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Role of Epithelial Apoptotic Force in Morphogenesis

Periodic Reporting for period 4 - EPAF (Role of Epithelial Apoptotic Force in Morphogenesis)

Reporting period: 2020-03-01 to 2021-02-28

Apoptosis was first identified as a way to get rid of abnormal cells. It came as a surprise to discover later on that apoptosis played an important role in morphogenesis, revealing the necessity to produce cells in excess before eliminating them. It was then proposed that the cell death at a precise location was sufficient to shape a tissue, thus reducing apoptosis to the simple elimination of cells.

Our project was to understand how apoptotic cells were influencing tissue morphogenesis and to identify, beyond cell elimination, how apoptosis is influencing the surrounding cells. We could identify that apoptotic cells, far from being passively eliminated, are actively pulling of their neighbors to make them change their shape and remodel the tissue. Since apoptosis is a process that is reactivated in cancer therapies, this discovery could have important repercussion on cancer treatments. We further show that the mechanical impact of apoptotic cells is not restricted to Drosophila tissue but mainly conserved in vertebrate as shown by the dynamics of apoptotic cells in the folding neural tube of chicken and quail embryos.

We also focused cell leaving epithelial sheet to migrate and integrate another tissue (epithelial-mesenchymal transition). We discovered that these epithelial cells, before leaving the epithelium, generate a force that increase tissue tension and by this way modify its shape, a behavior that is strikingly similar to the one observed in apoptotic cells. Since the formation of metastasis is though to rely on EMT, this work could reveal an important aspect of tissue mechanics at the onset of metastasis formation that could have an impact on potential treatment.
We initially focused on apoptotic cell dynamics. Using Drosophila developing leg as a model system to study apoptosis-dependent epithelium folding, we reveal that apoptotic cells, far from been eliminate without consequence on their surroundings, generate a traction force within tissues that drive tissue remodelling and folding (Monier et al, Nature, 2015). While apoptosis was known to contribute to tissue shape, it was generally assumed that it was a consequence of local elimination of cells that were no longer required. Our work identifies apoptotic cells as inductor of mechanical signals and brought apoptotic cells into the biomechanics field.

We next dissected the cellular mechanisms governing the generation of the apoptotic force. We found that it relies on the formation of a dynamic actomyosin contractile tether connecting the apical surface to a basally anchored nucleus, which constitutes an essential component of the force-producing machinery. This work reveals that the nucleus, in addition to its role in genome protection, actively participates in mechanical force production and connects the contractile actomyosin cytoskeleton to basal junctions (Ambrosini, Rayer et al, Developmental Cell, 2019).

Having discovered that apoptotic cells were generating forces, pulling on their neighbours and influencing tissue remodeling, we asked if this active role was conserved in higher organisms. Using chicken and quail embryos, we found that apoptosis mainly occurs shortly before or at the time of dorsal bending of the neural tube and is required for this bending. We further showed that apoptotic cells generate an apico-basal force before being extruded from the neuro-epithelium through the formation of a tensile acto-myosin cable that connect the nucleus to the apical surface of the cell. At the subcellular level, this force deforms the nucleus and precipitates its fragmentation. All together these data reveal that apoptotic cells influence their surroundings mechanically and strongly suggest their active contribution to neural tube bending. Furthermore, it suggests that the same force plays a role in tissue morphogenesis in fly and chicken embryos (Roellig et al, under review in Developmental Cell).

We further identified a network of genes orchestrating apoptosis dependent morphogenesis through a genetic screen. From around 1600 genes screened, we selected around 120 candidates required for leg morphogenesis. The rational was that these genes were potentially involved either in apoptosis regulation, apoptotic force generation and/or transmission, or apoptotic cell elimination (Barbaste, Schott et al, manuscript in preparation).

A new project in the team, regarding the amazing reproducibility of tissue shape from one individual to another originated from this screen. We could highlight a new role for mechanical forces in shape robustness. Indeed, in order to acquire a stereotyped 3D structure, a mechanical fence is built between different portions of the remodeling tissue ensuring the insensitivity to perturbations in the nearby environment. This mechanical fence relies on Arp2/3 complex, which drives Myosin II planar polarity and favors directional force transmission (Martin et al, Developmental Cell, 2021).

In parallel, we asked if the force exerted during apoptotic cell delamination was specific to apoptotic cells or share with other type of delaminating cells. To tackle this question we analyzed the dynamic of cell delamination during epithelial-mesenchymal transition (EMT) and combined state-of-the-art live imaging techniques, together with robust biophysical modelling.
EMT is an essential process in physiological and pathological contexts. While EMT is often associated with tissue invagination during development, the impact of EMT on tissue remodelling remains unexplored. We show that at the initiation of the EMT process, cells produce a force orthogonal to the surface of the epithelium that constitutes an essential driving force for tissue invagination. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if orthogonal forces are impaired, indicating that they constitute essential driving forces in the folding process. These data will help understand the initiation of metastasis (Gracia et al, Nature Communication, 2019).
Apoptosis or programmed cell death was mainly considered as a clean way to get rid of cells no longer required or potentially dangerous. The active participation of a dying cell to tissue remodelling has never been considered before this work. Similarly, the epithelio-mesenchymal transition was mainly view as a cell autonomous process allowing a cell to acquire migratory properties, but the impact of this delamination on the surrounding epithelial cells was never envisioned. Indeed, based on studies done in fixed sample, these two dynamic cellular processes were though to start with cell detachment from its neighbors, thus excluding the potential impact of the delaminating cell or dying cell on its neighbors. Our live imaging analysis, focusing on initial stage of apoptosis and EMT revealed on the contrary that these cells conserved strong adhesion until the last moment of their extrusion.

Thus, this work has brought new insights into these two fundamental cellular processes and open new avenues in the field of mechano-biology and will be of interest to an expanding community.
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