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

Periodic Reporting for period 3 - BRAINSTRUCT (Building up a brain: understanding how neural stem cell fate and regulation controls nervous tissue architecture)

Reporting period: 2018-07-01 to 2019-12-31

The brain is an extraordinary complex assembly of neuronal and glial cells that underpins cognitive functions. How adequate numbers of these cells are generated by neural stem cells in embryonic and early postnatal development, and how they distribute and interconnect within brain tissue are still debated questions. In particular, the potentialities of individual neural stem cells, their possible heterogeneity and the mechanisms regulating their function are still poorly characterized in situ; likewise, the clonal architecture of mature brain tissue and its influence on neural circuitry are only partially explored. Deciphering these aspects is essential to link neural circuit development, structure and function, and to understand the etiology of neurodevelopmental disorders. We have established transgenic strategies to simultaneously track the lineage of multiple individual neural stem cells in the intact developing brain and experimentally perturb their development. This project consists in applying these approaches in combination with new large-volume imaging methods for high-throughput analysis of individual neural and glial clones in the mouse cortex. Our aims are to assay neural progenitor potentialities and equivalence, characterize developmental changes occurring in the neurogenic niche, describe the clonal organization of the mature cortex and study its link with neural connectivity. To decipher intrinsic and extrinsic mechanisms regulating neural progenitor activity and understand how they produce appropriate numbers of cells, we will assay the outcome of functional perturbations targeting key steps of neural development, introduced in precursors or in their local environment. These experiments will reveal how neural stem cell output might be regulated by cell interactions and intercellular signals. This multidisciplinary project will set the basis for quantitative analysis of brain development with single-cell resolution in normal and pathological conditions.
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 interconnect together? Focusing primarily on the astroglial and pyramidal networks of the mouse cerebral cortex, the Brainstruct project aims at addressing these questions through an interdisciplinary approach combining genetic engineering and high-resolution imaging. During the first period of the project, we have focused on the establishment of methodologies to analyse neural progenitor output within intact neural tissue and have begun to apply these approaches to analyse cortical development. Progress has been made on the two main objectives of the project: characterizing the layout, interaction and connectivity of neural progenitor’s neuronal and glial descent, and deciphering the developmental sequence and regulation of progenitor cells:

1) Objective 1: Characterizing the clonal architecture of the neuronal and astroglial cell networks in the central nervous system

We have set two approaches to identify clones of cells generated by individual neural progenitors accurately and with high throughput. The first one stems from the MAGIC Markers strategy recently introduced by us (Loulier et al. Neuron 2014), which enables identification of neural clones with combinations of colour markers encoded by transposon-based vectors introduced in neural progenitors through electroporation. In the first part of our work, we have perfected this scheme by taking advantage of a new genetic switch which we have designed, in which expression of genes of interest is conditional to genomic integration. A publication describing this broadly applicable genetic switch is currently under preparation. The second approach uses new transgenic Brainbow mice generated in our laboratory with general expression of fluorescent proteins in the central nervous system. To analyse neural progenitor lineage in the cerebral cortex, we have crossed these mice with different lines expressing Cre/CreER recombinase in all or subsets of neural progenitors, and have verified that the expected expression patterns were obtained. To further increase the power of this multicolour lineage tracing scheme, new Brainbow transgenes expressing additional colour markers are also under development, using sets of mutually incompatible lox variants that we have designed and new transgene configurations explored by computer modelling in collaboration with the laboratory of R. Benosman (Institut de la Vision).

To image cortical clones labelled with Brainbow markers in their entirety in their intact environment, we have been developing a new multicolour 3D imaging setup in collaboration with the group of optic physicist E. Beaurepaire (LOB, Ecole Polytechnique). This scheme enables tri-dimensional imaging of virtually unlimited volumes of tissue without the need of sectioning the samples. This methodology will be available for the remaining of the project to characterize the layout and dispersion of neural clones.

Using the MAGIC Markers scheme (Loulier et al., 2014) in combination with the new imaging approach presented above, we are currently analysing the development of the astroglial network of the mouse cortex. The individual contribution of neural progenitors to the different compartments of the astroglial network, the kinetics of astrocyte proliferation and the layout of clonally-related astrocytes in mature nervous tissue are still incompletely known. Embryonic electroporation of MAGIC Markers in the dorsal telencephalon has enabled us to label and image large numbers (several hundreds) of cortical astrocyte clones. We determine that cortical astrocyte clones are heterogeneous in terms of cell number and composition, as well as spatial dispersion in the cortical parenchyma where they can spread out as the network matures.

To study the development of the pyramidal neuron network, as mentioned above, we have been setting conditions for through intercrossing of Brainbow mice with Cre drivers and establishment of homozygote colonies. Beyond the organization of pyramidal neuron clones, a major and debated question concerns the relation between ontogenic and functional neural units in the cortex. To probe this aspect, we are establishing strategies to analyse connectivity among clonally related neurons by combining our multicolour clonal identification scheme with functional analysis through Calcium imaging. To rapidly explore and validate combinations of fluorescent markers and calcium dyes with minimal animal usage, we are employing as a model the embryonic chicken in which progenitors of the neural tube and retina can be easily labelled through electroporation of genome-integrative transgenes. This approach will then be transposed in the mouse telencephalon for analysing the functional organization of clonally related neurons.

2) Objective 2: Deciphering the organisation, interaction, developmental sequence and regulation of neural progenitors

In this second part of the project, our aim is to characterize neural progenitor organization, dynamics and regulation in order to establish causal links between these early aspects and the final clonal architecture.

To probe the organization of the neurogenic niche and its evolution during development, we have tested approaches to induce colour markers at different times during the periods of progenitor amplification and neurogenesis. We have first validated such an approach in the chicken model, where it enables to follow the tangential dispersion of neural progenitors prior the start of neurogenesis. Applying it in the mouse cortex will enable us to assay progenitor intercalation and possible mutual interference during corticogenesis. Should this prove technically problematic, this objective could still be successfully conducted using the chicken retina as a model.

Beyond lineage tracing, we also aim at informing on the precise order of production of neural subtypes by progenitor cells. This is however challenging in the developing mouse cortex in which long term time-lapse imaging is not possible. To dissect neural progenitor developmental sequence, we have been testing different strategies. One of them uses a new type of reporter transgene designed by us, in which marker expression changes upon sequential recombination by two distinct site-specific recombinases expressed in a specific temporal order. We have validated this new reporter system in cultured cells and are currently generating lines of transgenic mice expressing it in a general manner. Using appropriate Cre and Flp driver lines that have been transferred at Institut de la Vision, we will apply this reporter system to reveal early and late-born fractions within individual neural clones.

We are also setting methodologies to functionally probe cellular interactions suggested to regulate neural progenitor cells, as is the case in varied non-neural models. Understanding the cell-autonomous vs. non cell-autonomous nature of neural progenitor/clone development is essential from a physiopathological point of view, in the light of studies demonstrating that somatic mutations occurring in neural progenitors can cause focal brain disorders. To probe neural progenitor interactions, we are testing approaches to create color-coded mosaic perturbations in neural progenitors or their progeny and analyse their long-term effects. We have generated transgenes to perturb either specific cell communication pathways or progenitor proliferation. We have verified that these transgenes induce the expected phenotypes when electroporated in the mouse or chicken neural tube. These results represent a first step towards assaying neural progenitor interactions and regulation.

Finally, the approaches that we develop have very general applicability to characterize stem/progenitor potentialities in development and homeostasis. We have collaborated on a study applying multicolour lineage tracing to analyse epidermal stem cell potentialities during skin renewal. The results have been published in EMBO J (Roy et al. 2016).

Loulier K et al., Neuron (2014) 81:505
Mahou P et al., Nature Methods (2012) 9:815-8
Ragan T et al., Nat Methods (2012) 9:255
Roy E et al., EMBO J (2016) 35:2658.
The neuronal and glial cells that compose brain circuits develop from seemingly homogenous neural progenitors through a series of events that include cell proliferation, migration and differentiation. Imbalance in these aspects can lead to a diverse host of invalidating neurodevelopmental pathologies that constitute a huge burden for society. Yet the link between early steps of brain development and the organization of mature neural circuits is still poorly understood, due to technical difficulties in tracking neural progenitor and their descent in their native environment. Major aspects of brain development are thus debated: i) the possible heterogeneity of neural progenitor cells in terms of their potentialities; ii) the stereotypy of neural progenitor behaviour and the regulatory mechanisms modulating their output; iii) the distribution and interconnection of their progeny. Understanding these aspects is essential to explain, better diagnose and design therapies for brain developmental disorders.
The aim of the Brainstruct project is in essence to understand how individual neural progenitors share the task of building neural tissue and how their behaviour is regulated in normal and pathologically relevant conditions. It is a highly interdisciplinary project which takes advantage of recent advances in genetic engineering, optics and developmental neuroscience. It uses new and complementary molecular and imaging approaches to assay the clonal architecture of the mouse cortex in an unbiased and comprehensive manner, and explore the regulation of neural progenitor development. These innovative approaches provide solutions to two major problems that have until now hindered quantitative analysis of brain structure and development: i) accessing the morphology and developmental history of the multiple individual cells that compose a brain area; and ii) imaging neural cells in intact brain tissue over large volumes and in live samples.
The Brainstruct projects shall thus open the way to system level, quantitative analysis of brain structure and development. The project is in particular bringing:
- New methodologies for multicolor imaging of large volumes of brain tissue with micrometer resolution.
- New genetic strategies, transgenes and mouse lines for high-information analysis of intact neural tissue development with individual cell precision.
- Combining the above approaches, our work will offer a novel, unbiased view of neural progenitor development, a better understanding of the associated regulatory mechanisms, and of their link with the organization of the mature brain.
Our work prepares the way for much needed answers in the medical field concerning cytoarchitecture and connectivity defects in pathological models. In particular, the project shall shed light on the aetiology of focal brain disorders recently demonstrated to result from somatic mutations occurring in neural progenitors during development. It should also have impact in the general scientific audience as our approaches have wide applicability in different biological contexts. It shall also raise interest in the general public through scientific image exhibitions and diffusion.