PAR-1 is required for the maintenance of oocyte fate in Drosophila

The PAR-1 kinase is required for the posterior localisation of the germline determinants in C. elegans and Drosophila, and localises to the posterior of the zygote and the oocyte in each case. We show that Drosophila PAR-1 is also required much earlier in oogenesis for the selection of one cell in a germline cyst to become the oocyte. Although the initial steps in oocyte determination are delayed, three markers for oocyte identity, the synaptonemal complex, the centrosomes and Orb protein, still become restricted to one cell in mutant clones. However, the centrosomes and Orb protein fail to translocate from the anterior to the posterior cortex of the presumptive oocyte in region 3 of the germarium, and the cell exits meiosis and becomes a nurse cell. Furthermore, markers for the minus ends of the microtubules also fail to move from the anterior to the posterior of the oocyte in mutant clones. Thus, PAR-1 is required for the maintenance of oocyte identity, and plays a role in microtubule-dependent localisation within the oocyte at two stages of oogenesis. Finally, we show that PAR-1 localises on the fusome, and provides a link between the asymmetry of the fusome and the selection of the oocyte.     

The Drosophila homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs oskar mRNA localization to the posterior pole

In C. elegans, the PAR-1 kinase is localized to the posterior of the zygote and is required for anterior-posterior axis formation. Here, we report that a Drosophila PAR-1 homolog localizes to the posterior of the oocyte with oskar mRNA. Furthermore, par-1 mutants show a novel polarity phenotype in which bicoid mRNA accumulates normally at the anterior, but oskar mRNA is redirected to the center of the oocyte, resulting in embryonic patterning defects. These phenotypes arise from a disorganization of the oocyte microtubule cytoskeleton, consistent with reports that mammalian PAR-1 homologs regulate microtubule dynamics. Thus, Drosophila PAR-1 may remodel the oocyte microtubule network to define the posterior as the site for oskar localization. These results identify a molecular parallel between anterior-posterior polarization in Drosophila and C. elegans.     

Bazooka and PAR-6 are required with PAR-1 for the maintenance of oocyte fate in Drosophila

The anterior-posterior axis of C. elegans is defined by the asymmetric division of the one-cell zygote, and this is controlled by the PAR proteins, including PAR-3 and PAR-6, which form a complex at the anterior of the cell, and PAR-1, which localizes at the posterior [1-4]. PAR-1 plays a similar role in axis formation in Drosophila: the protein localizes to the posterior of the oocyte and is necessary for the localization of the posterior and germline determinants [5, 6]. PAR-1 has recently been shown to have an earlier function in oogenesis, where it is required for the maintenance of oocyte fate and the posterior localization of oocyte-specific markers [7, 8]. Here, we show that the homologs of PAR-3 (Bazooka) and PAR-6 are also required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that develop 16 nurse cells and no oocyte. Furthermore, oocyte-specific factors, such as Orb protein and the centrosomes, still localize to one cell but fail to move from the anterior to the posterior cortex. Thus, PAR-1, Bazooka, and PAR-6 are required for the earliest polarity in the oocyte, providing the first example in Drosophila where the three homologs function in the same process. Although these PAR proteins therefore seem to play a conserved role in early anterior-posterior polarity in C. elegans and Drosophila, the relationships between them are different, as the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm.     

Cell polarity and oocyte determination in Drosophila melanogaster

During early oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte. Recent data suggest that the choice of this cell within the cyst is strongly biased as early as the cyst itself forms. However, it was further shown that, although selected, the oocyte fate needs to be maintained. The maintenance of the oocyte identity requires the activity of the Drosophila homologues of the Caenorhabditis elegans par genes. It was shown that the par genes are required for the first polarisation of the oocyte as early as in region 3 of the germarium. This reveals a striking conservation between the polarisation along the antero-posterior axis of the Caenorhabditis elegans one-cell embryo and the Drosophila oocyte.     

A Notch/Delta-dependent relay mechanism establishes anterior-posterior polarity in Drosophila

The anterior-posterior axis of Drosophila becomes polarized early in oogenesis, when the oocyte moves to the posterior of the germline cyst because it preferentially adheres to posterior follicle cells. The source of this asymmetry is unclear, however, since anterior and posterior follicle cells are equivalent until midoogenesis, when Gurken signaling from the oocyte induces posterior fate. Here, we show that asymmetry arises because each cyst polarizes the next cyst through a series of posterior to anterior inductions. Delta signaling from the older cyst induces the anterior polar follicle cells, the anterior polar cells signal through the JAK/STAT pathway to induce the formation of the stalk between adjacent cysts, and the stalk polarizes the younger anterior cyst by inducing the shape change and preferential adhesion that position the oocyte at the posterior. The anterior-posterior axis is therefore established by a relay mechanism, which propagates polarity from one cyst to the next.     

Observations on the polarity of mutant Drosophila follicles lacking the oocyte

Summary Homozygous females of the mutantsegalitarian andBicaudal-D ^R26produce follicles in which the oocyte is replaced by an additional nurse cell. Normal morphological markers for polarity can be identified in mutant follicles but the normal spatial organization of these markers is disturbed. For example, nurse-cell nuclei of different ploidy classes are present but, contrary to wild-type follicles, the nuclei show no anteroposterior ploidy gradient. The two cells with four intercellular bridges, one of which should have developed into the oocyte rather than a nurse cell, are located at the posterior pole only in young follicles (up to about stage 5), whereas during later stages they are more often found at lateral or intermediate positions. This disturbed polarity correlates with a variable aberrant pattern of extracellular ionic currents. Moreover, in the mutant follicles patches of columnar follicular epithelium differentiate locally although this type of epithelium forms normally only around the oocyte. The follicle cells at both follicle poles possess anterior quality since they migrate from both poles towards the centre of the follicle, as do the border cells restricted to the anterior pole in wild-type follicles. Our analysis indicates that in the mutants the follicular polarity is normal at first but becomes disturbed during stages 5 to 6. The secondary breakdown of polarity is likely to follow on from the absence of the oocyte.     

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte

Localization of the maternal determinant Oskar at the posterior pole of Drosophila melanogaster oocyte provides the positional information for pole plasm formation. Spatial control of Oskar expression is achieved through the tight coupling of mRNA localization to translational control, such that only posterior-localized oskar mRNA is translated, producing the two Oskar isoforms Long Osk and Short Osk. We present evidence that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. We show that Long Osk anchors both oskar mRNA and Short Osk, the isoform active in pole plasm assembly, at the posterior pole. In the absence of anchoring by Long Osk, Short Osk disperses into the bulk cytoplasm during late oogenesis, impairing pole cell formation in the embryo. In addition, the pool of untethered Short Osk causes anteroposterior patterning defects, owing to the dispersion of pole plasm and its abdomen-inducing activity throughout the oocyte. We show that the N-terminal extension of Long Osk is necessary but not sufficient for posterior anchoring, arguing for multiple docking elements in Oskar. This study reveals cortical anchoring of the posterior determinant Oskar as a crucial step in pole plasm assembly and restriction, required for proper development of Drosophila melanogaster.     

An oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte

The localization of oskar mRNA to the posterior of the Drosophila oocyte defines the site of assembly of the pole plasm, which contains the abdominal and germline determinants. oskar mRNA localization requires the polarization of the microtubule cytoskeleton, which depends on the recruitment of PAR-1 to the posterior cortex in response to a signal from the follicle cells, where it induces an enrichment of microtubule plus ends. Here, we show that overexpressed oskar mRNA localizes to the middle of the oocyte, as well as the posterior. This ectopic localization depends on the premature translation of Oskar protein, which recruits PAR-1 and microtubule-plus-end markers to the oocyte center instead of the posterior pole, indicating that Oskar regulates the polarity of the cytoskeleton. Oskar also plays a role in the normal polarization of the oocyte; mutants that disrupt oskar mRNA localization or translation strongly reduce the posterior recruitment of microtubule plus ends. Thus, oskar mRNA localization is required to stabilize and amplify microtubule polarity, generating a positive feedback loop in which Oskar recruits PAR-1 to the posterior to increase the microtubule cytoskeleton's polarization, which in turn directs the localization of more oskar mRNA.     

Strabismus requires Flamingo and Prickle function to regulate tissue polarity in the Drosophila eye

Tissue polarity in Drosophila is regulated by a number of genes that are thought to function in a complex, many of which interact genetically and/or physically, co-localize, and require other tissue polarity proteins for their localization. We report the enhancement of the strabismus tissue polarity phenotype by mutations in two other tissue polarity genes, flamingo and prickle. Flamingo is autonomously required for the establishment of ommatidial polarity. Its localization is dynamic throughout ommatidial development and is dependent on Frizzled and Notch. Flamingo and Strabismus co-localize for several rows posterior to the morphogenetic furrow and subsequently diverge. While neither of these proteins is required for the other's localization, Prickle localization is influenced by Strabismus function. Our data suggest that Strabismus, Flamingo and Prickle function together to regulate the establishment of tissue polarity in the Drosophila eye.     

Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires Centrosomin Motif 1

Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microtubule spindle apparatus at mitosis. Although the individual binding steps in centrosome maturation are largely unknown, Centrosomin (Cnn) is an essential mitotic centrosome component required for assembly of all other known pericentriolar matrix (PCM) proteins to achieve microtubule-organizing activity at mitosis in Drosophila. We have identified a conserved motif (Motif 1) near the amino terminus of Cnn that is essential for its function in vivo. Cnn Motif 1 is necessary for proper recruitment of gamma-tubulin, D-TACC (the homolog of vertebrate transforming acidic coiled-coil proteins [TACC]), and Minispindles (Msps) to embryonic centrosomes but is not required for assembly of other centrosome components including Aurora A kinase and CP60. Centrosome separation and centrosomal satellite formation are severely disrupted in Cnn Motif 1 mutant embryos. However, actin organization into pseudocleavage furrows, though aberrant, remains partially intact. These data show that Motif 1 is necessary for some but not all of the activities conferred on centrosome function by intact Cnn.     

Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell-cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.     

Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation

BACKGROUND: The cytoskeleton and associated motors play an important role in the establishment of intracellular polarity. Microtubule-based transport is required in many cell types for the asymmetric localization of mRNAs and organelles. A striking example is the Drosophila oocyte, where microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. RESULTS: Here, we show that mRNA localization and nuclear positioning at mid-oogenesis depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. CONCLUSION: The microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole.     

Stable anterior anchoring of the oocyte nucleus is required to establish dorsoventral polarity of the Drosophila egg

Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In the fruit fly Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Here we describe two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals.     

Lgl and its phosphorylation by aPKC regulate oocyte polarity formation in Drosophila

Specification of the anteroposterior (AP) axis in Drosophila oocytes requires proper organization of the microtubule and actin cytoskeleton. The establishment and regulation of cytoskeletal polarity remain poorly understood, however. Here, we show important roles for the tumor suppressor Lethal (2) giant larvae (Lgl) and atypical protein kinase C (aPKC) in regulating microtubule polarity and setting up the AP axis of the oocyte. Lgl in the germline cells regulates the localization of axis-specifying morphogens. aPKC phosphorylation of Lgl restricts Lgl activity to the oocyte posterior, thereby dividing the cortex into different domains along the AP axis. Active Lgl promotes the formation of actin-rich projections at the oocyte cortex and the posterior enrichment of the serine/threonine kinase Par-1, a key step for oocyte polarization. Our studies suggest that Lgl and its phosphorylation by aPKC may form a conserved regulatory circuitry in polarization of various cell types.     

Local actin-dependent endocytosis is zygotically controlled to initiate Drosophila cellularization

In early Drosophila embryos, several mitotic cycles proceed with aborted cytokinesis before a modified cytokinesis, called cellularization, finally divides the syncytium into individual cells. Here, we find that scission of endocytic vesicles from the plasma membrane (PM) provides a control point to regulate the furrowing events that accompany this development. At early mitotic cycles, local furrow-associated endocytosis is controlled by cell cycle progression, whereas at cellularization, which occurs in a prolonged interphase, it is controlled by expression of the zygotic gene nullo. nullo mutations impair cortical F-actin accumulation and scission of endocytic vesicles, such that membrane tubules remain tethered to the PM and deplete structural components from the furrows, precipitating furrow regression. Thus, Nullo regulates scission to restrain endocytosis of proteins essential for furrow stabilization at the onset of cellularization. We propose that developmentally regulated endocytosis can coordinate actin/PM remodeling to directly drive furrow dynamics during morphogenesis.     

poly is required for nurse cell chromosome dispersal and oocyte polarity in Drosophila

During Drosophila oogenesis, nurse cells undergo changes in chromosomal morphology, first from the polytenic form to a transient condensed phase known as the five-blob configuration, then into a diffuse polytenic-polyploid state for the remainder of oogenesis. The mechanism by which nurse-cell chromosome dispersal is regulated remains elusive. Mutations in several genes, including the heterogeneous ribonucleoprotein genes squid (sqd) and hrb27C, the alternative splicing factor gene poly U binding factor 68kDa (pUf68, also known as half-pint), and the germ-line-specific gene ovarian tumor (otu), that produce defects in nurse-cell chromosome dispersal also produce defects in oocyte polarity, suggesting a link between these two processes. Here, we characterize a novel gene named poly, which, when mutated in the germ line, disrupts nurse-cell chromosome dispersal, as well as localization of anteroposterior and dorsoventral determinants in the oocyte. We also show that poly interacts genetically with hrb27C and otu. We conclude that poly is required for nurse-cell chromosome dispersal and oocyte polarization in the Drosophila germ-line. In addition, our interaction data suggest that poly is probably a member of the characterized mRNP complex that mediates both processes.     

The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo

Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.