Phantom,a cytochrome P450 enzyme essential for ecdysone biosynthesis,plays a critical role in the control of border cell migration in Drosophila

The border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. Here we find that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wild-type. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wild-type. Interestingly, even small patches of wild-type follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd), encoding the last enzyme in the pathway that converts ecdysone to the active 20-hydroxyecdysone. Administration of high 20-hydroxyecdysone titers in the medium can also rescue the border cell migration phenotype in cultured egg chambers with an entirely phm mutant follicular epithelium. These results indicate that in normal oogenesis, the follicle cell epithelium of each individual egg chamber must supply sufficient ecdysone precursors, leading ultimately to high enough levels of mature 20-hydroxyecdysone to the border cells to initiate their migration. Neither the germline, nor the neighboring egg chambers, nor the surrounding hemolymph appear to provide threshold amounts of 20-hydroxyecdysone to do so. This “egg chamber autonomous” ecdysone synthesis constitutes a useful way to regulate the individual maturation of the asynchronous egg chambers present in the Drosophila ovary.     

Wnt signaling establishes the microtubule polarity in neurons through regulation of Kinesin-13

Neuronal polarization is facilitated by the formation of axons with parallel arrays of plus-end-out and dendrites with the nonuniform orientation of microtubules. In C. elegans, the posterior lateral microtubule (PLM) neuron is bipolar with its two processes growing along the anterior–posterior axis under the guidance of Wnt signaling. Here we found that loss of the Kinesin-13 family microtubule-depolymerizing enzyme KLP-7 led to the ectopic extension of axon-like processes from the PLM cell body. Live imaging of the microtubules and axonal transport revealed mixed polarity of the microtubules in the short posterior process, which is dependent on both KLP-7 and the minus-end binding protein PTRN-1. KLP-7 is positively regulated in the posterior process by planar cell polarity components of Wnt involving rho-1/rock to induce mixed polarity of microtubules, whereas it is negatively regulated in the anterior process by the unc-73/ced-10 cascade to establish a uniform microtubule polarity. Our work elucidates how evolutionarily conserved Wnt signaling establishes the microtubule polarity in neurons through Kinesin-13.     

The planar polarity pathway promotes coordinated cell migration during Drosophila oogenesis

Cell migration is fundamental in both animal morphogenesis and disease. The migration of individual cells is relatively well-studied; however, in vivo, cells often remain joined by cell-cell junctions and migrate in cohesive groups. How such groups of cells coordinate their migration is poorly understood. The planar polarity pathway coordinates the polarity of non-migrating cells in epithelial sheets and is required for cell rearrangements during vertebrate morphogenesis. It is therefore a good candidate to play a role in the collective migration of groups of cells. Drosophila border cell migration is a well-characterised and genetically tractable model of collective cell migration, during which a group of about six to ten epithelial cells detaches from the anterior end of the developing egg chamber and migrates invasively towards the oocyte. We find that the planar polarity pathway promotes this invasive migration, acting both in the migrating cells themselves and in the non-migratory polar follicle cells that they carry along. Disruption of planar polarity signalling causes abnormalities in actin-rich processes on the cell surface and leads to less-efficient migration. This is apparently due, in part, to a loss of regulation of Rho GTPase activity by the planar polarity receptor Frizzled, which itself becomes localised to the migratory edge of the border cells. We conclude that, during collective cell migration, the planar polarity pathway can mediate communication between motile and non-motile cells, which enhances the efficiency of migration via the modulation of actin dynamics.     

Rotating for elongation: Fat2 whips for the race

Dynamic rearrangements of the actin cytoskeleton are crucial for cell shape and migration. In this issue, Squarr et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508081) show that the cadherin superfamily protein Fat2 regulates actin-rich protrusions driving collective cell migration during Drosophila melanogaster egg morphogenesis through its interaction with the WAVE regulatory complex.Collective cell migration is a hallmark of tissue remodeling during embryonic development, as well as of tissue repair and cancer invasion (Rørth, 2012). A novel type of collective cell migration has emerged in recent years from studies of Drosophila follicles (Haigo and Bilder, 2011), highlighting what seems to be a potentially conserved, intrinsic property of epithelial cells growing in constricted environments. The Drosophila follicle or egg chamber is a spherical assembly of germ cells surrounded by an epithelium of somatic follicle cells. The egg chamber elongates to an ellipsoid during oogenesis, thereby conferring the egg its appropriate shape (Fig. 1). Egg chamber elongation is guaranteed by a molecular corset, formed by the follicle cells and the basal ECM, which directs the growth of the egg chamber along the anterior–posterior axis by constraining the central area of the egg (He et al., 2010). In particular, an ordered array of contractile actin filaments within the follicle cells, running perpendicular to the anterior–posterior axis of the egg chamber, contributes to this “corset” (He et al., 2010). Collective migration of follicle cells around the anterior–posterior axis of the egg chamber is required to promote the global polarization of these parallel actin bundles and of the follicular basement membrane (Haigo and Bilder, 2011; Cetera et al., 2014). This is a remarkable type of cell migration as it leads to a rotational movement of a group of cells within a constrained space and without an identified collective leading edge. Nonetheless, it turns out that actin-rich protrusions, which are typical of a leading edge, are present at the basal side of each individual follicle cell. These protrusions point toward the direction of rotation and are necessary to generate the collective rotational movement (Cetera et al., 2014).Open in a separate windowFigure 1.The actin-rich structures underlying egg chamber elongation. Maturing egg chambers contain germ cells (yellow), including the oocyte (D, brown), covered by a layer of follicle cells (light blue). During early maturation stages, the follicle cells drive the rotation of the egg chamber over the ECM (dark blue) and pull the germ cells along. This rotation promotes the elongation of egg chambers along the antero–posterior axis, observed from stage 7/8. (A) Schematic representation of an egg chamber at stage 5/6, during the collective rotation movement in the direction indicated by the arrow. The axis cross applies to A, C, and D. (B) A schematic of the follicle epithelium (transversal section) indicating its apical side, oriented toward the germ cells, and the basal membrane, laying over the ECM. Basal actin filaments are depicted in red. (C) Higher magnification of the follicle cells visualized from their basal side. Actin-rich structures are depicted in red. They include actin-rich protrusions at the leading edge of each cell and whip-like protrusions at tripartite junctions between cells. Within each cell, the bundles of actin filaments (basal actin) run parallel to the direction of follicle epithelium rotation (black arrow). (D) Illustration of the elongated egg chamber in the next maturation stages (7/8) in Drosophila development.Presence of these actin protrusions at the leading edge of follicle cells requires the WASP family verprolin homologous protein (WAVE) and the WAVE regulatory complex (WRC; Cetera et al., 2014), known activators of actin nucleation through the Arp2/3 complex, as depletion of WAVE or of the WRC component Abi eliminates all actin-based protrusions in follicle cells (Cetera et al., 2014).The details of the control of WAVE and WRC activation have been under scrutiny for many years (Stradal and Scita, 2006; Ismail et al., 2009; Chen et al., 2010; Mendoza, 2013). Nonetheless, a recent discovery opened the possibility of a novel and conserved mechanism for WRC activation (Chen et al., 2014). A combination of structural and biochemical approaches revealed that the WRC can be recruited to the membrane by an array of membrane proteins sharing a conserved peptide motif, the WRC interacting receptor sequence (WIRS). The binding surface for WIRS is provided by two WRC subunits, including Abi, and leads to activation of WAVE toward the Arp2/3 complex (Chen et al., 2014). Could a WIRS domain-containing molecule be involved in the localized activation of WAVE during the rotational movement of follicle cells? This attractive hypothesis is supported by the observation that disruption of the WIRS interface in the WRC leads to the generation of round eggs (Chen et al., 2014). The round-egg phenotype characteristically results from mutations in genes promoting the rotational movement of the follicle cells or organizing the underlying ECM (Gutzeit et al., 1991; Bateman et al., 2001; Frydman and Spradling, 2001; Deng et al., 2003; Schneider et al., 2006). One of these round-egg genes is the cadherin superfamily fat2/kugelei (Gutzeit et al., 1991). Individual follicle cells from fat2 mutants have parallel-arranged actin filaments. However, these actin filaments are no longer perpendicular to the anterior–posterior axis and their coordinated organization in the tissue is lost (Gutzeit et al., 1991; Viktorinová et al., 2009). Moreover, large clones of fat2 mutant follicle cells lead to uncoordinated arrangement of actin bundles in the wild-type neighboring cells (Viktorinová et al., 2009). Collectively, these previous results suggested that Fat2 coordinates actin organization in follicle cells.In this issue, Squarr et al. identified Fat2 as a novel WIRS domain–containing molecule that acts through the WRC to control collective cell migration during Drosophila oogenesis. Squarr et al. (2016) elegantly used live in vivo imaging and genetically encoded probes to characterize different types of actin-rich protrusions during egg chamber maturation. Their analysis showed that small filopodial protrusions extend at regular intervals in a polarized fashion at the basolateral cell border, whereas on the apical side filopodial protrusions are not polarized along the migration direction (Cetera and Horne-Badovinac, 2015; Squarr et al., 2016). Stunning time-lapse movies remarkably revealed an additional type of actin-rich whip-like protrusion at tricellular junctions (Fig. 1). All these types of actin protrusions depend on the WRC, as they are largely missing in follicle cells with impaired WRC function. At defined stages of egg chamber maturation, the WRC is prominently enriched at tricellular junctions, a localization that resembles that reported for Fat2 at the same stages (Viktorinová et al., 2009; Squarr et al., 2016). Motivated by this observation and in search of the molecular mechanisms driving the formation of the actin-rich structures, Squarr et al. (2016) asked whether Fat2 is functionally related to the WRC-dependent organization of actin. Indeed, they identified three conserved WIRS motifs in the cytoplasmic tail of Fat2 and demonstrated a direct interaction of Fat2’s cytoplasmic tail with the WRC through in vitro binding assays, establishing Fat2 as a novel WIRS ligand.They additionally proved that functional WIRS interactions are necessary for the formation of whip-like protrusions and polarized protrusions as well as egg chamber elongation by analyzing flies lacking the conserved WIRS binding surface in the WRC. To test the hypothesis that Fat2 is involved in recruiting the WRC, the researchers examined fat2 mutant cells, which displayed impaired WRC localization to the basal follicle side and to tricellular junctions as well as reduced actin-rich protrusions at the basal side. Thus, Fat2 contributes to the localization of the WRC in these egg chambers. Interestingly, analysis of additional mutant lines showed that neither loss of the WRC nor loss of the Fat2–WRC interaction affected the distribution of Fat2, suggesting it acts upstream of the WRC to control WRC localization and formation of polarized cell protrusions.Additional paths of WRC regulation might involve the ECM receptor phosphatase Dlar, as dlar mutants also exhibit a round-egg phenotype (Bateman et al., 2001). Indeed, Squarr et al. (2016) observed that Dlar and WRC subunit localization partially overlap during the early stages of egg chamber maturation and provide biochemical evidence for an indirect molecular interaction in vivo between Dlar and the WRC. Importantly, in dlar mutants, less WRC localized to the basal side and actin-rich protrusions along the membrane were also reduced. Nonetheless, the localization of the WRC at tricellular junctions is partially maintained and so is actin accumulation at these sites. Collectively, these data establish a model in which Fat2 and Dlar are linked in recruiting the WRC to induce the formation of polarized cell protrusions, contributing to different aspects of WRC regulation for collective follicle cell migration during Drosophila oogenesis.Squarr et al. (2016) combined genetic, biochemical, and live-imaging analyses in Drosophila to gain insight into molecular actin dynamics in vivo. Recent data from time-lapse imaging suggested that collective rotational movement is an intrinsic property of epithelial cells and a feature of developing glandular tissues in mammals (Ewald et al., 2008; Tanner et al., 2012). Squarr et al. (2016) confirmed that nonmalignant human breast epithelial cell lines form spheres and undergo multiple rotations (Tanner et al., 2012). To ask whether the actin-rich protrusions that they observed in fly follicle cells represent a conserved structure underlying epithelial rotational migration, they conducted live imaging of mammary epithelial cells expressing the actin marker LifeAct-EGFP to highlight actin organization. In this system, they observed actin accumulation at the basal side of the rotating spheres and actin-rich protrusions at basal intercellular junctions. The function of these actin whips and protrusions across species is not yet clear; Squarr et al. (2016) speculate that whip-like protrusions might interact with the ECM to synchronize directed cell migration and to drive the morphogenetic movement. The similar morphology of the protrusions in Drosophila and human epithelial cells additionally suggests that the molecular mechanisms driving tissue rotation might be similar, a hypothesis compatible with the known localization of the human homologue of Fat2 at intercellular epithelial junctions. It will be of great interest to address whether the Fat2-, Dlar-, and WRC-dependent molecular mechanisms described in this study are conserved in other rotational collective cell migration processes.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

Role of Notch pathway in terminal follicle cell differentiation during Drosophila oogenesis

 During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are required for this patterning. To understand how these pathways act in the process we have analyzed markers for anterior and posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression of Delta. We find that a constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. Our data are consistent with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway. Received: 5 November 1998 / Accepted: 14 December 1998     

The serine/threonine kinase dPak is required for polarized assembly of F-actin bundles and apical-basal polarity in the Drosophila follicular epithelium

During epithelial development cells become polarized along their apical-basal axis and some epithelia also exhibit polarity in the plane of the tissue. Mutations in the gene encoding a Drosophila Pak family serine/threonine kinase, dPak, disrupt the follicular epithelium that covers developing egg chambers during oogenesis. The follicular epithelium normally exhibits planar polarized organization of basal F-actin bundles such that they lie perpendicular to the anterior-posterior axis of the egg chamber, and requires contact with the basement membrane for apical-basal polarization. During oogenesis, dPak becomes localized to the basal end of follicle cells and is required for polarized organization of the basal actin cytoskeleton and for epithelial integrity and apical-basal polarity. The receptor protein tyrosine phosphatase Dlar and integrins, all receptors for extracellular matrix proteins, are required for polarization of the basal F-actin bundles, and for correct dPak localization in follicle cells. dpak mutant follicle cells show increased beta(Heavy)-spectrin levels, and we speculate that dPak regulation of beta(Heavy)-spectrin, a known participant in the maintenance of membrane domains, is required for correct apical-basal polarization of the membrane. We propose that dPak mediates communication between the basement membrane and intracellular proteins required for polarization of the basal F-actin and for apical-basal polarity.     

The Hippo pathway controls polar cell fate through Notch signaling during Drosophila oogenesis

During Drosophila oogenesis, the somatic follicle cells form an epithelial layer surrounding the germline cells to form egg chambers. In this process, follicle cell precursors are specified into polar cells, stalk cells, and main-body follicle cells. Proper specification of these three cell types ensures correct egg chamber formation and polarization of the anterior–posterior axis of the germline cells. Multiple signaling cascades coordinate to control the follicle cell fate determination, including Notch, JAK/STAT, and Hedgehog signaling pathways. Here, we show that the Hippo pathway also participates in polar cell specification. Over-activation of yorkie (yki) leads to egg chamber fusion, possibly through attenuation of polar cell specification. Loss-of-function experiments using RNAi knockdown or generation of mutant clones by mitotic recombination demonstrates that reduction of yki expression promotes polar cell formation in a cell-autonomous manner. Consistently, polar cells mutant for hippo (hpo) or warts (wts) are not properly specified, leading to egg chamber fusion. Furthermore, Notch activity is increased in yki mutant cells and reduction of Notch activity suppresses polar cell formation in yki mutant clones. These results demonstrate that yki represses polar cell fate through Notch signaling. Collectively, our data reveal that the Hippo pathway controls polar cell specification. Through repressing Notch activity, Yki serves as a key repressor in specifying polar cells during Drosophila oogenesis.     

Both contractile axial and lateral traction force dynamics drive amoeboid cell motility

Chemotaxing Dictyostelium discoideum cells adapt their morphology and migration speed in response to intrinsic and extrinsic cues. Using Fourier traction force microscopy, we measured the spatiotemporal evolution of shape and traction stresses and constructed traction tension kymographs to analyze cell motility as a function of the dynamics of the cell’s mechanically active traction adhesions. We show that wild-type cells migrate in a step-wise fashion, mainly forming stationary traction adhesions along their anterior–posterior axes and exerting strong contractile axial forces. We demonstrate that lateral forces are also important for motility, especially for migration on highly adhesive substrates. Analysis of two mutant strains lacking distinct actin cross-linkers (mhcA⁻ and abp120⁻ cells) on normal and highly adhesive substrates supports a key role for lateral contractions in amoeboid cell motility, whereas the differences in their traction adhesion dynamics suggest that these two strains use distinct mechanisms to achieve migration. Finally, we provide evidence that the above patterns of migration may be conserved in mammalian amoeboid cells.     

Role of Scrib and Dlg in anterior-posterior patterning of the follicular epithelium during Drosophila oogenesis

Background  

Proper patterning of the follicle cell epithelium over the egg chamber is essential for the Drosophila egg development. Differentiation of the epithelium into several distinct cell types along the anterior-posterior axis requires coordinated activities of multiple signaling pathways. Previously, we reported that lethal(2)giant larvae (lgl), a Drosophila tumor suppressor gene, is required in the follicle cells for the posterior follicle cell (PFC) fate induction at mid-oogenesis. Here we explore the role of another two tumor suppressor genes, scribble (scrib) and discs large (dlg), in the epithelial patterning.     

The receptor-like tyrosine phosphatase lar is required for epithelial planar polarity and for axis determination within drosophila ovarian follicles.

The follicle cell monolayer that encircles each developing Drosophila oocyte contributes actively to egg development and patterning, and also represents a model stem cell-derived epithelium. We have identified mutations in the receptor-like transmembrane tyrosine phosphatase Lar that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an indicator of oocyte anterior-posterior polarity. Alterations in actin filament organization correlate with these defects. Actin filaments in the basal follicle cell domain normally become polarized during stage 6 around the anterior-posterior axis defined by the polar cells, but mutations in Lar frequently disrupt polar cell differentiation and actin polarization. Lar function is only needed in somatic cells, and (for Oskar localization) its action is autonomous to posterior follicle cells. Polarity signals may be laid down by these cells within the extracellular matrix (ECM), possibly in the distribution of the candidate Lar ligand Laminin A, and read out at the time Oskar is localized in a Lar-dependent manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. We show that Lar acts somatically early in oogenesis, during follicle formation, and postulate that it functions in germarium intercyst cells that are required for polar cell specification and differentiation. Our studies suggest that positional information can be stored transiently in the ECM. A major function of Lar may be to transduce such signals.     

An InCytes from MBC Selection: Vimentin Regulates Scribble Activity by Protecting It from Proteasomal Degradation

Scribble (Scrib), Discs large, and Lethal giant larvae form a protein complex that regulates different aspects of cell polarization, including apical–basal asymmetry in epithelial cells and anterior–posterior polarity in migrating cells. Here, we show that Scrib interacts with the intermediate filament cytoskeleton in epithelial Madin-Darby canine kidney (MDCK) cells and endothelial human umbilical vein endothelial cells. Scrib binds vimentin via its postsynaptic density 95/disc-large/zona occludens domains and in MDCK cells redistributes from filaments to the plasma membrane during the establishment of cell–cell contacts. RNA interference-mediated silencing of Scrib, vimentin, or both in MDCK cells results in defects in the polarization of the Golgi apparatus during cell migration. Concomitantly, wound healing is delayed due to the loss of directional movement. Furthermore, cell aggregation is dependent on both Scrib and vimentin. The similar phenotypes observed after silencing either Scrib or vimentin support a coordinated role for the two proteins in cell migration and aggregation. Interestingly, silencing of vimentin leads to an increased proteasomal degradation of Scrib. Thus, the upregulation of vimentin expression during epithelial to mesenchymal transitions may stabilize Scrib to promote directed cell migration.     

Regulation of cell proliferation and patterning in Drosophila oogenesis by Hedgehog signaling

The localized expression of Hedgehog (Hh) at the extreme anterior of Drosophila ovarioles suggests that it might provide an asymmetric cue that patterns developing egg chambers along the anteroposterior axis. Ectopic or excessive Hh signaling disrupts egg chamber patterning dramatically through primary effects at two developmental stages. First, excess Hh signaling in somatic stem cells stimulates somatic cell over-proliferation. This likely disrupts the earliest interactions between somatic and germline cells and may account for the frequent mis-positioning of oocytes within egg chambers. Second, the initiation of the developmental programs of follicle cell lineages appears to be delayed by ectopic Hh signaling. This may account for the formation of ectopic polar cells, the extended proliferation of follicle cells and the defective differentiation of posterior follicle cells, which, in turn, disrupts polarity within the oocyte. Somatic cells in the ovary cannot proliferate normally in the absence of Hh or Smoothened activity. Loss of protein kinase A activity restores the proliferation of somatic cells in the absence of Hh activity and allows the formation of normally patterned ovarioles. Hence, localized Hh is not essential to direct egg chamber patterning.     

Follicle cell development is partly independent of germ-line cell differentiation in Drosophila oogenesis

Summary The developmental potential of the cells of the somatic follicular epithelium (follicle cells) was studied in mutants in which the differentiation of the germ-line cells is blocked at different stages of oogenesis. In two mutants, sn ^36a and kelch, nurse cell regression does not occur, yet the follicle cells around the small oocyte continue their normal developmental program and produce an egg shell with micropylar cone and often deformed operculum and respiratory appendages. Neither the influx of nurse cell cytoplasm into the oocyte nor the few follicle cells covering the nurse cells are apparently required for the formation of the egg shell. In the tumor mutant benign gonial cell neoplasm (bgcn) the follicle cells can also differentiate to some extent although the germ-line cells remain morphologically undifferentiated. Vitelline membrane material was synthesized by the follicle cells in some bgcn chambers and in rare cases a columnar epithelium, which resembled morphologically that of wild-type stage-9 follicles, formed around the follicle's posterior end. The normal polarity of the follicular epithelium that is characteristic for mid-vitellogenic stages may, therefore, be established in the absence of morphologically differentiating germ-line cells. However, the tumorous germ-line cells do not constitute a homogeneous cell population since in about 30% of the analyzed follicles a cell cluster at or near the posterior pole can be identified by virtue of its high number of concanavalin A binding sites. This molecular marker reveals an anteroposterior polarity of the tumorous chambers. In follicles mutant for both bgcn and the polarity gene dicephalic the cluster of concanavalin A-stained germ-line cells shifts to more anterior positions in the follicle.     

Functioning of the Drosophila orb gene in gurken mRNA localization and translation.

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, we have examined the role of the orb gene in the gurken (grk)-Drosophila epidermal growth factor receptor (DER) signaling pathway. During the previtellogenic stages of oogenesis, the grk-DER signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, the grk-DER pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. We show that orb functions in both the early and late grk-DER signaling pathways, and in each case is required for the localized expression of Grk protein. We have found that orb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Finally, we present evidence that Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10).     

Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension

Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila melanogaster, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. We show that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase–binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.     

Heading forwards: anterior visceral endoderm migration in patterning the mouse embryo

The elaboration of anterior–posterior (A–P) pattern is one of the earliest events during development and requires the precisely coordinated action of several players at the level of molecules, cells and tissues. In mammals, it is controlled by a specialized population of migratory extraembryonic epithelial cells, the anterior visceral endoderm (AVE). The AVE is a signalling centre that is responsible for several important patterning events during early development, including specifying the orientation of the A–P axis and the position of the heart with respect to the brain. AVE cells undergo a characteristic stereotypical migration which is crucial to their functions.