Developmental control of nuclear morphogenesis and anchoring by charleston, identified in a functional genomic screen of Drosophila cellularisation

Fanny Pilot Jean-Marc Philippe Cline Lemmers Jean-Paul Chauvin Thomas Lecuit ) Morphogenesis of epithelial tissues relies on the precise developmental control of cell polarity and architecture. In the early Drosophila embryo, the primary epithelium forms during cellularisation, following a tightly controlled genetic programme where specific sets of genes are upregulated. Some of them, for example, control membrane invagination between the nuclei anchored at the apical surface of the syncytium. We used microarrays to describe the global programme of gene expression underlying cellularisation and identified distinct classes of upregulated genes during this process. Fifty-seven genes were then tested functionally by RNAi. We found six genes affecting various aspects of cellular architecture: membrane growth, organelle transport or organisation and junction assembly. We focus here on charleston (char), a new regulator of nuclear morphogenesis and of apical nuclear anchoring. In char-depleted embryos, the nuclei fail to maintain their elongated shape and, instead, become rounded. In addition, together with a disruption of the centrosome-nuclear envelope interaction, the nuclei lose their regular apical anchoring. These nuclear defects perturb the regular columnar organisation of epithelial cells in the embryo. Although microtubules are required for both nuclear morphogenesis and anchoring, char does not control microtubule organisation and association to the nuclear envelope. We show that Char is lipid anchored at the nuclear envelope by a farnesylation group, and localises at the inner nuclear membrane together with Lamin. Our data suggest that Char forms a scaffold that regulates nuclear architecture to constrain nuclei in tight columnar epithelial cells. The upregulation of Char during cellularisation and gastrulation reveals the existence of an as yet unknown developmental control of nuclear morphology and anchoring in embryonic epithelia. INTRODUCTION The regulation of cell shape and cell polarity during development underlies the morphogenesis of tissues. In epithelia, tissues in which the cells exhibit an apicobasal polarity, both the cell surface, the organelles and cytoskeletal elements are precisely organised. Identifying the developmental pathways controlling cell shape at the cellular level is thus an important task for further our understanding of development. As with most developing embryos, the first morphogenetic process in Drosophila embryos is the formation of the primary epithelium, a process called cellularisation (Foe et al., 1993; Schejter and Wieschaus, 1993b). Cellularisation is a specialised form of embryonic cleavage that yields a polarised epithelium within 1 hour (Lecuit, 2004). Upon egg laying, the newly fertilised embryo undergoes a series of 13 synchronous nuclear divisions in a syncytium, producing about 6000 nuclei at the cell cortex. During cellularisation, the plasma membrane invaginates in a slow phase and a fast phase between the nuclei, thus packaging each nucleus, other organelles and cytoskeletal elements into about 6000 cells (Lecuit and Wieschaus, 2000). Cellularisation involves the polarised growth of the plasma membrane via the vectorial transport of vesicles through the Golgi and recycling endosomes and their insertion at specific sites of the plasma membrane (Lecuit and 1Institut de Biologie du Dveloppement de Marseille (IBDM) Laboratoire de Gntique et de Physiologie du Dveloppement (LGPD), UMR6545 CNRS-Universit de la Mditerranne. Campus de Luminy case 907, Marseille 13288 cedex9, France. 2Plateforme de microscopie lectronique, IBDM, France. Wieschaus, 2000; Papoulas et al., 2005; Pelissier et al., 2003; Sisson et al., 2000). Distinct plasma membrane domains are already established by this time. Polarised growth culminates in the formation of apical adherens junctions at the end of cellularisation and their subsequent stabilisation during gastrulation (Muller and Bossinger, 2003). Failure to form or stabilise apical junctions results in strong epithelial defects later on during gastrulation (Cox et al., 1996; Muller and Wieschaus, 1996; Tepass et al., 1996; Uemura et al., 1996). In addition, the formation of the primary epithelium involves the polarised organisation of the cytoskeleton and organelles. Microtubules (MTs) form an apicobasal network, with subpopulations of long MTs extending the plus ends basally around the nuclei and short MTs projecting towards the cortex. MTs control the apicobasal distribution of organelles, the nuclei being anchored apically, the Golgi apparatus mostly basal and lipid droplets undergoing basal and apical movements in two successive waves called clearing and clouding phases (Foe et al., 1993; Schejter and Wieschaus, 1993b; Sisson et al., 2000; Welte et al., 1998). The formation of the primary epithelium thus offers a good system with which to address how core cellular processes are developmentally regulated to produce a highly organised tissue exhibiting polarity at the cell surface and in the cytoplasm. Cellularisation is concomitant with zygotic genome activation and inhibition of zygotic transcription totally blocks cellularisation (Foe et al., 1993). However, only five zygotic genes have been reported for their role in cellularisation: nullo, Serendipity- (Sry- ) and slam, which are necessary for stabilisation of the membrane front called the furrow canal; bottleneck (bnk), which ensures the correct timing of basal closure of the cells; and frhstart (frs), required for the arrest in interphase 14 (Grosshans et al., 2003; Lecuit et al., 2002; Postner and Wieschaus, 1994; Rose and Wieschaus, 1992; Schejter and Wieschaus, 1993a; Schweisguth et al., 1990; Stein et al., 2002). Remarkably, these five genes are strongly induced during cellularisation. The fact that the expression of nullo, Sry- , bnk, frs and slam display a strong zygotic induction in cellularisation prompted us to screen for other genes induced during and required for cellularisation. It has become a major challenge to integrate into a global cellular network, the distinct pathways underlying the numerous aspects of epithelial polarity. Functional genomic approaches based on RNA interference (RNAi), mostly in Caenorhabditis elegans embryos and Drosophila cells, have contributed to the identification of many genes involved in cellular organisation, based on their knock-down phenotype (Boutros et al., 2004; Fraser et al., 2000; Gonczy et al., 2000; Kamath et al., 2003; Kiger et al., 2003; Sonnichsen et al., 2005). The major advantage of such RNAi screens is the direct association of a gene to a given biological function. Novel approaches using expression profiling have also proven successful in identifying genes whose expression correlates with specific cellular processes (Arbeitman et al., 2002; Stathopoulos et al., 2002; White et al., 1999). Here, we have sought to combine such genomic methodologies and functional screens (...truncated)