Summary: Collaborative work on the brain atlas describes how different cells are organized and connected throughout the mouse brain. Understanding what differentiates brain cells can lead to new research and potential therapies for brain disorders.
Source: Salk Institute
It takes billions of cells to make a human brain, and scientists have long struggled to map this complex network of neurons. Now, dozens of research teams around the country, led in part by Salk scientists, have made inroads into creating an atlas of the mouse brain as a first step toward a human brain atlas.
The researchers, collaborating as part of the National Institute of Health’s BRAIN Initiative Cell Census Network (BICCN), report the new data today in a special issue of the journal Nature. The results describe how different cell types are organized and connected throughout the mouse brain.
“Our first goal is to use the mouse brain as a model to really understand the diversity of cells in the brain and how they’re regulated,” says Salk Professor and Howard Hughes Medical Institute Investigator Joseph Ecker, co-director of the BICCN. “Once we’ve established tools to do this, we can move to working on primate and human brains.”
The NIH Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative is “a large-scale effort that seeks to deepen understanding of the inner workings of the human mind and to improve how we treat, prevent and cure disorders of the brain.” Since its initial funding in 2014, the BRAIN Initiative has awarded more than $1.8 billion in research awards.
The BICCN, one subset of the BRAIN Initiative, specifically focuses on creating brain atlases that describe the full plethora of cells—as characterized by many different techniques—in mammalian brains. Salk is one of three institutions that were given U19 awards to act as central players in generating data for the BICCN.
“This is not just a phone book for the brain,” says Margarita Behrens, a Salk associate research professor who helped lead the new BICCN papers. “In the long run, to treat brain diseases, we need to be able to hone in on exactly which cell types are having trouble.”
The special issue of Nature has 17 total BICCN articles, including five co-authored by Salk researchers that describe approaches to studying brain cells and new characterizations of subtypes of brain cells in mice. Some highlights include:
DNA Methylation Analysis
While other papers in the special issue relate to the function or structure of mouse brain cells, the work led by Ecker, Behrens and their colleagues largely focuses on the epigenomics of brain cells in mice. Every cell in a mouse brain contains the same sequence of DNA, but variations in how this DNA is regulated—its so-called “epigenome”—give cells their unique identity.
The arrangement of methyl chemical groups on the cytosine base in DNA (known as “cytosine methylation”), which specifies when genes are to be turned on or off, are one form of epigenomic regulation that may highly influence disease and health in the brain.
In one of the new papers, the Salk team analyzed 103,982 mouse brain cells using single-cell DNA methylation sequencing. This approach, developed in the Ecker lab, lets researchers study the pattern of methyl chemical groups on each strand of DNA in brain cells.
When they applied the technique to the thousands of cells collected from 45 different regions of the mouse brain, they were able to identify 161 clusters of cell types, each distinguished by their pattern of methylation.
“Before now, there have been a handful of ways to describe brain cells based on their location or their electrical activity,” says Hanqing Liu, a graduate student in the Ecker lab and co-first author of the paper. “We’ve really extended the definition of cell type here and used epigenomics to define hundreds of potential cell types.”
The team went on to show that the methylation patterns could be used to predict where in the brain any given cell came from—not just within broad regions but down to specific layers of cells within a region. This means that eventually, drugs could be developed that act only on small groups of cells, by targeting their unique epigenomics.
Neuron Destination Patterns
In another paper, co-authored by Ecker and Salk Professor Edward Callaway, researchers studied the association between DNA methylation and neural connections. The team developed a new way of isolating cells that connect regions of the brain, then studying their methylation. They used the approach on 11,827 individual mouse neurons, all extending outward from the mouse cortex. T
The patterns of methylation in the cells, they discovered, correlated with cells’ projection (destination) patterns. Neurons that led from the motor cortex to the striatum, for instance, had distinct epigenomics from neurons that connected the primary visual cortex and the thalamus.
“Neurons don’t function in isolation, they function by communicating with each other, so understanding how these connections are established and how they work is really fundamental to understanding the brain,” says Zhuzhu Zhang, a Salk postdoctoral fellow and a co-first author of the paper with graduate student Jingtian Zhou, both members of Ecker’s laboratory.
The researchers say that the new data on the mouse brain cells is merely the first step in creating a complete atlas of the mouse brain—let alone the human brain. But understanding what differentiates cell types is critical to future research and future brain therapeutics.
“In these foundational studies, we’re describing the ‘parts list’ for the brain,” says Callaway. “Having this parts list is revolutionary, and will open up a whole new set of opportunities for studying the brain.”
Hanqing Liu and Jingtian Zhou, both of Salk, were co-first authors on the DNA methylation atlas paper; Zhuzhu Zhang and Jingtian Zhou, also both of Salk, were co-first authors on the cortical projection paper.
Funding: The methylation atlas work was supported by the National Institutes of Mental Health (U19MH11483), the National Human Genome Research Institute (R01HG010634) and the Howard Hughes Medical Institute.
The cortical projection paper was supported by the National Institute of Mental Health (U19MH114831and R01MH063912), the National Eye Institute (R01EY022577 and F31 EY028853) and the Howard Hughes Medical Institute.
DNA Methylation Atlas of the Mouse Brain at Single-Cell Resolution
Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood.
Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas.
We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients.
Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data enabled prediction of high-confidence enhancer–gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments.
By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.
Epigenomic Diversity of Cortical Projection Neurons in the Mouse Brain
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 brain, 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.
A multimodal cell census and atlas of the mammalian primary motor cortex
Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis.
Our results advance the collective knowledge and understanding of brain cell-type organization.
First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps.
Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human.
Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex.
Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types.
We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function.
Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.