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Building up a brain: understanding how neural stem cell fate and regulation controls nervous tissue architecture

Periodic Reporting for period 4 - BRAINSTRUCT (Building up a brain: understanding how neural stem cell fate and regulation controls nervous tissue architecture)

Reporting period: 2020-01-01 to 2021-06-30

Neural progenitors of the embryonic brain have a major influence on nervous tissue architecture through the production of neuronal and glial cells. How these progenitors share the generation of neural cells and how their clonal progeny organizes in brain tissue are major questions in neuroscience, with direct impact on our understanding of neural circuits organization and neurodevelopmental disorders.
In the Brainstruct project, our overall objective has been to develop and apply new approaches based on transgenic labelling and optical imaging to explore these aspects in situ, focusing on the mouse cerebral cortex. We implemented schemes based on combinatorial fluorescent markers to track in parallel the fate of multiple individual neural progenitors over the long term in intact neural tissue, and imaged the resulting clonal patterns with a newly developed 3D high-resolution colour microscopy technique. To probe progenitor regulation, we introduced a new somatic transgenesis technique that facilitates the genetic manipulation of these cells. The results obtained with these new tools lay the ground for quantitative studies of neural development at the individual cell/clonal scale which should be transposable in a variety of biological contexts.
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).
In conclusion, we have developed complementary molecular and imaging approaches enabling to assay the clonal architecture of a brain region in a comprehensive manner, and to explore the regulation of its development. Progress made along the project include:
- A new methodology for 3D colour imaging of large volumes of brain tissue with micrometer resolution.
- A type of genome-integrating vector to assay stem cell output and regulation in normal and pathologically-relevant context, with broad potential beyond developmental studies to accelerate genetic engineering in eukaryotic cells.
- A comprehensive characterization of astrocyte development and clonal organization in the mouse cortex, evidencing the adaptability of these cells during their proliferation and differentiation.