A conserved differentiation program facilitates inhibitory neuron production in the developing mouse and human cerebellum

Jens Bager Christensen, Alex P.A. Donovan, Marzieh Moradi, Giada Vanacore, Mohab Helmy, Adam J. Reid, Jimmy Tsz Hang Lee, Omer Ali Bayraktar, Andrea H. Brand, N. Sumru Bayin

Posted on: 15 May 2025

Preprint posted on 10 April 2025

How do progenitor cells in the brain give rise to such a diverse range of cell types? ASCL1 and FOXO1 work together to create inhibitory neurons from bipotent progenitors

Selected by Jethro Lundie-Brown

Categories: cell biology, developmental biology

Background

The brain is a highly complex organ, both in terms of its structural organisation and its cellular diversity. Ensuring that the right cell types are made at the right time in the right place requires tightly regulated gene expression. Having the right genes expressed in the right cell types is coordinated by the activity of various transcription factors and developmental cues. The constituent parts of these regulatory networks remain somewhat poorly defined, particularly in regions of the brain like the cerebellum. In fact, the cerebellum is the region of the brain with the highest number of neurons, so understanding its development is crucial to understanding how the brain is made.

Key Findings

Christensen and colleagues used single cell RNA-seq and in vitro differentiation of progenitor cells from the cerebellum to elucidate the molecular pathways that govern neurogenesis.

Their single-cell data nicely reconstructed known lineages from a bipotent progenitor cell to either gliogenic or neurogenic fates. Using expression data of transcription factors, they pulled out FOXO1 as a predicted regulator of the transition from neural-fated progenitors to maturing inhibitory neurons. They confirmed the expression of FOXO1 in primary cerebellar cultures and showed that it follows ASCL1 expression.

To confirm the relationship between these proteins and neuronal differentiation, they used lentiviral overexpression to show that ASCL1 acts before FOXO1 in a hierarchical, stepwise progression to differentiation: ASCL1 overexpression induced both FOXO1 expression and neuronal differentiation, but FOXO1 overexpression did not affect ASCL1 levels and only strongly enhanced neuronal differentiation after 10 days in culture. Knockdown of FOXO1 impaired, but did not totally inhibit, neuronal differentiation. This might be due to convergent and redundant differentiation pathways, or a minority of cells that still expressed FOXO1.

Christensen and colleagues went on to confirm a direct interaction between ASCL1 and the Foxo1 locus using DamID, in which transcription factor binding is inferred from specific DNA methylation by an exogenous methylase. The binding data nicely supported the hierarchical relationship between ASCL1 and FOXO1 put forward in the earlier experiments. They used their transcriptomic data and in vitro differentiation assays to link Wnt signalling to a preference for neurogenesis over gliogenesis. Finally, the group used primary human cultures to show that these same pathways are relevant in the differentiation of progenitor cells in human as well as mouse, supporting a conserved model for inhibitory neuron differentiation in the cerebellum.

Preprint significance

I think this work deepens our understanding of how transcription factors work together to control cell fate. Establishing the relationship between these factors acting in sequence is important, particularly as a lot of previous studies have tended to focus on them one at a time. I like the use of both differentiation assays with primary cell cultures and analysis of tissue sections from the brain to compare these cell types in vitro and in vivo, and I also like the use of human cells to understand whether these mechanisms are conserved.

Questions for the authors & future direction

There is an interesting difference between the phenotype after ASCL1 overexpression compared to FOXO1. It would be interesting to know what changes ASCL1 induces that allow FOXO1 to function, beyond just direct upregulation of FOXO1. Are there epigenetic changes that make the cells more permissive for neuronal differentiation?
It would also be interesting to know what the cells are in culture that are ASCL1-negative, given that a relatively small percentage of the progenitor cells express this marker. Are these cells also competent for inhibitory neurons, or are they cells that are already specified for gliogenesis?
Wnt signalling is clearly implicated in driving the transition from ASCL1+ progenitors to FOXO1+, but it would also be interesting to know what controls the expression of ASCL1 and how the original bipotent progenitors become committed to becoming neurons versus glia.

Tags: brain, cerebellum, development, human, mouse, neurons

doi: https://doi.org/10.1242/prelights.40438

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