The process of red blood cell (RBC) production is named erythropoiesis. Erythroblasts are precursor cells of RBCs. In mammals, during their maturation process towards RBCs, erythroblasts lose their nuclei (enucleation). While studies identified regulators of the three stages of enucleation (nuclear polarization, extrusion, and detachment from the nascent reticulocyte), its detailed mechanisms are still unknown, especially in the final stage associated with reticulocytes passing through the narrow bone marrow and splenic inter-endothelial slits to reach the bloodstream. It is nowadays known that mechanical forces act in the enucleation process, but their role in not understood yet, essentially because we lack experimental approaches allowing to replicate in vitro the environmental conditions of the reticulocyte in the bone marrow (or spleen). We propose two hypotheses on these mechanical forces: 1) The bone marrow is a highly crowded environment in which erythroblasts collide with –and are squeezed by– their numerous neighbors, this external stress induces a mechano-sensitive response of the erythroblasts which in turn generate internal forces that drive nucleus expulsion; 2) When nascent reticulocytes pass through the inter-endothelial slits, they are subjected to other mechanical forces, generated both by the extreme confinement in the narrow slits and by the blood flow on the other side of the slits. To quantitatively elucidate the physical mechanisms at play in enucleation, our consortium proposes an interdisciplinary approach combining a physics component based on unique microfluidic device mimicking the bone marrow inter-endothelial slits, a cell biology component by comparing normal erythroblasts and erythroblasts deficient in mechanosensitivity actors, and a computational component to simulate the internal/external forces applied on the cell and its nucleus.

The intricate 3D structures of multicellular organisms emerge through genetically-encoded or self-organized spatio-temporal patterns of mechanical stress. Cell-scale maps of gene expression during embryogenesis are now available, but connecting these to mechanical design principles that govern the emergence of embryonic shape is hampered by our inability to measure mechanical stresses at single-cell resolution, across embryos, over time. In this project, we make use of a particularly tractable system, the ascidian embryo, and propose to construct a single-cell mechanical atlas, in 3D and in physical units, through gastrulation and up to the process of neurulation. We will then use this atlas to explore the regulatory logic of stress generation and its robustness to embryological and environmental perturbations. Aim 1: We will develop two complementary modeling approaches. We will first build an inverse computational approach, grounded in a new physical theory of multicellular aggregates, to measure 3D force patterns from advanced light-sheet-based imaging data and biophysical measurements of material parameters. We will also develop a novel 3D generalized vertex model, which will be used in the subsequent 2 aims to explore the mechanical design, modularity and robustness of ascidian embryonic development. Aim 2: We will combine this theory with biophysical measurements on live embryos to construct a dynamic atlas of mechanical forces in real units up to the neurula stages. Through the imaging of the dynamic patterns of myosin II activation and actin network organization embryos, we will decompose the inferred forces into their passive and active components. Aim 3: We will combine experimental and computational approaches to analyze the modularity of the mechanical design and the robustness of ascidian morphogenesis to genetic or environmental fluctuations. This project brings together 4 teams with complementary expertise in cell and developmental biology, theoretical and experimental physics, and computation, who will perform work that none can individually accomplish. It will push the frontiers of the theory of living matter and analyze the modularity and robustness of a particularly stereotyped and evolutionary conserved embryogenetic program. The mechanical atlas and computational models will be publicly available through the MorphoNet morphodynamic browser.