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1.
STAR Protoc ; 5(1): 102851, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38354083

ABSTRACT

Applying mechanical forces to tissues helps to understand morphogenesis and homeostasis. Additionally, recording the dynamics of living tissues under mechanical constraints is needed to explore tissue biomechanics. Here, we present a protocol to 3D-print a StretchCo device and use it to apply uniaxial mechanical stress on the Drosophila pupal dorsal thorax epithelium. We describe steps for 3D printing, polydimethylsiloxane (PDMS) strip cutting, and glue preparation. We detail procedures for PDMS strip mounting, tissue compaction, and live imaging upon force application. For additional details on the use and execution of this protocol, please refer to Cachoux et al. (2023)1 from which the StretchCo machine has been derived.


Subject(s)
Dimethylpolysiloxanes , Drosophila , Animals , Epithelium , Morphogenesis , Biomechanical Phenomena , Stress, Mechanical
2.
Curr Biol ; 33(22): 4807-4826.e6, 2023 11 20.
Article in English | MEDLINE | ID: mdl-37827152

ABSTRACT

Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, we know little about whether and how cell geometry controls apoptosis commitment in developing tissues. Here, we combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. We found that early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. We showed that these global and local geometric characteristics are sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, we established that the coupling between these two geometric features and apoptotic cells is dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, our work provides important insights into the spatial regulation of cell death in tissues and improves our understanding of the mechanisms that control cell number and tissue size.


Subject(s)
Apoptosis , Drosophila , Animals , Epithelium/physiology , Drosophila/genetics , Apoptosis/physiology , Cell Death , Mitosis , Epithelial Cells
3.
Nat Commun ; 10(1): 2951, 2019 07 04.
Article in English | MEDLINE | ID: mdl-31273212

ABSTRACT

Epithelial-mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. Here, we show that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila. When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.


Subject(s)
Drosophila melanogaster/embryology , Epithelial-Mesenchymal Transition , Epithelium/embryology , Morphogenesis , Animals , Cell Polarity , Computer Simulation , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Epithelium/metabolism , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Models, Molecular , Myosin Type II/metabolism
4.
Development ; 144(20): 3840-3846, 2017 10 15.
Article in English | MEDLINE | ID: mdl-28870988

ABSTRACT

Far from being passive, apoptotic cells influence their environment. For example, they promote tissue folding, myoblast fusion and modulate tumor growth. Understanding the role of apoptotic cells necessitates their efficient tracking within living tissues, a task that is currently challenging. In order to easily spot apoptotic cells in developing Drosophila tissues, we generated a series of fly lines expressing different fluorescent sensors of caspase activity. We show that three of these reporters (GFP-, Cerulean- and Venus-derived molecules) are detected specifically in apoptotic cells and throughout the whole process of programmed cell death. These reporters allow the specific visualization of apoptotic cells directly within living tissues, without any post-acquisition processing. They overcome the limitations of other apoptosis detection methods developed so far and, notably, they can be combined with any kind of fluorophore.


Subject(s)
Apoptosis , Drosophila melanogaster/genetics , Microscopy, Fluorescence/methods , Animals , Caspases/genetics , Cloning, Molecular , Fluorescent Dyes , Green Fluorescent Proteins/chemistry
5.
Mech Dev ; 144(Pt A): 33-42, 2017 04.
Article in English | MEDLINE | ID: mdl-27771340

ABSTRACT

It is now well established that apoptosis is induced in response to mechanical strain. Indeed, increasing compressive forces induces apoptosis in confined spheroids of tumour cells, whereas releasing stress reduces apoptosis in spheroids cultivated in free suspension (Cheng et al., 2009). Apoptosis can also be induced by applying a 100 to 250MPa pressure, as shown in different cultured cells (for review, see (Frey et al., 2008)). During epithelium development, the pressure caused by a fast-growing clone can trigger apoptosis at the vicinity of the clone, mediating mechanical cell competition (Levayer et al., 2016). While the effect of strain has long been known for its role in apoptosis induction, the reciprocal mechanism has only recently been highlighted. First demonstrated at the cellular level, the effect of an apoptotic cell on its direct neighbours has been analysed in different kinds of monolayer epithelium (Gu et al., 2011; Rosenblatt et al., 2001; Kuipers et al., 2014; Lubkov & Bar-Sagi, 2014). More recently, the concept of a broader impact of apoptotic cell behaviours on tissue mechanical strain has emerged from the characterisation of tissue remodelling during Drosophila development (Toyama et al., 2008; Monier et al., 2015). In the present review, we summarize our current knowledge on the mechanical impact of apoptosis during tissue remodelling.


Subject(s)
Apoptosis/genetics , Drosophila melanogaster/growth & development , Epithelial Cells/cytology , Gene Expression Regulation, Developmental , Morphogenesis/genetics , Abdomen/growth & development , Animals , Cell Division , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Epithelial Cells/metabolism , Extracellular Matrix/metabolism , Larva/genetics , Larva/growth & development , Larva/metabolism , Models, Biological , Pupa/genetics , Pupa/growth & development , Pupa/metabolism , Stress, Mechanical
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