zipper

DEVELOPMENTAL BIOLOGY

Embryonic

Dramatic changes in the localization of conventional non-muscle myosin characterize early embryogenesis in Drosophila. During cellularization, myosin is concentrated around the furrow canals that form the leading margin of the plasma membrane as the membrane plunges inward to package each somatic nucleus into a columnar epithelial cell. During gastrulation, there is specific anti-myosin staining at the apical ends of those cells that change shape in regions of invagination. Both of these localizations appear to result from a redistribution of a cortical store of maternal myosin. In the preblastoderm embryo, myosin is localized to the egg cortex (the part of the egg immediately underneath the surface membrane), sub-cortical arrays of inclusions, and, diffusely, to the yolk-free periplasm. At the syncytial blastoderm stage, myosin is found within cytoskeletal caps associated with the somatic nuclei at the embryonic surface. Following the final syncytial division, these myosin caps give rise to the myosin rings observed during cellularization. These myosin localizations and the coincident changes in cell morphology are consistent with a key role for non-muscle myosin in powering cellularization and gastrulation during embryogenesis (Young, 1991).

Peanut and Sep1, a second Drosophila septin identified based on its homolog to yeast septins, colocalize to the leading edge in cellularizing embryos. During the interphase between nuclear divisions 13 and 14, actin first reorganizes from the set of caps over the nuclei to form a hexagonal actin-myosin network at the embryo cortex. During the process of cellularization, the cleavage furrows move into the embryo around each nucleus, with actin and myosin concentrate at their leading edges, and spectrin concentrates slightly behind the leading edges. At the beginning of cellularization, Sep1 also assumes a hexagonal pattern similar to (but apparently less uniform than) that of actin and myosin. The nonuniformity of the Sep1 staining is maintained until the end of cellularization. As cellularization proceeds, Sep1 is concentrated at the leading edges of the advancing cleavage furrows, although some diffuse staining is still observed at the embryo cortex and in the underlying cytoplasm. Both Sep1 and Pnut co-localize at least at the resolution of the light microscope. Examination of double-stained embryos reveal that the septins co-localize with actin and myosin at the very leading edge of the cleavage furrows, in a position distinctly ahead of spectrin (Fares, 1995).

Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis. Drosophila embryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellularization, a specialized form of cytokinesis. During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton throughout phylogeny and in Drosophila. To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme from Clostridium botulinum or with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate downstream effectors. Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors. Of the 10 agents microinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests. Time-lapse video records of embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a dose-dependent fashion. Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do not proceed to gastrulation. It is concluded that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning (Crawford, 1998).

In mammalian cells, Rho and Cdc42 effectors function antagonistically. In competition are two distinct small GTPase protein-driven processes: the formation of stress fibers drive by Rho and the formation of filopodia driven by Cdc42. In Drosophila, Rho and Cdc42 effectors function antagonistically, but in contrast to the mammalian case the two phenotypes are indistinguishable. By this hypothesis, Rho and Cdc42 effectors function in independent pathways: Rho effector function is required for cellularization and maintenance of the actinomysin hexagonal array, whereas Cdc42 effector function antagonizes this process. Cdc42 effectors may inhibit Rho effector function directly, thereby phenocopying the disruption of Rho function generated by the microinjection of C3 exoenzyme or N19Rho. An alternative hypothesis that explains these data requires a mechanism of Rho subfamily regulation of the actomyosin cytoskeleton in a single pathway that depends only on Rho effector function during Drosophila cellularization. By this hypothesis, Rho and Cdc42 share a common factor that is required for Rho function, but is sequestered by GTP-bound Cdc42 (Crawford, 1998).

What is the role of myosin in cellularization? Since myosin function is necessary for cytokinesis and cellularization, a mechanism whereby myosin is regulated through phosphorylation by a Rho effector provides an attractive model. Because it is likely that force production for cytokinesis continuously requires activated myosin, it would be predicted that inhibition of Rho activity would block further progression of the cellularization front. Thus, agents that interfere with Rho activity would be expected to prevent the activation of myosin function and the formation of myosin bipolar filaments. It is possible that Rho effects on myosin can also explain the observed changes in the organization of actin. Indeed, Rho activity in the bundling of preexisting actin filaments may be directly or indirectly dependent on myosin function. Bipolar myosin filament formation can stimulate the formation and organization of filamentous actin; therefore, inhibition of myosin function may also explain the observed disruption of actin distribution (Crawford, 1998).

Cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms have been studied using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). These observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. These studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. Pseudocleavage furrow formation in the Drosophila blastoderm is analogous to how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and these findings are related to polar relaxation/global contraction mechanisms for furrow formation (Foe, 2000).

Laser scanning confocal microscope (hereafter LSCM) sectional views are provided of anterior ends of Drosophila embryos showing anaphase and interphase of cycle 9 (before centrosomes, microtubules and nuclei reach the cortex) and cycle 10 (the first round of bud formation and breakdown). In cycle 9, myosin II staining concentrates in a cortical rim during interphase but leaves the cortex during anaphase. Likewise, F-actin concentrates during interphase 9 in a cortical rim; the concentration attenuates greatly during anaphase 9. Throughout interphase 9, with no nuclei/asters near the cortex, cortical myosin II and F-actin co-localize. Similar waxing and waning of cortical F-actin and myosin II, co-localized and synchronized with globally synchronous mitotic cycles, is observed in cycle 8. Migrating nuclei with microtubule arrays reach the cortex 1 minute after interphase 10 begins. As telophase 9 ends and interphase 10 begins, F-actin and myosin II re-accumulate co-localized to high levels in a spatially uniform cortical rim. Two minutes after nuclei reach the cortex, cortical F-actin and myosin II are no longer co-localized but occur in the complementary patterns. Myosin II occurs at high levels between buds, but vacates the cortex where buds now protrude, while F-actin attains high levels precisely on the domes of the buds that myosin II has vacated. During anaphase 10, cortical levels of F-actin and myosin II are globally low. Cortical F-actin re-accumulation begins first near centrosomes at anaphase/telophase. Regardless of cortical fluctuations, high levels of myosin II staining occur throughout the embryo interior. When myosin dissociates from the cortex, it transiently boosts the concentration of myosin immediately beneath the cortex, but does not significantly boost the concentration of internal myosin globally, presumably because it is dispersing into an ocean of cytoplasmic myosin filling these large cells. Throughout the interior cytoplasm, F-actin occurs diffusely, and additionally in particles, but at lower levels than cortically (Foe, 2000).

When migrating nuclei with associated microtubule arrays first reach the periphery early in interphase 10, myosin II staining disappears from a small region immediately above the nuclei and, during the next 3 minutes of interphase and prophase, the holes vacated by myosin II staining enlarge into oblong holes. During metaphase and anaphase, the cortical myosin staining dims, then, during telophase, myosin staining returns brightly to the cortex except near centrosomes where it remains dim. Holes in the cortical myosin pattern, appearing in the cortex precisely when/where microtubule arrays first contact the cortex, are interpreted as implying an interaction between myosin II and microtubules (Foe, 2000).

Four hypotheses have been proposed about consecutive and simultaneous mitotic-cycle-modulated interactions between the cell cortex (the approximately 3 mm deep zone immediately underlying the plasma membrane), F-actin, myosin II, centrosomes and microtubules. When working together, they can explain these experimental results (Foe, 2000).

• Hypothesis H1: mitotic phase modulates the amount of myosin II associated with the cortex and the amount of actin polymerized into filaments at the cortex. Independent of the proximity of centrosomes and/or microtubules, both cortical myosin II and cortical F-actin increase from telophase through interphase then fall through metaphase and anaphase.
• Hypothesis H2: centrosomes, via the polymerization of the microtubule asters that they nucleate, boost the rate of actin polymerization nearby.
• Hypothesis H3: a kinesin-like activity attaches actin filaments to adjacent microtubules and transports actin filaments toward microtubule plus ends; this activity is weaker than the attachment of F-actin to the cortex.
• Hypothesis H4: a kinesin-like activity moves myosin II filaments towards microtubule plus ends; this is stronger than the attachment of myosin to the cell's cortex so that appropriately oriented microtubule arrays contacting the cortex strip myosin II off the plasma membrane.

The same four mechanical hypotheses that are proposed to explain pseudocleavage furrow and bud formation in the fly syncytial blastoderm, if operative in dividing mononucleate cells, could time and initiate localization of the actomyosin components of the contractile ring for cytokinesis. H1, by melting down prior interphase F-actin structures during metaphase, and H2 by causing F-actin to re-polymerize near centrosomes beginning in anaphase, in effect force a redeployment of the cell's actin just prior to beginning the specialized task of cytokinesis. By H1, the cortical concentrations of F-actin and myosin II, having fallen to low concentrations in metaphase-anaphase, rebuild in telophase. But the expanding telophase microtubule asters approaching the cortex at opposite poles of the cell would trigger, via H4, depletion of cortical myosin II filaments near the spindle poles where microtubules impinge, while simultaneously concentrating myosin II filaments by moving them through the cytoplasm towards the cell mid-zone. H4 will thus eventually concentrate myosin II in a three-dimensional disk whose perimeter will become the contractile ring. By H2, actin polymer will form coincidentally with microtubule outgrowth initially most concentrated near centrosomes, followed later in telophase by a migration of F-actin along astral microtubules away from centrosomes (by H3) and toward concentration in a three-dimensional disc-shaped volume centered at the equator where it will co-localize with myosin II. The 'rings' of cortical F-actin seen in late interphase, correspond to where these equatorial disc volumes intersect the cortex in the dividing mononucleate cell. The regions between buds where both F-actin and myosin II are present together at the cortex during interphase (though concentrations of F-actin may be higher elsewhere) constitute the so-called pseudocleavage furrows in the fly syncytial blastoderm. This region would be homologous to the cortex of the cleavage furrow, which constricts during cytokinesis (Foe, 2000 and references therein).

H1-H4 imply a bipolar 'global contraction-polar relaxation' mechanism for positioning the contractile apparatus for cytokinesis. Transient 'polar relaxation' would convert a global cortical contraction into a self-amplifying equatorial contraction. Computer simulations have shown how contracting an initially isotropic actomyosin meshwork into an equatorial belt aligns the filaments parallel to the equator, as in a contractile ring, positioning them to cleave a cell in two by a purse-string contraction. Plus-end transport along astral microtubules of cortical F-actin (H3) and of myosin II filaments (H4) could provide a mechanistic explanation for this oft-hypothesized polar relaxation, while simultaneously causing an increase in equatorial tension (assuming that cortical contractile strength is proportional to co-localized F-actin and myosin II concentrations). Either polar relaxation, or equatorial strengthening, or both together, can set the stage for the kind of actomyosin contraction-based cytokinesis that has been proposed (Foe, 2000 and references therein).

The establishment of the contractile furrow is a mechanism requiring two independent steps: the release in tension at the poles, preceded by (or coincident with) a global increase in cortical tensioning. The global loss of actin and myosin from the cortex during metaphase/anaphase and their return beginning in telophase, which is observed in Drosophila embryos (H1), if phylogenetically general, could underlie the cyclic changes in cortical contractility. Operating together, H1-H4 are potentially capable of implementing a 'global contraction-polar relaxation' mechanism of furrow initiation for cytokinesis. If cortical contractile strength is proportional to co-localized F-actin and myosin II concentrations, then H3 and H4 would bring about equatorial strengthening of the cortical actomyosin meshwork, in principal also implementing 'equatorial stimulation'. Note that, in flattened cells with small asters, the microtubules in the mid-zone between spindle poles are positioned to execute the same actomyosin rearrangements as astral microtubules in spherical cells. Note also that H3 plus H4 can cause equatorial concentration of whatever cytoplasmic actomyosin network a cell contains, focusing internal forces on the furrow cortex with the potential to aid furrow invagination in non-spherical cells. Homologies between cleavage and pseudocleavage, while attractive, come with the caveat that cortical tensioning and microtubule outgrowth both occur earlier in the mitotic cycle during cytokinesis in echinoderms than during pseudocleavage furrow formation in Drosophila syncytia (Foe, 2000 and references therein).

In summary, this study has aimed to deduce, from descriptions of wild-type and drug-perturbed cytoskeletal kinematics of microtubules, F-actin and myosin II in syncytial Drosophila embryos, the specific ways that these filament systems must be interacting. The speculative mechanistic hypotheses that were deduced, H1-H4, are consistent with a large body of circumstantial and partial evidence reviewed above. This machinery, if phylogenetically general, could unify old ideas about cytokinesis with new molecular findings, reconcile polar relaxation with equatorial stimulation models of furrow formation, and homologize cytoskeletal pseudocleavage furrow formation in syncytia with cleavage furrow formation in mononucleate cells. Future revelations can be expected of the molecular details by which the products of an ensemble of key genes (e.g. anillin, centrosomin, diaphanous, KLP-3A, pavarotti, polo kinase, Rac 1, septins, etc.) collaborate to bring about and regulate the interactions between F-actin, myosin II, centrosomes and microtubules (Foe, 2000).

The effects of the absence of puckered (pucE69) and its overexpression were compared on the levels and organization of the actin cytoskeleton and nonmuscle myosin. In pucE69 mutants, the expression of myosin and actin does not change dramatically in the periphery of the cells in lateral regions of the embryo, but these proteins fail to accumulate along the leading edge of the epidermis. Cell shape changes proceed almost normally. In contrast, epidermal cells of embryos overexpressing puc fail to change their shapes and accumulate low levels of spatially disorganized myosin at the leading edge. In these embryos, actin fails to be expressed in the amnioserosa and its levels are reduced in the epidermis. Actin and myosin tend to form clumps in these epidermal cells. Thus puc is an essential component in the control of the different steps of dorsal closure progression and acts by modulating the apical accumulation of actin and myosin at the leading edge. These results correlate with those of the effects of overexpression of DN-Drac1 and Djun mutants, and further suggest a role for Puc in the control of JNK activity over the cytoskeleton (Martin-Blanco, 1998).

To explore the nature of the defects seen in the absence of diaphanous function, wild-type and dia mutant embryos were stained at nuclear cycles 11-13 with the DNA dye DAPI and with an antibody directed against F-actin. In the wild type, nuclei are positioned at the embryo cortex at interphase of nuclear cycles 11-13; a structure referred to as the actin cap is situated between each nucleus and the plasma membrane. During the transition to prophase, filament reorganization results in a concentration of actin at the edge of the caps. At metaphase, the resulting rings of cortical actin, together with associated plasma membrane, invaginate to form metaphase furrows. As viewed from above, actin staining at these furrows appears as a hexagonal array over the embryonic surface. In the sagittal view, actin staining at the metaphase furrow appears as a line between the metaphase nuclei. In dia-deficient embryos, severe structural changes in the actin cytoskeleton are manifested after nuclear cycle 11. Formation of the hexagonal actin arrays is disrupted during prophase and metaphase and there is an absence of actin staining between the metaphase nuclei. Similar patterns of staining are obtained when dia embryos are stained with antibodies directed against anillin (Drosophila gene: Scraps) and Peanut, other components of the metaphase furrow. There is thus a failure in the formation of the metaphase furrow. Consistent with the known role of metaphase furrows in maintaining nuclear organization, the nuclei in dia mutant embryos frequently exhibit abnormal spacing and, in some cases, fuse in subsequent nuclear cycles. These irregularities are readily apparent in contrast to the uniform pattern observed in the wild type. In regions in which cortical actin staining is weak or absent, nuclei are frequently found displaced into the interior of the embryo, although the centrosomes remain at the surface (Afshar, 2000).

To investigate whether the absence of metaphase furrows results from a failure in membrane invagination, dia embryos were stained with antibodies directed against myosin. In wild-type embryos, myosin localizes to the embryonic cortex between the actin caps at each interphase, appears at the tip of the invaginating membrane at prophase and disappears at metaphase. In dia embryos, myosin staining, albeit very weak and irregular, is detected between the actin caps at the cortex during interphase. At prophase, myosin, where detectable, remains at the cortex, with no detectable membrane pinching or invagination. Therefore, despite the presence of myosin at the cortex between actin caps, the membrane invagination that precedes metaphase furrowing is absent in dia embryos (Afshar, 2000).

Immunolocalization was used to determine whether Diaphanous plays a role in the recruitment of anillin and Peanut, a Drosophila septin. In wild-type embryos both anillin and Peanut localize to the embryonic cortex, between the actin caps at interphase. During prophase and metaphase, they localize to the metaphase furrow and their pattern of staining is similar to that of actin. In dia embryos, the staining patterns of both anillin and Peanut are very weak during interphase. Similarly, in dia embryos the localization of both of these proteins is disrupted during prophase and metaphase, when the metaphase furrow is being formed in wild-type embryos. Diaphanous is thus required for recruitment and proper localization of anillin and Peanut as well as myosin to the regions of membrane invagination (Afshar, 2000).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Zipper and dorsal closure

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. Its role in the developing Drosophila embryonic epidermis during the process of dorsal closure has been investigated. By expressing the dominant negative DRhoA^N19 construct in stripes of epidermal cells, it has been confirmed that Rho function is required for dorsal closure and it is necessary to maintain the integrity of the ventral epidermis. Defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoA^N19 expression. Furthermore, these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoA^N19-expressing cells (Bloor, 2002).

Tension generated in the amnioserosa and the leading edge of the lateral epidermis independently contributes to the forces that drive dorsal closure. It has been proposed that nonmuscle myosin II activation generates tension in the leading edge and that this causes a leading edge intracellular actomyosin purse-string to shorten. Signaling downstream of RhoGTPase activates nonmuscle myosin II by modulating the level of myosin regulatory light chain phosphorylation. As such, expression of RhoA^N19 in epidermal stripes might disrupt contraction of the leading edge purse-string. Defects in actin and nonmuscle myosin II organization caused by RhoA^N19 expression are first observed at germband extension, up to 2 hours before purse-string formation. Thus, while actin and nonmuscle myosin II are localized at the leading edge in wild-type tissue, a purse-string structure is never formed in leading edge cells that express RhoA^N19. RhoA^N19 expression therefore effectively cuts the leading edge purse-string at multiple sites. This does not necessarily prevent progression of dorsal closure, confirming previous experiments which demonstrate that the integrity of the leading edge is not required for dorsal closure to continue to completion. It is concluded that small independent regions of leading edge in wild-type epidermal stripes can, in conjunction with contraction of the amnioserosa, migrate dorsally with relative normalcy (Bloor, 2002).

The question that arises is how do epidermal cells expressing RhoA^N19 move dorsally in the absence of a leading edge purse-string? These cells could hitchhike, i.e. they are pulled dorsally by the amnioserosa or dragged along with neighboring wild-type cells. Although spread and disorganized, dorsal RhoA^N19-expressing cells do maintain adhesion with wild-type neighbors and this might then allow passive RhoA^N19-expressing cells to move dorsally with wild-type tissue. This is consistent both with the inverse correlation between integrity of the ventral epidermis and the extent to which dorsal closure proceeds, as well as with observations on the distribution of tension at the embryo surface during dorsal closure. Thus, during the time that the epidermis lateral to the leading edge opposes dorsal closure, ventral failure of epidermal integrity (and hole formation) would release the tensional restraints on the remaining lateral epidermis, allowing it to move dorsally with more success. Similarly, in the absence of this release (i.e. the ventral epidermis retains its integrity and opposes dorsal movement of the epidermis), the leading edge is presumably no longer capable of generating sufficient force to drive dorsal closure to completion (Bloor, 2002).

Throughout development, a series of epithelial movements and fusions occur that collectively shape the embryo. They are dependent on coordinated reorganizations and contractions of the actin cytoskeleton within defined populations of epithelial cells. One paradigm morphogenetic movement, dorsal closure in the Drosophila embryo, involves closure of a dorsal epithelial hole by sweeping of epithelium from the two sides of the embryo over the exposed extraembryonic amnioserosa to form a seam where the two epithelial edges fuse together. The front row cells exhibit a thick actin cable at their leading edge. The function of this cable has been tested by live analysis of GFP-actin-expressing embryos in which the cable is disrupted by modulating Rho1 signaling or by loss of non-muscle myosin (Zipper) function. The cable serves a dual role during dorsal closure. It is contractile and thus can operate as a 'purse string,' but it also restricts forward movement of the leading edge and excess activity of filopodia/lamellipodia. Stripes of epithelium in which cable assembly is disrupted gain a migrational advantage over their wild-type neighbors, suggesting that the cable acts to restrain front row cells, thus maintaining a taut, free edge for efficient zippering together of the epithelial sheets (Jacinto, 2002).

To investigate the function of the actin cable during dorsal closure, advantage was taken of rho1 and also zip alleles that produce phenotypes in which the actomyosin cable disassembles part way through the dorsal closure process but in which the overall tissue movement typically does not fail. These mutants offered the opportunity to analyze the effects of cable loss at the cellular level without the complete disruption of tissue architecture. Also, they allowed the use of live GFP-actin embryos to analyze the dynamic cell behavioral response and determine how these behavioral changes influence the capacity of epithelial cells to participate in a coherent forward-sweeping movement (Jacinto, 2002).

Scanning electron micrographs of rho1 and zip embryos that complete dorsal closure reveal significant similarities. In both cases, combinations of amorphic or strong mutations, zip¹/zip^IIX62 or zip¹/zip¹ in the case of zip, and rho1^72O/rho1^72R in the case of rho1, were used. Both zip and rho1 mutant embryos exhibit the same dramatic defects in head involution, a tissue movement distinct from dorsal closure, but a clear, posterior dorsal hole is observed in none of the rho1 mutants, and in only 59% of the zip/zip^IIX62 or 8% of the zip¹/zip¹ embryos. The remaining zip mutants appear to complete dorsal closure successfully; although, frequently, like their rho1 counterparts, these embryos show puckering or segment misalignments along the closed midline seam. Interestingly, puckering has previously been observed in mutants in which epithelial movement is not properly downregulated at the midline (e.g., puckered), suggesting that the actin cable may be regulating cell spreading during some periods of dorsal closure. It is presumed that the phenotypic variation in zip embryos is due to differences in the maternal contribution to the myosin II RNA and protein pool, which must run out around the stage of dorsal closure (Jacinto, 2002).

Phenotypic similarities between zip embryos that complete closure of the dorsal hole and rho1 mutants are not surprising since rho1 and zip have previously been shown to interact genetically in Drosophila. Moreover, a signaling link has also been revealed in mammalian cell culture studies, with Rho-associated kinase (ROCK), a Rho effector, shown to regulate Myosin function, and thus the assembly and maintenance of cable-like stress fibers, by repression of Myosin Light Chain phosphatase and direct activation of the Myosin Light Chain. It is also likely that Rho1 regulates the cytoskeleton via alternative effectors. The kinase Protein kinase related to protein kinase N (see Rho1 Protein Interactions) has been shown to function downstream of Rho1 during Drosophila dorsal closure, and mDia, the murine homolog of Drosophila Diaphanous, has been shown to bind active Rho1 and contribute to the formation of stress fibers in mammalian cells by promoting actin polymerization. However, whether or not these signaling pathways regulate actomyosin contractility during dorsal closure has yet to be demonstrated. Interestingly, Rho1 also interacts with p120^ctn and regulates cadherin-based adherens junctions in the Drosophila embryo, suggesting that the leading edge disorganization seen in rho1 mutants may be not only a consequence of actin cable misregulation but also a result of defective adherens junctions (Jacinto, 2002).

Further similarities between the rho1 and zip embryos were revealed when higher-resolution SEM was used to look at the leading edge cells during the final stages of dorsal closure. In both mutants, the normally straight, and apparently taut, epithelial leading edge now appears to be relatively disorganized and to have lost tension, with front row cells extending increased levels of filopodial and lamellipodial protrusions compared to their wild-type counterparts (Jacinto, 2002).

To observe the dynamic behavior of these cells, time-lapse confocal microscopy was used to analyze the final stages of dorsal closure in living, wild-type embryos versus rho1 and zip embryos, expressing GFP-actin fusion proteins using the GAL4-UAS system. The fusion protein was driven in the epidermis by the epithelial driver e22cGAL4, and only embryos that subsequently closed the dorsal hole are described. As expected from SEM observations, the cytoskeletal architecture of the leading edge that normally characterizes wild-type dorsal closure is lost in both rho1 and zip mutants. The actin cable, which is clearly apparent in the wild-type leading edge from stage 13 onward, fails to assemble or disassembles during the dorsal closure process in rho1 and zip mutants. The disassembly of the actin cable is temporally coincident with a transition from an organized to disorganized leading edge. In both mutants, loss of cable is also coincident with more exuberant filopodial extensions than in wild-type leading edge cells, and these filopodia frequently coalesce to form lamellipodia. In wild-type embryos, lamellipodia are generally more a feature of the final stages of dorsal closure, as opposing epithelial fronts make contact with one another (Jacinto, 2002).

The coalescing of filopodia to form lamellipodia in mutant embryos leads to a broader extent of protrusion per unit length of leading edge cells and an increase of up to 300% in the total protrusion area extending from these cells. It is suggested that the increased level of actin-based protrusions seen in both rho1 and zip embryos at a time when the leading edge cable is disassembling may reflect that the cable serves some function in repressing excessive protrusive activity in these front row cells. The link could simply be due to a greater availability of free actin monomers, but it might also be a consequence of changes in membrane and cortical actin stiffness at the free edge in these cells. In this regard, myosin II has been shown to play a role in maintaining the integrity and stiffness of the cortical cytoskeleton during Dictyostelium morphogenesis. It is not clear from this data whether downregulation of Rho in any way feeds back on Cdc42 activity, but no increase in thickness or apparent contractility of the actin cable is observed in cells expressing dominant-negative versions of Cdc42 (Jacinto, 2002).

These observations indicate that maintenance of a fully formed and functioning actin cable is not an absolute requisite for closure of the dorsal hole. In order to test further how a failure to activate Rho1 and assemble an actin cable might influence a cell's capacity to participate in dorsal closure, the enGAL4 driver was used to express both GFP-actin and either dominant-negative Rho1 (Rho^N19) or constitutively active Rho1 (Rho^V14) in 4- to 5-cell-wide epithelial stripes. These embryos were then imaged live or, alternatively, fixed and costained with Alexa594-phalloidin to reveal the actin machinery of all the cells, including the intervening wild-type stripes that do not express GAL4. Initial live analysis of these embryos, from the earliest stages of dorsal closure, revealed differences between leading edge cells that are blocked in Rho1 signaling and their wild-type neighbors. Rho1^N19-expressing cells fail to assemble an actin cable but do express broad filopodia and lamellipodia. Without a cable, they do not constrict at their leading edge in the way that their wild-type neighbors clearly do. Subsequently, these mutant Rho1^N19 stripes of cells sweep forward, apparently released from their usual constraints, overspilling and displacing their wild-type neighbors. Frequently, the dorsal edges of intervening wild-type stripes are enveloped by adjacent Rho1^N19-expressing stripes, and this wild-type tissue is consequently trapped back from the leading edge. By the time that the dorsal hole is closed, most of the midline seam epithelium is taken up by Rho1^N19-expressing cells that clearly have a migration advantage over their wild-type, cable-assembling neighbors. In the converse experiment in which GFP-actin was expressed and a constitutively active Rho1 construct (Rho1^V14) was expressed using the enGAL4 driver, precisely the opposite effect is seen. Now, Rho1^V14-expressing cells are more constricted than their wild-type neighbors at early stages, and they are subsequently outcompeted during dorsal closure, such that wild-type stripes tend to dominate the leading edge during dorsal closure. Thus, when dorsal closure is complete, the midline seam epithelium is largely wild-type (Jacinto, 2002).

These results suggest that the actomyosin cable has a dual role to play during dorsal closure. It is a driver of leading edge cell contractility from the earliest stages of dorsal closure, but it is also required for restraining the leading edge epithelial cells and maintaining a coherent and taut epithelial margin during the later phases of dorsal closure, particularly as the epithelial faces are being zippered together. It is proposed that, in addition to the previously described 'purse string' function, actomyosin cables may have a more subtle, but equally important, support role during morphogenetic episodes such as dorsal closure. These cytoskeletal structures, regulated by Rho activity, function to maintain epithelial coherence during coordinated epithelial movements, particularly at free epithelial edges where a taut front enables efficient zippering together and alignment of cells at a zipper seam, in a process that is perhaps analogous to zippering closed a very full luggage bag (Jacinto, 2002).

In a sense, the restraining function of the cable during dorsal closure is analogous to that which operates for the band of actin at the periphery of a tissue culture clone of epithelial cells. In this case, the growth factor, scatter-factor, can trigger disruption of the restraining actin band as well as dissolution of cell:cell junctions, and cells at the periphery are consequently now able to break free of their neighbors (Jacinto, 2002).

The dual role for the actomyosin cable during dorsal closure, operating both as a 'purse string' and as a cell restrainer, is likely to be a finely balanced operation. Modulation of normal Rho1 activity, as in the experiments reported above, results in either overcontractility of cells or their release from leading edge constraints. These data support previous evidence suggesting that multiple cytoskeletal events drive dorsal closure but demonstrate that these events must be finely balanced to ultimately produce the precisely organized zippering required for perfect midline fusion of the two epithelial faces (Jacinto, 2002).

Zipper promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport

Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to ìpushî rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter (Barros, 2003).

Class II myosins are barbed end-directed motors that form bipolar filaments. The filaments bind actin and initiate contraction when the two ends of the bipolar filament pull in opposite directions. Myosin's mode of action makes it unlikely that myosin II could transport cargo from one side of the cell to the other, except perhaps by progressive contraction along the cortex. The lack of colocalization of myosin II and Miranda in neuroblasts further implies that myosin II does not transport Miranda directly. The data suggest, first, that myosin II is required to maintain an intact cortical actin cytoskeleton and, second, that active myosin modifies the actin cytoskeleton at the apical cortex to exclude Miranda binding. The C. elegans myosin II may act in a similar fashion, as it appears to limit PAR-3 to the anterior of the zygote (Barros, 2003).

Several alternative approaches were taken to inactivate myosin II in neuroblasts. First, germline clones of Sqh were analyzed. In severe sqh^1GLC embryos, levels of the regulatory light chain are greatly reduced from early development, and the heavy chain is found only in inactive aggregates. The actin cytoskeleton is disrupted, and neither Lgl nor Miranda localize to the cortex. Miranda concentrates instead at the spindle microtubules. Therefore, active myosin is necessary from early development to organize the actin cytoskeleton, which is in turn required for Lgl and Miranda to localize to the cortex (Barros, 2003).

Myosin II is activated by phosphorylation of its regulatory light chain by Rho kinase. Myosin was inactivated at the time of neuroblast cell division by inhibition of Rho kinase. Myosin II no longer localizes at the apical neuroblast cortex but instead spreads into the cytoplasm, and basal protein localization is disrupted. Although F-actin and Lgl remain uniformly at the cortex, cell fate determinants are now found around the entire cell cortex, demonstrating that apical cortical myosin is required to confine determinants to the basal half of the cell. Inhibition of Rho kinase also blocks cytokinesis, although the defect in basal protein localization is unlikely to be the consequence of mitotic arrest or a block in cytokinesis. First, basal protein localization is not disrupted in neuroblasts arrested in mitosis by colcemid treatment. Second, mitosis occurs without cytokinesis in pebble mutants, but the resultant polyploid neuroblasts still localize Numb and Prospero asymmetrically. Finally, the loss of asymmetry resulting from Rho kinase inhibition can be rescued by expression of a constitutively active form of the myosin II regulatory light chain (SqhE20E21). It is concluded that myosin II is required to restrict cell fate determinants to the basal cortex (Barros, 2003).

Myosin II localizes to the apical cortex of metaphase neuroblasts. Why is myosin localization/activity asymmetric? Lgl binds myosin II heavy chain directly and inhibits myosin filament formation. This binding is regulated by phosphorylation of Lgl; this phosphorylation inhibits its interaction with myosin II in vitro. If Lgl negatively regulates myosin activity and localization, then myosin should be uniformly distributed in an lgl mutant. Indeed, in lgl^1GLC mutants myosin II no longer concentrates apically but is found uniformly around the cortex. Most Miranda protein is released from the cortex and binds microtubules, again suggesting that myosin excludes Miranda from the cortex (Barros, 2003).

Myosin II localizes to the entire cortex in lgl mutants and thereby prevents Miranda binding basally. In Drosophila neuroblasts in which Lgl levels are reduced (zygotic lgl¹ mutants), Miranda is released from the cortex. Miranda localization can be rescued by simultaneously reducing the level of myosin II (zip¹ zygotic mutants). Reducing the level of active myosin may restore the balance between the levels of Lgl and myosin, enabling the remaining myosin to concentrate apically (Barros, 2003).

How does myosin II restrict neuroblast proteins to the basal side of the cell cortex? Myosin II and Miranda occupy primarily opposite sides at the neuroblast cortex: myosin II is concentrated at the apical cortex while Miranda localizes as a basal crescent. As myosin II shifts to the cleavage furrow, Miranda is segregated into the forming GMC. The apical F-actin compartment may be modified by myosin II to exclude binding of basal proteins like Miranda. Active myosin II requires Rho kinase activity and depends on inactivation of Lgl at the apical cortex by aPKC (Betschinger, 2003). Ectopic expression of a nonphosphorylatable form of Lgl, in which the conserved aPKC-dependent phosphorylation sites are mutated from Serines to Alanines (Lgl-3A), results in mislocalization of Miranda around the neuroblast cortex (Betschinger, 2003). The data support a spatially regulated interaction between myosin II and Lgl. Myosin is apically localized in wild-type neuroblasts, corresponding to the domain in which Lgl is inactivated by aPKC. In lgl mutants, myosin is no longer restricted apically but localizes around the entire cell cortex. Conversely, when nonphosphorylatable Lgl is expressed in neuroblasts, myosin is inhibited throughout the cell and drops off the cortex. It is proposed that myosin II is activated and can form filaments at the apical cortex, where phosphorylated Lgl is inactive and unable to bind myosin II. Myosin may then modify the actin cytoskeleton to prevent the binding of Miranda. At the basal cortex, in the absence of aPKC, Lgl is active and can bind and inhibit myosin. Myosin cannot form filaments, which are required for it to bind to the actin cortex. As a result, Miranda can bind to the basal cortex (Barros, 2003).

At anaphase, myosin II moves to the equator and appears to “push” cell fate determinants into the daughter cell. This movement is regulated in an Lgl-independent fashion and occurs whether myosin is restricted to the apical cortex or is uniformly cortical (as in lgl mutants). Cortical myosin is essential, however, to efficiently segregate determinants into the GMC at telophase (telophase rescue). In neuroblasts expressing Lgl-3A, myosin II is cytoplasmic, and determinants are not partitioned to the daughter cell. Nonetheless, at telophase, myosin seems to be recruited from the cytoplasm, since it still accumulates to the cleavage furrow. Thus three separate steps of myosin regulation in neuroblasts can be defined. First, myosin forms an apical crescent. This is positively regulated by Rho kinase and negatively regulated by Lgl. Second, cortical myosin moves to the equator. This movement occurs independently of Lgl. Third, cortical and cytoplasmic myosin accumulates at the cleavage furrow, a step that is also Lgl independent. Rho Kinase activation seems to be important for all three steps of myosin II regulation. When Rho kinase is inhibited, myosin falls into the cytoplasm, and there is no cleavage furrow formation (Barros, 2003).

In conclusion, these results demonstrate that myosin II acts downstream of Lgl and the apical protein complex to regulate the segregation of cell fate determinants. Myosin II does not negatively regulate basal protein targeting, as has previously been suggested nor does it transport determinants directly. Instead, it is proposed that myosin II acts in a novel fashion, excluding determinants from the apical cortex and 'pushing' them into the GMC at anaphase and telophase. Myosin II might modify the actin cytoskeleton to prevent determinants binding, although the actual structure formed and the physical change in the actin cytoskeleton remains to be determined (Barros, 2003).

Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis

The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. A collection of genes was examined that causes defects in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. These findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex (Dean, 2005).

This study has discovered three steps in the myosin II localization/activation process that involve distinct groups of genes: (1) an initial recruitment of myosin II to the equatorial cortex that is independent of F-actin and centralspindlin but requires rho1 signaling; (2) a secondary stabilization of myosin II at the midzone that requires F-actin and a second set of genes that are likely involved in building a specific type of actin network, and (3) the activation of furrowing once myosin II is localized that depends on centralspindlin (Dean, 2005).

Rho1, its activating guanidine nucleotide exchange factor pebble, and rho kinase are each required for the initial recruitment of myosin II to the equatorial cortex. Rho1 has been implicated in two pathways that are important for cytokinesis. In the first pathway, rho1 signals to F-actin through the formin diaphanous. However, proteins on this F-actin pathway, including F-actin itself, are not essential for the initial myosin II recruitment to the equatorial cortex. However, rho kinase, another downstream target of rho1, is essential. Because rho kinase phosphorylates the myosin II RLC, it is possible that phosphorylation of the RLC is essential for myosin II recruitment to the furrow. This hypothesis could not be directly tested, because the myosin II heavy chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).

Phosphorylation of the RLC both activates the motor domain and, in some myosins, increases bipolar thick filament formation. Because F-actin is not required for myosin II recruitment, activation of the motor is unlikely to be the mechanism by which phosphorylation of the RLC would cause recruitment of myosin II to the equatorial cortex. It is quite possible, however, that the rho kinase-mediated myosin II phosphorylation leads to thick filament assembly and that this assembly is important for localization of myosin to the equatorial cortex. Indeed, in Dictyostelium, it is clear that bipolar thick filament formation is sufficient for myosin II localization to the midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains unknown (Dean, 2005).

In contrast to the lack of F-actin involvement in the early recruitment of myosin II to the equatorial cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain myosin II in the equatorial region. Interestingly, the downstream rho1 effectors diaphanous/formin and chickadee/profilin are also necessary for myosin II maintenance at the equatorial midzone. Although the loss of these genes could deplete F-actin, phalloidin staining has shown that F-actin is still present in all of the RNAi-treated cells. In addition, these RNAi-treated cells still contract, unlike when F-actin is completely disrupted with LatA. Thus, myosin II appears to be interacting with F-actin in the cortex as it disperses in dynamic patches throughout the cortex of these diaphanous- or chickadee-depleted cells (Dean, 2005).

It is suggested that the role of diaphanous/formin and chickadee/profilin in maintaining the myosin II contractile ring is through the creation of specific F-actin structures. In particular, formin- and profilin-mediated nucleation results in unbranched actin filaments because profilin promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled filaments. These parallel filaments contrast with Arp2/3-mediated nucleation, which creates a highly branched actin filament network. Indeed, Arp2/3, although essential for lamellipodia formation, is not required for cytokinesis in Drosophila cells. The hypothesis here is that once myosin II is recruited to the equatorial cortex of the cell by a rho kinase-dependent mechanism, possibly localized activation of RLC phosphorylation, it is retained there because of its higher affinity for parallel, unbranched actin filaments than to branched actin networks. Consistent with this hypothesis, myosin II is depleted from the lamellipodia in migrating cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are more likely to be aligned in parallel bundles. Thus, it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow maintains a higher concentration of parallel actin filaments in this region compared with the rest of the cortex, and these parallel filaments serve to selectively retain myosin II at the equator to form a stable contractile ring. In the absence of these parallel actin filaments, myosin II can bind branched F-actin throughout the cortex, perhaps occasionally organizing them into parallel bundles that cause increased myosin recruitment corresponding to the flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).

Live-cell imaging shows that when pavarotti or racGAP50c are depleted, the cells do not display significant contractions despite recruiting myosin II to the equatorial cortex. Although there is some modest membrane contractile activity in these cells, it is clear that significant contraction or furrowing requires both components of the centralspindlin complex. It is surprising that only these proteins were found to be necessary for cortical contraction at sites of myosin II localization. Data from fixed cells, as well as earlier studies, indicated that Drosophila cells do not undergo equatorial contractions during mitosis when Diaphanous or Chickadee is depleted. However, live-cell imaging shows that when either of these two genes is depleted in S2 cells, not only is myosin II transiently localized to the equatorial cortex before dispersing, but cells do indeed display transient equatorial contraction. It is difficult to recognize these events in fixed cells because of their transient nature and the somewhat irregular shapes of cells depleted of these proteins. This work highlights the importance of live-cell imaging in the study of dynamic processes such as cytokinesis (Dean, 2005).

In addition to the suppression of furrowing, depletion of centralspindlin also leads to an inability to retain F-actin exclusively at the equatorial cortex during cytokinesis. This similar phenotype of the centralspindlin complex and the F-actin affecting proteins suggests that centralspindlin may be an upstream regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti have been shown to accumulate at the spindle poles and are associated with an abnormal accumulation of F-actin near the centrosomes. Centralspindlin may be acting indirectly by helping to localize an important actin-affecting protein at the central spindle, or it may act more directly on the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been hypothesized that centralspindlin affects the F-actin cortex through rho1 signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion, and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of centralspindlin may itself be important for signaling to the F-actin cortex. Finally, centralspindlin cannot be the major actomyosin ring positioning signal because myosin II is properly recruited in its absence (Dean, 2005).

A Rho GTPase signaling pathway, in conjunction with concertina and folded gastrulation, is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation

A single Rho GTPase family member is capable of initiating several different processes, including cell cycle regulation, cytokinesis, cell migration, and transcriptional regulation. It is not clear, however, how the Rho protein selects which of these processes to initiate. Guanine nucleotide exchange factors (GEFs), proteins that activate Rho GTPases, could be important in making this selection. In vivo, DRhoGEF2, a GEF that is ubiquitously expressed and specific for Rho1, is reiteratively required for epithelial folding and invagination, but not for other processes regulated by Rho. The limitation of DRhoGEF2 function supports the hypothesis that the GEF selects the outcome of Rho activation. DRhoGEF2 exerts its effects in gastrulation through the regulation of Myosin II to orchestrate coordinated apical cell constriction. Apical myosin localization is also regulated by Concertina (Cta), a Galpha12/13 family member that is thought to activate DRhoGEF2 and is itself activated by a putative ligand, Folded gastrulation (Fog). Fog and Cta also play a role in the morphogenetic events requiring DRhoGEF2, suggesting the existence of a conserved signaling pathway in which Fog, Cta, and DRhoGEF2 locally activate Myosin for epithelial invagination and folding (Nikolaidou, 2004).

If the guanine nucleotide exchange factor (GEF) is important in selecting the outcome of activating Rho, then its function should be limited to a subset of those associated with the GTPase. To address this possibility, the in vivo function of DRhoGEF2 was investigated. Two hypomorphic alleles, DRhoGEF2^PX6 and DRhoGEF2^PX10, in combination with null alleles of DRhoGEF2, give adults that have crumpled and/or blistered wings. Earlier in development, the DRhoGEF2^4.1/DRhoGEF2^PX6 wing discs appear buckled rather than conforming to the stereotypical folding pattern observed in the wild-type. This malformation is not a result of either improper patterning or loss of apico-basal polarity. It must therefore be caused by disruption of another mechanism -- for example, the propagation of a localized signal that brings about folding in specific places. To test this hypothesis, clones of DRhoGEF2^1.1 cells spanning a fold were generated (Nikolaidou, 2004).

In large mutant clones that are less influenced by physical constraints, the folds fail to follow the line of the fold in wild-type tissue. Bifurcation of folds does not occur in wild-type discs, supporting the idea that the mutant tissue is unable to respond to a localized signal to fold. Although the clonal and DRhoGEF2^4.1/DRhoGEF2^PX6 mutant tissues do appear folded, the irregularity of the folds indicates that this is probably a consequence of passive folding, as is seen in the gastrulation mutants and murine neurulation mutants that fail to invaginate tissue appropriately (Nikolaidou, 2004).

The possibility was investigated that other events involving epithelial invagination or folding might also require DRhoGEF2 activity. One such event is the invagination of a placode to form a salivary gland tube on both sides of the embryo. Combinations of dominant-negative alleles with a putative null allele of DRhoGEF2 showed that in 93% of embryos some or all of the salivary-gland cells fail to invaginate and instead remain on the outside. Because maternally provided DRhoGEF2 is vital for epithelial invagination in gastrulation, this and the above two phenotypes represent three examples of the requirement for DRhoGEF2 in epithelial-layer morphogenesis (Nikolaidou, 2004).

If DRhoGEF2 is participating in the selection of the cell's response to activated Rho, then its function should be limited. Rho is known to play a role in cytokinesis, cell cycle regulation and planar polarity. The large size of clones of DRhoGEF2, equivalent numbers of cells in twin wild-type and mutant clones, and normal polarity of mutant tissue indicate that unlike Rho, DRhoGEF2 is not required for any of these processes, nor is it required for apico-basal polarity. No significant defects were seen in the gross morphology of the nonepithelial tissues of muscles and neurons in late-stage DRhoGEF2^4.1/DRhoGEF2^6.5 and DRhoGEF2^4.1/DRhoGEF2^5.1 embryos. In addition, the normal cell cycle control shows that the convolution of DRhoGEF2 mutant wing discs is not a result of excessive proliferation (Nikolaidou, 2004).

Although the possibility exists that DRhoGEF2 has a function not addressed, it seems likely that its role is confined to the control of epithelial morphogenesis. This limit of DRhoGEF2 function suggests that it is important in selecting a role for Rho only in epithelial morphogenesis, whereas other GEFs would activate Rho in other processes; for example Pebble activates Rho primarily in cytokinesis, and Trio acts on Rac in neuronal outgrowth (Nikolaidou, 2004).

To study in more detail the mechanism by which DRhoGEF2 affects epithelial morphogenesis, the possible targets of DRhoGEF2 activation have been considered. One of these is myosin II. During gastrulation, Zipper (Zip), the heavy chain of myosin II, appears to accumulate on the apical side of the mesodermal precursors in the ventral furrow (VF). To address the possibility that apical myosin localization is required for other invagination events, salivary-gland formation was analyzed in embryos expressing the myosin light chain, Spaghetti squash (Sqh), as a fusion with green fluorescent protein (Sqh-GFP). Although Sqh-GFP is present at the cortex of all the cells, it is concentrated at the apical surface of salivary-gland precursors that are about to invaginate or are in the process of invaginating. Sqh-GFP does not accumulate apically until invagination, as demonstrated by the lack of apical localization in cells that are present more anteriorly in the placode but that will invaginate later (Nikolaidou, 2004).

It is not clear if this apical myosin accumulation is present in time to contribute to apical constriction. To resolve this question, the localization of Sqh-GFP was observed in the invaginating VF during gastrulation. In wild-type cells, Sqh-GFP is maintained at the tip of the growing membrane that forms between the nuclei during cellularization, the process immediately prior to gastrulation. At the end of cellularization, Sqh-GFP begins to decrease on the basal side and accumulate on the apical side of the ventral cells, i.e., only those that will constrict apically. This redistribution of myosin precedes apical cellular constriction, suggesting that it contributes to the process. Basally located Sqh-GFP is subsequently lost, and the apical levels increase (Nikolaidou, 2004).

In DRhoGEF2 germline clone-derived (GLC) embryos (i.e., those lacking maternal DRhoGEF2), Zip, the myosin heavy chain, is lost from the basal side of cells in the developing VF, but it accumulates at much lower levels on the apical side than it does in the wild-type. These results imply that a signal through DRhoGEF2 is needed in order for the ventral cells to induce apical Zip localization. In contrast, relocalization of β-heavy spectrin occurred normally in DRhoGEF2 GLC embryos, indicating that cell polarity is maintained in these cells and that at least some forms of protein relocalization, especially that of a protein that is found in close proximity to Zip (Nikolaidou, 2004).

The possibility was considered that myosin localization is also regulated by other components of the DRhoGEF2 signaling pathway. By analogy to the mammalian and C. elegans orthologs and as a result of genetic studies , DRhoGEF2 is thought to participate in a signal transduction pathway, which is called here the DRhoGEF2 signaling pathway, initiated by Folded gastrulation (Fog) and propagated by Concertina (Cta). Mutations in both these genes result in gastrulation defects. In embryos derived from cta mutant mothers, a low level of Zip accumulates on the apical side only of apically constricting cells in the invaginating VF. This is also true in DRhoGEF2 GLC embryos. In contrast, there is no apical myosin apparent in the cells that do not constrict their apical surface. These data clearly link the presence of apical myosin with apical constriction and indicate that in gastrulation this is controlled by the DRhoGEF2 signaling pathway. The link between DRhoGEF2 and Myosin is also supported by the documented genetic interactions between DRhoGEF2 and zip in leg and wing development (Nikolaidou, 2004).

It is not clear how DRhoGEF2 influences the apical accumulation of myosin. It could act via the Rho effector Rho kinase. When activated by Rho in mammalian cells, Rho kinase is responsible for revealing the actin binding site on the regulatory light chain of myosin II. Thus, in DRhoGEF2 mutants, a possible failure in the activation of Rho1 and Rho kinase would result in the inability of myosin to bind actin (Nikolaidou, 2004).

If DRhoGEF2 is required reiteratively for epithelial morphogenesis, it is hypothesized that Fog and Cta might also be used reiteratively. mRNA for fog is expressed in invaginating tissue during gastrulation and salivary-gland formation, suggesting that Fog also participates in invagination of the salivary gland. At present there are conflicting reports regarding the role of fog in salivary gland formation. This study finds that some or all cells fail to invaginate in 90% of the embryos. Because invagination in gastrulation is cell autonomous, it is considered more likely that this phenotype results from a lack of fog in these cells rather than because of earlier developmental defects (Nikolaidou, 2004).

The possibility was addressed that the pathway is also important in wing development. Initial descriptions indicate that Fog and Cta play no role in this process. However, demonstrating a previously undisclosed role for Fog and Cta, combinations of mutations in fog or cta and DRhoGEF2 result in synergistic effects on wing development. Together, these results point to the reiterative use of the DRhoGEF2 signaling pathway in development to bring about epithelial folding or invagination (Nikolaidou, 2004).

Preliminary data indicate that the folds in the wing disc are brought about by apical cell constriction. It is therefore proposed, because both gastrulation and salivary gland invagination also involve apical cell constriction, that this is a major aspect of DRhoGEF2 function. The location of the folds in wing discs is highly stereotypical, which would suggest that specific signals are activated in these locations to initiate folding. One candidate for this signal is Fog, which is perhaps acting in conjunction with a second signal to bring about epithelial folding in the wings. In gastrulation, fog and cta are essential, but their phenotypes are not as strong as that observed after the removal of maternal DRhoGEF2, again indicating the requirement for additional signals that activate DRhoGEF2. The nature of this additional signal, or signals, remains elusive (Nikolaidou, 2004).

Planar polarization of the denticle field in the Drosophila embryo: roles for Myosin II (zipper) and fringe

Epithelial planar cell polarity (PCP) allows epithelial cells to coordinate their development to that of the tissue in which they reside. The mechanisms that impart PCP as well as effectors that execute the polarizing instructions are being sought in many tissues. The epidermal epithelium of Drosophila embryos exhibits PCP. Cells of the prospective denticle field, but not the adjacent smooth field, align precisely. This requires Myosin II (zipper) function, and it was found that Myosin II is enriched in a bipolar manner, across the parasegment, on both smooth and denticle field cells during denticle field alignment. This implies that actomyosin contractility, in combination with denticle-field-specific effectors, helps execute the cell rearrangements involved. In addition to this parasegment-wide polarity, prospective denticle field cells express an asymmetry, uniquely recognizing one cell edge over others as these cells uniquely position their actin-based protrusions (ABPs; which comprise each denticle) at their posterior edge. Cells of the prospective smooth field appear to be lacking proper effectors to elicit this unipolar response. Lastly, fringe function was identified as a necessary effector for high fidelity placement of ABPs and it was shown that Myosin II (zipper) activity is necessary for ABP placement and shaping as well (Walters, 2006).

Since the prospective denticle field is clearly polarized, it was wondered if smooth field cells were similarly polarized but simply did not express a marker, such as the ABPs, that revealed that polarity. To test this, the formation of ABPs among prospective smooth cells was induced by ectopically expressing the transcription factor svb/ovo in small groups of cells in the ventral epidermis and these cells were marked by co-expression of GFP. Expression of svb/ovo is necessary and sufficient to induce the formation of ABPs, so by expressing svb/ovo in the smooth field, the localization of these protrusions can be visualized within the cell. When svb/ovo is induced in the smooth field, the ectopic ABPs did not preferentially localize to the posterior edge of cells. Instead, they showed a stochastic dispersal around the apical surface of the cell Both anti-phosphotyrosine and phalloidin stains label these misplaced ABPs, indicating that phospho-epitopes as well as actin are present. Out of the thirty-eight ectopic denticles scored, 63% were mis-positioned (nine on an anterior edge, fifteen placed centrally) and only 37% were localized to the posterior edge of the cell (Walters, 2006).

It is also possible that the stochastic ABP localization observed in the svb/ovo-positive cells was not due to the lack of polarity effectors in the smooth field, but instead a simple issue of developmental timing. Since a heat-shock-driven recombinase was used to induce svb/ovo expression, precise control over the timing of the recombination event or svb/ovo induction was not occuring. Attempts were made to remedy this concern by ectopically expressing svb/ovo using Ptc-GAL4. Since patched is expressed well before cell fate specification, any ectopic ABPs that are present should have had ample time to localize to the posterior cell edge. However, even when svb/ovo is expressed at this early time point, ABPs in the smooth field showed no preference for the posterior edge of cells and remained stochastically positioned. These data strongly suggest that smooth cells do not possess a latent ability to place ABPs with the unipolar asymmetry seen among prospective denticle field cells. At the minimum, an effector of unipolar asymmetry must be active only among prospective denticle field cells. Alternatively, unipolar asymmetry is established only late and is restricted to the prospective denticle field. To investigate the unipolar asymmetry further, the phenomena of ABP formation and eventual placement at the posterior edge of denticle field cells was studied (Walters, 2006).

Denticle field cells are aligned and exhibit posterior ABPs at late stages (Stage 14). Since the denticle field is specified between late stage 11 and stage 12, cell alignment and ABP formation were monitored from stage 11 onward. At late stage 12, there was little alignment among cells within each parasegment. However, cell contours evolved through stage 14 (Walters, 2006).

Turning to the development of ABPs, at late stage 12, there was little or no enhancement in actin accumulation at the apical surface of the prospective denticle field cells compared to cells within the smooth field. Early in stage 13, a diffuse actin meshwork appeared at the apical surface of prospective denticle cells. By late stage 13, the apically enriched actin had coalesced into several patches that appear to represent nascent protrusions. Surprisingly, these nascent protrusions were often located away from the posterior edge of a cell. Only later, during stage 14, were ABPs more uniformly at or near each cell's posterior edge. Note that more fully pointed and curved ABPs can be observed at stage 15 (Walters, 2006).

Quantification of actin accumulation within slices across the apical face of a cell throughout these developmental stages supported the notion of a progression to the posterior edge. The intensity of Rhodamine–phalloidin-labeled actin was measured by dividing a prospective denticle field cell into six domains (progressing from A to P) and recording the pixel intensities in each of the domains at the apical face of that cell. The process was repeated for 3 cells in 3 different animals at early stage 13, late stage 13 and stage 14. The results showed that, during early and late stage 13, actin was not biased to any cell edge. At stage 14, actin was enriched dramatically at the posterior edge. It is concluded that actin first accumulates stochastically on the apical face of the cell and then is later positioned at the posterior edge. A core PCP component, frizzled (fz), was tested for its potential role in establishing or maintaining unipolar asymmetry among prospective denticle cells (Walters, 2006).

The frizzled gene is important for polarizing the hairs on the wing and in the abdomen and for ommatidial orientation. Denticle cuticle pattern were examined in the progeny of fz^H51/fz^P21 mothers crossed with fz^H51/TM3 Ubx-LacZ fathers. Most larval cuticles appeared normal, though half of these were expected to be null for fz function. Two of thirty cuticles analyzed did exhibit patterning errors, but these were fusions among segments. This might have been caused by a partially penetrant deficit in canonical Wingless signaling since fz is also used in this pathway (where it is redundant with Dfrizzled2). The ABPs were examined directly in fz-deficient embryos; these were unambiguously identified by the lack of Ubx-LacZ expression. All fz-deficient embryos exhibited normal cell alignment of denticle field cells, and most mutants (six of eight) exhibited normal posterior placement of ABPs. Only two mutants exhibited misplaced protrusions, and in these embryos, the defects were restricted to row 1. Fully penetrant, but similarly restricted defects (to rows 1 and 2) have been reported for the core PCP mutants fz, strabismus, flamingo and even more weakly so for the PCP-specific mutant of disheveled (dsh¹. Collectively, the defects observed implicate core PCP genes in denticle field polarization, but the restriction of these defects solely to anterior-most rows suggests a more minor role than expected in executing unipolar asymmetry (Walters, 2006).

Denticle field cells elaborate ABPs, and this has allowed it to be established that these cells are polarized in the plane of the epithelium. Forcing smooth field cells to elaborate ABPs failed to reveal any latent unipolarity within this portion of the parasegment. Nevertheless, the evidence supports the proposition that both fields are polarized, albeit in different ways. This study establishes bipolar enrichment of Myosin II on prospective smooth and denticle field cells. This suggests that the whole ventral epithelium exhibits bipolar PCP, and all cells can discriminate their A/P from their D/V edges. Since this bipolar enrichment emerges during stage 12, it is possible that this reflects de novo establishment of polarity within the epithelium. However, at earlier stages, cells of this epithelium exhibit a strikingly similar bipolar distribution of Myosin II then used for convergence extension. Thus, the bipolar redeployment of Myosin II might reflect a memory of that earlier polarization. This possibility is particularly compelling given that Myosin II orchestrates the rearrangements of cell junctions necessary for convergence extension. Perhaps Myosin II is being engaged similarly at the later stages, and the re-emergence of a bipolar preference is a precondition to accomplish the necessary junctional re-organization for cell alignments. What signals direct this re-emergence are not yet known. In addition, since Myosin II is bipolar among both prospective denticle and smooth cells, though the latter do not align, there must either be cues or effectors specific to the denticle field that initiate the cell alignment process (Walters, 2006).

Note also that the bipolar enrichment of Myosin II yields no clues as to how the denticle field cells uniquely identify their 'P' cell edge and faithfully position the ABPs to this edge. One possibility is that only the prospective denticle field cells have proper effectors to transmute the global bipolarity into asymmetric unipolarity. Since it is difficult to imagine how this might occur, a second possibility is that unipolarity is imparted locally, only across the prospective denticle field. The failure to observe proper ABP placement after ectopic expression of svb/ovo in the prospective smooth field supports this possibility. If unipolarity is imparted locally, then this also places constraints on the timing of the signals for unipolarity. The epithelium is sorted into smooth and denticle fields only late in development as a consequence of the antagonism between Wingless signaling and EGF receptor activation. This results in the establishment of the domain of expression for Svb. Thus, the denticle field is only established after this time, and the idea is favored that unipolarity is assigned after this stage. It is believed that analysis of Fringe supports this contention: fringe comes to be expressed within denticle field cells only after this stage and mutation of fringe interferes with unipolarity across the whole denticle field. This identifies fringe as, at the minimum, an effector of denticle field unipolarity (Walters, 2006).

Among a set of core genes involved in the establishment and maintenance of polarity in other tissues is frizzled, and surpriskingly it did not play a major role in denticle field polarization. Only two of eight embryos exhibited any defects, and in these, only row 1 cells appeared affected. Yet, a role for frizzled signaling in denticle field unipolarity cannot be ruled out due to possible redundancy with DFrizzled 2. In fact, recent work has implicated several core PCP genes in denticle field unipolarity. Mutation of frizzled, dsh, flamingo or strabismus lead to mild defects restricted to rows 1 and 2 reminiscent of what are report here for the minority of fz embryos. In addition, enrichment for Flamingo, Fz and Dsh occurs on the edges of prospective denticle field cells. In aggregate, these data are very suggestive for a role of core PCP genes in ABP polarity. However, the restriction of the phenotype to the very anterior rows of the denticle field leaves open the possibility that these genes do not play as major of a role as they do in, for example, the wing. In addition, while it has been shown that Fz and Dsh are enriched on certain cell edges, it will be of interest to know whether the enrichment is uniquely to one edge of cells, as it is in wing cells. For example, the data show that Zipper and Squash-GFP are enriched to cell edges but that these are likely both A and P edges of each cell. In denticle field cells, if Fz and Dsh exhibit such bipolar enrichment, rather than the unipolar asymmetry observed in wing cells, then their role in the denticle field would be quite distinct from that currently proposed for wing cells (Walters, 2006). It will be difficult to establish definitively whether Fz or Dsh play more extensive roles in denticle field polarity, especially since this data strongly suggest that effectors for this polarity are likely established late, after the epithelium is sorted into smooth versus denticle field cells. It would not be possible to easily interpret the removal of all function for dsh and frizzled (which likely would entail the removal of both frizzled and Dfrizzled2) because both proteins play earlier and essential roles in Wingless signal transduction. Removing both maternal and zygotic dsh (or fz Dfz2) function might well lead to polarity phenotypes, but these may be secondary to earlier deficits in Wg signaling. This is especially the case as Wg (and Hedgehog as well) plays a major role in establishing the denticle versus smooth field and subdividing parasegments into smaller signaling territories as well as establishing the responses of the cells to those signals (Alexandre, 1999, Gritzan, 1999, Hatini and DiNardo, 2001b and Wiellette and McGinnis, 1999). These considerations also raise a caution in drawing the conclusion from Wg or Hh null mutants that these pathways play any direct role in polarization (Walters, 2006).

Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis

Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper testing of this hypothesis. Loss- and gain-of-function approaches were used to examine the role of Diaphanous in Drosophila morphogenesis. Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. Constitutively active Diaphanous was used to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. This hypothesis was tested by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis (Homem, 2008).

Previous analysis of Diaphanous-related formins (DRF) function in morphogenesis was hampered by mammalian DRF redundancy and the key role of DRFs in cytokinesis. The genetic tools available in Drosophila were used to partially circumvent these difficulties. Dia is known to regulate cellularization. The analysis revealed new roles during morphogenesis. In embryos with severely reduced Dia function, gastrulation is disrupted. Apical constriction of central furrow cells is delayed or blocked in a subset of cells, suggesting a possible role for Dia in regulated apical constriction. Previous work suggested that an unknown Rho effector regulates actin during this process; perhaps this is Dia. Adjacent cells, which are stretched during invagination, become massively multinucleate in dia⁵M mutants. This may reflect the fact that the cells of the blastoderm undergo an incomplete form of cytokinesis, remaining connected to the underlying yolk by actin-lined yolk canals. These canals normally close at the onset of gastrulation in ventral furrow cells; this may prevent cell membrane rupture initiated at the yolk canal under stress. Dia may regulate yolk canal closure; it localizes there at the end of cellularization and similar defects are seen in mutants lacking the septin Peanut, a yolk canal component. Alternately, Dia may stabilize cortical actomyosin or its connections to AJs, with its absence weakening cortical integrity under stress (Homem, 2008).

Dia also plays a key role during germband retraction. The most striking effect of reduced Dia/Rho1 function is altered amnioserosal cell protrusiveness. They normally make stable AJs and form a tight tissue boundary with the epidermis; this is destabilized in dia/Rho1 and dia⁵M/Z mutants. Altered protrusive activity was seen in other contexts: dia⁵M/Z ectoderm exhibited cortical blebbing, and dia/Rho1 and dia⁵M/Z amnioserosal cells had altered protrusiveness during dorsal closure. Reduced Dia function destabilized AJs, and the striking increase in cell protrusiveness in dia/Rho1 mutants was substantially rescued by increasing Myosin phosphorylation. These data support the hypothesis that Dia normally helps restrict actomyosin contractility to AJs, and in its absence, AJs are destabilized and myosin activity outside AJs stimulates protrusiveness. Interestingly, in cultured cells, fully assembled AJs inhibit Rac1 and lamellipodial activity while increasing Rho and contractility, and myosin is required for mouse Dia1-induced AJ strengthening (Homem, 2008).

Given these morphogenetic roles, the mechanisms of action of Dia was explored, initially hypothesizing that it would primarily affect actin. Dia^CA expression did elevate F-actin levels (reducing Dia did not dramatically decrease F-actin, but residual Dia may suffice). However, surprisingly, Dia activation also increased Myosin accumulation and cell contractility, and some effects of reduced Dia function were rescued by increasing Myosin activation. This suggests a surprising role for Dia in regulating Myosin levels/activity. The data also suggest that Dia stabilizes AJs, supporting earlier work in cultured cells. There are intimate connections between AJs and cortical actomyosin: AJs help organize the apicolateral actin ring while cortical actin stabilizes AJs. Interestingly, in cultured cells Myosin-mediated contraction of actin filaments is essential for cell-cell contact expansion, and mouse Dia1 AJ stabilization requires myosin activity, providing further evidence that contractility, AJ stability and Dia activity are mechanistically linked. The actomyosin network may also stabilize AJs by preventing endocytosis. Perhaps Dia, like formin 1, directly binds AJs. Linear actin filaments promoted by Dia at AJs might recruit α-catenin, which then might inhibit Arp2/3-induced actin branching, facilitating formation of a belt of unbranched actin. Thus, Dia is poised to act at the interface between AJs, cortical actin and contractility (Homem, 2008).

The Yin-Yang relationship between adhesion and protrusiveness is a very interesting one, underlying epithelial-mesenchymal transitions. Stable AJs probably actively inhibit protrusiveness, and Dia may assist this, acting in a reinforcing loop that concentrates actomyosin activity at AJs. There are interesting parallels with the role of Dia in cytokinesis. Dia stabilizes Myosin at the midzone. In its absence Myosin is delocalized around the cortex, leading to abnormal protrusiveness. Future work will reveal how Dia fits into the regulatory network coordinating adhesion and cytoskeletal regulation (Homem, 2008).

From these data, a mechanistic model was developed for the regulation of actin, Myosin and AJs by Dia during cell shape change, in which Dia activation stabilizes actin and active Myosin at AJs. In radially symmetric amnioserosal cells, Dia promotes organization/activation of an apical actomyosin network linked to AJs, inducing precocious apical cell constriction. Activation of endogenous Dia may regulate normal amnioserosal constriction: this now needs to be tested. Dia activation in cells where AJs are planar polarized, such as epidermal cells, promotes actomyosin organization preferentially at cell borders where AJs are enriched. This leads to cell widening or helps cells resist elongation, generating groove-cell-like morphology. Once again, Dia may be normally activated specifically in groove cells to modulate their shape (Homem, 2008).

How does Dia activation activate Myosin? It does not occur primarily through SRF, which triggers Myosin accumulation but not cell shape changes. Myosin activation via MLCK partially mimicked Dia^CA, suggesting that Myosin activation is an important part of the process. However, MLCK did not precisely mimic Dia^CA, suggesting that Dia acts preferentially at specific sites such as AJs rather than globally activating Myosin. The dual effects on actin and Myosin suggests the speculative possibility that feedback mechanisms exist to coordinate the Rho-regulated actin and Myosin pathways. Recent work revealed that active Dia1 can activate RhoA by binding the Rho-GEF LARG, and strong genetic interactions were seen between dia and the LARG relative RhoGEF2, making this idea more plausible. Future work is needed to test this hypothesis (Homem, 2008).

Larval and Pupal development

Zygotically expressed myosin II is required for leg disc eversion. Depletion of Spaghetti squash, the myosin regulatory light chain, results in curved, thickened upper legs and defective wings. Depletion of SQH often results in a complete disorder in the usually perfect hexagonal packing of the ommatidia of the eye, as well as the development of an anterior notch on the eye. The time at which SQH is withheld is critical in full penetrance of the eye phenotype (Edwards, 1996).

Adult

Stable intercellular bridges called ring canals form following incomplete cytokinesis, and interconnect mitotically or meiotically related germ cells during spermatogenesis. Ring canals in Drosophila melanogaster males are surprisingly different from those previously described in females. Mature ring canal walls in males lack actin and appear to derive directly from structural proteins associated with the contractile ring. Ring canal assembly in males, as in females, initiates during cytokinesis with the appearance of a ring of phosphotyrosine epitopes at the site of the contractile ring. Following constriction, actin and myosin II disappear. However, at least four proteins present at the contractile ring remain: the three septins (Pnut, Sep1 and Sep2) and anillin. In sharp contrast, in ovarian ring canals, septins have not been detected, anillin is lost from mature ring canals and filamentous actin is a major component. In both males and females, a highly branched vesicular structure, termed the fusome, interconnects developing germ cells via the ring canals and is thought to coordinate mitotic germ cell divisions. In males, unlike females, the fusome persists and enlarges following cessation of the mitotic divisions, developing additional branches during meiosis. During differentiation, the fusome and its associated ring canals localize to the distal tip of the elongating spermatids (Hime, 1996).

Rho-associated kinase indirectly affects the activity of myosin II in the development of planar cell polarity

Drosophila Rho-associated kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain (Winter, 2001), and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).

Rok signaling regulates the phosphorylation of nonmuscle myosin regulatory light chain (MRLC), and hence the activity of myosin II. Does the phosphorylation state of MRLC modify the multiple hair phenotype of dishevelled mutants? Use was made of a series of mutant spaghetti squash (sqh) transgenes (sqh codes for the Drosophila MRLC) with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine. Can the phosphorylation state of MRLC also modulate Fz/Dsh signaling? An examination was made to determine whether the phosphomimetic and nonphosphorylatable forms of MRLC could directly modify the dsh¹ multiple hair phenotype. Introducing one copy of sqhE20E21 reduces the number of multiple hair cells in dsh¹ mutants by 5-fold. sqhE21, or sqhA20E21, also suppresses the dsh¹ phenotype by more than 2-fold. In contrast, introduction of sqhA21 into the dsh¹ background enhances the multiple hair phenotype. The involvement of MRLC in the Fz/Dsh pathway was also examined using the Fz-overexpression assay. Reducing the wild-type sqh gene dosage from two to one, by introducing a single copy of the sqh^AX3 null allele, results in a 2-fold suppression of the multiple hair phenotype caused by Fz overexpression. These results support the notion that MRLC functions in the PCP pathway to restrict F-actin bundle assembly to a single site (Winter, 2001).

MRLC phosphorylation in response to Rok activation would be predicted to modify the conformation and elevate the catalytic activity of its associated heavy chain, Zipper (Zip). Does Zip also participate in regulating actin distribution/wing hair number in response to Fz/Dsh? Loss of one copy of the zip gene enhances the dsh¹ phenotype by 4.5-fold, consistent with the genetic interaction data between fz/dsh and sqh. These results suggest that myosin II functions positively downstream of Fz/Dsh in regulating actin prehair development (Winter, 2001).

The localization of Zip protein in wing cells further supports its role downstream of Fz/Dsh. At the apical surface of the pupal wing cell, Zip is asymmetrically localized to the distal portion of the cell, where prehair growth initiates. This distal localization appears to coincide, temporally, with prehair initiation. To test whether Zip localization could be modified by Fz/Dsh signaling, Zip distribution at the apical surface was examined in dsh¹ mutants. Instead of being concentrated in the distal region of the cell, Zip is concentrated in the center of the cell, where prehairs form in dsh¹ mutants (Winter, 2001).

Does reduction in myosin II/Zip activity also result in the multihair phenotype? Use was made of the hypomorphic zip⁰²⁹⁵⁷, since zip and sqh null mutations appear to be cell lethal in the wing. As is the case with rok, some homozygous zip⁰²⁹⁵⁷ wing cells possess multiple F-actin prehairs (Winter, 2001).

Tests were performed to see if the gene crinkled (ck) is involved in the Fz/Dsh signaling pathway regulating wing hair number because (1) ck mutant cells in the wing lead to multiple hair and split hair phenotypes, and (2) ck encodes the Drosophila myosin VIIA protein. Mutations in mouse myosinVIIA lead to stereocilia disorganization and the formation of multiple bundles of stereocilia (Winter, 2001 and references therein).

Reduction of ck activity potently suppresses the dsh¹ multiple hair phenotype. This result contrasts with the result that zip¹ enhances the dsh¹ multiple hair phenotype, and suggests that the two myosin heavy chains have opposing effects in regulating prehair assembly (Winter, 2001).

Both myosin heavy chain genes were tested for their ability to interact with the hs-fz induced multiple hair phenotype, and again it was found that they have opposing effects. Surprisingly, loss of one copy of zip slightly but significantly enhances the late hs-fz multiple hair phenotype, while loss of one copy of ck markedly suppresses this phenotype. These results are the reverse of what one would expect based on their interactions with dsh¹, and suggests the possibility that there is a signal from Fz to Ck that is independent of Dsh, or that the multiple hair phenotypes resulting from hypo- or hyper-activity of the Fz/Dsh pathway arise via distinct biochemical mechanisms (Winter, 2001).

To further assess the nature of the relationship between the two myosins, the effect of raising or lowering the activity of MRLC on the ck phenotype was tested. The multiple hair phenotype in animals homozygous for a weak ck mutation is enhanced by one copy of the sqhE20E21 transgene (and hence, a probable increase in myosin II activity), but not by a sqhA20A21 transgene. Taken together, these experiments suggest that a balance between the activities of myosin II and myosin VIIA is important in regulating wing hair number (Winter, 2001).

Unlike other characterized PCP mutants that affect both orientation and number of wing hairs, the primary defect in Drok² clones appears to be the presence of multiple hairs per cell, with little or no wing hair orientation defect. This suggested that Rok and what lies downstream are involved in transmitting a subset of the Fz/Dsh signal. Supporting this idea, it was found that tubP-Drok and sqhE20E21 suppress the multiple hair phenotype of dsh¹, but not the hair misorientation phenotype. Additional data supporting this conclusion comes from observing the site of prehair initiation. Prehairs emerge aberrantly from the center of dsh¹ mutant cells, rather than from the distal vertex as seen in wild type cells. Such mispositioning of prehair initiation correlates with the failure to acquire the proper distal orientation. While tubP-Drok expression suppresses multiple prehair formation, it does not affect the site of F-actin initiation in dsh¹. Finally, the hair orientation defect resulting from Fz overexpression (via hs-fz) at 24 hours is suppressed by reducing dsh gene dosage but not that of RhoA, rok, sqh or ck. Taken together, these observations suggest that separate mechanisms allow Fz/Dsh to independently regulate the number and the orientation of prehairs, and that only the former involves Rok signaling (Winter, 2001).

The data presented in this study suggest that the Rok/myosin II pathway is involved in regulating the number -- but not orientation -- of the wing hair. What then are the components that regulate wing hair orientation? One possibility is that a bifurcation of the pathway occurs at the level of RhoA, with a separate effector pathway regulating wing hair orientation. In the eye, the JNK pathway has been implicated in functioning downstream of RhoA in regulating ommatidial polarity. However, the function of the JNK pathway in the wing has not been described, and a signaling pathway that regulates transcription is unlikely to encode the requisite spatial information necessary for selection of the site of prehair initiation. Therefore, it is likely that a separate signal from or upstream of RhoA may control the selection of the F-actin assembly site, and therefore the orientation of the wing hair (Winter, 2001 and references therein).

By what mechanism do myosins restrict F-actin bundle formation? In light of the finding that myosin II is concentrated at the site of prehair formation, it seems plausible that myosin II is actively involved in either the recruitment of F-actin to the prehair site, or that it directly participates in the assembly of actin bundles, or both. Studies of mammalian myosin II provide a precedent for a role in the formation of F-actin bundles. Phosphorylation of MRLC promotes a conformational change in myosin II from a folded to an extended state that readily forms multivalent bipolar filaments capable of binding multiple actin filaments. This is thought to result in F-actin bundling and stress fiber formation (Winter, 2001 and references therein).

It appears that in the developing wing, the level of MRLC phosphorylation/myosin II activity must be within an optimal range to establish the formation of a single hair. It is possible that the efficiency of F-actin bundle formation is regulated by MRLC phosphorylation in a manner similar to the control of stress fiber formation. If one further assumes that there are certain bundling substrates present only at limiting concentrations (e.g., F-actin itself), then one would predict that the assembly of one F-actin bundle would reduce the probability of forming a second bundle. When MRLC phosphorylation falls below some threshold level (e.g., in rok mutant cells), the efficiency of primary bundle formation is reduced, and thus the concentration of the limiting substrate remains at sufficient levels to support the assembly of secondary bundles/prehairs. Conversely, if MRLC is hyperphosphorylated (e.g., in Fz-overexpressing cells), the bundling efficiency may increase such that the threshold concentration for bundle formation would be reduced, thereby increasing the probability of assembling multiple bundles/prehairs. Future studies will be required to determine the detailed mechanisms involved (Winter, 2001).

In addition to nonmuscle myosin II, which resembles the myosin II from skeletal muscle, there exists a large class of unconventional myosins that have different properties and potential functions in nonmuscle cells. For instance, several different classes of unconventional myosins are expressed in inner ear epithelium with different subcellular localization. Mutations in three of the unconventional myosins, myosin VI, VIIA, and XV, cause hearing/balancing defects in mice, two of which when mutated in humans result in deafness. Of particular interest in the context of this study is myosin VIIA, mutations of which are responsible for mouse shaker-1 and human Usher's syndrome 1B. Loss-of-function ck (Drosophila Myosin VIIA) mutants exhibit a multiple hair and split wing hair phenotype. ck exhibits strong genetic interactions with components of the signal transduction pathway defined in this study, and has the opposite effects as that of myosin II. The seemingly antagonistic relationship between myosin II and myosinVIIA may suggest a mechanism in which the balance of the activities or stoichiometry of these two myosins is critical for the common process they regulate. For example, myosin II and myosin VIIA may share some common, limiting component(s) required for their activity. Thus, by reducing the myosin VIIA dose, myosin II has a larger share of the common component(s) and thus its activity is upregulated (Winter, 2001 and references therein).

The role of the actomyosin cytoskeleton in coordination of tissue growth during Drosophila oogenesis

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

To analyze the correlation between cyst growth and follicle cell division, dividing cells in the follicular epithelium were counted. Within the first 56 hr that are required to form a stage 3 egg chamber, cell-division rates are very low. In the 14 hr period between stage 4 and stage 6, however, cell-division rates continuously increase. During this time, the volume of the cyst increases approximately 11-fold. This parallel increase in mitosis and cyst growth reflects how the growth of the inner cyst is compensated by cell division in the outer follicular epithelium. After stage 7, the follicle cells stop dividing and undergo diverse morphological changes (Wang, 2007).

Newly formed egg chambers are round and change their shape to ellipsoid during early oogenesis. After stage 7, the process of egg-chamber elongation, which is mediated by a polarized actin cytoskeleton within the follicular epithelium, starts. Actin fibers at the basal cortex of the follicle cells run perpendicular to the anterior-posterior axis of the egg chamber, and their contraction leads to an axis expansion. The mechanisms that shape the egg chamber before elongation takes place are unknown. The simultaneous and rapid growth of cyst and epithelium after stage 3 indicates that the development of the two tissues is precisely coordinated. It is, however, unclear how epithelial morphogenesis and proliferation are coupled to the growth of the cyst (Wang, 2007).

The actin cytoskeleton is central for the cell shape, and is thus a possible candidate involved in a controlled epithelial response to the cyst growth. The activity in the actomyosin cytoskeleton was examined by using an antibody specific for the activated form of the regulatory light chain of nonmuscle myosin II (RMLC; Spaghetti squash). The phospho-specific RMLC antibody binds to phosphoSerine21 and reveals myosin in its active state. Around stage 3 of oogenesis, myosin activity restricts to the apical cortex of the follicle cells, where it is maintained until late oogenesis. After stage 7, myosin activity is also present at the basal cortex of the follicle cells in the actin bundles required for egg chamber elongation. Optical confocal sections reveal a pattern of myosin activity in these long parallel bundles that is reminiscent of stress fibers. In contrast, at the apical cortex, myosin is active in short fibers with random orientation reminiscent of a web (Wang, 2007).

The membrane domains of the follicle cells are established before myosin activity restricts to the apical cortex at stage 3. To examine how apical myosin activation relates to follicle cell polarity, mutants affecting epithelial polarity were examined. To avoid perdurance of the wild-type protein after clone induction, focus was placed on large clones, or clones spanning the whole epithelium. The adherence junctions are central for the organization of the apical actin cytoskeleton, and the domain of myosin activity extends into the region where they localize. Therefore null mutants of armadillo (arm), which encodes Drosophila β-catenin, were examined. It has been shown that the adherence junctions are disrupted in arm follicle cell clones since neither DE- or DN-cadherin are detectable. As a result, arm mutant cells exhibit strong cell-shape defects. Surprisingly, it was found that myosin activity is clearly restricted to the apical membrane in arm follicle cell clones. Thus, myosin activity restricts apically in the absence of adherence junctions (Wang, 2007).

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

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

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

To investigate how myosin is activated at the apical cortex, the function of Rok, which has been shown to regulate myosin phosphorylation, was analyzed. Myosin phosphorylation is greatly reduced but still detectable in rok mutant follicle cell clones. This confirms that Rok phosphorylates myosin in the follicular epithelium, but also indicates that Rok is not the only kinase involved in myosin activation. A HA-Rok fusion protein accumulates in particles at the apical cortex of the follicle cells, which are in close proximity to the web-like myosin fibers. Thus, localized Rok activates myosin in the follicular epithelium (Wang, 2007).

Because RMLC phosphorylation is strongly reduced in rok mutant cells, rok clones were used to examine the function of apical myosin activity. rok mutant follicle cells divide normally, form a monolayered follicular epithelium, and retain polarity. However, rok mutant cells fail to adopt a normal shape. As a con