Understanding brain development and disease: from embryos to organoids

The neocortex consists of two main classes of neurons - excitatory and inhibitory neurons. Excitatory cells represent 80% of the neurons in the neocortex. They are long-projecting neurons that connect different cortical areas to one another or to subcortical structures, brainstem, and spinal cord. There is a great diversity of excitatory cell types, which are primarily distinguished by their projection patterns. These cells are generated by progenitors through several rounds of division; post-mitotic cells then migrate radially to progressively form the neocortex. Although the main steps of corticogenesis are unveiled, several questions remain unanswered:

• When do post-mitotic cells acquire their excitatory cell type identity?
• Which molecular programmes regulate their differentiation as well as identity diversification?

Prof. Arlotta’s group addressed these questions in a recent study (Di Bella et al., 2021), through single-cell RNA sequencing of the developing neocortex over the entire period of corticogenesis. This consists in high-throughput profiling of the RNAs carried by individual cells, including progenitors, post-mitotic excitatory cells, and glial cells that originate from the same progenitors. This approach not only provides a molecular atlas of the developing neocortex, but also underlines the different developmental trajectories leading to cell type diversity.

Interestingly, all types of excitatory neurons arise from a common type of progenitor, indicating that neuronal diversification is only observed post-mitotically. Indeed, post-mitotic cells progressively bifurcate towards an identity or another, and this high-throughput RNA sequencing highlighted the transcriptional programmes mediating this identity divergence. These included known and novel regulators of excitatory cell fate. These findings were further supported by assessment, at the DNA level, of the gene accessibility for transcription. Finally, all these results were verified using spatial transcriptomics, where the spatial localization of candidate genes is determined on brain sections.

This access to the gene regulatory programmes controlling the acquisition of different cell identities during development, will also help to understand the origin of developmental disorders. As the combined action of several regulators dictates fate divergence and neuronal diversification, a further step would be to simultaneously manipulate these regulators and assess the consequences on corticogenesis.

The Arlotta Laboratory recently developed such a technique, called Perturb-Seq (Jin et al., 2020). In this study, mutations of 35 candidate genes associated with autism spectrum disorders and developmental delay, were simultaneously introduced in dividing progenitor cells from the developing mouse neocortex using Crispr/Cas9. These perturbations were followed by single-cell RNA sequencing and analysis at postnatal ages. Some neurons, derived from the perturbed progenitor cells, received a combination of mutations, while other neurons received a single mutation among the 35 possible mutations. This allows for the simultaneous assessment of the impact of different mutations on gene regulatory programmes in individual cells.

Prof. Arlotta’s research also focuses on building-up experimental models of human brain development (Quadrato et al., 2017; Velasco et al., 2019). Her laboratory recently demonstrated that human 3D brain organoids constitute very promising and reproducible models to study long-term human development, and the generation of cellular diversity. Indeed, the cellular diversity of up to 69 individual organoids was assessed by single-cell RNA sequencing and showed high consistency between organoids, at different ages (1 month, 3 months, 6 months, over 9 months), similar to that found in human embryos.

Human organoids can thus be used to model and understand the onset of neurodevelopmental disorders, using for instance cells derived from patients.

Overall, Prof. Arlotta’s research unravels the molecular mechanisms shaping and maintaining neuronal diversity during cortical development, and develops promising assays to study and model neocortical human neurodevelopment in health and disease.

References

Di Bella, D.J. et al. (2021). Molecular logic of cellular diversification in the mouse cerebral cortex. Nature.

Jin, X. et al. (2020). In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes. Science.

Quadrato, G. et al. (2017). Cell diversity and network dynamics in photosensitive human brain organoids. Nature.

Velasco, S. et al. (2019). Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature.

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