By Leo Barolo
Exploring the spatial distribution of cell types and transcriptional profiles is important for understanding how organs and tissues work. This is especially true for understanding the brain and how it functions.
Now, the Waisman Center at UW-Madison has purchased new technology to study the spatial organization of the brain and how that system is disrupted by disease. The technology will soon be available to all campus investigators through Waisman’s fee-for-service facilities.
Guided by the mission to advance knowledge of human development, intellectual and developmental disabilities, and neurodegenerative diseases, the Waisman Center is investing heavily in functional genomics on campus, including a “cluster” hiring initiative that brought in three new faculty.
The latest addition to their toolkit is the MERFISH system for spatial transcriptomics.
“We need to always stay at the cutting edge of technology,” explains Dr. Qiang Chang, Director of the Waisman Center and Professor of Medical Genetics and Neurology. “This is the logical next step to expand our functional genomics capability into spatial transcriptomics.”
MERFISH technology
MERFISH works through sequential rounds of single molecule RNA Fluorescence in situ hybridization (smFISH). A thin tissue section is exposed to fluorescently tagged probes that label RNA transcripts of interest. Counting the number of fluorescent spots can determine the number of molecules of that RNA in each region of the sample and enable spatially resolved, single-cell gene expression profiling. By using a combinatorial labeling approach and sequential rounds of smFISH imaging, the system can spatially resolve up to 1,000 different transcript sequences at single-cell resolution.
Advancing brain research on campus
Chang and others have a broad vision for the technology. Chang’s research on Rett syndrome will use MERFISH to expand upon information he previously obtained using bulk single-cell approaches.
He explains that by using standard single-cell RNA-seq technologies, “You know Cell Type A expressed Gene X, but you don’t know where in the tissue that Cell Type A is located. That is an important piece of information as it relates to function. Not only is cell type important, but also the location of a cell in the tissue has a direct implication for the function of gene expression.”
Dr. Xinyu Zhao, professor in the Department of Neuroscience and investigator in the Waisman Center, explains that spatially resolved information is particularly important for brain research, as the nervous system is hierarchically organized.
“The location is super important because there’s local connectivity and there’s also long or short-range connectivity. Where the gene expression happens, in what cells, and the location of these cells really makes a big difference in terms of their function,” she illustrates.
Zhao explains that spatial information can help inform which cells are talking to one another through ligand-receptor interactions.
“If when two cells are next to each other, one expresses a receptor and the other expresses its ligand – but cells located with a larger distance don’t express [those genes] even though they’re the same type of cells, then you can speculate that the local interaction [between the two adjacent cells] is important” she explains.
Zhao has multiple projects on mouse genetics in which she plans to incorporate MERFISH. One investigates how mutations in epigenetic regulators alter gene expression and impair neuronal development in the context of autism. MERFISH technology will allow her to know precisely where in the brain the gene expression differences are located.
Another project investigates how experiences such as chronic stress affect the parvalbumin interneurons, a cell type controlling brain network activities that is important for cognitive functions. However, these neurons only make up less than 2% of the total cells in the brain. Zhao explains that researchers can detect those cells in homogenized brain tissue analyzed by single-cell RNA sequencing, but the spatial information for those cells is largely missing.
“It turns out parvalbumin interneurons have several subtypes [that are indistinguishable by single-cell sequencing], but their locations and functions are different,” she clarifies. MERFISH will reveal stress-responsive mRNA expression patterns in those cells and their location in the brain.
“The location of the cells with changes in gene expression can have a significant impact on the development, circuit, and function of the brain.”
Dr. Xinyu Zhao
Comparing MERFISH to other campus technologies in spatial genomics
MERFISH adds to other spatial transcriptomics technologies on campus, including the 10X Genomics Visium system.
“The only caveat [of MERFISH] is you need to know which genes you want to look for. It’s not an unbiased profiling,” explains Dr. André Sousa, Assistant Professor in the Department of Neuroscience and part of the Waisman Center functional genomics cluster hiring initiative.
Since MERFISH is hybridization-based, probes must be designed for each gene of interest. Current panels can analyze up to 500 genes simultaneously, with the promise of 1000 in the upcoming months. Meanwhile, sequencing–based approaches such as Visium are unbiased and can cover larger tissue areas, but they lack single-cell resolution compared to MERFISH. “There are advantages and disadvantages in each of the methods,” concludes Sousa.
Sousa’s lab investigates the molecular and cellular mechanisms that govern human brain development and how that development evolved between humans and nonhuman primates. The lab creates functional-genomic profiles of post-mortem human brains analyzed at various stages of development and compares them with the profiles of nonhuman primates.
Once differentially expressed genes between humans and nonhuman primates are identified, Sousa looks for the differences in the regulation of those genes.
“A lot of the genes that have human-specific expression profiles are also associated with psychiatric disorders, so we are also interested in understanding how these differential expressions might make us more vulnerable to certain disorders,” he adds.
Sousa explains that spatial information will significantly expand our understanding of psychiatric disorders, as it’s currently unknown if dysfunction in those diseases is broad throughout the brain or localized to certain cell types in specific brain regions.
The Waisman Center expects the platform to be available campus-wide in the Spring at an affordable rate. The technology adds to several other fee-for-service facilities housed by the Center that advance brain studies and campus research more broadly.
Researchers interested in using the technology should connect to Karla Knobel (kknobel@wisc.edu), the Intellectual and Developmental Disabilities Models Core manager.