Developmental and evolutionary dynamics of cis-regulatory elements in mouse cerebellar cells

INTRODUCTION

The cerebellum contributes to many complex brain functions, including motor control, language, and memory. During development, distinct neural cells are generated at cerebellar germinal zones in a spatiotemporally restricted manner. Cis-regulatory elements (CREs), such as enhancers and promoters, and the transcription factors that bind to them are central to cell fate specification and differentiation. Although most CREs undergo rapid turnover during evolution, a few are conserved across vertebrates.

RATIONALE

Bulk measurements of CRE activity have provided insights into gene regulation in the cerebellum, as well as into the evolutionary dynamics of CREs during organ development. However, they lack the cellular resolution required to assess cell-type differences in regulatory constraint and unravel the regulatory programs associated with the specification and differentiation of cell types.

RESULTS

Here, we generated a single-cell atlas of gene regulation in the mouse cerebellum spanning 11 developmental stages, from the beginning of neurogenesis to adulthood. By acquiring snATAC-seq (single-nucleus assay for transposase accessible chromatin using sequencing) profiles for ~90,000 cells, we mapped all major cerebellar cell types and identified candidate CREs.

Characterization of CRE activity across the cerebellum development highlights the cell- and time-specificity of gene regulation. Many of the differentially accessible CREs are specific to a single cell type and state, but we also identified a fraction of CREs with pleiotropic (shared) activity. At early developmental stages, temporal changes in CRE activity are shared between progenitor cells from different germinal zones, supporting a model of cell fate induction through common temporal cues. Pleiotropic CREs in major cerebellar neuron types (granule cells, Purkinje cells, and inhibitory interneurons) are more active at early differentiation states, and the regulatory programs gradually diverge as differentiation proceeds.

Based on comparisons to vertebrate genomes, we observed a decrease in CRE sequence conservation during development for all cerebellar cell types, a pattern that is largely explained by differentiation as well as by additional temporal differences between cells from matched differentiation states. Across cell types, differences in regulatory conservation are most pronounced in the adult, where microglia—the immune cells of the brain—show the fastest evolutionary turnover. By contrast, mature astrocytes harbor the most conserved intergenic CREs, not only in the cerebellum but also across a wide range of cell types in adult mouse organs.

To evaluate the conservation of CRE activity, we acquired snATAC-seq profiles for ~20,000 cerebellar cells from the gray short-tailed opossum, a marsupial separated from mouse by ~160 million years of evolution. Our comparative analysis of CRE activity in the two therian species reinforced our sequence-based conclusions regarding differences in CRE constraint across cell types and developmental stages and also revealed that despite the overall high turnover of CREs, radical repurposing of spatiotemporal CRE activity is rare, at least between cell types in the same tissue.

CONCLUSION

This study reveals extensive temporal differences in CRE activity across cerebellar cell types and a shared decrease in CRE conservation during development and differentiation. Given that the cerebellum has been successfully used as a model system to study cell fate specification, neurogenesis, and other developmental processes, we expect that our observations regarding the developmental and evolutionary dynamics of regulatory elements, and their interplay, are also applicable to mammalian organs in general.