Expansion of the neocortex during evolution has played a key role in the appearance of higher cognitive functions in humans. Neocortex development occurs via proliferation of neural stem cells that generate all neocortical neurons, and developmental defects can lead to severe cortical malformations. The central goal of this proposal is to unravel how human neural stem cells proliferate and self-renew to expand their pool and sustain the development of the greatly enlarged human neocortex. In mice, the major neural stem cells are the apical Radial Glial (aRG) cells, which are highly elongated cells extending long apico-basal cytoplasmic processes. The remarkable expansion of the human neocortex is thought to arise from an additional pool of neural stem cells, the basal Radial Glial (bRG) cells. Because of their extreme rarity in rodent models, the mechanisms controlling proliferation and self-renewal of bRG cells remain largely unexplored. Combining microfluidics, generation of cerebral organoids, culture of human fetal brain slices and state-of-the-art live imaging techniques we will: Aim1: Examine how the dynein molecular motor enables expansion of the human bRG cell pool. Mutations in dynein and dynein-associated factors are highly associated with human cortical malformations. We hypothesise that dynein controls critical aspects of human bRG cell behavior, including cell migration, mitotic spindle positioning and proliferation. Aim2: Identify polarised cell fate regulators in mouse and human aRG and bRG cells. The fate of RG cells and their ability to self-renew has been tightly associated with the maintenance of their basal process. We hypothesise that critical cell fate regulators are asymmetrically localised to this cytoplasmic process. With this project we will identify key molecular mechanisms controlling human neurogenesis, which is fundamental to understand cerebral malformations such as genetically or pathogen-induced microcephaly.

Non-canonical DNA and RNA structures, such as G-quadruplexes (G4) as well as hairpin-like, slipped-strand structures formed by tri-, tetra- and oligonucleotide repeats in DNA and RNA, represent therapeutically important targets whose biological functions can be modulated using small-molecule ligands. However, most known ligands, developed by rational design or combinatorial chemistry approaches, suffer from insufficient selectivity, in particular in terms of differentiation between structurally related targets (e.g., various G4-DNA and G4-RNA polymorphs; structurally similar RNA repeat structures). DYCONAS aims the development of a flexible dynamic combinatorial chemistry (DCC) methodology for target-guided discovery of novel, highly affine and selective ligands for non-canonical secondary structures of nucleic acids. The key feature of DYCONAS lies in the implementation of highly versatile acylhydrazone exchange chemistry for generation of dynamic combinatorial libraries that can be easily adapted to various DNA and RNA targets, covering a broad range of putative ligand structures. The perimeter of this project includes: (i) implementation, validation and optimization of the acylhydrazone-based DCC approach for nucleic acid targets; (ii) exploitation of this methodology for identification of novel potent ligands for therapeutically important nucleic acid targets, followed by a biophysical characterization of their interaction with the target in vitro; as well as (iii) exploration of acylhydrazone chemistry for the development of target-guided “in situ” synthesis of fluorescent or chromogenic ligands, aiming selective optical detection of nucleic acid targets.

During inflammation, monocytes are rapidly recruited and differentiate in situ into monocyte-derived macrophages (mo-Mac) and monocyte-derived dendritic cells (mo-DC). During immune responses, mo-Mac are generally involved in clearance of pathogens, while mo-DC can stimulate T cells or transport antigens to local lymph nodes. However, in chronic inflammatory diseases and autoimmune disorders, monocyte-derived cells fuel the inflammation and are major contributors to tissue damage. This phenomenon has been evidenced in various pathological models in the mouse, and in humans in Crohn's disease, psoriasis and rheumatoid arthritis. While the role of monocyte-derived cells in inflammation and inflammatory diseases is becoming well documented, how monocytes differentiate into mo-DC or mo-Mac is still poorly characterized. In particular, what stimuli orient monocyte fate towards mo-Mac versus mo-DC remains to be established. Moreover, what transcription factors drive the differentiation program of mo-Mac and mo-DC remains unknown. Given their central role in mediating tissue damage in inflammatory disorders, monocytes and monocyte-derived cells have emerged in the past few years as a promising target for therapies. A better understanding of the molecular ontogeny of monocyte-derived cells would provide novel molecular targets for the therapy of inflammatory diseases. In this project, we will address these questions using a new culture model of human mo-DC and mo-Mac that we have developped during preliminary work. In this culture model, we can generate DC and macrophages that closely resemble human inflammatory DC and macrophages found in vivo. Moreover, we have identified in preliminary work transcription factors involved in mo-DC versus mo-Mac differentiation. We have also evidenced that signals derived from pathogens influence monocyte differentiation. The objectives of the project are: 1) to identify how signals derived from pathogens impact monocyte differentiation at the molecular level 2) to unravel transcriptional networks involved in mo-Mac versus mo-DC differentiation. 3) to determine whether the transcription factors we identified are involved in lineage commitment or maintenance of mo-DC and mo-Mac. These results will be instrumental in the design of novel strategies for therapeutic intervention on the differentiation of monocyte-derived cells.

In complex multi-organ animals cells can migrate between distant tissues. This trans-organ migration is commonly used to distribute cells trough the body. This occurs, for example, when cell precursors are sent to target tissues to repopulate organs and maintain body homeostasis. This type of motility is also important for leukocytes, immune cells that upon infection migrate between organs to perform and coordinate adaptive immune responses. Trans-organ migration is also observed in pathological situations such as autoimmune diseases and cancer metastasis, highlighting the relevance and therapeutic potential of this function. The general objective of this proposal is to understand the mechanisms that make cells efficient in migration between distant organs. This proposal is focused in the study of leukocytes, immune cells that promptly colonize secondary tissues to ensure adaptive immune protection. Adaptive immunity starts by antigen recognition at the periphery of the body. This function is performed by dendritic cells (DCs), leukocytes that randomly scan tissues searching for harmful particles. After encountering with a pathogenic element such as bacterial products, DCs change their motile properties. They transit from a random migration to a more persistent and directional mode of locomotion. This change in motility is accompanied by chemokine guidance that drives DCs to lymphatic vessels, the pathway to lymph nodes. To enter the lymphatics DCs deform and expand preexisting portals localized at the surface of the vessels. Once inside, they get exposed to a flat shaped environment. At the end of their journey, DCs reach the draining lymph node, exit the lymphatics and then migrate towards the center of this secondary organ to encounter and activate cognate naïve T lymphocytes (TLs). After activation by DCs, TLs proliferate and exit the lymph node. They enter into blood vessels and home to the inflamed tissue where they perform the effector function of the adaptive immune system. Migration and colonization of a secondary tissue by DCs and TLs is extremely efficient, taking place in only few hours. However, the cellular machinery used by these leukocytes to migrate between distant organs remains elusive. The aim of this project is to take advantage of the professional capacity of leukocytes to colonize secondary tissues to decipher the cellular requirements that facilitate migration of cells between distant organs. I propose a multidisciplinary project that combines micro-fabrication, cell biology and immunology to identify cellular mechanisms that naturally evolved in immune cells to facilitate exchange of cells between tissues. Using novel in vitro micro-fabricated tools I will identify molecules that control different stages of trans-organ migration. The function of these molecules will be validated in physiological environments. This proposal focuses in the role of calcium channels and cytoskeleton rearrangements, two key cellular functions that combined regulate cell contractility, main requirement for cell motility in complex environments. The success of this project might open new possibilities in the treatment of pathologies in which cell motility is altered such as autoimmune diseases or cancer metastasis.

Most methods in modern cell biology are implicitly based on the assumption that the behavior of a cell is predominantly determined by its individual biochemical and genetic properties. Moreover, complex processes such as tumorigenesis, are studied by methods that make it quite difficult if not impossible to take into account the diversity of the participating cells and their mutual interactions. Nevertheless, it is now widely acknowledged that cellular diversity and microenvironmental effects are key determinants for the emergence of collective processes such as the homeostasis of healthy tissues or their destabilization during dysplasia or tumor growth and metastatic escape. In the prokaryotic world as well, several collective phenomena have been observed, such as quorum sensing between bacteria or the formation of bacterial biofilms. In all these collective phenomena, each individual cell seems to obey external signals that guide its individual behavior. Conversely, many observations indicate that altered collective behaviors in a complex microenvironment, e.g. the loss of tissue homeostasis, are key steps in the process of cancer development, leading to pathological types of organization : benign or malignant solid tumor, metastatic foci. Nevertheless, we do not know of any quantitative model to quantitatively study collective aspects of tumorigenesis by controling each participating individual cell and the microenvironment. How project here is to construct 3D microstructures to organize an ensemble of cells –first as a 2D lattice– and control the interactions between them, by providing each individual with a geometrically, mechanicaly and chemicaly defined position template. We will study in a quantitative and reproducible fashion, how collective properties emerge either spontaneously or as a response to local perturbations : tissue stability, dysplasic destabilization, tumor growth, mesenchymal transition and metastatic escape. We will also study the local and collective conditions for the nucleation and growth of bacterial biofilms. 3D microstructures will be fabricated by two-photon induced photopolymerization, and the unique features of that method will be very useful for the present project : 1/ microstructure can be made with virtually no constraint on the geometry, provided it can be described with submicroscopic voxel –typically 0.2x0.2x0.7 μm3–, 2/ a broad range of materials can be used – hydrogels and resins-, offering a wide range of chemical derivatizations and elasticity, 3/ microstructures can be constructed sequentially using different chemistries and Young moduli. Since elasticity can be controlled, the envisaged microstructures will also be used to assess intercellular forces by measuring microstructure strains. The two participating teams focus on chemistry and microbiology on the one hand, and cell biology and non-linear imaging and optics on the other hand. Therefore, the collaboration is very important to develop the microfabrication methodology between optics and chemistry, but two distinct biological models and questions will be then studied with our microstructures : 1/ construction of a controlled lattice of epithelial cells to study the stability of the unperturbed cell lattice, and the emergence of the order-disorder transition during the induction of a dysplasic-like behavior or the stimulation of an epithelium-mesenchyme transition, 2/ fabrication of various templates to study the condition for the nucleation and growth of biofilms. Microstrutures compatible with cell culture should be more generally useful as "3D multicellular chips" for cell biology, pharmacology, and tissue engineering at the single-cell level. For those reasons, we want to set-up a two-photon photopolymerization microfabrication station ; we also ask for a technical support salary, because that equipment will be open to collaborations.