bazooka
Several proteins, including Numb and Miranda, segregate into the basal daughter cell and are needed for the determination of its correct cell fate. Both the apical-basal orientation of the mitotic spindle and the localization of Numb and Miranda to the basal cell cortex are directed by Inscuteable, a protein that localizes to the apical cell cortex before and during neuroblast mitosis. The apical localizaton of Inscuteable requires Bazooka, a protein containing a PDZ domain that is essential for apical-basal polarity in epithelial cells. Bazooka localizes with Inscuteable in neuroblasts and binds to the Inscuteable localization domain in vitro and in vivo. In embryos lacking both maternal and zygotic bazooka function, Inscuteable no longer localizes asymmetrically in neuroblasts and is instead uniformly distributed in the cytoplasm. Mitotic spindles in neuroblasts are misoriented in these embryos, and the proteins Numb and Miranda fail to localize asymmetrically in metaphase. These results suggest that direct binding to Bazooka mediates the asymmetric localization of Inscuteable and connects the asymmetric division of neuroblasts to the axis of epithelial apical-basal polarity (Schober, 1999).
In Drosophila neuroblasts, inscuteable controls both spindle orientation and the asymmetric localization of the cell-fate determinants Prospero and Numb. Inscuteable itself is localized in an apical cortical crescent and thus reflects the intrinsic asymmetry of the neuroblast. Localization of Inscuteable depends on Bazooka, a protein containing three PDZ domains with overall sequence similarity to Par-3 of Caenorhabditis elegans. Bazooka and Inscuteable form a complex that also contains Staufen, a protein responsible for the asymmetric localization of Prospero messenger RNA. It is proposed that, after delamination of the neuroblast from the neuroepithelium, Bazooka provides an asymmetric cue in the apical cytocortex that is required to anchor Inscuteable. Since Bazooka is also responsible for the maintenance of apical-basal polarity in epithelial tissues, it may be the missing link between epithelial polarity and neuroblast polarity (Wodarz, 1999).
A direct interaction between Partner of Inscuteable and Baz in yeast two hybrid and GST pull-down experiments could not be demonstrated. However, Baz complexes with Insc in vivo, and directly interacts with Insc in vitro (Schober, 1999; Wodarz, 1999). Since Pins interacts with Insc, these observations suggest that Insc may be acting to link Baz to Pins. Several observations support this view. (1) For NBs and cells of mitotic domain 9, Pins does not localize apically in the absence of insc function. (2) Also supportive is the apparent temporal order in which these genes are recruited to the apical cortex of NB: Baz (while part of the epithelia), followed by Insc (during delamination), followed by Pins (after delamination). (3) In epithelial cells that do not express Insc, Pins and Baz do not colocalize; Baz is found on the apical cortex and Pins shows lateral cortical distribution, yet the ectopic expression of Insc (which localizes apically) is sufficient to recruit Pins to the apical cortex of these cells. All of the available data are consistent with the model that the formation and maintenance of an apical protein complex that imparts apical/basal polarity in NBs comprises the following events: cells in the neuroepithelium destined to become NBs have apical/basal polarity as evidenced by the apical localization of Baz; as these interphase cells delaminate, Insc is recruited to the apical complex in a Baz-dependent manner; Pins is in turn recruited to this complex and this involves interaction with Insc. Some as yet undefined events must occur between delamination (interphase) and mitosis that change the nature of this complex such that its maintenance becomes codependent on these three molecules (Yu, 2000).
Both insc and pins are required for the execution of the more downstream processes associated with asymmetric cell divisions and the relative roles of the two genes are at present unclear. However, some interesting distinctions can be made between the two genes. insc was originally isolated on the basis of its expression in neural precursors. Insc expression is restricted, conforms to the prepattern-proneural-neurogenic-panneural cascade, and links general neuronal differentiation programs to lineage information; Pins shows a wider expression pattern and becomes involved in asymmetric cell divisions only when a signal (i.e., insc) is active. Pins and Insc also appear to follow different routes to reach the apical cortex -- Pins apparently transiting via the membrane but not Insc, which suggests that other interactors may be involved in linking Pins to the cortex. Finally, the only known direct links to downstream events associated with asymmetric cell divisions appear to be mediated through Insc (Yu, 2000).
Atypical protein kinase C (aPKC) from Drosophila shows very high sequence similarity to PKClambda and PKCzeta from vertebrates and PKC-3 from C. elegans. Drosophila aPKC and Baz coimmunoprecipitate and directly bind to each other in a yeast two-hybrid assay. In embryos, both proteins colocalize in the apical cortex of almost all epithelial tissues and in neuroblasts. Apical localization of DaPKC in epithelia and neuroblasts is abolished in baz mutants, and vice versa: Baz is delocalized in DaPKC mutants. The phenotype of aPKC loss-of-function mutants resembles that of baz mutants, consistent with a close functional interdependence of both proteins. Together, these data provide in vivo evidence for an essential role of an atypical protein kinase C isoform in establishment and maintenance of epithelial and neuronal polarity (Wodarz, 2000).
To test whether aPKC and Baz colocalize, double-label immunofluorescence stainings of embryos was performed. aPKC and Baz are clearly colocalized in the epidermis and in neuroblasts. To determine the precise subcellular localization of aPKC and Baz with respect to the ZA, double-label immunofluorescence stainings were performed with antibodies against Arm, a component of the ZA and Baz. The merged image shows that Baz is localized apically to Arm. The same is true for aPKC. At the resolution of the confocal microscope, the possibility that the localization of Baz and DaPKC partially overlaps with Arm in the ZA cannot be ruled out (Wodarz, 2000).
Binding studies showing a physical association of aPKC and Baz, and colocalization of these two proteins suggests that they may functionally interact with each other. In stainings of baz mutant embryos derived from germ line clones (baz null embryos) with anti-aPKC antibody, apical localization of aPKC could not be detected in epithelia and neither could apical localization be detected in neuroblasts. Instead, aPKC was distributed in a diffuse fashion in the cytoplasm. baz null embryos also show a loss of membrane polarity that is evident by mislocalization of the basolateral transmembrane protein Nrt. In contrast to wild type, Nrt is not excluded from the apical plasma membrane. Moreover, the monolayered structure of the epidermis is lost and cells pile up on top of each other, as has been described before for sdt;baz double mutants (Wodarz, 2000).
To test whether mislocalization of Baz is sufficient to induce mislocalization of aPKC, Baz was overexpressed by means of the GAL4 system. Under these conditions, Baz is not confined to the apical cytocortex anymore and is found in more lateral and basal positions in epithelia and neuroblasts. Concomitantly, aPKC is also mislocalized and colocalized in ectopic positions with ectopic Baz, confirming that ectopic Baz can recruit aPKC to ectopic sites in the cytocortex (Wodarz, 2000).
It has been shown before that Baz is required for apical localization of Insc in neuroblasts and that Insc is required for stabilization of Baz in neuroblasts after delamination. A test was performed to see whether Baz and Insc are also required for localization of aPKC in neuroblasts. aPKC localization is indistinguishable from wild type in neuroblasts of inscP49/CyO heterozygous embryos, but is neither cortical nor apical in neuroblasts of inscP49 homozygous mutant embryos. In embryos lacking maternal Baz but carrying a paternal wild-type allele of baz (partial paternal rescue), asymmetric cortical localization of aPKC is detected in most neuroblasts at metaphase. However, aPKC crescents and metaphase plates are often misoriented with respect to the surface of the embryo, a phenotype that has also been observed at low penetrance in embryos lacking only zygotic expression of Baz. In embryos lacking both maternal and zygotic expression of Baz (baz null), aPKC is completely delocalized in neuroblasts and epithelial tissues. These results indicate that Baz is absolutely required for apical localization of aPKC in neuroblasts and epithelial tissues, while Insc is required for localization of aPKC only in neuroblasts. Baz levels are strongly reduced in neuroblasts of insc mutant embryos, most likely because Insc is required for stabilization of Baz. Thus, the effect of Insc on DaPKC localization is probably indirect and can be explained by the loss of Baz in insc mutant neuroblasts (Wodarz, 2000).
In the case of aPKC and Baz, the situation is more complicated. Consistent with a function as a scaffold, Baz is required for localization of the signaling protein aPKC. However, Baz itself is not properly localized in the absence of aPKC. It is easy to imagine how a structural multi-PDZ domain protein like InaD or Baz can localize a protein kinase, but how can aPKC be responsible for localization and stabilization of Baz? Baz possesses a PKC consensus phosphorylation site that is conserved between Baz, Par-3, and ASIP. Phosphorylation of this site by aPKC could be important to regulate binding of Baz to other proteins or to protect Baz from proteolytic degradation. It is also possible that aPKC binds simultaneously to Baz and another protein that may be required for localization of Baz. A detailed structure-function analysis of both Baz and aPKC will be necessary to clarify this issue (Wodarz, 2000).
To test whether there is a physical interaction between Bazooka and par-6, Drosophila embryo extracts were incubated with beads containing maltose-binding protein (MBP) or an MBP-Bazooka fusion protein. A significant amount of Par-6 protein can be detected by immunoblotting in the proteins bound to MBP-Bazooka, but not in the control. Whether this was due to direct binding of the two proteins was tested by incubating MBP and MBP-Bazooka beads with in vitro translated Par-6 protein. Par-6 binds to MBP-Bazooka, but not to MBP alone, and this interaction is not significantly altered in the presence of in vitro translated Inscuteable protein. These results suggest that Par-6 can directly bind to Bazooka. Despite this in vitro interaction, the two proteins could not be co-immunoprecipitated in vivo using anti-Bazooka or any of the different Par-6 antibodies generated. Thus, interaction between the two proteins maybe weak and not stable under the conditions needed to solubilize Bazooka. In vitro translated Par-6 protein does not bind to MBP–Inscuteable, which, together with the colocalization data and the binding of Bazooka to both Inscuteable and Par-6, suggests that binding of Par-6 to Inscuteable is indirect and occurs through Bazooka (Petronczki, 2001).
Par-6 is apically localized in asymmetrically dividing neuroblasts. To test whether the protein is required for asymmetric cell division, the distribution of Bazooka and Inscuteable were analyzed in neuroblasts of Par-6GLC embryos (embryos deficient for both maternal and zygotic par-6). Seventy-three per cent of the Par-6GLC mutant neuroblasts revealed homogeneous cytoplasmic distribution of Bazooka. In 27% of the mutant neuroblasts, Bazooka still shows some weak apical localization, but the strong apical crescents that are observed in 97% of the control neuroblasts were never seen. Whereas Inscuteable localizes asymmetrically at the apical cortex in 94% of the control neuroblasts, only 23% of the Par-6GLC mutant neuroblasts show clear Inscuteable crescents. In 44% of the mutant neuroblasts, the protein is partially delocalized, and in 32% Inscuteable is cytoplasmic. Thus, Par-6 is required for correct localization of both Inscuteable and Bazooka, even though the effect on Bazooka localization is stronger. Both Bazooka and Inscuteable are required for spindle orientation and asymmetric localization of Numb and Miranda (Petronczki, 2001).
Whether Par-6 is required in these processes was examined by staining Par-6GLC embryos for DNA and Miranda or Numb. Metaphase plates are frequently misoriented indicating a defect in spindle orientation. Statistical analysis showed that 25% of the neuroblast metaphase plates were misoriented by more than 60° relative to the horizontal plane, and 37% of the metaphase plates were misorientated between 30° and 60°. Although in control embryos Miranda localizes into a basal cortical crescent in 100% of all metaphase neuroblasts, no signs of asymmetric localization were detected in 80% of metaphase neuroblasts from Par-6GLC embryos. In 20% of Par-6 mutant metaphase neuroblasts, Miranda was excluded from the apical-most quarter of the neuroblast cortex, but a basal cortical crescent was never detected in these mutants. During anaphase and telophase, Miranda maintained its basal localization and segregated into the basal daughter cell in 100% of the control neuroblasts. In Par-6 mutant anaphase neuroblasts, Miranda concentrated at the cleavage furrow (77% or was actually indistinguishable from wild type (23%), indicating that there is a second, Par-6-independent mechanism involved in Miranda localization during late mitosis. Similar observations were made for Numb. Thus, Par-6 is required in neuroblasts for spindle orientation, for apical localization of Bazooka and Inscuteable, and for basal localization of Numb and Miranda during mitosis (Petronczki, 2001).
In the dorsal thorax (notum) of the Drosophila pupa, the pI cell divides unequally with its spindle axis aligned with the anterior-posterior (a-p) axis of the fly body. It produces two different daughter cells, pIIa and pIIb. During this division, Numb and its adaptor protein Partner of Numb (Pon) form an anterior crescent and segregate unequally into the anterior pIIb cell. In fz mutant pupae, the division of the pI cell is oriented randomly relative to the a-p axis and the Numb crescent does form, but at a random position. Thus, Fz is not required to establish planar asymmetry per se, but is necessary to orient the axis of the asymmetric cell division. This indicates that additional genes may be required for establishing, rather than orienting, planar asymmetry in the pI cell (Bellaïche, 2001 and references therein).
Fz organizes the actin cytoskeleton at the site of hair formation. Planar polarity in the pI cell is established by a mechanism that involves a remodeling of the previously established apical-basal polarity. During the pI cell division, Baz and DaPKC relocalize from the apical cortex to the posterior lateral cortex, while Dlg and Pins accumulate asymmetrically at the anterior lateral cortex. This redistribution along the a-p axis leads to the formation of two complementary planar domains at the cell cortex. This mechanism of polarity establishment is distinct from the one described in Drosophila neuroblasts. In these cells, Pins is recruited via Insc by Baz to the apical cortex, and acts in a Dlg-independent manner to maintain the Baz/DmPAR-6/DaPKC/Insc complex at the apical cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell (Bellaïche, 2001).
In the dividing pI cell, Numb and Pon colocalize at the anterior pole of the lateral cortex, marked with Fasciclin3 (Fas3), below the adherins junction (AJ), marked with DE-Cadherin (Shotgun). In epithelial cells in interphase, Baz colocalizes with Shotgun at the AJ around the apical cortex. In the pI cell, Baz accumulates at the posterior cortex during mitosis. Prior to chromosome condensation, this accumulation is seen at the level of the AJ. Then, during prophase and metaphase, Baz forms a posterior crescent below AJ and opposite to Numb. At telophase, the pIIa cell inherits a higher level of Baz than its sister cell. DaPKC shows a similar distribution to Baz in the pI cell (Bellaïche, 2001).
In neuroblasts, a key function of the Baz/DaPKC/DmPAR-6 complex is to recruit the Insc and the Pins proteins. However, in the pI cell, Insc is not expressed and Pins does not colocalize with Baz at the posterior cortex. Rather, it localizes to the anterior pole in early prophase and colocalizes with Numb at the anterior lateral cortex at metaphase (Bellaïche, 2001).
Because DaPKC and Baz have a dual function in epithelial polarity and asymmetric neuroblast division, it was hypothesized that genes required for epithelial polarity might also regulate planar polarity in the pI cell. To test this hypothesis, the planar distribution of various proteins known to be distributed asymmetrically along the apical-basal axis of epithelial cells was examined. Of these, only Dlg was identified as a protein localizing asymmetrically along the planar axis in the pI cell. Dlg overlaps with Fas3 below the AJ in interphase cells. In dividing pI cells, Dlg redistributes in part along the planar a-p axis. From late prophase onward, Dlg becomes enriched at the anterior cortex, where it colocalizes with Numb and Pins. During this time, Dlg does remain detectable at the posterior lateral cortex. At telophase, a higher level of Dlg segregates into the pIIb cell. Thus, the accumulation of Dlg/Pins and Baz at opposite poles of the cell defines two complementary cortical domains oriented along the a-p planar axis of the pI cell. The position of the mitotic spindle at metaphase correlates with the localization of these two cortical domains. The posterior spindle pole is positioned near the accumulation of Baz, and the anterior spindle pole lies near the accumulation of Dlg. In both pI and epidermal cell, the mitotic spindle poles are found below the AJ, which appear to remain functional since they retain their ability to recruit Arm (Bellaïche, 2001).
To determine the possible function of Baz in the planar polarization of the pI cell, clones of baz mutant cells were studied in the notum. Loss of baz activity does not affect the localization of Shotgun and Dlg, indicating that apical-basal polarity in the notal epithelium is maintained in the absence of Baz. In the dividing pI cell, Numb either does not localize asymmetrically or forms a weak crescent at the anterior cortex at prometaphase. In contrast, Pins localizes asymmetrically at the cortex of the pI cell during division. Moreover, baz mutant pI cells divide within the plane of the epithelium with a normal a-p orientation with Pins localizing at the anterior cortex. This shows that baz is required for the asymmetric localization of Numb but is not essential to establish asymmetry nor to orient polarity along the a-p axis (Bellaïche, 2001).
The role of Pins and Dlg in localizing Baz asymmetrically was examined. In pins mutant pI cells, Baz accumulates at the posterior cortex at metaphase, but the asymmetry is less pronounced than in wild-type cells. This raises the possibility that Pins participates in the asymmetric localization of Baz. In dlg1P20 mutant pupae, Baz is correctly localized to the apical posterior cortex prior to chromosome condensation, but does not form a cortical crescent below the AJ during late prophase and prometaphase. Instead, Baz accumulates in the cytoplasm and remains cortical only at the level of the AJ. Thus, the initial posterior localization of Baz at the level of the AJ does not depend on the activity of the GUK domain of Dlg, but its cortical localization below the AJ does require dlg activity. It is concluded that planar polarization of the pI cell cannot be maintained without Dlg activity (Bellaïche, 2001).
To test whether the initial Dlg-independent localization of Baz at the posterior cortex depends on Fz signaling, the distribution of Baz was studied in fz mutant pupae. In wild-type pupae, a clear accumulation of Baz is seen at the level of the AJ in 61% of the interphase pI cells. By contrast, an asymmetric distribution of Baz at the apical cortex is detected in only 19% of the interphase pI cells in fz mutant pupae. In the remaining 81% of the cells, the asymmetric accumulation of Baz is either weak or similar to that seen in the surrounding epithelial cells. This indicates that Fz signaling regulates the initiation of the asymmetric localization of Baz at the posterior cortex. At metaphase, however, Baz and Pins form misoriented crescents relative to the a-p axis that localize at opposite poles in fz mutant pI cells. It is concluded that the formation of the two opposite Baz and Pins domains does not depend on fz activity, and that planar asymmetry can be established in the absence of Fz signaling. However, as previously seen for pins, the asymmetric distribution of Baz is less pronounced in fz mutant pI cells than in wild-type cells. Moreover, Dlg is distributed around the entire cell cortex, indicating that Fz signaling is required for the anterior accumulation of Dlg (Bellaïche, 2001).
Since Pins localizes asymmetrically in a Fz-independent manner, it was asked whether Pins is necessary to localize Baz at one pole of the pI cell in the absence of Fz. It was found that Baz localizes uniformly around the basal-lateral cortex in 82% of the fz;pins double mutant pI cells at metaphase. Moreover, although Numb forms a crescent at anaphase in pI cells mutant for pins or fz, no Numb crescent is seen at either metaphase or anaphase in fz;pins double mutant pI cells. Consistently, loss of fz activity enhances the pins bristle loss phenotype. These data show that Pins and Fz act in a redundant manner to exclude Baz from the anterior cortex and to establish planar asymmetry in the pI cell (Bellaïche, 2001).
These results show that Pins localizes to the anterior cortex in a Baz-independent manner, in an orientation opposite that of Baz, as does Numb. Pins cooperates with Fz to exclude Baz from the anterior cortex of the pI cell. In contrast, in neuroblasts, Pins localizes in a Baz-dependent manner to the apical pole, opposite Numb, and stabilizes the Insc/Baz/DmPAR-6/DaPKC complex. Nevertheless, Pins promotes the localization of Numb in both cell types (Bellaïche, 2001).
One important difference between pI cells and neuroblasts is the lack of insc expression in pI cells. To test the functional significance of this lack of Insc, Insc was expressed in the pI cell. Under these circumstances, Insc and Pins localize at the anterior cortex. Insc triggers the anterior relocalization of Baz, while Numb forms a posterior crescent at anaphase. The pI cell division remains planar. This contrasts with the effect of Insc in epithelial cells. In these cells, Insc localizes apically and orients the spindle along the apical-basal axis. This further indicates that the apical-basal polarity is remodeled in the pI cell. It is concluded that the ectopic expression of Insc is sufficient to reverse the planar polarity axis of the pI cell and to modify the activity of Pins relative to Baz. In the absence of Insc, the Dlg/Pins complex excludes Baz, while expression of Insc leads to the formation of a Pins/Insc/Baz complex. In both cases, Numb localization is opposite that of Baz (Bellaïche, 2001).
The PAR-3/PAR-6/aPKC complex is required to establish polarity in many different cell types, including the C. elegans zygote and epithelial and neuronal cells in Drosophila and mammals. In each context, the components of this complex display a mutually dependent asymmetric cortical localization. PAR-6 is a direct effector of Rho family GTPases and binds to and regulates aPKC. Mammalian PAR-3 (mPar3) can associate with transmembrane proteins and may link the complex to the membrane, but this can account for only part of the requirement for this protein in the complex. The function of a novel conserved domain, CR1, of PAR-3 has been investigated using computational, biochemical, and genetic approaches. Sequence-structure comparison by FUGUE predicts that CR1 has the same structural fold as a bacterial oligomerization domain. CR1 of the Drosophila homolog, Bazooka (BAZ), mediates oligomerization in vitro and in vivo. Furthermore, deletion of CR1 disrupts BAZ localization in both epithelial cells and the germline and strongly impairs BAZ function in epithelial polarity. These results indicate that this domain is important for the localization and activity of the PAR-3/PAR6/aPKC complex and define a new role for PAR-3 in assembling higher order protein complexes (Benton, 2003).
CR1 is not homologous to any known protein domain, but Position-Specific Iterated BLAST searches reveal significant similarity to an N-terminal region of unknown function in mammalian histidine ammonia lyases (HAL). Since this primary structural analysis was uninformative, the identification of higher order structural homologs of CR1 was undertaken using the FUGUE program. FUGUE aligns a given query sequence with a database of proteins whose structures have been determined to calculate a statistical score (Z score) of the likelihood of the query adopting a particular known fold, using environment-specific amino acid substitution tables and structure-dependent gap penalties. The former are derived from the analysis of the occurrence of each amino acid in each type of structural environment, and the latter take into account the possible variability in the lengths of surface turns and loops in otherwise structurally homologous proteins. The sensitivity and accuracy of the search is enhanced by generating a consensus query sequence from a multiple alignment of all primary sequence homologs (in this case, all PAR-3 and HAL proteins). With BAZ CR1 (amino acids 1–83) as a query, FUGUE identifies one 'likely' match: E. coli DinI. DinI is similar in length to CR1 and forms a ßalphaßßalpha structure, but does not show significant primary sequence homology to CR1. The similarity in the predicted structure of CR1 to DinI is therefore unlikely to reflect a common evolutionary origin of these proteins, but may reflect a shared biochemical property. Notably, both DinI and a homologous protein in coliphage 186, Tum, have been reported to form dimers or tetramers in vitro. Furthermore, two enzymes (cytokine D-dopachrome tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that have, like CR1, a similar fold but no obvious sequence homology to DinI can also form multimers. These observations suggest that the protein fold adopted by DinI may represent a common oligomerization domain (Benton, 2003).
CR1 mediates oligomerization. Using the yeast two-hybrid system, it was found that both BAZ and mPar3 CR1 can indeed self-associate. This interaction is direct, since a purified MBP-fusion protein of the BAZ N terminus precipitates full-length BAZ translated in vitro. To determine whether BAZ self-associates in vivo, a full-length BAZ:GFP fusion protein was immunoprecipitated from embryonic extracts using an anti-GFP antibody and a Western blot of the immunoprecipitate was probed with an anti-BAZ antibody. Endogenous BAZ was found to efficiently copurify with this fusion protein. The amount of endogenous protein in the immunoprecipitate is in fact about twice that of the tagged protein, suggesting that each molecule of BAZ binds to several other molecules to form higher order complexes. In contrast, a GFP-tagged truncated version of BAZ, lacking the oligomerization domain (BAZ-deltaN:GFP), fails to coimmunoprecipitate endogenous BAZ, indicating that this domain is essential for BAZ oligomerization in vivo (Benton, 2003).
To assess the importance of this property, the subcellular localization of these GFP-fusion proteins was first analyzed, as well as a tagged version of the BAZ N terminus containing CR1 (BAZ-N:GFP). BAZ:GFP displays an identical distribution to endogenous BAZ in follicular epithelial cells: it colocalizes with aPKC along the apical cortex and concentrates at the zonula adherens marked by Armadillo (ARM; Drosophila ß-catenin). Almost no GFP fluorescence is detected elsewhere in these cells, indicating that BAZ is very efficiently recruited to these sites. In contrast, BAZ-deltaN:GFP is largely diffuse in the cytoplasm of epithelial cells, although a small proportion of the protein is detected at the apical cortex. These observations indicate that the ability of BAZ to oligomerize is important to concentrate it apically. The distribution of BAZ-N:GFP is similar to that of BAZ-deltaN:GFP: most of the protein is cytoplasmic, but a very small fraction localizes to the zonula adherens. Since BAZ-N:GFP contains the oligomerization domain, this localization is likely to be mediated by its association with the endogenous full-length protein (Benton, 2003).
When expressed in the germline, BAZ:GFP is efficiently recruited to the cortex of germ cells. The protein does not localize uniformly along the cortex, however, but forms discrete foci, which are reminiscent of the 'clusters' of PAR-3, PAR-6, and aPKC around the anterior cortex of the C. elegans embryo. BAZ-deltaN:GFP does not concentrate in such foci and, as in follicle cells, is largely diffuse within the cytoplasm. BAZ-N:GFP displays a striking distribution in the germline, in huge spherical cytoplasmic aggregates of up to 10 microm diameter. Such aggregates, albeit smaller, are also observed in follicle cells, and these may reflect the ability of CR1 to self-associate to form oligomeric complexes (Benton, 2003).
To determine the functional importance of BAZ oligomerization, the ability of these truncated proteins to rescue baz mutant phenotypes was assessed. When full-length BAZ:GFP is expressed in baz mutant follicle cell clones, it efficiently rescues epithelial polarity, as assessed by the localization of aPKC, the formation of the zonula adherens (detected by ARM staining), and cell morphology. BAZ-deltaN:GFP displays partial rescuing activity and can be detected, albeit weakly, at the zonula adherens in morphologically normal cells, indicating that its recruitment to this site must depend upon interactions with proteins other than endogenous BAZ. In approximately half of the egg chambers, however, mutant cells display either abnormal polarity and morphology or, more frequently, appear to have been lost from the epithelium (no gaps are ever seen in baz clonal egg chambers that express BAZ:GFP). Thus, the N terminus of BAZ is important, but not absolutely essential, for its function in the follicular epithelium. As expected, BAZ-N:GFP does not significantly rescue baz mutant phenotypes, and almost all egg chambers contain gaps in the epithelium (Benton, 2003).
The functional properties of these proteins was analyzed by scoring their ability to rescue the embryonic lethality of baz zygotic mutants. The full-length protein efficiently rescues embryonic lethality, and the cuticles of the few unhatched embryos are almost wild-type, containing only a very small dorsal hole. In contrast, the truncated proteins are either severely (BAZ-deltaN:GFP) or completely (BAZ-N:GFP) compromised in their ability to rescue lethality. In both cases, the cuticles of the dead embryos are fragmented or contain large dorsal holes, similar to control baz mutant embryos. These results indicate that the oligomerization domain of BAZ is important in vivo (Benton, 2003).
These data demonstrate that oligomerization is critical for the efficient apical localization of BAZ in epithelial cells; this is essential for its function in establishing apical-basal polarity. This property is likely to be conserved, since CR1 of mPar3 also self-associates. Indeed, these results provide an explanation for the inhibitory effect of overexpression of an N-terminal portion of mPar3 on the polarization of cultured hippocampal neurons. This region lacks the PDZ domains and the aPKC binding site, but still contains the CR1 domain, and may therefore exert its dominant-negative effect by associating with and inhibiting the oligomerization of endogenous mPar3. No dominant-negative effects of BAZ-N:GFP were detected, but this could reflect the fact that most of the protein is sequestered in apparently harmless cytoplasmic aggregates. CR1 may therefore have a higher affinity for itself than for the full-length protein, and the ectopic expression levels of this domain achieved in Drosophila cells may be insufficient for it to exert the inhibitory effects observed in the mammalian system (Benton, 2003).
It is unclear how the BAZ/PAR-6/aPKC complex is targeted to the apical membrane in epithelia, but evidence from other organisms indicates that this is likely to be mediated by the association of BAZ with the cytoplasmic tail of an apical transmembrane protein. The observation that the removal of CR1 disrupts its localization suggests that this domain is essential to stabilize the association of the complex with the apical membrane. Since CR1 is not sufficient for significant localization, it seems unlikely that it binds directly to an apical anchor. A more probable explanation for its role is that it increases the avidity of the interaction with the apical membrane by generating oligomeric complexes that contain multiple binding sites for such anchors. The self-association of CR1 may also contribute to the function of the complex by allowing a single membrane-tethered molecule of BAZ to nucleate the formation of higher order BAZ/PAR-6/aPKC complexes along the cortex. This may serve as an amplification mechanism by generating, for example, a high local concentration of aPKC activity in response to an upstream spatial cue. Moreover, the ability of BAZ to form an interconnected protein network could underlie its function in establishing a physical barrier to prevent the lateral movement of membrane or cortical proteins (Benton, 2003).
Two other proteins involved in epithelial polarity, Discs Lost (DLT: now redefined as Drosophila Patj) and Lethal(2) Giant Larvae (LGL), have also been observed to self-associate in vitro and/or in vivo. These proteins are thought to be components of distinct protein complexes that act in conjunction with the BAZ/PAR-6/aPKC complex to establish apical-basal asymmetries in Drosophila epithelia. While the biological significance of oligomerization has not yet been demonstrated for either DLT or LGL, these observations, together with this functional analysis of BAZ, suggest that the ability to form interconnected protein networks or complexes is fundamental to the establishment of cell polarity (Benton, 2003).
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).
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 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).
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 EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).
Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins 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 Pon, but not spindle orientation. Bazooka also regulates cell proliferation in the SOP lineage; loss of bazooka function results in supernumerary cell divisions and apoptotic cell death (Roegiers, 2001).
For Bazooka to be involved in every asymmetric division of the
adult SOP lineage one might expect Bazooka to be expressed in every
precursor cell. Indeed, Bazooka is asymmetrically localized in every dividing cell of the SOP lineage.
Starting with a strong accumulation at the apical surface of interphase
pI cells, specifically at junctions with neighboring epithelial cells, Bazooka becomes enriched at the posterior cortex
during mitosis and shows no overlap with the anterior Numb crescent at
metaphase. By early anaphase, Bazooka forms a smooth posterior crescent. At
anaphase B, Bazooka is localized to the posterior cortex, although a
significant amount remains anterior to the cleavage furrow. The pI
division is followed by division of the pIIb cell, which exhibits an
apical-posterior crescent of Bazooka at mitosis. Subsequently, in the mitotic
pIIa, Bazooka accumulates in the cell cortex and a strong patch of
Bazooka is detected at the anterior cortex, a region that coincides
with the position of the anterior-most centrosome of the mitotic
spindle. Finally, in the mitotic pIIIb cell, Bazooka forms an
apical-posterior crescent similar to the one observed in the pIIb cell. In
the pI, pIIb, and pIIIb divisions, Bazooka is localized opposite the
Numb crescent in mitosis; whereas in the mitotic pIIa cell, the
anterior accumulation of Bazooka may colocalize with Numb. Following
completion of these asymmetric divisions, Bazooka expression is
enriched at the apical borders in cells of the developing es organ,
specifically the hair and socket cells. Based on these observations, it is
concluded that Bazooka is expressed in all precursor cells within the
SOP lineage; it is asymmetrically enriched during each cell division of
the SOP lineage, and its expression is maintained in the postmitotic
cells that will give rise to the external structures of the es organ,
the hair and socket cells (Roegiers, 2001).
During embryonic neuroblast divisions, Bazooka is required not only to
localize Inscuteable to the apical cortex and Numb, Miranda, Prospero,
and Pon to the basal cortex, but also to orient the mitotic spindle
along the apical-basal axis. To determine the requirement
of bazooka in the asymmetric divisions of the adult SOP
lineage, the MARCM system was used to generate baz
mutant clones expressing both Pon-GFP (as a reporter for Numb localization) and Tau-GFP (as a reporter for spindle orientation) under
the control of scabrous-GAL4, which is strongly expressed in the SOP cell and in the SOP lineage. The movements of Pon-GFP
and Tau-GFP were monitored in live tissue throughout all asymmetric
divisions of the SOP lineage. In bazxi106
or bazEH171 null mutant clones, pI cells
underwent mitosis at ~15 h APF as in wild type. However, in all
mutant pI cells observed, Pon-GFP remained
uniformly distributed and never formed an anterior crescent as seen in
dividing wild-type pI cells. Nor did Pon-GFP crescents form in the subsequent
divisions in the lineage. Thus, although only the pIIb resembles the embryonic neuroblast in its orientation of division and requirement for
Inscuteable, Bazooka is required for the asymmetric Pon/Numb
localization in the pI division, as well as all subsequent divisions (Roegiers, 2001).
Because Numb functions as an asymmetrically localized cell-fate
determinant in the SOP lineage, the absence of Numb crescents in
baz mutant clones could lead to cell-fate transformations in the daughters of the pI cell. Thus the anterior daughter cell of the pI in bazooka mutant clones (the pIIb
cell in the wild type) is referred to as pIIbb, and the
posterior daughter cell as pIIab. It is worth
noting, however, that either loss-of-function or misexpression of
numb causes cell-fate transformation only in a subset of
sensory organs, presumably because the Notch-mediated mutual
inhibition may still allow the two daughter cells to adopt different
cell fates, albeit without a bias set by the Numb crescent.
Transformation of pIIa to pIIb cell fate is known to alter the timing
of mitosis of the transformed pIIa cell. Timing of the pIIbb, pIIab, and pIIIbb
divisions is indistinguishable from wild-type pIIb, pIIa, and pIIIb
cells. In addition, the pI and pIIab spindles
align along the A-P axis in all mutant clones. And in eight of the nine clones examined the pIIbb spindles were oriented along the
apical-basal axis as in wild type (the remaining
pIIbb cell divided before the
pIIab, but had its spindle oriented along the
anterior-posterior axis). Because an apically localized Inscuteable is
required for mitotic spindle positioning in the pIIb cell, Inscuteable localization was also examined in the pIIbb cell in bazooka mutant clones. Inscuteable is localized to an
apical stalk in pIIbb, similar to the wild-type
pIIb (n = 12). Thus the great majority of
pIIbb and pIIab cells
resemble wild-type pIIb and pIIa cells in their timing and orientation
of division, as well as the expression of Inscuteable in the
pIIbb. It therefore appears that these
bazooka mutations do not cause detectable cell-fate
transformation in most of the pIIbb and
pIIab cells, although it remains possible that
there are partial transformations and cell-fate changes in a subset of
these cells. In light of these observations, the complete loss of
Pon-GFP crescents in every mitotic pIIbb and
pIIab cell examined strongly supports a model wherein
Bazooka controls Pon/Numb asymmetric localization in not only pI but
also pIIb and pIIa cells (Roegiers, 2001).
Some cell-fate changes apparently take place in the progeny of pIIa and
pIIb. On adult nota, mutant clones contain patches of bald cuticle
and regions with small bumps that may represent lost ES organs, and
sockets without hairs. Ectopic
Prospero-expressing cells were found in baz mutant clones, indicating that additional sheath and/or
glial cells are present because of cell-fate transformations in the
pIIbb lineage. Interestingly, in six of nine
mutant SOP lineages examined, ectopic divisions in the
pIIab cell lineage were found.
Whereas the wild-type pIIa cell divides only once to give rise to two
external cells of the ES organ (the hair and socket cells), in
baz mutant clones each daughter of the
pIIab cell undergoes another round of division,
causing the SOP lineage to produce a cluster of seven cells, as opposed
to the normal five cells (Roegiers, 2001).
In the wild-type SOP lineage, shortly after the last division of
pIIIb (~24 h after pupa formation) one of the pIIIb daughters
forms a neuron and extends an axon. Within an hour after completion of
the pIIIb mitosis, the small glial cell migrates away from the cluster
along the axon. By following the development of the ES organs
in vivo in baz mutant clones to observe their
morphogenesis, no axon extension or glial cell migration was found in 16 of 20 ES organs examined. Moreover, within 3-6 h
after the last mitosis, clusters of cells underwent apoptosis in ten of twenty clones examined. Apoptotic bodies formed and dispersed rapidly. Cell death of ES organ cells is specific to the bazooka mutants, because apoptosis was never observed in wild-type ES cells (Roegiers, 2001).
Bazooka and its homolog Par-3 in C. elegans are known to
be required for asymmetric divisions, in embryonic neuroblasts in
Drosophila and in the zygote and early
blastomeres of the worm embryo, respectively. In
Drosophila neuroblasts, Bazooka forms a complex with Pins
and localizes Inscuteable, which coordinates the asymmetric
localization of Numb and spindle orientation. In the
mitotic pI cell, an anterior Pins/Dlg complex has been shown to be
required for Bazooka localization, and Bazooka is required for
Numb localization. This study of the adult SOP lineage reveals
several functions for Bazooka. (1) Bazooka is the first
molecule to be required for the asymmetric localization of Pon, the
adapter protein for Numb, in every division of the SOP lineage, even
though only the pIIb division resembles embryonic neuroblast division
both in its orientation along the apical-basal axis and its dependence on Inscuteable. It thus appears that Bazooka may localize Pon
and Numb in a pathway independent of Inscuteable. (2) Bazooka is
not required for proper spindle orientation in the asymmetric
divisions of the SOP lineage. The function of Bazooka in the SOP
lineage, therefore, concerns only determinant localization but not
spindle orientation. Unlike the neuroblast, in the pIIb cell
Inscuteable orients spindles along the apical-basal axis in the
absence of Bazooka, indicating that pIIb cells may have a
Bazooka-independent mechanism for Inscuteable localization. (3) Although bazooka mutations did not cause detectable
cell-fate transformation in most pIIb and pIIa cells, there is apparent cell-fate transformation occurring in the pIIbb
lineage, and possibly partial cell-fate transformations of the pIIab lineage leading to formation of cells of
indeterminate cell fates, such as sockets without hairs or the bumps in
bazooka mutant clones. (4) Loss of Bazooka function
leads to apoptosis. The possibility that
inadequate cell-fate specification results in apoptosis cannot be ruled out.
However, the cell-fate transformations in the SOP lineage in various
mutants reported thus far have not been associated with
apoptosis, indicating that cell-fate transformation per
se does not necessarily lead to apoptosis. The terminal
fates of mutant es cell clusters are difficult to determine with
certainty, because most undergo apoptosis rather than
differentiation. (5) Bazooka appears to limit the number of cell
cycles of the pIIa to one; the pIIab daughters
in bazooka mutant clones often proceed with mitosis instead
of differentiating into hair and socket cells. Similarly, antiproliferative activity has been found in follicle cells of the
ovary. (6) Finally, loss of Bazooka function leads to failure of ES
neuron axonal outgrowth and glial cell migration. These defects could reflect cell-fate changes in the pIIbb lineage or a requirement for bazooka in differentiation of ES organ cells (Roegiers, 2001).
In summary, Bazooka has a much broader spectrum of
function in the SOP lineage than previously suspected. In
bazooka mutant clones the Numb-anchoring protein Pon fails
to form a crescent in every division of the SOP lineage, regardless of
the requirement for Inscuteable. The function of Bazooka in the SOP lineage also differs from that in embryonic neuroblasts because Bazooka
controls spindle orientation in neuroblasts but not in the SOP lineage.
The pI, pIIb, and pIIa cells show little evidence of cell-fate
transformation in bazooka mutant clones, and yet exhibit a
total loss of Pon-GFP crescent formation. It thus appears that Bazooka
controls Pon/Numb crescent formation in these precursors with
different planes of division. Although in bazooka mutant clones there appears to be partial cell-fate transformation, in later
divisions in the lineage it is striking that asymmetric localization
of determinants is abolished in all divisions. The loss of Pon-GFP
crescent is fully penetrant, in contrast to the variable and partially
penetrant cell-fate transformation phenotype. These observations
suggest that Bazooka is the general link between polarity cue and the
localization of cell-fate determinants in all asymmetric cell
divisions. Other previously uncharacterized functions uncovered in this
study include the ability of Bazooka to restrict the number of
divisions in the SOP lineage and to promote differentiation instead of
apoptosis. It is worth noting that the function of Bazooka in
the central nervous system has been previously studied
only for the neuroblast division. Based on these findings in the SOP
lineage, it will be interesting to learn whether in the CNS Bazooka
also has an Inscuteable-independent role in controlling asymmetry of
subsequent divisions, as well as in regulating proliferation and
apoptosis. Given that Bazooka/Par-3 is part of an
evolutionarily conserved gene cassette, these findings of a myriad
of previously uncharacterized functions of Bazooka in the sensory organ
lineage raise the possibility that Bazooka/Par-3 may have a similarly
wide range of functions in vertebrates (Roegiers, 2001).
Inscuteable is the founding member of a protein complex localized to the apical cortex of Drosophila neural progenitors that controls progenitor asymmetric division. Aspects of asymmetric divisions of all identified apicobasally oriented neural progenitors characterized to date, in both the central and peripheral nervous systems, require inscuteable. The generality of this requirement has been examined. Many identified neuroblast lineages, in fact, do not require inscuteable for normal morphological development. To elucidate the requirements for apicobasal asymmetric divisions in a context where inscuteable is not essential, focus was placed on the MP2 ---> dMP2 + vMP2 division. For MP2 divisions, asymmetric localization and segregation of Numb and the specification of distinct dMP2 and vMP2 identities require bazooka but not inscuteable. It is concluded that inscuteable is not required for all apicobasally oriented asymmetric divisions and that, in some cellular contexts, bazooka can mediate apicobasal asymmetric divisions without inscuteable (Rath, 2002).
Two obvious candidate molecules that might be responsible for mediating the MP2 asymmetric division are Baz and Partner of Inscuteable (Pins). pins appears not to be a major player since, in embryos lacking both maternal and zygotic pins, only 5.7% of the hemisegments show dMP2 duplication, as demonstrated by three odd-positive cells. Assessing the role of baz is problematic since loss of zygotic function has no effect, and removing both the maternal and zygotic baz results in embryos with severe morphological defects that prevent scoring of dMP2 and vMP2 fates in older embryos. These problems were circumvented by performing RNAi with baz double-stranded RNA on AJ96 embryos, which yielded ~25% embryos with reduced Baz protein but without the severe morphological defects that prevented scoring of vMP2 and dMP2 identities. In such embryos, vMP2>dMP2 transformations are observed in the great majority of hemisegments (57/60), as demonstrated by the presence of two cells double positive for ß-Gal and Odd. Moreover, localization of Pon and Numb becomes cortical in dividing MP2 (38/40). However, there does not appear to be a dramatic defect on the orientation of the cell division, with almost all of the MP2 divisions oriented within 45° of the A/B axis (32/34). These defects associated with baz RNAi cannot merely be due to secondary effects associated with a disruption of the epithelium since, in crumbs loss of function, Baz (12/12) and Pon (10/10) remain correctly localized to the MP2 apical and basal cortex, respectively. These results indicate that Baz is required for the asymmetric localization of Pon and Numb in the MP2 asymmetric division (Rath, 2002).
Although apical complex members, like Baz, Insc and Pins, are expressed and apically localized in both MP2 and NBs, their behavior appears to differ somewhat in the two cellular contexts. For example, when baz function is attenuated, Insc is in the cytosol of NBs while Insc remains localized as an apical crescent in most dividing MP2 cells; similar results are seen in dividing MP2s of embryos derived from bazXi106 germline clones, which lack both maternal and zygotic function. Moreover, it is interesting to note that in the absence of insc function, Baz and Pins can be localized to the apical cortex of metaphase MP2s, although the intensity of staining is always reduced compared with WT MP2s, and in some cases the weak apical crescent of Baz can be difficult to detect. These observations suggest that even a small amount of apically localized Baz is sufficient to mediate basal localization of Pon and direct the MP2 asymmetric division. Strikingly, Baz, but not Insc and Pins, seems to play a dominant role in mediating Pon/Numb basal localization in MP2. When baz function is attenuated, Pon/Numb become cortically localized even though Insc and Pins can remain apically localized. These observations indicate that the precise requirements for asymmetric protein localization differ between MP2s and NBs (Rath, 2002).
MP2 appears to be the only known A/B-oriented asymmetric division that does not require insc. Although MP2 delaminates from the neuroectoderm and divides in an apico-basal fashion like NBs, there are unique features that set MP2 apart from other neuroblasts. Unlike NBs that divide in a stem-cell-like mode, MP2 undergoes one differentiative division, making it more like a GMC or a pIIb division. Insc is present in both GMC/SOP lineages. A/B-oriented asymmetric GMC divisons, like those of GMC4-2a, require insc. While the first SOP division (the anterior-posterior pI > pIIa + pIIb) does not require insc, recent work has shown that the spindle orientation of the strikingly GMC-like A/B division of the pIIb cell is dependent on Insc. Finally, unlike NBs, Pros shows nuclear localization in MP2. There is evidence supporting the view that Pros acts to terminate cell proliferation during Drosophila neurogenesis. It is plausible that both GMCs and the MP2 precursor use nuclear Pros as a cue to reduce their mitotic potential and undergo a single differentiative division. Two recent reports have shown that planar asymmetric divisions undertaken by pI in the peripheral nervous system and epithelial cells with disrupted adherens junctions both require baz and not insc. It has been demonstrated in this study that insc is not required for all A/B-oriented asymmetric divisions. These findings further support the view that baz is a more general mediator of asymmetric divisions than insc, and can act to promote both A/B and planar asymmetric divisions in the absence of insc (Rath, 2002).
Cell division often generates unequally sized daughter cells by off-center cleavages, which are due to either displacement of mitotic spindles or their asymmetry. Drosophila neuroblasts predominantly use the latter mechanism to divide into a large apical neuroblast and a small basal ganglion mother cell (GMC), where the neural fate determinants segregate. Apically localized components regulate both the spindle asymmetry and the localization of the determinants. Asymmetric spindle formation depends on signaling mediated by the Gβ subunit of heterotrimeric G proteins. Gβ13F distributes throughout the neuroblast cortex. Its lack induces a large symmetric spindle and causes division into nearly equal-sized cells with normal segregation of the determinants. In contrast, elevated Gβ13F activity generates a small spindle, suggesting that this factor suppresses spindle development. Depletion of the apical components also results in the formation of a small symmetric spindle at metaphase. Therefore, the apical components and Gβ13F affect the mitotic spindle shape oppositely. It is proposed that differential activation of Gβ signaling biases spindle development within neuroblasts and thereby causes asymmetric spindles. Furthermore, the multiple equal cleavages of Gβ mutant neuroblasts accompany neural defects: this finding suggests indispensable roles of eccentric division in assuring the stem cell properties of neuroblasts (Fuse, 2003).
During mitosis, neuroblasts localize the cell fate determinants Prospero and Numb to the basal cortex and orient the mitotic spindle along the apical-basal axis to segregate the determinants into GMCs. These processes are regulated by the apical protein complex that includes Inscuteable, Bazooka, atypical protein kinase C (DaPKC), the G protein subunit Gαi, and Partner of Inscuteable (Pins). The depletion of any single apical component does not severely affect the cell size difference between the neuroblast daughters. However, a recent study shows that the two signaling pathways, Bazooka/DaPKC and Pins/Gαi, within the apical complex control in parallel the production of unequal-sized daughters (Fuse, 2003).
During a mutational screen with Miranda, the adaptor protein of Prospero, the f261 mutant, which is defective in unequal-sized neuroblast divisions, was obtained. In germline clone embryos that are both maternally and zygotically mutant for f261 (f261 mutant), the neuroblasts produce nearly equal-sized daughters, although the GMC is still slightly smaller than the sibling neuroblast after the initial divisions. Nevertheless, after a slight delay in crescent formation, Miranda localizes normally in f261 neuroblasts and segregates to the GMC. Consequently, Prospero is inherited by the GMCs. The abnormal division in f261 causes neuroblasts to be smaller and smaller after each succeeding division. The f261 mutant turned out to be a protein null mutant of the Gβ13F gene that encodes a β subunit of heterotrimeric G proteins. In wild-type neuroblasts, this protein distributes uniformly at the cell cortex. A deletion mutant of Gβ13F has been reported (Schaefer, 2001) to show delayed localization of Miranda and randomized orientation of neuroblast division, as well as gastrulation defects, all of which occur in f261 embryos, but cell size defects have not been described. Deletion mutants lacking the entire Gβ13F coding sequence have been created. Such a mutant, Gβ13FΔ15, as well as the deletion mutant reported previously (Schaefer, 2001) indeed show the same neuroblast phenotypes as those of f261 embryos. Therefore, the loss of Gβ13F activity affects cell size asymmetry but essentially does not affect the segregation of the cell fate determinants. The neuroblast phenotypes observed in the Gβ13F mutants are not consequences of morphological defects before neuroblast formations because the neuroblast-specific expression of Gβ13F rescues the phenotype of cell size asymmetry (Fuse, 2003).
In canonical heterotrimeric G protein signaling, the Gβ and Gγ complex (Gβγ) associates with the GDP form of Gα, but the conversion of GDP to GTP releases Gβγ from Gα; both Gβγ and Gα can then signal downstream. In Drosophila neuroblasts, it is unlikely that GTP-Gαi acts as a signal. Instead, it has been suggested that the GDP form of Gαi binds to Pins to release the Gβγ subunit (Schaefer, 2001). According to this model, Pins-dependent activation of Gβ signaling occurs at the apical cortex, where Pins and Gαi are colocalized. Unlike the Gβ13F mutants, defects in unequal-sized divisions are observed only in a small fraction of pins mutant neuroblasts, probably because of the bazooka/DaPKC activity that functions in parallel to form asymmetric spindles. Therefore the effects of pins and Gβ13F on microtubule development were compared under conditions in which bazooka activity is simultaneously depleted. In the absence of both Gβ and bazooka, metaphase neuroblasts form a large symmetric spindle resembling that seen in f261. In contrast, the simultaneous loss of pins and bazooka activities results in the formation of a small symmetric spindle at metaphase, which is rather similar to the basal half of the wild-type spindle. Therefore, Gβ and Pins exert opposite effects on spindle formation during metaphase in the absence of bazooka. This reciprocal effect of Pins and Gβ on spindle development is not straightforwardly deduced from the model that shows that Pins induces the free and active Gβγ. These states of the mitotic spindle in the double mutants appear to persist throughout mitosis because the midbody, the bundled central spindle at telophase, is notably narrower in the pins-bazooka double mutant than in the Gβ-bazooka mutant. In comparison, astral microtubules develop to a similar extent from anaphase onward under those two mutant conditions. The asters in these double mutants develop more than the basal half of wild-type but less than that seen in f261 and appear at an intermediate level. The differential influence of the mutations on the mitotic spindle (or central spindle) and asters may originate from different mechanisms that regulate these microtubule structures. This possibility has been suggested by the existence of asterless mutants, in which asters are apparently absent, whereas the mitotic spindle appears to develop normally. The role of astral microtubules in cell size asymmetry is controversial because asterless mutant neuroblasts still bud off small GMCs by forming an asymmetric central spindle (Fuse, 2003).
Drosophila neuroblast asymmetric divisions generate two daughters of unequal size and fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apicobasal orientation of the mitotic spindle, but how daughter cell size is controlled has remained unclear. Mitotic spindle geometry and unequal daughter cell size were shown to be controlled by two parallel pathways (Bazooka/DaPKC and Pins/Galphai) within the apical complex. While the localized activity of either pathway alone is sufficient to mediate the generation of an asymmetric mitotic spindle and unequally sized neuroblast daughters, loss of both pathways results in symmetric divisions. In sensory organ precursors, Bazooka/DaPKC and Pins/Galphai localize to opposite sides of the cortex and function in opposition to generate a symmetric spindle (Cai, 2003).
Thus members of the NB apical protein complex control the generation of daughter cells of unequal size. There are two redundant pathways: (1) Baz/DaPKC/ (and presumably DmPar6) as well as (2) Pins/Gαi, either of which, when asymmetrically localized to the NB cortex, can lead to the formation of an asymmetric mitotic spindle through the preferential elongation of the proximal spindle arm and the displacement of the spindle toward the distal cell cortex, resulting in the production of unequal-sized daughter cells. In addition, in NBs, Insc is required for the function of the Baz/DaPKC/(DmPar6) pathway. When both pathways are inactivated/attenuated, spindle asymmetry and displacement fail to occur and equal-sized daughter cells are produced at high frequency. In the PNS progenitor, pI, where Baz/DaPKC are localized to the posterior cortex and Pins/Gαi are localized to the anterior cortex, the mitotic spindle is symmetric. Consistent with this hypothesis that both pathways can act to cause the preferential elongation of the proximal spindle arm relative to the distal spindle arm, removing posterior baz function without abolishing the localization and function of the anterior components results in the production of an asymmetric spindle with an anterior bias; removing anterior pins function without affecting the function of the posterior components results in a posteriorly biased asymmetric spindle; if components of both pathways are localized to the anterior cortex through the ectopic expression of Insc, an anteriorly biased asymmetric spindle results. These findings suggest that DaPKC and hetrotrimeric G protein signaling work in conjunction in the NB to produce an asymmetric spindle and in opposition in pI to produce a symmetric spindle (Cai, 2003).
Several lines of evidence suggest that localized signaling is essential to generate an asymmetric spindle and daughter cells of unequal size. (1) When both signaling pathways are abolished/attenuated (e.g., in insc/pins double mutant) or when signaling is uniform, which is assumed to be the case when Baz/DaPKC/Pins/Gαi are all uniformly localized throughout the cell cortex (e.g., in the case of Gαi overexpression in wt NBs), equal-sized daughters are generated. (2) When pins function is removed and DaPKC/Baz is asymmetrically localized (e.g., in pins mutant NB) or when Pins/Gαi are uniformly cortical but DaPKC/Baz are asymmetrically localized (e.g., in 69% [n = 51] of wt NBs overexpressing C-Pins), the site of the DaPKC/Baz localization coincides with the position where the larger daughter forms. (3) When Pins/Gαi is asymmetrically localized but baz/DaPKC function has been compromised (e.g., in insc mutant) or when Pins/Gαi is asymmetrically localized but Baz/DaPKC is uniformly cortical (in the case of NBs with basal Pins-C-Pon crescents), the site of localization coincides with the larger daughter and the extended spindle arm. These observations indicate that just one localized signal source, mediated presumably by either heterotrimeric G protein or DaPKC, is sufficient to cause proximal spindle arm elongation and the generation of unequal-sized daughters (Cai, 2003).
The situation is different in pI where Baz/DaPKC/(DmPar6) and Pins/Gαi act in opposition and where Insc is not required for the function of the Baz/DaPKC/(DmPar6) with respect to spindle elongation. Here, a distinction can be made between two possible models for explaining how spindle asymmetry/geometry is mediated. The first model is that the presence of either asymmetrically localized Baz/DaPKC/(DmPar6) or Pins/Gαi on one side of the cell is sufficient to cause elongation of the proximal spindle arm, regardless of what occurs on the other side of the cell. A second model would be that the signals from the opposite sides of the cortex are integrated and the bias in the spindle geometry depends on the relative magnitude of the two signals. The simplest prediction of the first model would be that the distance from the cleavage furrow to the spindle pole of wt telophase pI should be equivalent to the longer of the two spindle arms in telophase pI mutant for either baz or pins. This appears not to be the case. The average length of the longer spindle arm in telophase pI mutant for pins or baz is greater than that of a wt spindle arm and the length of the shorter of the spindle arms in mutant pI is less than that of a wt spindle arm . An equivalent analysis is difficult to do with NBs, since the size of the 30 or so NBs found in each hemisegment is more variable. Nevertheless, based on these observations the second type of model is favored (Cai, 2003).
Previous work has shown that Pins binds to the GDP bound form of Gαi and can cause Gαi to dissociate from Gβ13F; moreover, some phenotypes seen when Gαi is overexpressed in wt NBs (e.g., equal size divisions) are not seen when GαiQ205L, an activated form of Gαi lacking GTPase activity that should be in the GTP bound form, is overexpressed, or when Gβ13F function is abolished. These phenotypes therefore are unlikely to be induced by GTP bound Gαi or by depletion of Gβγ, suggesting that the GDP bound form of Gαi may be responsible for the equal size NB divisions seen when wt Gαi is overexpressed. These findings clearly support the view that the Pins/GDP-Gαi complex has a role for generating the signal associated with spindle asymmetry. (1) Equal size divisions seen when Gαi is overexpressed in wt NBs is drastically reduced when overexpression is performed in the absence of Pins. (2) Whenever unequal size division occurs when Baz/DaPKC function is compromised, Pins and Gαi are always colocalized to the side of the cell where the future larger daughter is formed (Cai, 2003).
Although in the nematode embryo generation of unequal-sized daughters involves only the posterior displacement of a symmetric spindle, there appears to be some parallels between the two model systems. In the wt nematode P0 division, the magnitude of the forces acting on the two spindle poles apparently depend on the character of the anterior and posterior cortex. In wt P0, PAR-3 and PAR-2 localize to the anterior and posterior cortex, respectively, and the mitotic spindle is displaced toward the posterior pole, correlating with a greater net posterior force acting on the posterior spindle pole relative to the net anterior force acting on the anterior spindle pole. In par-2 mutants, PAR-3 expands to occupy the whole of the cortex, imparting anterior character throughout, and the net force acting on both spindle poles has a magnitude equivalent to that of the wt force acting on the anterior spindle pole. Conversely in par-3 P0, PAR-2 becomes cortical, imparting posterior character to the entire cortex, and the magnitude of both forces acting on the spindle poles is equivalent to that of the wt posterior acting force. In both par-2 and par-3 mutants, the forces acting on the spindle poles are equalized, mitotic spindle is no longer displaced, and equal-sized daughters result (Cai, 2003).
In Drosophila NBs, although spindle displacement occurs, the generation of an apically biased mitotic spindle mediated by either asymmetrically localized Baz/DaPKC or Pins/Gαi makes the major contribution to the difference in daughter cell size. It is proposed that the asymmetric localization of components of either of these pathways can make the region of the cell cortex they occupy different from the cortical regions that they don't occupy through localized DaPKC or heterotrimeric G protein signaling mediated through Pins/Gαi. In wt NBs, the components of either pathway would impart apical character to the cell cortex where they are localized. One effect of the asymmetric signaling is to generate the preferential elongation of the spindle arm closest to the site of the localized signal. If signaling is symmetric, for example either when Baz/DaPKC and Pins/Gαi are all uniformly cortical, or when Baz/DaPKC and Pins/Gαi are localized to opposite sides of a dividing progenitor, as in pI, a symmetric spindle results. Hence, in both the nematode P0 and in Drosophila NBs the generation of unequal-sized daughters is regulated by asymmetrically localized cortical components. In the nematode there is compelling evidence that differential forces acting on the two spindle poles mediate spindle displacement and the generation of unequal daughters. However, NBs of Drosophila asterless mutants are apparently devoid of functional centrosomes and astral microtubules, yet they form functional asymmetric anastral mitotic spindles and undergo unequal cytokinesis to generate unequal size daughters. It remains to be seen how the localized properties of the NB cell cortex influences its spindle geometry (Cai, 2003).
The anterior-posterior axis of C. elegans is defined by the asymmetric division of the one-cell zygote, and this is
controlled by the PAR proteins, including PAR-3 and PAR-6, which form a complex at the anterior of the cell,
and PAR-1, which localizes at the posterior. PAR-1 plays a similar role in axis formation in Drosophila: the protein localizes to the posterior of the oocyte and is necessary for the localization of the
posterior and germline determinants. PAR-1 has recently been shown to have an earlier function in
oogenesis, where it is required for the maintenance of oocyte fate and the posterior localization of
oocyte-specific markers. The homologs of PAR-3 (Bazooka) and PAR-6 are also
required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that
develop 16 nurse cells and no oocyte. Furthermore, oocyte-specific factors, such as Orb protein and the
centrosomes, still localize to one cell but fail to move from the anterior to the posterior cortex. Thus, PAR-1,
Bazooka, and PAR-6 are required for the earliest polarity in the oocyte, providing the first example in Drosophila where the three homologs function in the same process. Although these PAR proteins therefore seem to play a conserved role in early anterior-posterior polarity in C. elegans and Drosophila, the relationships between them are different, since the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm (Huynh, 2001).
Drosophila oogenesis begins in region 1 of the germarium when a germline stem cell divides asymmetrically to give rise to a new stem cell and a cystoblast, which then undergoes four rounds of division with incomplete cytokinesis to produce a cyst of 16 cells interconnected by ring canals. A vesicle-rich organelle called the fusome ensures that the pattern of divisions is invariant by anchoring one pole of each spindle at every division to give rise to a cyst that contains two cells with four ring canals, two with three, four with two, and eight with one. The two cells with four ring canals both start to develop as oocytes and are therefore referred to as pro-oocytes. One of them then becomes a nurse cell along with the other 14 cells in the cyst, while the other differentiates as the oocyte. The determination of the oocyte requires the activity of BicD and Egalitarian (Egl) proteins, which both localize to this cell. How this cell is chosen remains unclear, but several lines of evidence suggest that this depends on the asymmetric segregation of the fusome during the cyst divisions, since one of the pro-oocytes always inherits more fusome than the other cells (Huynh, 2001).
PAR-1 localizes to the fusome in regions 1 to 2 of the germarium and is required for the determination of the oocyte, since all 16 cells become nurse cells in par-1 null mutant germline clones. Although this has provided the first example of a fusome component that plays a specific role in oocyte determination, a detailed analysis of the par-1 phenotype reveals that it is not required for the initial selection of the oocyte but for the maintenance of its fate. The determination of the oocyte can be followed with three kinds of markers: (1) oocyte-specific cytoplasmic proteins, such as Orb, BicD, and Egl, accumulate first in the pro-oocytes and then in the oocyte in a microtubule-dependent manner; (2) the centrosomes migrate along the fusome from the other cells of the cyst into the oocyte in a process that is not disrupted by microtubule-depolymerizing drugs; and (3) the oocyte is the only cell of the cyst to remain in meiosis, and this can be followed by the formation of the synaptonemal complex as the chromosomes pair during pachytene. The paired chromosomes then condense to form a hollow sphere called the karyosome, whereas the 15 nurse cells endoreplicate their DNA to become polyploid. Oocyte-specific proteins and the centrosomes still accumulate in one cell in par-1 null mutant germline clones, and this cell remains in meoisis longer than the other 15 cells. However, the centrosomes and oocyte-specific cytoplasmic proteins fail to translocate from the anterior to the posterior of the oocyte, and this cell soon exits meiosis and becomes a polyploid nurse cell. Thus, the par-1 phenotype identifies a new step in oocyte determination, which involves an anterior-posterior movement within the oocyte (Huynh, 2001 and references therein).
To investigate whether this early anterior-posterior polarity in the Drosophila oocyte shows further similarities with the anterior-posterior polarization of the first cell division in C. elegans, whether the Drosophila homologs of other PAR proteins play a role in oogenesis was examined. The best characterized of these is Bazooka, the homolog of PAR-3, which localizes to the apical side of ectodermal cells and neuroblasts in the embryo and is required for epithelial polarity and the localization of cell fate determinants during asymmetric neuroblast divisions. To test for a requirement for bazooka during oogenesis, germline clones of the strongest available allele, baz815-8, were induced; these clones were marked by the loss of green fluorescent protein (GFP). The majority of baz mutant egg chambers stop growing at stage 5 and adopt an oval shape. Furthermore, none of the cells in the cyst accumulate the higher levels of cortical actin that are normally found in the oocyte, although the two cells with four ring canals lie at the posterior of mutant egg chambers, as they do in wild-type. This phenotype suggests that baz mutants fail to form an oocyte, and therefore another oocyte marker, Staufen protein, was stained examined. Staufen accumulates in wild-type oocytes at around stage 5, but the majority of baz mutant cysts show no asymmetric accumulation of Staufen, even when they are older than wild-type cysts that have already localized the protein. Wild-type egg chambers always develop 15 polyploid nurse cells and an oocyte with its DNA compacted into a karyosome. In contrast, staining of baz mutant egg chambers with the DNA stain Hoechst reveals that they usually contain 16 polyploid cells. Thus, the loss of Bazooka activity from the germline gives rise to egg chambers in which all 16 cells develop as nurse cells (Huynh, 2001).
When germline clones of baz815-8 are generated using the ovoD technique to arrest the development of nonmutant cysts, the females produce a few fertilized eggs, indicating that baz mutant egg chambers sometimes develop a normal oocyte. The penetrance of the baz oogenesis phenotype was examined by scoring the frequency of egg chambers with an identifiable oocyte at two different times after the clones were induced. When females are dissected 2 days after eclosion, 14% of the mutant cysts contain a normal oocyte, whereas 86% arrest at stage 5 with the 16 nurse cell phenotype. The frequency of normal egg chambers falls to about 3%, however, when the females are dissected after 6 days. This suggests that Bazooka is essential for oocyte determination but that some egg chambers escape because the wild-type protein perdures for a long time after the clones are induced (Huynh, 2001).
Egg chambers mutant for egl, BicD, or par-1 also contain 16 nurse cells and no oocyte, but, in egl and BicD cysts, oocyte-specific proteins, such as Orb, never become restricted to one cell, whereas these proteins transiently localize to the anterior of the oocyte in par-1 mutant cysts. To test when Bazooka is required in oocyte determination, the localization of Orb protein was examined in baz815-8 germline clones. In wild-type cysts, Orb localizes to the oocyte in late region 2a of the germarium, where it accumulates at the anterior of the cell before translocating to the posterior in region 3. Orb protein still localizes to the anterior of the oocyte in baz mutant cysts, although this is slightly delayed compared to wild-type. The protein never relocalizes to the posterior, however, and is no longer enriched in the oocyte by stage 3 (Huynh, 2001).
The determination of the oocyte can also be followed by the migration of the centrosomes, which move along the fusome to cluster at the anterior of the oocyte in region 2b, and then translocate to the posterior of the oocyte in region 3. In baz mutant cysts, the centrosomes accumulate at the anterior of the oocyte but never move to the posterior cortex. Furthermore, alpha-tubulin stainings of mutant cysts indicate that microtubules remain focused on the anterior of the oocyte and fail to rearrange. These phenotypes are identical to those produced by par-1 mutants, indicating that Bazooka and PAR-1 act in the same step in oocyte determination. Neither is necessary for the initial selection of the oocyte, because Orb and the centrosomes still become restricted to one cell, but they are required for the maintenance of oocyte fate, since the oocyte soon dedifferentiates and becomes a nurse cell. This failure to maintain oocyte identity correlates with a block in the movement of oocyte-specific factors and the centrosomes from the anterior to the posterior of the cell, suggesting that this early polarization is important for the further development of the oocyte (Huynh, 2001).
PAR-6 has been shown to localize to the same protein complex as PAR-3 in C. elegans, Drosophila, and mammalian cells and is essential both for the localization and the function of this complex. In Drosophila, Bazooka and PAR-6 colocalize to the apical side of the embryonic ectoderm, where they are necessary for the maintenance of epithelial polarity, and both proteins are also inherited by the neuroblasts when they delaminate and are required for the basal localization of cell fate determinants during their asymmetric divisions. To test if Drosophila PAR-6 also functions with Bazooka during oogenesis, germline clones were generated of the par-6Delta226 allele, which is a deletion of the promoter, the start codon, and the first 121 amino acids of the protein and is therefore a strong loss of function mutation if not a null. The majority of mutant egg chambers appear small, oval-shaped, and contain 16 polyploid nurse cells and no oocyte, indicating that PAR-6 is also required for oocyte determination. Furthermore, Orb and the centrosomes accumulate in one cell at the posterior of the cyst, although with a slight delay compared to wild-type. Both remain at the anterior of the oocyte, however, and fail to translocate to the posterior pole. Thus, the loss of PAR-6 from the germline gives an identical phenotype to Bazooka and PAR-1. As is the case for bazooka germline clones, some of the par-6 mutant egg chambers escape the early arrest and go on to produce normal eggs. When the females are scored 2 days after eclosion, half of the egg chambers form a normal oocyte, and about a quarter still do so after 10 days. This increase in the penetrance of the phenotype with age shows that PAR-6 protein perdures for many days after the clones are produced. Consistent with this, PAR-6 appears to be unusually stable in the embryo; the protein can be detected throughout embryogenesis in zygotic par-6 null embryos, at levels that are only slightly lower than in wild-type. However, the continued presence of escapers after 10 days suggests that PAR-6 may not be essential for oocyte determination in all cases and that there may be redundant pathways that can partially compensate for its absence (Huynh, 2001).
During the asymmetric divisions of the neuroblasts, the Bazooka/PAR-6 complex recruits Inscuteable to the apical side of the cell, where it plays a role in directing the basal localization of Miranda protein. Germline clones of null mutants in inscuteable or miranda cause no visible defects in oocyte determination or the posterior localization of Orb, however, and give rise to normal eggs that can be fertilized. Furthermore, neither protein shows any asymmetric localization in early egg chambers. Thus, some of the downstream effectors of early oocyte and neuroblast polarity are different, despite the similar roles of Baz and PAR-6 in the two processes (Huynh, 2001).
To investigate the relationships between Bazooka, PAR-6, and PAR-1 during oocyte determination, their localizations were analyzed in both wild-type and mutant germaria. In region 2a to region 3 of the germarium, Bazooka localizes around the ring canals, in a ring that is about twice the diameter of that formed by actin. This localization is very similar to that of the adherens junction components Shotgun (E-cadherin) and Armadillo. A double staining was therefore performed for Arm and Baz. Although Arm localizes to these rings before Bazooka in early region 2a, the two proteins colocalize from the middle of region 2a until region 3, when they both disappear. Bazooka also colocalizes with Shotgun and Armadillo in the zonula adherens of the embryonic epithelium, which provides a boundary between the apical and basolateral membrane domains. This raises the possibility that the Shotgun, Armadillo, and Bazooka rings in the germarium perform a similar function by marking the separation between an anterior and a posterior domain within the oocyte. It is unclear whether PAR-6 also localizes to these rings, since none of the available antibodies give any significant staining that disappears in par-6 null germline clones (Huynh, 2001).
In C. elegans, the PAR-3/PAR-6 complex is required for the posterior localization of PAR-1. This is not the case during Drosophila oogenesis, however, since PAR-1 shows a wild-type localization to the fusome in baz and par-6 germline clones. Furthermore, the localization of Bazooka around the ring canals does not require PAR-6, since it is unaffected in mutant germline clones. This is in marked contrast to both the C. elegans zygote and Drosophila neuroblasts and epithelia, where the localizations of PAR-3/Baz and PAR-6 depend on each other. Bazooka and PAR-6 also localize to the apical sides of the somatic follicle cells of the egg chamber, and mutants in either gene disrupt the localization of both proteins and cause the cells to overproliferate and lose their apical-basal polarity. Thus, the relationship between Bazooka and PAR-6 is different in the germline and the somatic follicle cells, where they appear to have a similar role to that described in other epithelia (Huynh, 2001).
These results show that PAR-1, Bazooka, and PAR-6 act in the same step in oocyte determination, providing the first example in Drosophila where these three homologs of C. elegans PAR proteins participate in the same process. Furthermore, mutants in all three genes disrupt the movement of oocyte-specific proteins and the centrosomes from the anterior to the posterior of the oocyte, which is the earliest visible sign of polarity within the oocyte. Given the role of these PAR proteins in other systems, it seems very likely that their primary function in the germarium is in the anterior-posterior polarization of the oocyte, and that the failure to maintain oocyte fate is a consequence of this defect (Huynh, 2001).
It is intriguing that this very early anterior-posterior polarity of the Drosophila oocyte requires three of the PAR proteins that mediate the anterior-posterior polarization of the first cell division in C. elegans. Although this suggests that these proteins act in a conserved pathway for generating cell polarity in these two systems, the relationships between the localizations of these proteins are quite different in the Drosophila oocyte and C. elegans zygote. Thus, at least some aspects of their function are not conserved, and it will therefore be interesting to determine whether the downstream pathways that generate other cellular asymmetries in response to this polarity are related (Huynh, 2001).
The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote,
subsequently have been shown to regulate cellular polarity in diverse
cell types by means of an evolutionarily conserved protein complex
including PAR-3, PAR-6, and atypical protein kinase C (aPKC).
The Drosophila homologs (in parentheses) of C. elegans par-1, par-3 (bazooka), par-6 (DmPar-6), and pkc-3 (aPKC; DaPKC) each are known to play
conserved roles in the generation of cell polarity in the germ line as
well as in epithelial and neural precursor cells within the embryo. In
light of this functional conservation, the potential role
of baz and DaPKC in the regulation of
oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of
initial anterior-posterior polarity within germ-line cysts and
maintenance of oocyte cell fate. Germ-line clonal analyses indicate
both proteins are essential for two key aspects of oocyte
determination: the posterior translocation of oocyte specification
factors and the posterior establishment of the microtubule organizing
center within the presumptive oocyte. Baz and DaPKC
colocalize to belt-like structures between germarial cyst cells.
However, in contrast to their regulatory relationship in the
Drosophila and C. elegans embryos, these
proteins are not mutually dependent for their germ-line localization,
nor is either protein specifically required for PAR-1 localization to
the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).
To examine the potential oogenic function of baz and
DaPKC, protein null germ-line mutant clones were generated for
both baz and DaPKC. Germ-line clones, identified
by the absence of nuclear GFP expression, were counterstained with the
chromatin marker propidium iodide to examine the number and ploidy of
the germ-line nuclei. In contrast to wild-type egg chambers, which invariably contain 15 nurse cells and a single oocyte, baz mutant
germ-line clones, while containing the normal complement of germ-line
nuclei, fail to differentiate an oocyte, resulting in a 16-nurse cell
phenotype as revealed by the polyploid state of all 16 germ-line nuclei.
Similarly, DaPKCk06403 germ-line clones
also fail to differentiate an oocyte as indicated by the presence of 16 polyploid nurse cell nuclei in germ-line mutant egg chambers. These results reveal a germ-line autonomous requirement for DaPKC and confirm the
role of Baz in oocyte differentiation and/or maintenance (Cox, 2001).
These analyses further reveal that germ-line depletion of either
DaPKC or baz function from the follicle cells leads to their
multilayering, which disrupts the normal partitioning of germ-line
nuclei to successively mature egg chambers caused by mispositioning of
mutant follicle cells. These mispartitioned
baz+ nurse cell nuclei, into an otherwise
baz null germ-line clone, may provide the threshold of
germ-line Baz required to rescue the oocyte differentiation defect and
allow production of a mature egg. Alternatively, the Baz protein may
exhibit a long perdurance after mitotic clone induction, which depletes
over a period of days, resulting in the cessation of egg production in
these mosaic females. These results, however, in no way invalidate the
previous conclusions that maternally provided Baz masks the severity of the embryonic polarity phenotype in both epithelial cells and neuroblasts. Rather, the results indicate that these embryos are not likely to represent a complete maternal depletion for Baz (Cox, 2001).
Oocyte differentiation requires the polarized accumulation of oocyte
specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in
the localization of these factors, mutant germ-line clones
for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst
cells in region 2a, and then both molecules are targeted first to the
two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In
contrast, ORB fails to translocate from the anterior to a posterior
crescent in both baz and DaPKC null germ-line cysts in germarial
region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in
baz and DaPKC null germ-line clones in germarial
region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).
Furthermore, in contrast to wild-type germ-line cysts in which DHC64C
localizes to a single posterior cell, in DaPKC null germ-line clones DHC64C fails to localize to a single cell posteriorly, but rather accumulates in the two posterior-most presumptive
pro-oocytes of the mutant germ-line cyst.
Therefore, baz and DaPKC display essentially
identical phenotypes in germ-line mutant clones with regards to oocyte
differentiation and the establishment of initial A-P polarity within
the oocyte. The failure to maintain oocyte identity in either
baz or DaPKC mutant cysts can therefore be directly correlated with defects in the A-P translocation of oocyte specification factors within a single posterior cell of a germ-line cyst, suggesting oocyte differentiation depends on this early polarization event (Cox, 2001).
The posterior assembly of a functional MTOC has been directly
implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the
failure to translocate these factors to a posterior crescent in region
3 baz or DaPKC mutant cysts may result from a defect
in microtubule reorganization within these mutant cysts. In contrast to
wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of
oocyte specification factors in these mutants are likely caused, at
least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).
In addition to the microtubule network, both ring canals and the fusome
play critical roles in cyst polarization and oocyte differentiation. The formation and spatial distribution of ring canals as well as fusome morphogenesis was examined in both baz and
DaPKC mutant germ-line cysts. In baz mutant cysts, ring canal formation and spatial distribution are indistinguishable from wild-type germ-line cysts. Furthermore, the wild-type
spatial arrangement of ring canals in baz null germ-line
cysts suggests there is no apparent disruption in germ cell adhesion
within the cyst. Similarly, no defects were observed in ring canal
formation or spatial distribution in DaPKC mutant cysts (Cox, 2001).
These analyses indicate baz mutant cysts display relatively normal fusome morphology, although mutant fusome branches appear slightly thinner when compared with wild-type fusomes within the same germarium. Similarly, no apparent defect is observed in fusome morphology in DaPKC null germ-line cysts, indicating DaPKC
is dispensable in the germ line for proper fusome morphogenesis (Cox, 2001).
To investigate the mechanism by which Baz and DaPKC exert their effects
on oocyte differentiation, the localization of these
proteins was analyzed in the germ line and soma during oogenesis. The specificity of the Baz and DaPKC
antibodies was verified by using mosaic ovaries containing
baz or DaPKC null mutant clones. Baz is first detected in the germarium as a belt-like specialization on germ cell membranes at sites of germ cell interconnection. These belt structures are
reminiscent of ring canals with respect to their position between
germ-line cyst cells; however, in contrast to ring canals these "Baz
belts" are approximately 2-fold greater in diameter. Germaria
double-labeled for Baz and rhodamine phalloidin reveal that the Baz belts
localize adjacent to ring canals and further reveal an approximate 1:1 ratio between the two structures within germ-line cysts. The microtubule cytoskeleton as well as the fusome were observed projecting through individual cystocytes coincident with
the site of Baz belt expression on the germ cell membrane. Later in
oogenesis, Baz is transiently enriched in the oocyte cytoplasm at
stages 5-6 before the onset of vitellogenesis and is subsequently undetectable in the germ line of
vitellogenic egg chambers. As with Baz localization to the apical
junctional zone in the embryonic epithelium, tight apical localization of Baz is also observed in follicular epithelia (Cox, 2001).
DaPKC localizes to the Baz belts in the germarium, whereas in follicle cells DaPKC is apically constricted consistent with Baz localization in these cells (Cox, 2001).
In embryonic epithelia, Baz, DmPAR-6, and DaPKC apically colocalize and
partially overlap with the apico-laterally enriched Arm and DE-cadherin
proteins in the region of the apical zonula adherens. Furthermore,
these proteins are required to maintain epithelial cell polarity and
are mutually dependent for their proper localization. DE-cadherin is localized to belt-like structures adjacent to ring canals in region 2 germarial cysts reminiscent of Baz belt localization, suggesting that these adherens junction components may similarly colocalize in the germ line. To further investigate the molecular and functional nature of Baz belt expression in the
germarium, germaria were labeled with anti-Baz, anti-Arm, and
anti-DE-cadherin antibodies and their potential colocalization
within the germarium was examined. These
analyses reveal Baz colocalizes with both DE-cadherin and Arm to the Baz belts within the
germarium. Furthermore, consistent with the colocalization of Baz and
DaPKC to Baz belts within the germarium, partial
colocalization of DaPKC with DE-cadherin is observed in wild-type germarial cysts. To assay whether these proteins are mutually dependent for their germ-line localization, the localization of DE-cadherin and Arm was examined in baz null germ-line clones. In contrast to their mutual dependence in embryonic epithelia, these analyses indicate germ-line Baz function is dispensable for the localization of either DE-cadherin or Arm to the Baz belts in
the germarium. DaPKC is dispensable for the localization of either DE-cadherin or Arm
to these structures. These results
indicate Baz and DaPKC function are not required for the formation of
these structures because both Arm and DE-cadherin localization to these
belts in baz or DaPKC mutant cysts is indistinguishable from
that observed in wild-type cysts. Previous studies have revealed that germ-line clones of a strong allele of
shotgun (shgIG29), the gene
encoding DE-cadherin, disrupts the arrangement of germ cells in region
2b germarial cysts, suggesting DE-cadherin may mediate germ cell
adhesion. In contrast, these analyses of ring canal spatial distribution
in baz and DaPKC mosaic cysts strongly suggests
Baz and DaPKC function are dispensable for normal germ cell adhesion.
Furthermore, germ-line clonal analyses of either shg or
arm reveal neither gene is required for oocyte
differentiation, whereas both
baz and DaPKC are essential in oocyte determination. These results suggest the components of the Baz belts
likely mediate diverse cellular functions essential for germ-line cyst
development, which may include cell adhesion, cell signaling, and cyst
polarization (Cox, 2001).
In the C. elegans zygote, the PAR-3/PAR-6/PKC-3
complex is localized to the anterior where it is required for the
posterior localization of PAR-1. To investigate the potential regulatory relationship between baz and par-1 in the Drosophila germ line,
baz null germ-line clones were generated and PAR-1 expression and
localization was analyzed. In wild-type germ-line cysts, PAR-1 is localized to the spectrosome and fusome in germarial regions 1 and 2a and subsequently
is down-regulated on fusomes in regions 2b and 3. In
baz mutant germ-line cysts, PAR-1 localization to the
spectrosome is unaffected and is likewise present on fusomes although somewhat weaker localization is observed on fusome branches of baz mutant germ-line cysts when compared with wild-type germ-line cysts. Taken together, these results suggest germ-line baz is dispensable in the germarium for
normal PAR-1 expression and localization (Cox, 2001).
To determine whether par-1 may regulate Baz expression, par-1 null germ-line clones were generated and Baz
localization was examined. No defect was observed in Baz belt expression in
par-1 mutant germ-line cysts. Furthermore, in contrast to wild-type stage 5-6 egg
chambers in which Baz is transiently enriched in the oocyte cytoplasm, in par-1 mutant egg chambers Baz localization is abolished from the posterior presumably due to the
defect in oocyte differentiation observed in par-1 mutant egg chambers. These results indicate
germ-line par-1 is dispensable for normal Baz belt
expression and localization (Cox, 2001).
To assay the regulatory relationship between baz and
DaPKC the localization of DaPKC was analyzed in
baz mutant germ-line cysts, as well as
the localization of Baz in DaPKC mutant germ-line cysts. In contrast to their mutual dependence for
localization in the embryo, both Baz and DaPKC localization within the germ line is mutually independent. These results indicate that
despite the apparent functional conservation of Baz, DaPKC, and PAR-1
in generating oocyte polarity, the regulatory relationships among these
genes are not conserved in the germ line (Cox, 2001).
The colocalization of Baz, DaPKC, Arm, and DE-cadherin to
the Baz belt structures in the germarium strongly suggests the components of
the Baz belts are capable of mediating a multiplicity of functions in
germ-line cysts. In embryonic epithelia, these proteins function in the formation of the apical zonula adherens junction and are mutually dependent for their apical localization. The germ-line colocalization of these molecules to the Baz
belts suggests these structures may represent a potential polarity cue
on the germ cell plasma membrane. The restricted localization of Baz
belt components to germ cell membranes at points of cell-cell contact
represents an asymmetry on the plasma membrane of germ-line cyst cells
with regard to the A-P axis of the cyst and stage 1 oocyte. The
asymmetric localization of these molecules to one side of the germ cell
plasma membrane may act as a polarity cue in defining anterior versus
posterior within individual cystocytes of the 16-cell germ-line cyst
and thus contribute to the establishment of an initial A-P axis and to
subsequent germ-line cyst polarization. These results further suggest
that Baz and DaPKC likely function in a signaling capacity, rather than
a structural one, to mediate oocyte differentiation, whereas
DE-cadherin and Arm are more likely to function in maintaining germ
cell adhesion and cyst integrity (Cox, 2001).
Consistent with par gene function in other systems, these results indicate that Baz, DaPKC, and PAR-1 are required for the
establishment and maintenance of cellular polarity in the
Drosophila germ line; however, the regulatory relationships
observed between these genes in the germ line versus that observed in
embryonic blastomeres, epithelial cells, and neural precursor cells
indicates that, while these molecules are functionally conserved, the
mechanisms by which these genes act appear to be less well conserved.
In contrast to their mutual dependence for localization in the embryo,
Baz, DaPKC, and PAR-1 each are mutually independent for their
localization in the germ line. Taken together, these results underscore
the functional utility of the par genes and their effectors
as a molecular module for generating cellular polarity in diverse cell
types. Furthermore, the independence of Baz, DaPKC, and PAR-1 in their germ-line localization provides a unique opportunity to probe new
mechanisms by which these highly conserved proteins function in
regulating diverse processes such as cellular polarity, asymmetric cell
division, or growth control (Cox, 2001).
After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Par-1 is shown to become restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. baz is required for Par-1 relocalization within the oocyte and the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization (Vaccari, 2002).
It has been reported that, like Par-1, the D. melanogaster homolog of Par-3 Bazooka (Baz) is necessary for oocyte maintenance. Germline clones of a baz null allele are defective in oocyte polarization, establishing a functional parallel with C. elegans, in which the PAR genes polarize the early embryo. In the worm, restriction of Par-1 activity to the posterior cortex of the embryo crucially depends on Par-3, but the reverse is not the case. In the Drosophila germarium, Par-1 localization to the fusome appears to be independent of Baz. To assess whether the subsequent localization of Par-1 in the oocyte requires Baz, Par-1 distribution was further evaluated in baz germline clones. Par-1 is initially detected at the anterior of the oocyte in region 2b, but the protein disappears in region 3, revealing that Par-1 relocalization is baz dependent (Vaccari, 2002).
The relative distributions of Par-1 and Baz were investigated during later stages of oogenesis in wild-type ovaries. Par-1 is detected at the posterior of the oocyte as of germarial region 3 and becomes tightly associated with the cortex between oogenesis stages 3 and 5. Concomitantly, Baz becomes transiently localized to the anterior of the oocyte. The anterior localization of Baz is specific; it is not observed in germline clones of a baz null allele. Interestingly, maximal expression of Baz at the anterior coincides with the apparent tightening of Par-1 signal at the posterior cortex, suggesting a role for Baz in sharpening Par-1 localization. At this stage, the localization of the two proteins appears to be mutually exclusive. The anterior enrichment of Baz between stages 2 and 5 is absent in germline clones of the par-1 null allele (Vaccari, 2002).
The mutually exclusive distribution of Par-1 and Baz in the Drosophila oocyte is strikingly reminiscent of that observed in the C. elegans embryo. The ability to visualize the two proteins has allowed their respective roles in achieving this distribution to be genetically evaluated. The seeming dependence of Baz localization on par-1 is in contrast to results in C. elegans, in which localization of Par-3 is independent of Par-1. However, the absence of Baz in par-1 germline clones may well reflect the loss of oocyte fate and the onset of its degeneration that occurs at stage 1. Nonetheless, the anterior localization of Baz during stages 2–5 suggests that, after acting in oocyte polarization in region 2b/3, baz may be required again during oogenesis. The existence and nature of such a second requirement for baz after oocyte polarization is not yet clear (Vaccari, 2002).
Research has shown that the fusomal localization of Par-1 is unaffected in baz mutants, suggesting a difference between the Drosophila oocyte and the C. elegans embryo, in which Par-1 localization depends on par-3. Remarkably, at the time when oocyte polarization takes place, baz is in fact required for Par-1 localization within the oocyte. Hence, it appears that a similar relationship exists between Par-1 and Baz at the time when their activities are critical in the two organisms (Vaccari, 2002).
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date revised: 20 July 2004
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