Tag: RAUZI

Master2 internship: interdisciplinary project at the interface between live microscopy, developmental biology, biophysics and image analysis
Master2 internship: interdisciplinary project at the interface between live microscopy, developmental biology, biophysics and image analysis
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Master2 internship: interdisciplinary project at the interface between live microscopy, developmental biology, biophysics and image analysis

Studying the mechanisms and mechaincs of epithelial tube formation in the sea urchin P. lividus embryo

Epithelial folding is a vital process during embryo development. Defects in folding can impair neurulation or gastrulation leading to major birth defects (e.g., spina bifida) or death. In mature tissues, folding is also pathologically relevant: tissues can for instance buckle before cancer invasion. Understanding the cell mechanisms and mechanics of tissue folding is thus of major importance. In the lab we use the Paracentrotus lividus sea urchin embryo as a model system and focus on the process of tissue folding during sea urchin gastrulation that leads to the formation of the gut of the sea urchin larva. By implementing 4D multi-view light sheet microscopy, micro-indentation, micro-pipette aspiration, infra-red femtosecond ablation to perturb the cytoskeleton and molecular inhibition, this work will shine new light on the cell mechanisms of epithelial folding at both the molecular and mechanical level.

Novelty of the project. The sea urchin is historically among the first model systems used to study embryo development. The sea urchin gastrula combines a number of outstanding features making this model system a unique opportunity to study the mechanisms and mechanics of tissue folding: (1) the sea urchin gastrula is a 1000 cells spherical monolayer epithelium: a very simple thus appealing model for experimentation and modelling; (2) tissue folding can be nicely imaged since the sea urchin gastrula is transparent; (3) the signaling factors controlling sea urchin vegetal plate folding are known and can be knocked down to dissect their function; (4) the gastrula is a mechanically accessible tissue: it can be partitioned, cells can be transplanted, micro-indentation and micro-pipetting techniques can be applied to measure tissue mechanical properties on both tissue apical and basal side. The sea urchin gastrula is thus a perfect playground for biologists and biophysicists focus in understanding the mechanisms and mechanics controlling and driving tissue morphogenesis.

The project that we propose is a breakthrough in the study of epithelial folding using the P. lividus model organism. We are now able, by using the sea urchin gastrula, to extract 3D quantitative cell morphology information with sub-cellular resolution. We devised a fluorescent live imaging and processing pipeline that allows to (1) image reliably the gastrulation of the sea urchin embryo in 4D with 200 nm isotropic resolution at a frequency of 1 image/min, (2) segment all 1000 cells constituting the gastrula in 3D and (3) track the 3D segmented cells over time. In this way we can extract precise morphological and cinematic information and use them to rule out or advance hypotheses supporting potential mechanisms driving vegetal plate folding. Hypothesis are tested by using advanced 4D image processing and analysis, mechano- techniques (e.g., in-plane micro-indentation, infra-red (IR) femtosecond (fs) laser dissection coupled to multi- view light-sheet microscopy) and back tested theoretically with mathematical modelling.

Seeking a talanted and very motivated candidate to work on 4D live imaging, molecular biology and biophysics. Send a CV, a motivation letter, master scores/ranking and reference letters to matteo.rauzi@univ-cotedazur.fr

Visit the RAUZI LAB web page: http://ibv.unice.fr/research-team/rauzi/

Interdisciplinary Doctoral Project
Interdisciplinary Doctoral Project
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Interdisciplinary Doctoral project in the RAUZI lab (University Côte d’Azur, IBV, Nice) and in the ETIENNE lab (Univ Grenoble Alpes, LIPHY, Grenoble), at the interface between computational physics and biology

Studying the mechanisms and mechanics driving tissue folding

Mophogenesis builds living shapes. A key morphogenetic transformation that shapes tissues and organs is epithelial invagination: a tissue bends and it is eventually internalized transforming the physiological topology of the system. The invagination of epithelial tissues is a vital transformation during embryo development since it is pivotal during embryo gastrulation and neurulation. While much is known of the mechanisms and mechanics driving epithelial flattening (first phase) and bending (second phase), how a tissue is eventually internalized (third phase) is still poorly understood. To tackle this, we propose to use the Drosophila embryo that provides the most advanced genetic tools and study the process of mesoderm internalization. On the computational physics side, we will develop a formal physical framework that can theoretically reproduce morphogenetic processes and predict features of the system that are then back tested experimentally. More specifically, we will design a mechanical model based on active viscoelastic shells and use numerical simulations based on existing tools (e.g., Surface Evolver in 3D) to calculate shell deformations. On the biology side, we will implement multi-view light sheet microscopy coupled to optogenetics and plasma-based laser ablation and image data analysis to characterize and synthetically modulate tissue shape changes to test numerical predictions. The student will be trained on these multiple approaches and techniques to develop an interdisciplinary project focused on uncovering the fundamental principles governing epithelial folding. This knowledge could be used in the future to synthetically build and shape functional organs.The project will be developed in both the Rauzi lab (http://ibv.unice.fr/research-team/rauzi/) and the Etienne lab (http://www-liphy.univ-grenoble-alpes.fr/pagesperso/etienne).

We are seeking a highly motivated and talented candidate to develop this interdisciplinary PhD project. Send a CV, a motivation letter, master scores/ranking and reference letters to matteo.rauzi@univ-cotedazur.fr and jocelyn.etienne@univ-grenoble-alpes.fr

The egg puts on a corset to get the right shape
The egg puts on a corset to get the right shape
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Thanks to an interdisciplinary and exciting collaboration between the iBV and the CBI in Toulouse, the Rauzi and the Wang teams unveiled a Cdc42 dependent mechanical process responsible for tissue elongation in the developing Drosophila egg chamber. By engineering new optogenetic techniques and infra-red laser based surgery, Anna Popkova and others show that a polarized supracellular actomyosin network, working as a molecular corset, generates tissue scale forces directing egg chamber elongation. Finally, this study opens new avenues to better understand how supra-cellular cytoskeletal networks emerge to drive embryo-scale morphogenesis during development. This work was published in Nature Communications.

Understanding the mechanisms that are responsible to shape tissues and organs is an important and exciting challenge in developmental biology. The actomyosin cortex often plays a key role in generating the forces necessary to shape individual cells. Nevertheless, the actomyosin architecture can extend beyond the size of a single cell: cytoskeletal networks emerge at the scale of a tissue. These supra-cellular contractile structures can generate large forces that can rapidly shape epithelia.

In this study, scientists from the Rauzi and the Wang lab use the Drosophila egg chamber as a model system to study the origin and the mechanics of supra-cellular actomyosin networks. Popkova and others show that a supra-cellular array of parallel actin bundles, emerging from interdigitating filopodia, envelopes the follicle tissue. Filopodia radiate in a polarized way from basal stress fibers and extend by penetrating the neighboring cell cortexes. Filopodia can be mechanosensitive and function as anchors between cells. The small GTPase Cdc42 governs the formation of intercellular filopodia and stress fibers in the follicular cells. Thus, a Cdc42-dependent supracellular cytoskeletal network provides a scaffold integrating local oscillatory actomyosin contractions at the tissue scale to drive global polarized forces and tissue elongation.

Inset

Actin filaments assemble into diverse protrusive and contractile networks to generate forces in diverse cellular processes. Stress fibers are contractile higher order cytoskeletal structures composed of actomyosin bundles. Stress fibers play a key role in generating forces along the fiber direction and have important implication in cell adhesion to the extracellular matrix.

Filopodia are dynamic, finger-like plasma membrane protrusions that act as antennae to sense the mechanical and chemical environment. They are often regarded as “sensory organelles”. Filopodia are involved in many biological processes, such as growth cone guidance, cell migration, wound closure, and macrophage-induced cell invasion. These thin membrane protrusions are 60–200 nm in diameter and contain parallel bundles of 10–30 actin filaments held together by actin-binding proteins. 

Laser dissection is a useful tool in developmental biology to probe the mechanical forces from the subcellular to the tissue/embryo scale. During tissue morphogenesis, cells are equipped with actomyosin networks generating forces. In this study, researchers used near-infrared (NIR) femtosecond (fs) pulsed laser surgery to dissect the actomyosin cytoskeleton with subcellular precision.  This technique allows to selectively ablate actomyosin networks while preserving the cell plasma membrane. The resulting recoil of the remaining network, after laser dissection, is imaged and analyzed to deduce local forces responsible for tissue morphogenesis.

Optogenetics allows to control, via light stimulation, protein conformation changes and thus protein activity with spatial and temporal specificity. By using molecular engineering, proteins of interest are fused to photo-activatable proteins than can be expressed in specific cells. Using the optogenetic tool PA-Cdc42 which leads to the expression of a light-activatable Cdc42 protein, researchers were able to precisely determine the role of Cdc42 in follicular cells.

To read more: 

A Cdc42-mediated supracellular network drives polarized forces and Drosophila egg chamber extension.
Popkova A, Stone OJ, Chen L, Qin X, Liu C, Liu J, Belguise K, Montell DJ, Hahn KM, Rauzi M@, Wang X@.
Nat Commun. 2020 Apr 21;11(1):1921. doi: 10.1038/s41467-020-15593-2.

 

Press release : Actualités scientifiques de l’INSB

Movie: Time-lapse of a representative mCD8GFP-expressing egg chamber labelled with MyoII-mCherry. Laser dissection of the supra-cellular actomyosin network was performed along the AP axis of the egg chamber. Scale bar 10 μm.