Studying cytokinesis in Drosophila epithelial tissues.

Epithelial tissue cohesiveness is ensured through cell-cell junctions that maintain both adhesion and mechanical coupling between neighboring cells. During development, epithelial tissues undergo intensive cell proliferation. Cell division, and particularly cytokinesis, is coupled to the formation of new adhesive contacts, thereby preserving tissue integrity and propagating cell polarity. Remarkably, the geometry of the new interfaces is determined by the combined action of the dividing cell and its neighbors. To further understand the interplay between the dividing cell and its neighbors, as well as the role of cell division for tissue morphogenesis, it is important to analyze cytokinesis in vivo. Here we present methods to perform live imaging of cell division in Drosophila epithelial tissues and discuss some aspects of image processing and analysis.

Comparative study of non-invasive force and stress inference methods in tissue.

In the course of animal development, the shape of tissue emerges in part from mechanical and biochemical interactions between cells. Measuring stress in tissue is essential for studying morphogenesis and its physical constraints. For that purpose, a possible new approach is force inference (up to a single prefactor) from cell shapes and connectivity. It is non-invasive and can provide space-time maps of stress in a whole tissue, unlike existing methods. To validate this approach, three force-inference methods, which differ in their approach of treating indefiniteness in an inverse problem between cell shapes and forces, were compared. Tests using two artificial and two experimental data sets consistently indicate that our Bayesian force inference, by which cell-junction tensions and cell pressures are simultaneously estimated, performs best in terms of accuracy and robustness. Moreover, by measuring the stress anisotropy and relaxation, we cross-validated the force inference and the global annular ablation of tissue, each of which relies on different prefactors. A practical choice of force-inference methods in different systems of interest is discussed.

Subcellular control of Rac-GTPase signalling by magnetogenetic manipulation inside living cells.

Many cell functions rely on the coordinated activity of signalling pathways at a subcellular scale. However, there are few tools capable of probing and perturbing signalling networks with a spatial resolution matching the intracellular dimensions of their activity patterns. Here we present a generic magnetogenetic approach based on the self-assembly of signalling complexes on the surface of functionalized magnetic nanoparticles inside living cells. The nanoparticles act as nanoscopic hot spots that can be displaced by magnetic forces and trigger signal transduction pathways that bring about a cell response. We applied this strategy to Rho-GTPases, a set of molecular switches known to regulate cell morphology via complex spatiotemporal patterns of activity. We demonstrate that the nanoparticle-mediated activation of signalling pathways leads to local remodelling of the actin cytoskeleton and to morphological changes.

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Lineage diversity in the Drosophila nervous system.

The detailed descriptions of cellular lineages in the Drosophila nervous system have provided the foundations for an in-depth genetic analysis of the mechanisms that regulate fate decisions at every cell cycle.

Lineage, cell polarity and inscuteable function in the peripheral nervous system of the Drosophila embryo.

The stereotyped pattern of the Drosophila embryonic peripheral nervous system (PNS) makes it an ideal system to use to identify mutations affecting cell polarity during asymmetric cell division. However, the characterisation of such mutations requires a detailed description of the polarity of the asymmetric divisions in the sensory organ lineages. We describe the pattern of cell divisions generating the vp1-vp4a mono-innervated external sense (es) organs. Each sensory organ precursor (SOP) cell follows a series of four asymmetric cell divisions that generate the four es organs cells (the socket, shaft, sheath cells and the es neurone) together with one multidendritic (md) neurone. This lineage is distinct from any of the previously proposed es lineages. Strikingly, the stereotyped pattern of cell divisions in this lineage is identical to those described for the embryonic chordotonal organ lineage and for the adult thoracic bristle lineage. Our analysis reveals that the vp2-vp4a SOP cells divide with a planar polarity to generate a dorsal pIIa cell and a ventral pIIb cell. The pIIb cell next divides with an apical-basal polarity to generate a basal daughter cell that differentiates as an md neurone. We found that Inscuteable specifically accumulated at the apical pole of the dividing pIIb cell and regulated the polarity of the pIIb division. This study establishes for the first time the function of Inscuteable in the PNS, and provides the basis for studying the mechanisms controlling planar and apical-basal cell polarities in the embryonic sensory organ lineages.

Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division.

Cell-fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell divisions. In the Drosophila pupa, the pI sense organ precursor cell is polarized along the anterior-posterior axis of the fly and divides asymmetrically to generate a posterior pIIa cell and an anterior pIIb cell. The anterior pIIb cell specifically inherits the determinant Numb and the adaptor protein Partner of Numb (Pon). By labelling both the Pon crescent and the microtubules in living pupae, we show that determinants localize at the anterior cortex before mitotic-spindle formation, and that the spindle forms with random orientation and rotates to line up with the Pon crescent. By imaging living frizzled (fz) mutant pupae we show that Fz regulates the orientation of the polarity axis of pI, the initiation of spindle rotation and the unequal partitioning of determinants. We conclude that Fz participates in establishing the polarity of pI.

Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan.

Hedgehog (Hh) molecules play critical roles during development as a morphogen, and therefore their distribution must be regulated. Hh proteins undergo several modifications that tether them to the membrane. We have previously identified tout velu (ttv), a homolog of the mammalian EXT tumor suppressor gene family, as a gene required for movement of Hh. In this paper, we present in vivo evidence that ttv is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, suggesting that HSPGs control Hh distribution. In contrast to mutants in other HSPG biosynthesis genes, the activity of the HSPG-dependent FGF and Wingless signaling pathways are not affected in ttv mutants. This demonstrates an unexpected level of specificity in the regulation of the distribution of extracellular signals by HSPGs.

Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell.

The bristle mechanosensory organs of the adult fly are composed of four different cells that originate from a single precursor cell, pI, via two rounds of asymmetric cell division. Here, we have examined the pattern of cell divisions in this lineage by time-lapse confocal microscopy using GFP imaging and by immunostaining analysis. pI divided within the plane of the epithelium and along the anteroposterior axis to give rise to an anterior cell, pIIb, and a posterior cell, pIIa. pIIb divided prior to pIIa to generate a small subepithelial cell and a larger daughter cell, named pIIIb. This unequal division, oriented perpendicularly to the epithelium plane, has not been described previously. pIIa divided after pIIb, within the plane of the epithelium and along the AP axis, to produce a posterior socket cell and an anterior shaft cell. Then pIIIb divided perpendicularly to the epithelium plane to generate a basal neurone and an apical sheath cell. The small subepithelial pIIb daughter cell was identified as a sense organ glial cell: it expressed glial cell missing, a selector gene for the glial fate and migrated away from the sensory cluster along extending axons. We propose that mechanosensory organ glial cells, the origin of which was until now unknown, are generated by the asymmetric division of pIIb cells. Both Numb and Prospero segregated specifically into the basal glial and neuronal cells during the pIIb and pIIIb divisions, respectively. This revised description of the sense organ lineage provides the basis for future studies on how polarity and fate are regulated in asymmetrically dividing cells.

I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila.

As a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms.

Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion.

Hedgehog (Hh) proteins act through both short-range and long-range signalling to pattern tissues during invertebrate and vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects are not understood. Here we identify a new Drosophila gene, named tout-velu, that is required for diffusion of Hedgehog. Characterization of tout-velu shows that it encodes an integral membrane protein that belongs to the EXT gene family. Members of this family are involved in the human multiple exostoses syndrome, which affects bone morphogenesis. Our results, together with the previous characterization of the role of Indian Hedgehog in bone morphogenesis, lead us to propose that the multiple exostoses syndrome is associated with abnormal diffusion of Hedgehog proteins. These results show the existence of a new conserved mechanism required for diffusion of Hedgehog.

Computer assisted retraining of attentional impairments in patients with multiple sclerosis.

OBJECTIVE: To evaluate the efficacy of a computer based retraining of specific impairments of four different attentional domains in patients with multiple sclerosis. METHODS: Twenty two outpatients with multiple sclerosis received consecutively a specific training comprising 12 sessions in each of the two most impaired attention functions. The baseline of attentional deficits, the performance after each training period, and the course of performance in the next nine weeks was assessed by a computerised attention test battery. Additionally, the impact of the training on daily functioning was evaluated with a self rating inventory. RESULTS: Subgroups of patients with multiple sclerosis showing different patterns of attentional impairment could be separated. Significant improvements of performance could almost exclusively be achieved by the specific training programmes. The increase of performance remained stable for at least nine weeks. For quality of life patients reported less attention related problems in everyday situations. CONCLUSIONS: In patients with multiple sclerosis it seems worthwhile to assess attentional functions in detail and to train specific attention impairments selectively.

Neither the homeodomain nor the activation domain of Bicoid is specifically required for its down-regulation by the Torso receptor tyrosine kinase cascade.

Bicoid (Bcd) is a maternal morphogen responsible for patterning the head and thorax of the Drosophila embryo. Correct specification of head structure, however, requires the activity of the Torso receptor tyrosine kinase cascade, which also represses expression of Bcd targets at the most anterior tip of the embryo. Here, we investigate the role of both the homeodomain (HD) and the activation domain of Bcd in the anterior repression of its targets. When a Bcd mutant protein whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein, whose activation domain has been replaced by a generic activation domain, indicates that the activation domain of Bcd is also not specifically required for its down-regulation by Torso. We propose that Torso does not affect the ability of Bcd to bind DNA, but instead directs modification of Bcd or of a potential Bcd co-factor, which renders the Bcd protein unable to activate transcription.

[Multiple sclerosis: therapy with recombinant beta-1b interferon: initial results with 30 multiple sclerosis patients in northwest Switzerland]. / Multiple Sklerose: Therapie mit rekombinantem interferon beta-1b: Erste Erfahrungen mit 30 Multiple-Sklerose-Patienten in der Nordwestschweiz.

Recombinant interferon beta-1b has been registered with the Swiss health authorities since August 1995. Due to a special arrangement with health insurances it has been possible to prescribe this medication since spring 1995. We report on our experience with the first 30 consecutively treated multiple sclerosis patients. Indication, adverse event profile and clinical response to treatment are described. The most common side effects were local injection site reactions (63%), influenza-like symptoms (50%) and fatigue (33%). As compared to the prestudy period we observed a 49% reduction in the exacerbation rate. Compliance was excellent, possibly due to strict selection and extensive information about possible effects and side effects.