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.

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.

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.

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.

Cell surface receptors coupled to the activation of phospholipase C-g (receptor-type tyrosine kinases, immunoreceptors) or phospholipase C-b (G protein-coupled receptors) lead to the increase in intracellular calcium ion concentration through the sequential opening of specific calcium channels. The increase in intracellular calcium concentration induces the activation of calcium- and calmodulin (CaM)-dependent protein kinases including CaMKII, CaMKIV and that of calcineurin, a CaM-dependent serine/threonine-specific protein phosphatase. Calcineurin (PP2B) plays a pivotal role in many important biological processes, including the development and function of the immune and nervous systems, patterning of the embryonic vasculature, muscle growth and development and heart valve morphogenesis. Sustained signaling through calcium/calcineurin result in the activation of transcription factors of the NFAT family (NFAT : Nuclear Factors of Activated T cells), and mouse genetic studies have demonstrated a strong epistatic relationship between calcineurin and NFATs activation and function in many developmental processes. In lymphoid cells, the calcineurin/NFAT signaling pathway is esssential to specific aspects of T cell development and plays a central role in the activation of the immune response and in its homeostatic control. The critical role of the calcineurin/NFAT signaling module in the immune response is illustrated (i) by the fact that the two most effective immunosuppressants used in human medicine, FK506 (Prograf) and cyclosporin A (CsA) are inhibitors of calcineurin; (ii) by the fact that patients with a rare form of hereditary severe combined immunodeficiency (SCID) show a selective defect in calcineurin/NFAT activation. Despite their central role in T cell development, immune function and homeostasis, deregulation of calcineurin and of the calcineurin/NFAT pathway have not been shown so far to have a pro-oncogenic role in lymphomagenesis. We have recently shown that (i) several pre-clinical mouse models of human lymphoma present the persistent activation of the calcineurin/NFAT pathway through mechanism(s) that require the maintenance of lymphoma cells in their in vivo cellular context ; (ii) that treatment of mice carrying lymphoma induced by completely distinct initiating mechanisms (e.g. TEL-JAK2- and NotchIC-induced lymphoma) with either CsA or FK506 result both in inactivation of the calcineurin/NFAT pathway in lymphoma cells and in severe inhibition of tumor progression. These observations show that the calcineurin/NFAT pathway is critical to lymphomagenesis and is a target of therapeutic interest in the control of these diseases. The objectives of this research proposal are, mostly through the use of genetically modified mice (i) to demonstrate that calcineurin is required in a tumor cell autonomous fashion in lymphomagenesis ; (ii) to analyze whether, in keeping with its essential role as a calcineurin effector in normal T cells, NFATs are also essential effectors of calcineurin in lymphomas ; (iii) to analyze whether the different factors of the NFAT family found to be activated in these cells (NFAT1, NFAT2, NFAT4) function in a specific or redundant fashion to favour lymphomagenesis ; (iv) to identify the calcineurin/NFAT-specific transcriptome in lymphomas to gain insight into the molecular pathway(s) controled by calcineurin/NFAT in these pathological situations.