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First published online 18 January 2006
doi: 10.1242/dev.02251

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

¹ Institut de Biologie du Développement de Marseille (IBDM) Laboratoire de Génétique et de Physiologie du Développement (LGPD), UMR6545 CNRS-Université de la Méditerrannée. Campus de Luminy case 907, Marseille 13288 cedex9, France.
² Plateforme de microscopie électronique, IBDM, France.

View larger version (30K): [in a new window]   Fig. 1. Different categories of gene expression corresponding to successive stages of early development. (A) Early Drosophila development showing representative embryos of the five successive stages of epithelium formation and remodelling selected for transcriptome analysis (T0-T4, DIC images, the dorsal side is towards the top and anterior towards the left). (B-D) Examples of genes exhibiting a developmental regulation of expression. Increased and decreased expression compared with the mean expression of the five time points (set to 0) for each gene are shown in red and green, respectively. The colour scale ranges from saturated green for log2 ratios -2.0 and below, to saturated red for log2 ratios +2.0 and above. Cluster of genes with a predominant maternal expression (T0, B). Clusters of genes displaying an increased expression in slow phase of cellularisation (T1, C) or throughout cellularisation (T1+T2, D). (E) Schematic representation of the different categories of gene expression profiles identified. The variations are only qualitative. The number of genes associated with each category is indicated. Some genes are absent from T0 to T4 (7232, bright green), or display a uniform expression from T0 to T4 (278, dark green).

View larger version (51K): [in a new window]   Fig. 2. Cluster representation of the 57 selected genes for the RNAi screen together with five genes already known for their role during cellularisation using standard genetic screens (blue). The three different clusters are characterised by a peak induction at early middle and late stages of cellularisation (from top to bottom, respectively). Clustering was made on the mean values of the triplicate experiments for each time-point. The colour scale is the same as in Fig. 1. The genes that showed a distinct phenotype in the RNAi screen are highlighted in orange.

View larger version (77K): [in a new window]   Fig. 3. CG5175/char is required for nuclei organisation and apical anchoring. (A,B) Phase-contrast views of living Drosophila embryos at different time intervals. In a control embryo (A), the nuclei (white) elongate and keep a regular organisation until gastrulation (arrow). In a char RNAi embryo (B), the nuclei progressively lose their proper apical alignment when the membrane invagination front reaches the basal extent of the nuclei (top, middle and arrows in insets) and acquire an irregular round morphology later (arrow). Blue arrowheads indicate the position of the membrane invagination front in the insets. (C,D) Confocal images at successive time points of cellularisation in control embryos (C) and char RNAi embryos (D). Nuclei are labelled with Hoechst (green) and PatJ (blue) highlights the membrane invagination front. (E,F) Nuclear envelope (marked with a Lamin antibody, red) and nucleus (green) in control (E) and char RNAi embryos (F) at early and late stages of cellularisation and viewed from the top.

View larger version (138K): [in a new window]   Fig. 4. Defects in epithelial organisation associated with unconstrained nuclear shape in char RNAi embryos. Sections showing phalloidin, a marker of F-actin (red), the apical protein Patj (green) and the nuclear dye Hoechst (blue) in control (A,B) and char RNAi (C,D) embryos at the end of cellularisation, viewed from the side (A,C) and from the top (B,D). The white lines define the cell contour determined by the localisation of phalloidin and PatJ. In B and D, z1 and z2 are, respectively, apical and more lateral sections indicated in A and C. The apicobasal morphology of cells is regular in control embryos. In char RNAi embryos, cell shape is irregular: many cells display a small apical section (D, arrows) and others have a larger apical section when the nuclei are located apically (D, arrowheads). Apical markers (PatJ) are abnormally present in the deeper section z2 (D), indicating defects in junctional organisation in char RNAi embryos. Scale bar: 5 µm.

View larger version (101K): [in a new window]   Fig. 5. Microtubules organisation in control and char RNAi embryos. (A) Nuclear localisation during cellularisation after injection of colcemid to depolymerise microtubules (MTs). Neurotactin (green) marks the plasma membrane, Even-skipped (blue) labels the nuclei. Arrows indicate misaligned nuclei. (B-H) MTs organisation in control (B,D,F) and char RNAi (C,E,E',G,H) embryos, during early (B,C) and late (F-H) cellularisation. MTs (-tubulin) are in green, nuclei (Hoechst) are in blue and the membrane invagination front (Patj) is in red. Arrowheads show apical astral microtubules. The white arrow indicates a falling nucleus in char RNAi embryo in H. (D-E') Grazing sections showing astral MTs in the control (D) and char RNAi (E,E') embryos. Section planes (z1-z3) are schematised in I. (E'') Detail of a grazing section at z3 of the bottom nucleus shown in H.

View larger version (91K): [in a new window]   Fig. 6. char controls centrosomes association with nuclei. Sagittal sections of control (A-A'') and char RNAi embryos (B-B'') at successive time points of cellularisation showing centrosomes (-tubulin) in green, membrane invagination front (Patj) in red and nuclei (Hoechst) in blue. Arrows indicate centrosomes detached from the nuclei in char RNAi embryos.

View larger version (123K): [in a new window]   Fig. 7. Char localises to the nuclear envelope. (A,B) Char antibody staining (green) highlights the nuclear envelope during cellularisation in a control embryo (A) but is absent in a char RNAi embryo (B). (C-F) Char staining in embryos from heterozygous flies for a deficiency covering the char locus (Dfchar), viewed from the top (C,D, insets) and in sagittal sections (E,F). Two categories of embryos, inferred to be, respectively, homozygous DfChar embryos and heterozygous siblings, are observed: in the first, the nuclei have a normal morphology and high levels of Char at the NE (C,E); in the second, the nuclei display the distinct charRNAi nuclear envelope phenotype together with a low expression of Char (D,F). Scale bars: 5 µm.

View larger version (65K): [in a new window]   Fig. 8. Char is farnesylated. (A,B) HA-Char localises to the nuclear envelope in a cellularising embryo (A), whereas a Char mutant protein deleted of the farnesylation motif CSIM (HA-CharCSIM) is mostly present in the cytoplasm and the nucleoplasm, although traces are detected at the NE (B). (C) Western blot showing the different migration on SDS-PAGE of HA-Char and HA-CharCSIM from the lysate of Drosophila S2 cells in the absence or presence of 10 to 40 µM of the farnesyl-transferase inhibitor FTI-277. The arrow indicates the position of the fast migrating, non-farnesylated fraction of Char (lower band). (D) Injection of FTI-277 60 minutes prior to cellularisation causes a `char-like' phenotype: the nuclei round up and fall from the cortex. The inset shows a detailed view of the boxed area. The nuclei (in white) are not properly anchored apically (arrows). (E) Confocal section from the top showing the nuclear morphology and position with Hoechst (green) and Lamin (red). z-stack projections are shown at the top and to the right showing the abnormal positions of the nuclei viewed from the side. (F) In FTI-injected embryos during cellularisation (right), Char is no longer present at the NE compared with control water-injected embryos (left), whereas Lamin localisation is not affected yet.

View larger version (121K): [in a new window]   Fig. 9. Char is localised at the inner nuclear membrane. (A,B) Localisation of Char (red) and Lamin (green) in the NE of embryos viewed in sagittal sections (A) and from the top (B). (C,D) WGA (green) and Char (red) localisation at the NE of embryos viewed from the top (C-C'') and in sagittal sections (D,D'). C shows a detail of C' and D' a detail of D. Scale bars: 5 µm, except in C,D' (300 nm). (E-H) Immunogold localisation of HA-Char (black arrows) in an early embryo. The nucleoplasm (N) and cytoplasm (C) of three different cells are indicated and have very different electron density. The white arrows indicate contacting cell surfaces. HA-Char is concentrated at the NE. High magnification views of representative localisation at the NE are shown in F, where the white line defines the position of the NE. (G) Representative localisation of Lamin. (H) Quantification of the localisation of HA-Char and Lamin. We positioned the NE at the boundary between the nucleoplasm and cytoplasm (white lines in F and G show examples) and determined the localisation of gold particles (15 nm) at the boundary, or the inner (in) or outer (out) side of the NE. Lamin and HA-Char are distributed similarly, and are particularly biased towards the inner side of the NE. (I,J) S2 cells stained with Char (green), Lamin (red), Hoechst (blue) and Phalloidin (white) to mark F-actin at the cell cortex after permeabilisation with Triton (I) or digitonin (J). Char staining is absent from the NE when the plasma membrane but not the NE is permeabilised (with digitonin).