An important morphogenetic event of mammalian embryogenesis is the formation of a blastocyst with a fluid-filled cavity, blastocoel, and the establishment of three cell types essential for implantation. Morphogenesis of the blastocyst begins with the emergence of multiple nascent cavities, which progressively coalesce to form one cavity segregating the cavity-facing primitive endoderm from the epiblast within the inner cell mass. While cell-to-cell gene expression heterogeneity is well characterised during this lineage specification, little is known about the physical principles governing self-organized blastocyst morphogenesis and patterning. In particular, changes in fluid pressure, cell shape and polarity during blastocyst formation remain uncharacterized. In this project, I will study the roles of fluid cavities in coordinating tissue mechanics, polarity and lineage specification. I will establish a novel micropressure technique to quantify the growth of luminal pressure during blastocyst development. Combining micropipette aspiration with high-resolution live-embryo imaging, I will characterize the impact of fluid pressure on trophectoderm fate specification through dynamic changes in cell shape and adhesion, and cytoskeletal remodeling. To assess the impact of fluid pressure on inner cell mass, I will study if cavity expansion induces apical polarisation and enhances primitive endoderm differentiation in cavity-facing cells. Combining laser ablation with light-sheet microscopy, we will build a spatio-temporal map of intercellular forces in vivo during blastocyst development. We will further manipulate the cavity size to study if fluid pressure is functionally required and sufficient for driving lineage segregation. This interdisciplinary and quantitative study will establish the novel role of fluid cavities and elucidate their interplay with biochemical signaling within the multi-cellular self-organization process.

Meiotic divisions in the oocyte have been shown to be surprisingly error-prone compared to the reliable chromosome segregation that takes place in dividing somatic cells. The high frequency of chromosomal abnormalities found in pre-implantation embryos in mammals coupled with the fact that the first divisions of the embryo resembles meiosis in several aspects suggests that the mechanisms controlling chromosome segregation, most importantly the spindle assembly checkpoint (SAC), only become fully operational after the transition from meiosis to mitosis during early development. Despite the importance for early embryonic development, the sensitivity of mammalian embryos to light and the absence of a functional reporter of the SAC in mice have precluded real-time imaging of chromosome segregation and its control in the first embryonic divisions. Recent advances in light sheet microscopy in the Ellenberg lab now allow me to study chromosome segregation. In addition, in collaboration with the EMBL Transgenic Facility I will be able to rapidly generate the first SAC reporter mice that will permit me to test the checkpoint functionality up to the blastocyst stage. Taking advantage of this unique opportunity to combine new technology with a novel reporter animal model, I plan to study how the SAC changes from meiosis to the first embryonic divisions of blastocysts. To this end, I will analyze SAC signalling and dynamics and assess whether the robustness of the SAC increases with development. My project aims to improve our understanding of chromosome segregation during mammalian pre-implantation development, and therefore the results of my research will be important to shed light on the molecular causes of aneuploidy in the early embryo, fundamental for our understanding of infertility and to improve the process of in vitro fertilization.

Mycobacterium abscessus (Mab) is an opportunistic-multidrug-resistant non-tuberculous mycobacteria responsible for multiple clinically-acquired infections both pulmonary and extrapulmonary. Unlike many rapidly growing mycobacteria (RGM), Mab is able to survive and multiply within macrophages, similar to slow growing mycobacteria (SGM) such as M. tuberculosis (Mtb). In Mtb, five T7SS (ESX-1-5) have been identified and shown to be essential for intracellular survival (ESX-1), virulence (ESX-1 and ESX-5) or growth (ESX-3). T7SS are composed of five protein components essential for function: EccB, EccC, EccD, EccE and MycP. Except for a low-resolution structure of the holo ESX-5 complex from the host lab at 13 Å resolution, no structural data on any T7SS have been published to date, rendering structural work timely and eagerly awaited by relevant communities. Deemed inactive due to its lack of one of the established T7SS components EccE4, ESX-4 has been considered an ancestral T7SS form. However, Mab possess a fully intact and functional ESX-4, essential for its intracellular survival, rendering it a highly attractive target for an in-depth characterization. Here, I propose an interdisciplinary project that includes both functional and structural investigation. As the 2 M Dalton-holo-complex crosses the Mab inner membrane, experimental structural work will be challenging and require an integrative modeling approach to combine diverse experimental data sets. Complementary infection biology experiments including microbiology, genetics and cell biology will be carried out by collaborators. With this work, I aim to respond to central questions related to T7SS in general and Mab ESX-4 specifically, such as: what is the mechanism of T7SS-mediated secretion? What makes ESX-4 specific and different from other T7SS? What is the specific role of EccE4 to establish a functionally active ESX4? and What are the substrates and specific mechanism of ESX-4 substrate recognition?