Eli Zunder’s New National Institutes of Health RO1 Award
Our somatosensory nervous system enables people to gauge their relationship to the world around them. Thanks to the messages it carries, we feel sensations such as temperature, pressure, heat, and pain. It is the key not only to our physical interactions, but also to our social ones.
Much about how the somatosensory nervous system develops remains mysterious, however. We know that the sensory neurons of this system emerge from a pool of neural crest progenitor cells that migrate during embryonic development and coalesce into dorsal root ganglia—nerve clusters on the left and right side of each vertebrae along the spinal cord. Ultimately, axons from these sensory neurons will infiltrate virtually every part of the body and help us sense the world around us.
A key question that remains unanswered is which factors control the development of specific sensory neuron types. It has long been assumed that developing sensory neurons are pre-programmed from the start, but Eli Zunder, an assistant professor of biomedical engineering, is among those challenging this assumption.
This is important, because the number of touch, pain, heat and cooling and position receptors we end up with calibrates our sensory perception. Dysregulation of this development process contributes to pain and sensory disorders.
Zunder hypothesizes that secreted molecules from the nerve target tissue called neurotrophic factors can contribute to cell specification decisions in the dorsal root ganglia. “These factors are known to control cell death and survival decisions, but we think they contribute to cell lineage decisions as well,” said Zunder.
Zunder recently received a $2.5 million grant from the National Institute of Neurological Disorders and Stroke in the National Institutes of Health to investigate how the interplay of different trophic factors and death signaling factors contributes to sensory neuron specification. The findings from this study could ultimately help researchers find better ways to regenerate damaged nerve tissue and produce more effective non-opioid painkillers
Teasing out the process of sensory neuron differentiation is complicated by the fact that each dorsal root ganglia is packed with several different cell types in close proximity to each other. To overcome this issue and study the genesis of each cell type individually, Zunder and his colleagues perform single cell analysis on embryonic and postnatal tissues. “Our goal is to identify all the cell types present in the dorsal root ganglia and then track how their molecular profiles change over the course of development,” Zunder said.
A key advantage of their experimental approach is the ability to monitor cell signaling pathway activation simultaneously with markers of cell identity. This multiplexed approach promises to create a molecular roadmap of dorsal root ganglia development, while also providing the underlying signaling behavior that controls these cell fate decisions.
“We use things like transcription factors, neural filaments and cell surface proteins to define cell identity,” Zunder said. “What’s really interesting is to look at internal cell state at the same time. We know that many of the signaling pathways activated by cell surface receptors are interconnected with each other, and we have the ability to visualize this interplay in every cell type across development.”
Zunder and colleagues are particularly interested in understanding the interplay between neurotrophic factor signaling and death factor signaling—an axis they term a trophic “rheostat”—under different cell contexts in the dorsal root ganglia.
To monitor how the balance of these trophic rheostats changes in different cellular contexts, Zunder will rely on single-cell mass cytometry, a mass spectroscopy technique capable of measuring over 40 molecular markers per cell at the rate of one million cells per hour. This technique is ideally suited to analyze blood, where cells are individually suspended in fluid. It’s more problematic for solid tissues like dorsal root ganglia, because the cells are so tightly interwoven that it is difficult to physically separate individual neurons.
As a postdoctoral fellow at Stanford University, Zunder helped develop a protocol that uses digestive enzymes and mechanical forces to tease neurons apart and a fixative to “freeze” their molecular status.
“One of the challenges we faced in this project was that typical dissociation protocols for this type of tissue take around an hour,” Zunder said. “We worried that by the time we applied fixative, the stress signaling that came from pulling the cells apart might have already obscured the relevant developmental signaling.” The team’s solution is to culture dorsal root ganglia cells in vitro, which allows them to dissociate and freeze individual neurons in just 30 seconds.
As they develop their map of in vivo dorsal root ganglia cell development trajectories, Zunder and his colleagues will test their conclusions against in vitro dorsal root ganglia cells to confirm if the cell signaling states they observe actually produce specific cell differentiation patterns. Toward this end, they will use knockout mouse models and specific signaling pathway inhibitors to confirm which pathways actually affect cell differentiation in the developing dorsal root ganglia.
“The ultimate test of our work is learning enough so that we can control the development of specific sensory neurons,” Zunder said. “Our hope is that this work will provide a strong foundation for future medical breakthroughs in fields like pain management and regenerative medicine.”