The mammalian cerebral cortex is a complex laminar structure with a variety of neuronal and non-neuronal cell types that develop in a finely orchestrated and stereotypic manner. Final laminar position and synaptic specificity of most cortical cell types are well described. Strikingly, any alteration in the developmental unfolding of one of these processes, even for a single cell type among tens, can be sufficient to generate neurodevelopmental disorders. However, how the establishment of this precise cellular architecture is regulated at the molecular level remains largely unknown. Several lines of evidence suggest a role of cell-cell communications via ligand-receptor (LR) interactions. Using a single cell RNA-seq (scRNA-seq) approach in mice, we have generated a bioinformatic atlas that infers LR based cellular communications across all cell types over somatosensory (SS) cortex development. Querying our atlas for known LR interactions has demonstrated its validity, but new LR-mediated cell-cell interactions remain to be discovered to interrogate its power as a hypothesis generator. In parallel, a technique called Multiplexed-Error Robust Fluorescence In Situ Hybridization (MERFISH) has been recently developed and implemented by us, which images single cell transcriptomes in situ and thereby adds precious information about spatial expression. Here, we will: (i) test some LR interactions predicted by our scRNA-seq atlas for a role in SS microcircuit development, (ii) build on the MERFISH technique to complement our atlas with spatial information and to characterize SS cortex cellular development with unprecedented resolution and (iii) use MERFISH to interrogate altered developmental processes in the SS cortex of a mouse model of neurodevelopmental disorder, the Neurod2 KO mouse.

We propose to decipher the mechanisms of action of neuroendothelial N-methyl-D-aspartate receptors (NMDAR). In addition to neurons, where they drive glutamatergic neurotransmission, NMDAR are expressed in a variety of cell types. In particular, brain endothelial cells express NMDAR, which could be involved in blood-brain barrier maintenance and alteration. In a recent paper, we identified an unexpected population of NMDAR in endothelial cells, expressed at the luminal side of microvessels and located at the vicinity of blood/spinal cord barrier-forming tight junctions. We developed a monoclonal antibody (Glunomab®), directed against a specific site of NMDAR (aminoacids 176-180), which blocked the entry of leukocytes to the inflamed spinal cord, thus providing therapeutic effects in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Nevertheless, the downstream targets which link endothelial NMDAR function to leukocyte migration across the blood/brain and blood/spinal cord barriers are not fully understood yet. Interestingly, our current studies show that these receptors have an unconventional composition, including the presence of the rare GluN3 subunit, which provides response to glycine (in addition to glutamate) and metabotropic signaling (in addition to ionotropic function). Interestingly, naturally occurring circulating auto-antibodies against NMDAR are present in ~10% of human subjects and are overexpressed in neuropsychiatric and neurological diseases. Beyond these quantitative data, qualitative information are needed concerning the regions of NMDAR recognized by these antibodies. In fact, circulating autoantibodies against NMDAR could provide either beneficial or deleterious effects, depending on the region that they target. In line with this, we postulate that identifying the regions targeted by NMDAR auto-antibodies could have prognosis value in neurological diseases. Given our recent work concerning NMDAR in animal models of MS, we believe that investigating circulating antibodies would be particularly relevant for prognosis of MS. Thus the goals of this project are i) To characterize the signaling pathways and downstream targets triggered by NMDAR activation in endothelial cells, in relation to leukocyte penetration towards the spinal cord, ii) To identify the repertoire of NMDAR antibodies in MS patients (based on their target regions on NMDAR) and to determine whether different clusters of antibodies are associated with different outcomes, iii) To investigate the effects of these different clusters on the function of endothelial NMDAR and in animal models of MS, and iv) To bridge experimental data and clinical observations.

This project intends to design, fabricate and study advanced III-V semiconductor nanostructures-based devices for the generation of coherent optical frequency combs (OFCs) with controllable transverse patterns dynamics. Our approach consists in an optically-injected vertical-emission Kerr Gires-Tournois Interferometer (KGTI) integrated in a compact free-space cavity. The KGTI shall consist in a (Al)GaAs/InGaAs metasurface-based VCSEL, with controlled light confinement and phase dispersion, to enhance fast nonlinear light-matter interaction. The coupled-cavity system will be designed to reach the bistable regime and achieve coherent light states with properties overcoming current limitations for telecom and imaging applications. This new experimental framework will be complemented by the development and bifurcation analysis of a hybrid time-delayed, partial differential equations 3D theoretical model, that includes both transverse 2D diffraction and on-axis temporal dynamics. The external cavity design will allow to pass from a single transverse mode to a highly transverse degenerate (self-imaging) system. In that latter case, we envision the possibility to generate multiple, spatially independent, OFCs. We expect this project to yield as a final product, a first experimental demonstrator of vertically-emitted 1D and 3D OFCs in a mature planar III-V semiconductor based platform. Our vertical KGTI will allow to produce combs with high coherence, low power consumption, GHz repetition rates, and containing hundreds of lines in the near infrared spectral domain, with, thanks to the planar vertical architecture, potentially 10 × 10 transversally multiplexed and reconfigurable beams.These results will have groundbreaking applications for instance in massively parallel comb generation or for double comb sensing application and it will help to overcome several limitations for telecom applications. On the technological and experimental sides, the technical barriers to be lifted consists in developing a microcavity containing a nonlinear material having a very high value of the Kerr nonlinear coefficient. For this objective we plan on using the almost untapped potential of AlGaAs-based semiconductor materials operated below their band-gap. The nonlinear interaction will benefit from the strong light confinement in the microcavity. The microcavity design shall find a compromise between the width of the frequency comb targeted as well as the value of the optical power one wishes to inject into the KGTI system. Critical power threshold for the formation of Kerr combs can be controlled via the external cavity reflectivity and imaging configuration, and detuning of the optical pumping with respect to the microcavity resonance; the sign of the latter allowing also to explore both anomalous and normal dispersion regimes. On the theoretical side, the modeling of the system we wish to realize necessitates using Delay Algebraic Equations (DAEs). While the latter have a great potential for the modeling of dispersive phenomena in photonic systems, their studies is comparatively less developed than those of partial differential equations (PDEs). In addition, if DAEs are the natural choice for studying temporal dispersive dynamics, the diffractive propagation of light in the transverse plane of the cavity as well as field curvature effects induced by lenses and mirrors require using PDEs. As such, a full 3D model shall consists of a hybrid DAE-PDE system whose analysis is way beyond the state of the art and represents an exciting and challenging endeavor. The theoretical aspects of KOGIT will also significantly advance the study of spatio-temporal phenomena in nonlinear media. The proposed experimental framework will be complemented by the development of bifurcation analysis method of a hybrid DAE-PDE system that will constitute a qualitative jump in the state of the art.

Membrane contact sites (MCSs) enable specific lipid exchange between organelles. Our recent results on the archetypical OSBP/VAP complex suggest that the architecture and dynamics of MCS are influenced by intrinsically disordered regions (IDRs). These overlooked structural attributes enable formation of MCS of adjustable thickness and reduce protein crowding. These effects are likely to be general given the abundance of IDRs in MCS proteins. We posit that IDRs guarantee the lateral and/or vertical flexibility of proteins. We will dissect the link between these characteristics and the function, dynamics and organization of MCSs, by using a multidisciplinary and multi-scale approach involving quantitative and super-resolution cell imaging, in vitro reconstitution of membrane systems, structural analysis by cryo-electron tomography, and the development of innovative pharmacological tools. This project will offer new perspectives on the efficiency, plasticity and specificity of MCSs.

The superfamily of TGF-ß ligands represents one of the most prominent families of morphogens. These factors have profound effects on many aspects of embryonic development, cell behaviour and homeostasis and malfunction of the pathways associated with these cytokines can lead to a variety of pathologies. Despite intensive research, there are still large gaps in our knowledge regarding the specificity of these ligands, the regulation of the activity of their receptors and the interactions between the TGF-ß pathways and other signalling pathways. In particular, how sources of TGF-ß morphogens are generated and how the resulting morphogen gradients can be translated into patterns of gene expression during early development remain central questions in current developmental biology. This proposal attempts to fill these gaps in our knowledge by characterising novel regulators of dorsal-ventral axis formation upstream and downstream of Nodal and by modelling the gene regulatory network (GRN) activated by this factor. We address this question within the sea urchin model, an organism phylogenetically close to vertebrates but with many advantages for the analysis of regulatory networks in early development. Our first aim is to characterize the early events that shape the Nodal gradient and initiate the downstream GRN that controls dorsal-ventral (D/V) axis formation. Our laboratory has recently discovered several key factors involved in D/V axis formation. We identified a maternal TGF-ß ligand, a transmembrane protein, an ETS domain transcriptional repressor and the JNK kinase as factors critically required to restrict the spatial expression of nodal. The similarity of the phenotypes caused by inactivation of either this maternal TGF-ß ligand, the transmembrane protein or this ETS factor strongly suggests that they act in the same pathway to specify the D/V axis. However, the relationships between these factors and the mechanisms by which they antagonize nodal expression are presently completely enigmatic. To clarify the relationships between these factors we will identify the binding partners of the TGF-ß ligand and perform biochemical analyses and epistasis experiments. In this first aim, we will also determine whether Nodal and/or BMP2/4 work as morphogens, i.e. as long-range, concentration-dependent signalling factors in the sea urchin embryo by using a combination of treatments with recombinant Nodal and BMP2/4 proteins and ectopic expression of mRNAs encoding these ligands or the activated forms of their receptors. The second aim of this project is to extend and model the gene regulatory network activated by Nodal. We recently identified and validated about fifteen novel genes regulated by Nodal encoding a variety of regulatory proteins including transcription factors, cytokines, and secreted proteins most of which have never been characterized. The expression patterns of these genes identify novel regulatory domains and boundaries along both the animal-vegetal and dorsal-ventral axes, revealing an unsuspected complexity in patterning of the ectoderm. We propose to dissect the regulatory mechanisms establishing these new domains, to characterize these novel Nodal target genes and to analyze their function and position in the GRN. Finally, to further test the role of individual components of this network and understand how it achieves both robustness to environmental perturbations such as regulative development, and plasticity to evolutionary scenarios, we will start to construct a logical model of this GRN. Our third and last aim is to start dissecting the mechanisms that allow responding cells to read different levels of Nodal or BMP2/4. We will start to investigate how thresholds of response are encoded in the genome. We will perform detailed bioinformatics analyses on an extended set of Nodal and BMP2/4 target genes to identify and dissect the architecture of the cis-regulatory modules of these selected target genes.