Veit Riechmann

Axis formation during Drosophila oogenesis

Recent advances shed light on the cellular processes that cooperate during oogenesis to produce a fully patterned egg, containing all the maternal information required for embryonic development. Progress has been made in defining the early steps in oocyte specification and it has been shown that progression of oogenesis is controlled by a meiotic checkpoint and requires active maintenance of the oocyte cell fate. The function of Gurken signalling in patterning the dorsal-ventral axis later in oogenesis is better understood. Anterior-posterior patterning of the embryo requires activities of bicoid and oskar mRNAs, localised within the oocyte. A microtubule motor, Kinesin, is directly implicated in localisation of oskar mRNA to the posterior pole of the oocyte.

A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation.

A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation

Par-1 regulates stability of the posterior determinant Oskar by phosphorylation

Par-1 kinase is critical for polarization of the Drosophila melanogaster oocyte and the one-cell Caenorhabditis elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Here we show that Drosophila Par-1 phosphorylates the posterior determinant Oskar (Osk) and demonstrate genetically that Par-1 is required for accumulation of Osk protein. We show in cell-free extracts that Osk protein is intrinsically unstable and that it is stabilized after phosphorylation by Par-1. Our data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk.

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 [1, 2]. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. Here we identify a role for the actomyosin cytoskeleton in this process. We show that myosin activity is restricted to the epitheliums apical surface, which is facing the growing cyst. We demonstrate that the epithelium collapses in the absence of myosin activity and show that 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, our data indicate that cyst growth induces cell division in the epithelium. In addition, we show how 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 PP1beta9C. In addition, our data indicate that active myosin is apically anchored by the Baz/Par-6/aPKC complex.

Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia.

The Ser/Thr kinase Par-1 is required for cell polarisation in diverse organisms such as yeast, worms, flies and mammals. During Drosophila oogenesis, Par-1 is required for several polarisation events, including localisation of the anterior determinant bicoid. To elucidate the molecular pathways triggered by Par-1, we have performed a genome-wide, high-throughput screen for Par-1 targets. Among the targets identified in this screen was Exuperantia (Exu), a mediator of bicoid mRNA localisation. We show that Exu is a phosphoprotein whose phosphorylation is dependent on Par-1 in vitro and in vivo. We identify two motifs in Exu that are phosphorylated by Par-1, and show that their mutation abolishes bicoid mRNA localisation during mid-oogenesis. Interestingly, exu mutants in which Exu phosphorylation is specifically affected can to some extent recover from these bicoid mRNA localisation defects during late oogenesis. These results demonstrate that Par-1 establishes polarity in the oocyte by activating a mediator of bicoid mRNA localisation. Furthermore, our analysis reveals two phases of Exu-dependent bicoid mRNA localisation: an early phase that is strictly dependent on Exu phosphorylation and a late phase that is less phosphorylation dependent.

Stepwise polarisation of the Drosophila follicular epithelium

The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, beta-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm.

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.

Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis

Tao initiates morphogenesis of a squamous epithelium by promoting the endocytosis of the adhesion molecule Fasciclin 2 from the lateral membrane.

A genome-scale in vivo RNAi analysis of epithelial development in Drosophila identifies new proliferation domains outside of the stem cell niche.

ABSTRACT The Drosophila oogenesis system provides an excellent model to study the development of epithelial tissues. Here, we report the first genome-scale in vivo RNA interference (RNAi) screen for genes controlling epithelial development. By directly analysing cell and tissue architecture we identified 1125 genes, which we assigned to seven different functions in epithelial formation and homeostasis. We validated the significance of our screen by generating mutants for Vps60, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. This analysis provided new insights into spatiotemporal control of cell proliferation in the follicular epithelium. Previous studies have identified signals controlling divisions in the follicle stem cell niche. However, 99% of cell divisions occur outside of the niche and it is unclear how these divisions are controlled. Our data distinguish two new domains outside of the stem cell niche where there are differing controls on proliferation. One domain abuts the niche and is characterised by ESCRT, Notch and JAK/STAT-mediated control of proliferation. Adjacent to this domain, another domain is defined by loss of the impact of ESCRT on cell division. Thus, during development epithelial cells pass through a variety of microenvironments that exert different modes of proliferation control. The switch between these modes might reflect a decrease in the ‘stemness’ of epithelial cells over time.

Three mechanisms control E-cadherin localization to the zonula adherens

E-cadherin localization to the zonula adherens is fundamental for epithelial differentiation but the mechanisms controlling localization are unclear. Using the Drosophila follicular epithelium we genetically dissect E-cadherin transport in an in vivo model. We distinguish three mechanisms mediating E-cadherin accumulation at the zonula adherens. Two membrane trafficking pathways deliver newly synthesized E-cadherin to the plasma membrane. One is Rab11 dependent and targets E-cadherin directly to the zonula adherens, while the other transports E-cadherin to the lateral membrane. Lateral E-cadherin reaches the zonula adherens by endocytosis and targeted recycling. We show that this pathway is dependent on RabX1, which provides a functional link between early and recycling endosomes. Moreover, we show that lateral E-cadherin is transported to the zonula adherens by an apically directed flow within the plasma membrane. Differential activation of these pathways could facilitate cell shape changes during morphogenesis, while their misregulation compromises cell adhesion and tissue architecture in differentiated epithelia.