Epigenomic diversity of cortical projection neurons in the mouse brain

Zhuzhu Zhang ,Conor Fitzpatrick ,Yan Pang ,Matthew W Jacobs ,Xin Jin ,Joseph R Ecker ,Carolyn O’connor ,Paula Assakura Miyazaki ,Elora Williams ,Joseph R Nery ,Jingtian Zhou ,Eran A Mukamel ,Minh Vu ,Angeline Rivkin ,Rosa Castanon ,Bertha Dominguez ,Sheng-Yong Niu ,Cheng-Ta Lee ,Andrew Aldridge ,Anna Bartlett ,Lara Boggeman ,Tony Ito-Cole ,Alexis D Franklin ,Mohammad Rashid ,Kuo‐Fen Lee ,Jared B Smith ,Pengcheng Tan ,Megan A Kirchgessner ,M Margarita Behrens ,António Pinto-Duarte ,Hanqing Liu ,Edward M Callaway

doi:10.1038/s41586-021-03223-w

Abstract

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

Highlights

Previous single-cell analyses have revealed transcriptomic clusters and linked them to neuron types with different projection patterns in a few particular brain regions[2,3,4,5]
The results suggest that agranular insular cortex (AI)–pons neurons are more distinct from AI–superior colliculus neurons and are similar to AI–thalamus neurons
We identified differences between both IT neurons projecting to different cortical areas and between L5 ET neurons projecting to different ET targets

Summary

Transcription factors

For each target pair, the performance of the predictive model varied among neurons from different source cortical regions (Fig. 2a, Extended Data Fig. 4a–d). These analyses suggest that epigenetic differences between CC projection neurons depend on a combination of both the specific targets to which neurons project and the sources where the neurons reside. Neurons projecting to the MOp versus neurons projecting to the ACA were more distinguishable (that is, had higher AUROC scores) than neurons projecting to the SSp versus those projecting to the ACA, we observed variation of the AUROC scores across different source regions for both target pairs (Fig. 2f, g).

Target g

Discussion

Methods

Findings

Code availability