The development of the neural networks of the brain raises two major questions: how are their cellular components generated in appropriate numbers and types by neural progenitor cells during embryonic and early postnatal development? How do these cells distribute within nervous tissue and interact together? The Brainstruct project aimed at probing these questions through an interdisciplinary approach combining genetic engineering and high-resolution imaging.
To identify clones of cells generated by individual neural progenitors with high throughput and accuracy, we took advantage of combinatorial labelling schemes enabling to multiplex single-cell lineage analysis with fluorescent colour markers. A major challenge of the project was to image neural clones labelled in this manner in their entirety and with sufficient precision to resolve juxtaposed cells and colour marker combinations. To solve this problem, in collaboration with the group of optics specialist Emmanuel Beaurepaire (Laboratory for Optics and Biosciences, Ecole polytechnique, Palaiseau, France), we introduced a new multicolour 3D imaging scheme that associates trichromatic excitation by two-photon wavelength mixing with serial block-face sectioning and image acquisition. This technique termed ChroMS microscopy opens the way to micrometer-scale 3D trichromatic imaging of transgenic labels over virtually unlimited tissue volumes. It provides unprecedented color images that can span entire brain regions or even the whole mouse brain (Abdeladim et al. Nat Commun 2019).
Using combinatorial clonal markers and our new 3D imaging approach, we performed a detailed analysis of astrocyte development and clonal organization in the mouse cerebral cortex. Embryonic electroporation enabled us to label and image large numbers of cortical astrocyte clones. The variable composition and semi-dispersed organization of these clones suggest that they expand in a plastic manner, initially intermixing with neighboring clones prior to adopting a more cohesive mode of development. Our results also unambiguously demonstrated that two subtypes of astrocyte observed in the cerebral cortex are not generated by distinct specialized progenitors. Altogether, this work suggests that local environment likely determine astrocyte clonal expansion and final morphotype (Clavreul et al. Nat Commun 2019).
Beyond the analysis of neural progenitor lineage, another challenge of the project was to probe how their output may be regulated at the individual cell level. To this aim, we implemented a new technology opening the way to functional mosaic analysis in the developing vertebrate nervous system by simple somatic transfection. This new expression scheme termed iOn conditions exogenous transgene expression to integration into the host cell genome, thus ensuring that expression of a marker by a cell accurately reflects its lineage. The application of this approach in the neural tube indicates that interactions among progenitors could regulate their proliferation, as is the case in several non-neural models. Beyond neurodevelopmental studies, the iOn expression strategy also has considerable potential in eukaryotic models to simplify and accelerate additive transgenesis (Kumamoto et al. Neuron 2020; Patent application EP18305623).
Finally, we also applied our multicolour lineage tracing approaches in collaborative studies on the potentialities of stem cell in the skin epidermis (Roy et al. EMBO 2016) and the lineage of ependymal cells in the developing brain (Ortiz-Álvarez et al. Neuron 2019).
