General research interests

Living tissues have a remarkable plasticity illustrated by their capacity to regenerate and develop normal organs despite dramatic perturbations. This is based on the amazing capacity of every single cell to adapt its behavior to local information (e.g.: paracrine signal, contact dependant signaling, adhesive forces) and tissue scale information (e.g: tissue size, tissue density).  Our laboratory is generally interested by the plasticity of cell behavior and more specifically by the adjustment and regulation of cell death in epitheliums.  Epitheliums are two dimensional layers of adhesive cells that form barriers in the organism. Our group is currently focusing on two aspects of the regulation of cell death in epithelial context :

1. The influence of mechanical forces and cell shape on death induction in physiological and pathological contexts

2. The orchestration of epithelial cell clearance by effector caspases and the commitment to apoptosis

 

We use the fruitfly (Drosophila melanogaster) to tackle these questions through a combination of genetics, live imaging, quantitative image analysis, optogenetics and biophysics.

Competition for Space

Despite our deep knowledge of the pathways regulating cell proliferation, growth and survival, we still do not understand how single cells can match their behavior to properties of the entire tissue (including its size and shape).  Mechanical forces can convey tissue scale information (tissue size, density) to single cells. While the effects of mechanical forces on cell proliferation have been widely studied, their influence on cell survival have been poorly studied in vivo. Recently, it was shown that a local increase of cell density in an epithelium can induce cell extrusion and elimination[1][2][3]. We recently showed that caspase activation is necessary for cell elimination in the pupal notum (a single layer epithelium) [3]. We are currently trying to identify new regulators of  apoptosis induction which are sensitive to cell deformation and tissue crowding. 

Cell elimination (purple) in the developing pupal notum (E-cad::GFP)

Timelaps of a Drosophila pupal notum showing  local tissue compaction (right side, warm colours) extracted by PIV (left orange arrows) and cell delamination events (green spots). 

We are also testing how spatial constrains could affect cell survival in various developmental contexts and test their contribution to morphogenesis and tissue size regulation. We use a combination of live imaging (particule image velocimetry, tissue segmentation and cell tracking) and clonal analysis to understand how this could help to fine tune the proportion of dying cells in growing tissues, during programmed tissue clearance and during tissue homeostasis.

Finally, we are trying to understand how crowding induced death can contribute to the competitive interactions between different cell types. Cell competition is a process inducing the elimination of slow proliferating cells by faster proliferative cells through apoptosis (reviewed in [4]). Cell competition is a conserved mechanism required for the correction of developmental errors, to fine tune tissue size and could contribute to tumor expansion through the elimination and replacement of neighbouring healthy cells by pretumoral cells. We and others [5][3] have recently shown that fast growing clones resistant for apoptosis can promote WT cell eliminations though their compaction and induction of apoptosis. We use a combination of genetics, live imaging, laser nanodissection and theory to understand how spatial constrains can contribute to cell competition and how confrontation of two cell populations can lead to cell compaction and elimination.

Timelaps in the Drosophila pupal notum showing the compaction and elimination of WT cells near a fast growing clone (UAS-RasV12 ,purple)

Commitment to apoptosis and the orchestration of cell clearance

Despite the detailed characterisation of the molecular players of apoptosis, its orchestration and fine regulation in multicellular contexts is not well understood. While apoptosis is often considered as a simple binary process, there are nowadays many evidences showing that apoptosis is a complex decision making process, where cells can undergo transient caspase activation without proceeding to death. This is in agreement with the multiple non-apoptotic functions of caspases and the architecture of the apoptotic pathway which includes several negative feedback loops. This complexity increases further in epithelial cells, where cell clearance requires orchestration of successive remodelling events necessary to extrude the cell from the epithelial layer without impairing tissue barrier function.

 

 We recently showed that cell delamination in the Drosophila pupal notum always requires effector caspases activity and that caspase activation precedes delamination[3]. Moreover, we measured very various lagtimes between the onset of caspase activation and delamination (30 min to several hours) and also observed transient caspase activation which do not lead to cell delamination. These observations suggest that the commitment in delamination and apoptosis is a complex decision making process. Moreover, our results suggest that yet uncharacterized substrate(s) of effector caspases are required for delamination and that the same molecular player (the effector caspase) orchestrates all the remodelling events of apoptosis (such as delamination, cytoplasmic compaction, DNA condensation, nucleus and cell fragmentation). We are currently studying the orchestration of cell death and extrusion in Drosophila epithelium in order to :

1) find new caspase substrates required for cell delamination 

2) dissect the mechanism that regulates the successive remodelling events 

3) understand the cell decision making process leading to irreversible apoptosis

4) study the consequences of impaired cell delamination for epithelial homeostasis.

Examples of transient caspase activation visualised in the Drosophila pupal notum with Apoliner (GFP relocation to the nucleus, green).

Visualisation of caspase activity with a FRET marker (Scat3) in the Drosophila pupal notum. A diminution of FRET (right side, cold colours) indicates activation of caspases.

For this, we combine quantitative live imaging, proteomics, genetic, optogenetic, cell biology and theoretical approaches to build a predictive framework that will help to understand the orchestration of apoptosis in a living epithelium.

References and relevant litterature :

1. Eisenhoffer, G. T. et al. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 484, 546-549, doi:10.1038/nature10999 (2012).

2. Marinari, E. et al. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. Nature 484, 542-545, doi:10.1038/nature10984 (2012).

3. Levayer, R., Dupont, C. & Moreno, E. Tissue Crowding Induces Caspase-Dependent Competition for Space. Current biology : CB 26, 670-677, doi:10.1016/j.cub.2015.12.072 (2016).

4. Levayer, R. & Moreno, E. Mechanisms of cell competition: themes and variations. The Journal of cell biology 200, 689-698, doi:10.1083/jcb.201301051 (2013).

5. Wagstaff, L. et al. Mechanical cell competition kills cells via induction of lethal p53 levels. Nature communications 7, 11373, doi:10.1038/ncomms11373 (2016).

© 2016 by Romain Levayer

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