Picking brains with new single-cell techniques

The brain is an extraordinarily complex organ, controlling physiological functions ranging from movement to thought. When it comes to brain biology, neurons are often the stars of the show. In fact, decades of research have characterized the genetic and functional diversity of neurons, giving scientists important insights into neurological mechanisms underlying normal and disease-associated brain functions. Despite the impacts of this work, the field has almost entirely neglected the role of glial cells in the brain. Glia make up nearly half the cells in the brain, functioning as support staff for neurons. Glial function varies widely – they can impact behavior ranging from learning and memory to motor control. In humans, glia can be broken down into subtypes like astrocytes, oligodendrocytes, and microglia. In the past, researchers have tried to tie glial function to their anatomical differences, but anatomically identical glia can exhibit very different patterns of gene expression depending on where they are located in the brain. Because of these distinctions, categorizing glial function based on anatomy alone has led to slow progress in the field.

To gain more insight into the function of glia, Drs. Aakanksha Singhvi and Manu Setty in the Basic Sciences division teamed up to tackle a massive project: sequencing the RNA in every glial cell in C. elegans, a nematode model for neurobiology. In C. elegans, animals with female chromosomes (technically hermaphrodites) have 56 glia and male animals have 82 glia. Only a few of these cells had been studied prior to Singhvi and Setty’s collaboration. “When we started this project…there were no molecular markers, no knowledge, no insight into most of the remaining glia,” explains Singhvi. Because of the sheer lack of knowledge and heterogeneity in glia, the team felt that interrogating the function of each glia one by one would be too slow. Therefore, they chose to perform single-nuclear RNA sequencing of each glial cell in male and female C. elegans animals.

The choice to profile male and female animals was very intentional. Prior to this work, there was much debate in the field on whether the same glial cell had different functions in men and women. Profiling glia from the two sexes could have important implication for understanding human disease. Singhvi highlights that the occurrence of neurological disorders varies highly between women and men. For example, men are twice as likely to develop Parkinson’s, and women are more likely to develop Alzheimer’s. This high-throughput approach to characterizing glia across sexes could give researchers new insights into molecular regulators of glial function.

Once the pair had isolated and sequenced the RNA of all C. elegans glia, they encountered an analysis problem. Typically, RNA sequencing data is clustered according to which cells express similar transcripts. These clusters are then named based on which transcripts are present or absent. For most cell types, decades of literature have established which genes distinguish them from other cell types, but this in-depth analysis simply does not yet exist for glial cells. “In this context, we did not have that kind of literature base because the glial cells are so understudied in general,” says Singhvi, “when you go in completely unbiased into a field, how do you derive meaning from the atlas you created?”

The answer came from Singhvi’s collaboration with Setty. Setty’s team was able to create a new analysis tool called PairDEx. Existing computational methods to cluster cells based on which genes they express are generally good at identifying which cells express which genes, but they are unable to determine if a particular gene is specific to a particular type of cell. “If you want to do any in vivo experiments, you want to know which genes are expressed and which genes are specifically expressed in a given cell type,” says Setty. In other words, for any follow up work to be guided by their single-cell atlas, they needed to be able to tie specific glial cells to a molecular signature. “Existing tools do well, but they don’t necessarily guarantee you specificity of expression. When you compare one cell type to others, you get a bunch of genes that are [expressed differently] in this cell type, but not specific to the cell type,” explains Setty. Instead of comparing one cluster to all other cells, PairDEx compares one cluster to all of the other clusters, one by one. This approach allows researchers to rank the genes that are specific to a particular glial cell type.

Once the team had their glial molecular signatures in hand, they validated their findings extensively in vivo. As predicted, they were able to identify different molecular signatures between male and female animals. Outside of the sex differences, they identified the expression of a molecule called NLP-16 in glial cells. NLP-16 is a member of an important family of signaling molecule in the brain called neuropeptides. While the glial cells produced NLP-16, they lacked the machinery that neurons typically use to release neuropeptides into the brain. This suggests that there are other release mechanisms for neuropeptides in glia that researchers have not yet identified. This is one surprising finding, but the team anticipates many more to come. They published their atlas for other researchers to interrogate, and this resource is already pushing science forward across institutions. Since the atlas was published, many scientists have reached out to tell the team that this resource helped them gain insights into targets that were previously dead ends. “I’m hoping that it spurs more people to have the resources to do what they need to do,” says Singhvi.

This work was supported by funding from the Washington Research Foundation, the National Institutes of Health, the National Science Foundation, the Simons Foundation, the Esther A. & Joseph Klingenstein Fund, the Brain Research Foundation, the Glenn Foundation, the American Federation for Aging Research, and philanthropic donors.

Fred Hutch/UW/Seattle Children’s Cancer Consortium members Drs. Manu Setty and Aakanksha Singhvi contributed to this work.

Purice MD, Quitevis EJA, Manning RS, Severs LJ, Tran NT, Sorrentino V, Finkbeiner C, Wu F, Zager M, Setty M, Singhvi A. 2025. Molecular profiling of adult C. elegans glia across sexes by single-nuclear RNA-seq. Dev Cell. S1534-5807(25)00324-7. doi: 10.1016/j.devcel.2025.05.013.