LA JOLLA (December 14, 2023)—Salk Institute researchers, as part of a worldwide initiative to revolutionize scientists' understanding of the brain, analyzed more than 2 million brain cells from mice to assemble the most complete atlas ever of the mouse brain. Their work, published December 14, 2023 in a special issue of Nature, not only details the thousands of cell types present in the brain but also how those cells connect and the genes and regulatory programs that are active in each cell.
The efforts were coordinated by the National Institutes of Health's Brain Research Through Advancing Innovative Neurotechnologies® Initiative, or the BRAIN Initiative®, which ultimately aims to produce a new, dynamic picture of mammalian brains.
"With this work, we have not only gained a lot of information about what cells make up the mouse brain, but also how genes are regulated within those cells and how that drives the cells' functions," says Salk Professor, International Council Chair in Genetics, and Howard Hughes Medical Institute Investigator Joseph Ecker, who contributed to four of the new papers. "When you take this epigenome-based cell atlas and start to look at genetic variants that are known to cause human disease, you get new insight into what cell types may be most vulnerable in the disease."
The NIH BRAIN Initiative was launched in 2014 and has provided more than $3 billion in funding to researchers to develop transformative technologies and apply them to brain science.
In 2021, researchers supported by the BRAIN Initiative—including teams at Salk—unveiled the first draft of the mouse brain atlas, which pioneered new tools to characterize neurons and applied those tools to small sections of the mouse brain. Earlier this year, many of the same techniques were used to assemble an initial atlas of the human brain. In the latest work, researchers expanded the number of cells studied and which areas of the mouse brain were included, as well as used new, single-cell technologies that have only emerged in the last few years.
"This is the entire brain, which hasn't been done before," says Professor Edward Callaway, a senior author on two of the new papers. "There are ideas and principles that come out of looking at the whole brain that you don't know from looking at one part at a time."
To help assist other researchers studying the mouse brain, the new data is publicly available through an online platform, which can not only be searched through a database but also queried using the artificial intelligence tool ChatGPT.
"There is an incredibly large community of people who use mice as model organisms and this gives them an incredibly powerful new tool to use in their research involving the mouse brain," adds Margarita Behrens, a Salk research professor who was involved in all four new papers.
The special issue of Nature has 10 total NIH BRAIN Initiative articles, including four co-authored by Salk researchers that describe the cells of the mouse brain and their connections. Some highlights from these four papers include:
Single-cell DNA methylation atlas
To determine all the cell types in the mouse brain, Salk researchers employed cutting-edge techniques that analyze one individual brain cell at a time. These single-cell methods studied both the three-dimensional structure of DNA inside cells and the pattern of methyl chemical groups attached to the DNA—two different ways that genes are controlled by cells. In 2019, Ecker's lab group pioneered approaches to simultaneously make these two measurements, which lets researchers work out not only which genetic programs are activated in different cell types, but also how these programs are being switched on and off.
The team found examples of genes that were activated in different cell types but through different ways—like being able to flip a light on or off with two different switches. Understanding these overlapping molecular circuits makes it easier for researchers to develop new ways of intervening in brain diseases.
"If you can understand all the regulatory elements that are important in these cell types, you can also begin to understand the developmental trajectories of the cells, which becomes critical to understanding neurodevelopmental disorders like autism and schizophrenia," says Hanqing Liu, a postdoctoral researcher in Ecker's lab and first author of this paper.
The researchers also made new discoveries about which areas of the brain contain which cell types. And when cataloguing those cell types, they additionally found that the brain stem and midbrain have far more cell types than the much larger cortex of the brain—suggesting that these smaller parts of the brain may have evolved to serve more functions.
Other authors of this paper include Qiurui Zeng, Jingtian Zhou, Anna Bartlett, Bang-An Wang, Peter Berube, Wei Tian, Mia Kenworthy, Jordan Altshul, Joseph Nery, Huaming Chen, Rosa Castanon, Jacinta Lucero, Julia Osteen, Antonio Pinto-Duarte, Jasper Lee, Jon Rink, Silvia Cho, Nora Emerson, Michael Nunn, Carolyn O'Connor, and Jesse Dixon of Salk; Yang Eric Li, Songpeng Zu, and Bing Ren of UC San Diego; Zhanghao Wu and Ion Stoica of UC Berkley; Zizhen Yao, Kimberly Smith, Bosiljka Tasic, and Hongkui Zeng of the Allen Institute; and Chongyuan Luo of UC Los Angeles.
Single-cell chromatin maps
Another way of indirectly determining the structure of DNA, and which stretches of genetic material are being actively used by cells, is testing what DNA is physically accessible to other molecules that can bind to it. Using this approach, called chromatin accessibility, researchers led by Bing Ren of UC San Diego—including Salk's Ecker and Behrens—mapped the structure of DNA in 2.3 million individual brain cells from 117 mice.
Then, the group used artificial intelligence to predict, based on those patterns of chromatin accessibility, which parts of DNA were acting as overarching regulators of the cells' states. Many of the regulatory elements they identified were in stretches of DNA that have already been implicated in human brain diseases; the new knowledge of exactly which cell types use which regulatory elements can help pin down which cells are implicated in which diseases.
Other authors of this paper include co-first authors Songpeng Zu, Yang Eric Li, and Kangli Wang of UC San Diego; Ethan Armand, Sainath Mamde, Maria Luisa Amaral, Yuelai Wang, Andre Chu, Yang Xie, Michael Miller, Jie Xu, Zhaoning Wang, Kai Zhang, Bojing Jia, Xiaomeng Hou, Lin Lin, Qian Yang, Seoyeon Lee, Bin Li, Samantha Kuan, Zihan Wang, Jingbo Shang, Allen Wang, and Sebastian Preissl of UC San Diego, Hanqing Liu, Jingtian Zhou, Antonio Pinto-Duarte, Jacinta Lucero, Julia Osteen, and Michael Nunn of Salk; and Kimberly Smith, Bosiljka Tasic, Zizhen Yao, and Hongkui Zeng of the Allen Institute.
Neuron projections and connections
In another paper, co-authored by Behrens, Callaway, and Ecker, researchers mapped connections between neurons throughout the mouse brain. Then, they analyzed how these maps compared to patterns of methylation within the cells. This let them discover which genes are responsible for guiding neurons to which areas of the brain.
"We discovered certain rules dictating where a cell projects to based on their DNA methylation patterns," says Jingtian Zhou, a postdoctoral researcher in Ecker's lab and co-first author of the paper.
The connections between neurons are critical to their function and this new set of rules may help researchers study what goes awry in diseases.
Other authors of this paper include co-first author Zhuzhu Zhang of Salk; May Wu, Hangqing Liu, Yan Pang, Anna Bartlett, Wubin Ding, Angeline Rivkin, Will Lagos, Elora Williams, Cheng-Ta Lee, Paula Assakura Miyazaki, Andrew Aldridge, Qiurui Zeng, J. L. Angelo Salida, Naomi Claffey, Michelle Liem, Conor Fitzpatrick, Lara Boggeman, Jordan Altshul, Mia Kenworthy, Cynthia Valadon, Joseph Nery, Rosa Castanon, Neelakshi Patne, Minh Vu, Mohammed Rashid, Matthew Jacobs, Tony Ito, Julia Osteen, Nora Emerson, Jasper Lee, Silvia Cho, Jon Rink, Hsiang-Hsuan Huang, António Pinto-Duarte, Bertha Dominguez, Jared Smith, Carolyn O'Connor, and Kuo-Fen Lee of Salk; Zhihao Peng of Nanchang University in China; Zizhen Yao, Kimberly Smith, Bosiljka Tasic, and Hongkui Zeng of the Allen Institute; Shengbo Chen of Henan University in China; Eran Mukamel of UC San Diego; and Xin Jin of East China Normal University in China and New York University Shanghai.
Comparing mouse, monkey, and human motor cortexes
The motor cortex is the part of the mammalian brain involved in the planning and carrying out of voluntary body movements. Researchers led by Behrens, Ecker, and Ren studied the methylation patterns and DNA structure in more than 200,000 cells from the motor cortexes of humans, mice, and nonhuman primates to better understand how motor cortex cells have changed throughout human evolution.
They were able to identify correlations between how particular regulatory proteins have evolved and how, in turn, the expression patterns of genes evolved. They also discovered that nearly 80 percent of the regulatory elements that are unique to humans are transposable elements—small, mobile sections of DNA that can easily change position within the genome.
Other authors of this paper include co-first authors Nathan Zemke and Ethan Armand of UC San Diego; Wenliang Wang, Jingtian Zhou, Hanqing Liu, Wei Tian, Joseph Nery, Rosa Castanon, Anna Bartlett, Julia Osteen, Jonathan Rink, and Edward Callaway of Salk; Seoyeon Lee, Yang Eric Li, Lei Chang, Keyi Dong, Hannah Indralingam, Yang Xie, and Michael Miller of UC San Diego; Daofeng Li, Xiaoyu Zhuo, Vincent Xu, and Ting Wang of Washington University in Missouri; Fenna Krienen of Princeton University and Harvard Medical School; Qiangge Zhang and Guoping Feng of the Broad Institute and MIT; Steven McCarroll of Harvard Medical School and the Broad Institute; and Naz Taskin, Jonathan Ting, and Ed Lein of the Allen Institute and University of Washington in Seattle.
Summary
"I think in general this whole package serves as a blueprint for other people's future studies," says Callaway, also the Vincent J. Coates Chair in Molecular Neurobiology at Salk. "Someone studying a particular cell type can now look at our data and see all the ways those cells connect and all the ways they're regulated. It's a resource that allows people to ask their own questions."
The work was supported by the National Institutes of Health BRAIN Initiative (U19MH11483, U19MH114831-04s1, 5U01MH121282, UM1HG011585, U19MH114830).
About the Salk Institute for Biological Studies:
Unlocking the secrets of life itself is the driving force behind the Salk Institute. Our team of world-class, award-winning scientists pushes the boundaries of knowledge in areas such as neuroscience, cancer research, aging, immunobiology, plant biology, computational biology, and more. Founded by Jonas Salk, developer of the first safe and effective polio vaccine, the Institute is an independent, nonprofit research organization and architectural landmark: small by choice, intimate by nature, and fearless in the face of any challenge. Learn more at www.salk.edu.
