Analyse du développement et de la myélinisation des circuits neuronaux par de nouvelles stratégies transgéniques – Brainbow

Visualizing the cellular interactions driving complex processes such as synaptogenesis or myelination is a major issue in developmental biology. Because of technical limitations, this analysis has mostly been limited to either whole cellular populations, or single cells. Because they offer the advantage of a live staining, genetically encodable dyes such as GFP are probably the best way to label nerve cells. Dr Jean Livet, who has recently joined the Vision Institute, has developed a transgenic system for stochastic expression of multiple genes, the Brainbow, based on CRE/Lox recombination. In mice expressing Brainbow constructs under the control of the neuronal promoter Thy1, cells express random combinations of three or more spectrally distinct fluorescent proteins (CFP, YFP RFP…). Dozens of hues are created by mixing these three primary colors, providing a spectrum of color labels allowing one to distinguish multiple neighboring cells and their processes in a single tissue sample. Because connectivity data are based on sparse labeling techniques, the quantitative features of neural circuits such as the numbers of axons that contact a postsynaptic cell or the number of postsynaptic cells an axon innervates are largely inaccessible. As well, it is known that neurons belonging to a same level of a circuit are often arranged in a topographic manner, but the precision of this organization can only be indirectly inferred in absence of methods to label and distinguish adjacent and potentially overlapping axonal arbors. Thus our understanding of neuronal circuit organization at the cellular level relies mostly on qualitative rather than quantitative data. Likewise, our knowledge gap concerning many developmental aspects of myelination is still striking. Oligodendrocytes are the myelinating cells of the central nervous system whose processes enwrap axons except in some restricted domains named the nodes of Ranvier, allowing the rapid and saltatory conduction of action potentials. Many myelin proteins have been identified and antibodies raised against these allow visualizing myelinated axons and myelinating oligodendrocytes on brain sections and in vitro cultures, in normal or pathological conditions. However, while it has been shown that precise rules regulate competition between developing axons, it is still unknown if these rules also apply to myelinating oligodendrocytes. Other unanswered key questions are: how do individual oligodendrocytes myelinate several axons in vivo? How do neighboring oligodendrocytes share a set of axons to myelinate or remyelinate? Does the myelination process involve competitive interactions and mutual repulsion between oligodendrocytes in particular at the nodes? Filling these fundamental knowledge gaps concerning oligodendrocytes organization and development will require a technique to visualize oligodendrocyte morphology at the cellular level. This problem would be solved if one could perform live imaging of oligodendrocytes with multiple distinct labels, such that the identity of their processes would be made evident. Our main purpose is to study cellular interactions at the fine scale level, in adult mice and during development. Our approach will use a combination of molecular approaches, to identify the genes involved in these phenomena, and cellular approaches, to visualize cellular morphology and interactions. We will use the Brainbow imaging technique, to visualize simultaneously many individual cells within a brain region. We will also perfect the Brainbow approach and develop new tools and techniques that will circumvent the drawbacks of that technique. Our specific aims are 1) to implement the Brainbow technique and develop new tools for visualizing cells and their interactions; 2) to investigate at the cellular and molecular level the organization and postnatal development of a neural circuit: the binaural pathway of the auditory system; 3) to characterize the cellular interactions occurring during myelin formation. This project will use a multidisciplinary approach, combining state of the art molecular and imaging technologies to understand the multicellular architecture of a neuronal circuit and identify the genes controlling its development. We expect that the tools we will develop will be broadly applicable to the study of cellular interactions in various cell types and systems.