead, residual myosin filaments are retained between pulses, and may act as intracellular ratchets to harness the pulsed contractions. By contrast, residual myosin filaments were rarely observed between actomyosin pulses in amnioserosa cells, possibly explaining their relaxation after each cell constriction. It has been proposed that the leading edge actomyosin cable of the surrounding epidermis acts as an extracellular ratchet to harness amnioserosa contractility. However, the ability of myosin expression in the amnioserosa alone to drive DC suggests that other mechanisms contribute. Indeed, DC is a robust process with redundant contributions from both amnioserosa and epidermis. At later stages, filopodia-based epidermal zippering at the canthi could provide another extracellular ratchet. In addition, each amnioserosa cell has a persistent circumferential actin belt that might act as an intracellular ratchet, and other uncharacterized processes, such as membrane trafficking or basal activities, could also contribute (David, 2010).
Actomyosin activity also appears to be linked between cells. The networks display preferential D-V movement, and a network in one cell appears to promote network formation in neighbors. Overall amnioserosa cell shape changes are also coordinated between neighbors. Moreover, myosin activity in isolated amnioserosa cells can elicit cortical myosin accumulation in neighboring epidermal cells. It is speculated that feedback from epidermal cells might orient the D-V movement of amnioserosa actomyosin networks. Interestingly, amnioserosa cells also preferentially contract along the D-V axis. Although the actomyosin networks move in this direction, it is unlikely that they are solely responsible for the directional cell shape changes -- the networks affect the cell circumference both along the axis of their trajectory and perpendicular to it, and, as discussed, both effects are transient. Thus, forces from the epidermis might be needed for the biased D-V amnioserosa cell contraction, and they might also direct the D-V movement of amnioserosa actomyosin networks to facilitate DC (David, 2010).
As the actomyosin networks assemble and disassemble, they translocate across a persistent PAR protein patch. These transient associations and lack of specific colocalization between the actomyosin networks and the PAR proteins argue against PAR proteins being integral parts of the actomyosin networks. However, the current results show that the PAR proteins regulate the networks. Genetic interaction tests indicate that Baz, Par-6 and aPKC support myosin activity for proper DC. Strikingly, the live imaging revealed that Baz and Par-6/aPKC regulate distinct phases of the myosin assembly/disassembly cycle. Together, the loss-of-function and gain-of-function studies show that Baz promotes network durations, whereas Par-6 and aPKC promote lull times between pulses. Baz overexpression also decreased lull times, which could result indirectly from increased network durations or from more direct inhibition of the lull phase. Importantly, overexpression experiments indicate that the effects occur specifically in amnioserosa cells, and analyses of cell polarity and AJs indicate that the PAR proteins have relatively direct effects on the actomyosin networks. However, it remains possible that the PAR proteins have additional functions in the amnioserosa (David, 2010).
A number of molecular interactions must control PAR protein activity in the apical domain of amnioserosa cells. The PAR proteins often, but not exclusively, colocalize in amnioserosa cells, suggesting a dynamic relationship consistent with separate Baz and Par-6/aPKC functions. They also show colocalization with Crb, an apical transmembrane protein at the core of the Crb polarity complex. Interestingly, Crb is known to regulate DC, and Par-6 and aPKC can bind Crb complex components. Thus, Crb might be one anchor for PAR proteins at the apical surface of amnioserosa cells (David, 2010).
Molecular mechanisms connecting PAR proteins to myosin and actin have been implicated in a number of studies. For example, aPKC phosphorylates and inhibits mammalian myosin IIB, although these sites are not present in Drosophila Myosin II (Zipper). Par-6/aPKC also inhibits Rho by activating the ubiquitin ligase Smurf1 in mammalian cells. Additionally, Baz and aPKC immunoprecipitate with Sqh from Drosophila egg chambers. Analogous to amnioserosa morphogenesis, mammalian Par-3 and Par-6/aPKC regulate distinct aspects of cell shape change through different cytoskeletal regulators during dendritic spine morphogenesis: Par-3 inhibits cell protrusions by inhibiting Rac through sequestering the RacGEF Tiam1, whereas Par-6/aPKC promotes protrusions by inhibiting Rho via p190 RhoGAP (David, 2010 and references therein).
Amnioserosa cell apical constriction has similarities to endoderm precursor cell apical constriction during C. elegans gastrulation. Here, myosin activity drives cell ingression. Similar to in amnioserosa cells, the PAR complex and myosin accumulate at the center of the apical surface of these cells and of earlier cells as well. However, these C. elegans actomyosin networks do not appear to undergo full assembly/disassembly cycles and instead progressively accumulate or display continual network flows. Interestingly, apical myosin enrichment requires PAR-3 in C. elegans endodermal precursor cells. Apical myosin enrichment also requires Baz, Par-6 and aPKC in Drosophila egg chamber follicle cells. These results suggest that the PAR complex initiates actomyosin network assembly, contrasting with the amnioserosa, in which networks can assemble without detectable Baz and are inhibited by Par-6/aPKC. Perhaps, actomyosin networks with full assembly/disassembly cycles are regulated distinctly. In the one-cell C. elegans embryo, PAR protein puncta move with a multifaceted cortical myosin network to the embryo anterior. Each facet of the network assembles and disassembles with durations similar to those of the amnioserosa actomyosin networks. The network can also form without the PAR proteins, but the overall flow of the network fails with loss of PAR-3, PAR-6 or aPKC. It would be interesting to test whether PAR-3, PAR-6 and aPKC have distinct effects on the individual facets of these networks (David, 2010).
What triggers actomyosin network assembly in amnioserosa cells? It appears to be independent of Baz, and must overcome Par-6/aPKC inhibition. The Rho pathway triggers actomyosin contractility in many contexts. However, amnioserosa-targeted expression of dominant-negative Rho does not appear to block DC. Alternatively, actin assembly might trigger the networks. Actin networks appear larger and last longer than myosin networks as both start forming during germband retraction. This suggests that actin might organize these networks during germband retraction and possibly DC. Intriguingly, Rac inhibition disrupts DC and reduces amnioserosa actin levels. The trigger might also involve intercellular forces from networks in neighboring cells (David, 2010).
How is the actomyosin assembly/disassembly periodicity regulated? Since more than one network per cell is rarely observed, network assembly might require disassembly of the existing network. Disassembly might begin a cascade that ultimately triggers formation of the next network. For cycling, assembly might likewise elicit disassembly. The data indicate that the PAR proteins are important elements of the regulatory network that is involved. Once a network is triggered, Baz prolongs it, but as the network persists, trigger and maintenance signals must be overcome for network disassembly. With disassembly, Par-6/aPKC activity appears to inhibit new assembly, promoting lull times. With time, this Par-6/aPKC activity must diminish and/or be overwhelmed by the trigger mechanism for new network assembly to occur. Identifying trigger and feedback mechanisms within this cycle will be key for understanding how pulsed actomyosin contractions are regulated in the amnioserosa (David, 2010).
To study the consequences of aPKC loss-of-function on epithelial polarity and asymmetric division of neuroblasts, the phenotype of mutants in the aPKC locus was analyzed. BLAST searches with the genomic sequence of the aPKC gene revealed that the P-element insertion l(2)k06403 is located in the third intron of the aPKC gene. This insertion line is homozygous lethal, contains a single PlacW P-element, and does not complement Df(2R)Jp1, which removes the cytological interval 51C3;52F5-9. Staining intensity with anti-PKCzeta antibody C20 was reduced to background level in embryos homozygous mutant for l(2)k06403. The P-element was mobilized using the transposase source P[delta2-3] and several revertants lacking the w+ marker of the original P-element insertion were recovered. Of seven revertant lines tested, 3 were homozygous viable, demonstrating that insertion of the P-element into the aPKC locus was the cause of lethality and of the observed phenotype. It was thus conclude that l(2)k06403 is an allele of aPKC and it was renamed DaPKCk06403 (Wodarz, 2000).
Homozygous mutant DaPKCk06403 embryos do not produce any cuticle. The reason for this phenotype is early embryonic lethality before the onset of cuticle secretion. Transheterozygous DaPKCk06403/Df(2R)Jp1 embryos show exactly the same cuticle phenotype, suggesting that DaPKCk06403 is a strong hypomorphic or null allele. The analysis of the aPKC loss-of-function phenotype was complicated by the fact that aPKC mutants show maternal haploinsufficiency with incomplete penetrance. The evidence for this conclusion comes from the following observations: it was noticed that, in egg collections from the DaPKCk06403/CyO stock, significantly more than 50% of embryos (including CyO/CyO homozygotes) fail to hatch, which means that even embryos with a zygotic wild-type allele of aPKC frequently show developmental defects. This finding was confirmed in crosses of heterozygous DaPKCk06403/CyO females to wild-type males, where considerable embryonic lethality was observed. The reciprocal cross does not show an increase of embryonic lethality compared with wild-type controls. It is therefore concluded that the decreased viability of the progeny from DaPKCk06403/CyO mothers is caused by the reduction of the maternal DaPKC level during oogenesis (Wodarz, 2000).
To produce embryos with the wild-type maternal contribution of aPKC, but lacking any zygotic aPKC expression, C(2)v females were crossed to DaPKCk06403/CyO males and the cuticle phenotype of their progeny was analyzed. One quarter of the embryos derived from that cross completely lacked zygotic expression of aPKC and produced cuticles with characteristic defects. In most cases, head structures were missing and the ventral cuticle either showed large holes or was missing altogether. These phenotypes are strikingly similar to those of baz mutants. baz null embryos derived from germ-line clones produce very little or no cuticle and resemble DaPKCk06403 mutant embryos derived from heterozygous mothers. baz mutant embryos lacking only the zygotic expression produce cuticles with characteristic head defects and ventral holes that are very similar to DaPKCk06403 mutant embryos derived from C(2)v mothers (Wodarz, 2000).
The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote,
subsequently have been shown to regulate cellular polarity in diverse
cell types by means of an evolutionarily conserved protein complex
including PAR-3, PAR-6, and atypical protein kinase C (aPKC).
The Drosophila homologs of par-1, par-3 (bazooka [baz]), par-6 (DmPar-6), and pkc-3
(Drosophila aPKC; DaPKC) each are known to play
conserved roles in the generation of cell polarity in the germ line as
well as in epithelial and neural precursor cells within the embryo. In
light of this functional conservation, the potential role
of baz and DaPKC in the regulation of
oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of
initial anterior-posterior polarity within germ-line cysts and
maintenance of oocyte cell fate. Germ-line clonal analyses indicate
both proteins are essential for two key aspects of oocyte
determination: the posterior translocation of oocyte specification
factors and the posterior establishment of the microtubule organizing
center within the presumptive oocyte. Baz and DaPKC
colocalize to belt-like structures between germarial cyst cells.
However, in contrast to their regulatory relationship in the
Drosophila and C. elegans embryos, these
proteins are not mutually dependent for their germ-line localization,
nor is either protein specifically required for PAR-1 localization to
the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).
To examine the potential oogenic function of baz and
DaPKC, protein null germ-line mutant clones were generated for
both baz and DaPKC. Germ-line clones, identified
by the absence of nuclear GFP expression, were counterstained with the
chromatin marker propidium iodide to examine the number and ploidy of
the germ-line nuclei. In contrast to wild-type egg chambers, which invariably contain 15 nurse cells and a single oocyte, baz mutant
germ-line clones, while containing the normal complement of germ-line
nuclei, fail to differentiate an oocyte, resulting in a 16-nurse cell
phenotype as revealed by the polyploid state of all 16 germ-line nuclei.
Similarly, DaPKCk06403 germ-line clones
also fail to differentiate an oocyte as indicated by the presence of 16 polyploid nurse cell nuclei in germ-line mutant egg chambers. These results reveal a germ-line autonomous requirement for DaPKC and confirm the
role of Baz in oocyte differentiation and/or maintenance (Cox, 2001).
These analyses further reveal that germ-line depletion of either
DaPKC or baz function from the follicle cells leads to their
multilayering, which disrupts the normal partitioning of germ-line
nuclei to successively mature egg chambers caused by mispositioning of
mutant follicle cells. These mispartitioned
baz+ nurse cell nuclei, into an otherwise
baz null germ-line clone, may provide the threshold of
germ-line Baz required to rescue the oocyte differentiation defect and
allow production of a mature egg. Alternatively, the Baz protein may
exhibit a long perdurance after mitotic clone induction, which depletes
over a period of days, resulting in the cessation of egg production in
these mosaic females. These results, however, in no way invalidate the
previous conclusions that maternally provided Baz masks the severity of the embryonic polarity phenotype in both epithelial cells and neuroblasts. Rather, the results indicate that these embryos are not likely to represent a complete maternal depletion for Baz (Cox, 2001).
Oocyte differentiation requires the polarized accumulation of oocyte
specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in
the localization of these factors, mutant germ-line clones
for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst
cells in region 2a, and then both molecules are targeted first to the
two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In
contrast, ORB fails to translocate from the anterior to a posterior
crescent in both baz and DaPKC null germ-line cysts in germarial
region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in
baz and DaPKC null germ-line clones in germarial
region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).
Furthermore, in contrast to wild-type germ-line cysts in which DHC64C
localizes to a single posterior cell, in DaPKC null germ-line clones DHC64C fails to localize to a single cell posteriorly, but rather accumulates in the two posterior-most presumptive
pro-oocytes of the mutant germ-line cyst.
Therefore, baz and DaPKC display essentially
identical phenotypes in germ-line mutant clones with regards to oocyte
differentiation and the establishment of initial A-P polarity within
the oocyte. The failure to maintain oocyte identity in either
baz or DaPKC mutant cysts can therefore be directly correlated with defects in the A-P translocation of oocyte specification factors within a single posterior cell of a germ-line cyst, suggesting oocyte differentiation depends on this early polarization event (Cox, 2001).
The posterior assembly of a functional MTOC has been directly
implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the
failure to translocate these factors to a posterior crescent in region
3 baz or DaPKC mutant cysts may result from a defect
in microtubule reorganization within these mutant cysts. In contrast to
wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of
oocyte specification factors in these mutants are likely caused, at
least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).
In addition to the microtubule network, both ring canals and the fusome
play critical roles in cyst polarization and oocyte differentiation. The formation and spatial distribution of ring canals as well as fusome morphogenesis was examined in both baz and
DaPKC mutant germ-line cysts. In baz mutant cysts, ring canal formation and spatial distribution are indistinguishable from wild-type germ-line cysts. Furthermore, the wild-type
spatial arrangement of ring canals in baz null germ-line
cysts suggests there is no apparent disruption in germ cell adhesion
within the cyst. Similarly, no defects were observed in ring canal
formation or spatial distribution in DaPKC mutant cysts (Cox, 2001).
These analyses indicate baz mutant cysts display relatively normal fusome morphology, although mutant fusome branches appear slightly thinner when compared with wild-type fusomes within the same germarium. Similarly, no apparent defect is observed in fusome morphology in DaPKC null germ-line cysts, indicating DaPKC
is dispensable in the germ line for proper fusome morphogenesis (Cox, 2001).
To investigate the mechanism by which Baz and DaPKC exert their effects
on oocyte differentiation, the localization of these
proteins was analyzed in the germ line and soma during oogenesis. The specificity of the Baz and DaPKC
antibodies was verified by using mosaic ovaries containing
baz or DaPKC null mutant clones. Baz is first detected in the germarium as a belt-like specialization on germ cell membranes at sites of germ cell interconnection. These belt structures are
reminiscent of ring canals with respect to their position between
germ-line cyst cells; however, in contrast to ring canals these "Baz
belts" are approximately 2-fold greater in diameter. Germaria
double-labeled for Baz and rhodamine phalloidin reveal that the Baz belts
localize adjacent to ring canals and further reveal an approximate 1:1 ratio between the two structures within germ-line cysts. The microtubule cytoskeleton as well as the fusome were observed projecting through individual cystocytes coincident with
the site of Baz belt expression on the germ cell membrane. Later in
oogenesis, Baz is transiently enriched in the oocyte cytoplasm at
stages 5-6 before the onset of vitellogenesis and is subsequently undetectable in the germ line of
vitellogenic egg chambers. As with Baz localization to the apical
junctional zone in the embryonic epithelium, tight apical localization of Baz is also observed in follicular epithelia (Cox, 2001).
DaPKC localizes to the Baz belts in the germarium, whereas in follicle cells DaPKC is apically constricted consistent with Baz localization in these cells (Cox, 2001).
In embryonic epithelia, Baz, DmPAR-6, and DaPKC apically colocalize and
partially overlap with the apico-laterally enriched Arm and DE-cadherin
proteins in the region of the apical zonula adherens. Furthermore,
these proteins are required to maintain epithelial cell polarity and
are mutually dependent for their proper localization. DE-cadherin is localized to belt-like structures adjacent to ring canals in region 2 germarial cysts reminiscent of Baz belt localization, suggesting that these adherens junction components may similarly colocalize in the germ line. To further investigate the molecular and functional nature of Baz belt expression in the
germarium, germaria were labeled with anti-Baz, anti-Arm, and
anti-DE-cadherin antibodies and their potential colocalization
within the germarium was examined. These
analyses reveal Baz colocalizes with both DE-cadherin and Arm to the Baz belts within the
germarium. Furthermore, consistent with the colocalization of Baz and
DaPKC to Baz belts within the germarium, partial
colocalization of DaPKC with DE-cadherin is observed in wild-type germarial cysts. To assay whether these proteins are mutually dependent for their germ-line localization, the localization of DE-cadherin and Arm was examined in baz null germ-line clones. In contrast to their mutual dependence in embryonic epithelia, these analyses indicate germ-line Baz function is dispensable for the localization of either DE-cadherin or Arm to the Baz belts in
the germarium. DaPKC is dispensable for the localization of either DE-cadherin or Arm
to these structures. These results
indicate Baz and DaPKC function are not required for the formation of
these structures because both Arm and DE-cadherin localization to these
belts in baz or DaPKC mutant cysts is indistinguishable from
that observed in wild-type cysts. Previous studies have revealed that germ-line clones of a strong allele of
shotgun (shgIG29), the gene
encoding DE-cadherin, disrupts the arrangement of germ cells in region
2b germarial cysts, suggesting DE-cadherin may mediate germ cell
adhesion. In contrast, these analyses of ring canal spatial distribution
in baz and DaPKC mosaic cysts strongly suggests
Baz and DaPKC function are dispensable for normal germ cell adhesion.
Furthermore, germ-line clonal analyses of either shg or
arm reveal neither gene is required for oocyte
differentiation, whereas both
baz and DaPKC are essential in oocyte determination. These results suggest the components of the Baz belts
likely mediate diverse cellular functions essential for germ-line cyst
development, which may include cell adhesion, cell signaling, and cyst
polarization (Cox, 2001).
In the C. elegans zygote, the PAR-3/PAR-6/PKC-3
complex is localized to the anterior where it is required for the
posterior localization of PAR-1. To investigate the potential regulatory relationship between baz and par-1 in the Drosophila germ line,
baz null germ-line clones were generated and PAR-1 expression and
localization was analyzed. In wild-type germ-line cysts, PAR-1 is localized to the spectrosome and fusome in germarial regions 1 and 2a and subsequently
is down-regulated on fusomes in regions 2b and 3. In
baz mutant germ-line cysts, PAR-1 localization to the
spectrosome is unaffected and is likewise present on fusomes although somewhat weaker localization is observed on fusome branches of baz mutant germ-line cysts when compared with wild-type germ-line cysts. Taken together, these results suggest germ-line baz is dispensable in the germarium for
normal PAR-1 expression and localization (Cox, 2001).
To determine whether par-1 may regulate Baz expression, par-1 null germ-line clones were generated and Baz
localization was examined. No defect was observed in Baz belt expression in
par-1 mutant germ-line cysts. Furthermore, in contrast to wild-type stage 5-6 egg
chambers in which Baz is transiently enriched in the oocyte cytoplasm, in par-1 mutant egg chambers Baz localization is abolished from the posterior presumably due to the
defect in oocyte differentiation observed in par-1 mutant egg chambers. These results indicate
germ-line par-1 is dispensable for normal Baz belt
expression and localization (Cox, 2001).
To assay the regulatory relationship between baz and
DaPKC the localization of DaPKC was analyzed in
baz mutant germ-line cysts, as well as
the localization of Baz in DaPKC mutant germ-line cysts. In contrast to their mutual dependence for
localization in the embryo, both Baz and DaPKC localization within the germ line is mutually independent. These results indicate that
despite the apparent functional conservation of Baz, DaPKC, and PAR-1
in generating oocyte polarity, the regulatory relationships among these
genes are not conserved in the germ line (Cox, 2001).
The colocalization of Baz, DaPKC, Arm, and DE-cadherin to
the Baz belt structures in the germarium strongly suggests the components of
the Baz belts are capable of mediating a multiplicity of functions in
germ-line cysts. In embryonic epithelia, these proteins function in the formation of the apical zonula adherens junction and are mutually dependent for their apical localization. The germ-line colocalization of these molecules to the Baz
belts suggests these structures may represent a potential polarity cue
on the germ cell plasma membrane. The restricted localization of Baz
belt components to germ cell membranes at points of cell-cell contact
represents an asymmetry on the plasma membrane of germ-line cyst cells
with regard to the A-P axis of the cyst and stage 1 oocyte. The
asymmetric localization of these molecules to one side of the germ cell
plasma membrane may act as a polarity cue in defining anterior versus
posterior within individual cystocytes of the 16-cell germ-line cyst
and thus contribute to the establishment of an initial A-P axis and to
subsequent germ-line cyst polarization. These results further suggest
that Baz and DaPKC likely function in a signaling capacity, rather than
a structural one, to mediate oocyte differentiation, whereas
DE-cadherin and Arm are more likely to function in maintaining germ
cell adhesion and cyst integrity (Cox, 2001).
Consistent with par gene function in other systems, these results indicate that Baz, DaPKC, and PAR-1 are required for the
establishment and maintenance of cellular polarity in the
Drosophila germ line; however, the regulatory relationships
observed between these genes in the germ line versus that observed in
embryonic blastomeres, epithelial cells, and neural precursor cells
indicates that, while these molecules are functionally conserved, the
mechanisms by which these genes act appear to be less well conserved.
In contrast to their mutual dependence for localization in the embryo,
Baz, DaPKC, and PAR-1 each are mutually independent for their
localization in the germ line. Taken together, these results underscore
the functional utility of the par genes and their effectors
as a molecular module for generating cellular polarity in diverse cell
types. Furthermore, the independence of Baz, DaPKC, and PAR-1 in their germ-line localization provides a unique opportunity to probe new
mechanisms by which these highly conserved proteins function in
regulating diverse processes such as cellular polarity, asymmetric cell
division, or growth control (Cox, 2001).
Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype; reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes (Rolls, 2003).
aPKCk06403 zygotic mutant embryos die before gastrulation and all epithelial and neuroblast polarity is lost. A much later onset phenotype is found: survival of aPKCk06403 homozygotes to second larval instar with no significant embryonic phenotype. Although surprising, it is felt that these results reflect the authentic zygotic aPKCk06403 mutant phenotype for several reasons. (1) Recent work from the Wodarz lab confirms that aPKCk06403 homozygotes survive to at least late embryonic stages without significant epithelial or neuroblast defects (Wodarz, A., personal communication to Rolls, 2003), presumably due to the absence of deleterious second site mutations that were on the original aPKCk06403 chromosome. (2) PCR was used to verify the presence of the aPKCk06403 allele in all of of the stocks used in this study, and dominantly marked balancer chromosomes were used to independently confirm the identity of every aPKCk06403 homozygote analyzed. (3) aPKCk06403 homozygous second instar larvae were shown to have very low levels of aPKC mRNA and no detectable full-length protein; thus, a very strong or null aPKC mutant phenotype was examined. (4) It was shown that aPKCk06403/aPKCk06403 and aPKCk06403/Df(2R)JP1 larvae have indistinguishable lethal phases and quantitative neuroblast phenotypes; thus, aPKCk06403 behaves as a genetic null allele. (5) aPKCk06403 homozygous embryos have persistent maternal aPKC protein, explaining the lack of an embryonic phenotype in these mutants (Rolls, 2003).
One of the more unexpected findings is that aPKC mutant neuroblasts show normal Baz/Par3 apical localization. The Baz/Par3-Par6-aPKC complex has been suggested to form a functional unit that is interdependent for localization in C. elegans, mammals, and Drosophila. Baz/Par3 shows normal apical localization in aPKC mutant neuroblasts, showing that normal Baz/Par3 localization can occur without being part of the Par3-Par6-aPKC complex. In addition, neuroblasts lacking apical aPKC and Par6 still form a molecularly defined apical cortical domain containing Baz/Par3, Insc, Pins, and Dlg. These results lead to the proposal of a hierarchy for apical protein localization in neuroblasts: Baz/Par3-Insc-Pins-Dlg --> aPKC --> Par6-Lgl. This hierarchy is consistent with recent biochemical analyses in which a protein complex was isolated containing Par6-aPKC-Lgl, but not Baz/Par3. It is suggested that aPKC may be required to anchor the Par6-aPKC-Lgl complex at the apical cortex of the neuroblast (Rolls, 2003).
Both aPKC and Lgl are required for Miranda basal localization in neuroblasts, and all available data support a model in which Lgl is required for targeting Miranda to the neuroblast cortex, whereas aPKC blocks Lgl function on the apical side of the neuroblast: (1) lgl mutants have little or no Miranda at the cortex; (2) aPKC mutants show uniform cortical Miranda localization; (3) a weak lgl phenotype can be suppressed by reducing aPKC levels, showing that aPKC activity antagonizes Lgl activity; (4) aPKC and Lgl physically interact; (5) an overexpressed nonphosphorylatable Lgl protein is uniformly cortical and able to induce uniform cortical Miranda localization, whereas phospho-Lgl is preferentially released from the cell cortex. This has led to a model in which Lgl acts as an anchor for Miranda at the basal cortex, but is absent from the apical cortex due to aPKC-mediated phosphorylation. Although this simple model is attractive, it is noted that Lgl has never been observed colocalized with Miranda in a basal cortical crescent, and a role for cytoplasmic Lgl in Miranda localization has not been definitively ruled out (Rolls, 2003).
The Baz/Par3-Par6-aPKC complex has a well-characterized role in regulating neuroblast spindle orientation. Spindle orientation can be measured relative to extrinsic landmarks around the neuroblast (e.g., perpendicular to the overlying ectoderm) or relative to intrinsic cues within each neuroblast (e.g., perpendicular to the Baz/Par3-Par6-aPKC apical crescent). Mutations in baz or par6 genes randomize embryonic neuroblast spindle orientation relative to the overlying ectoderm, but it is not clear whether these phenotypes are due to disruption of the ectodermal layer or to a cell-autonomous defect in the neuroblast. The function of aPKC in embryonic neuroblast spindle orientation cannot be assayed due to high levels of maternal aPKC protein present in aPKC zygotic mutant embryos. However, aPKC is not required for intrinsic spindle orientation in larval neuroblasts; the mitotic spindle is always perpendicular to the Baz/Par3-Insc-Pins apical crescent (Rolls, 2003).
The Baz/Par3 and Par6 proteins are required to establish epithelial polarity in Drosophila, and aPKC is also shown to be required for normal apical/basal epithelial cell polarity. The similar phenotype in baz, par6, and aPKC mutants may indicate that these proteins function together as a complex to provide a single function in epithelia, despite the evidence that they have independent functions in neuroblasts. One primary function may be the inhibition of Lgl activity because it is found that the lgl epithelial polarity defects can be strongly suppressed by reducing aPKC levels. Lgl-Dlg-Scrib activity can also antagonize Baz/Par3-Par6-aPKC activity, and it is tempting to speculate that aPKC inactivates Lgl by phosphorylation, whereas Lgl can inactivate aPKC by sequestering it into an Lgl-Par6-aPKC complex and out of the Baz/Par3-Par6-aPKC complex (Rolls, 2003).
The role of aPKC in cell proliferation has not been previously investigated in Drosophila. There are three lines of evidence showing that aPKC promotes cell proliferation in neuroblasts and epithelia. The number of cells in aPKC mutant mushroom body neuroblast clones is significantly lower than the number in wild-type clones. There appears to be a normal number of early-born gamma neurons in these clones, followed by only a few later-born alpha neurons. The normal number of early-born gamma neurons suggests that loss of aPKC does not lead to cell death in this population; moreover, no decrease is seen in the number of neuroblasts per brain lobe in aPKC mutant larvae. It is concluded that cell death is not contributing to the reduction in neurons observed in the clones, but rather, that the neuroblast stops dividing near the time the neuroblast switches over to generating alpha and ß neurons. The neuroblast may become arrested at some point in the cell cycle, or it may undergo a terminal division to generate a pair of GMCs (perhaps due to both daughter cells inheriting Miranda and Prospero GMC determinants). A second indication that aPKC promotes cell proliferation is that far fewer epithelial cells are observed in aPKC mutant eye imaginal discs compared with the wild type, even with an additional day of growth as second instar larvae. Finally, a 50% reduction in aPKC levels (aPKC/+) can strongly suppress the epithelial and brain overproliferation phenotypes of lgl mutants. Together, these data show that aPKC positively regulates cell proliferation in epithelia and neuroblasts. Interestingly, reduction in the function of the mammalian atypical PKCzeta (using overexpression of a dominant-negative kinase) can suppress Rac1/cdc42-induced overproliferation. Thus, aPKC may have an evolutionarily conserved role in promoting cell proliferation, as well as in the establishment of cell polarity (Rolls, 2003).
Although aPKC and Lgl act antagonistically to regulate many aspects of epithelial and neuroblast cell polarity and cell proliferation, they may share a common positive function in regulating neuroblast apical cell size. lgl zygotic mutants have some embryonic telophase neuroblasts with an abnormally small apical cortical domain, apical spindle pole, and neuroblast size. These defects are not suppressed by reducing aPKC levels, and in fact may be enhanced. Thus, it is proposed that Lgl and aPKC both act positively to promote large apical cell and spindle pole size. It has been reported that Baz/Par3-Par6-aPKC and Pins-Galphai act in parallel pathways to promote large apical cell size and apical spindle size; Lgl could be acting as part of the Pins-Galphai pathway, or as a third input promoting apical cell and spindle size (Rolls, 2003).
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date revised: 15 December 2011
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