Biology subjects

Keeler, A. B.

Publications and source records attributed to Keeler, A. B..

3 recordsLinked to original sources

bioRxiv2022

A developmental atlas of the mouse brain by single-cell mass cytometry

Development of the mammalian brain requires precisely controlled differentiation of neurons, glia, and nonneural cells. To investigate protein-level changes in these diverse cell types and their progenitors, we performed single-cell mass cytometry on whole brain (E11.5/E12.5) and microdissected telencephalon, diencephalon, mesencephalon, and rhombencephalon (E13.5-P4) collected at daily timepoints from C57/BL6 mice. Measuring 24,290,787 cells from 112 sample replicates with a 40-antibody panel, we quantified 85 molecularly distinct cell populations across embryonic and postnatal development, including microglia putatively phagocytosing neurites, neural cells, and myelin. Differentiation trajectory analysis also identified two separate pathways for producing oligodendrocyte precursor cells. Comparison with previous studies revealed considerable discrepancies between protein and mRNA abundances in the developing brain, demonstrating the value of protein-level measurements for identifying functional cell states. Overall, our findings demonstrate the utility of mass cytometry as a high-throughput, scalable platform for single-cell profiling of brain tissue.

neuroscience↗

bioRxiv2022

A developmental atlas of somatosensory diversification and maturation in the dorsal root ganglia by single-cell mass cytometry

Precisely controlled development of the somatosensory system is essential for detecting pain, itch, temperature, mechanical touch, and body position. To investigate the protein-level changes that occur during somatosensory development, we performed single-cell mass cytometry on dorsal root ganglia from C57/BL6 mice, with litter replicates collected daily from E11.5 to P4. Measuring nearly 3 million cells, we quantified 30 molecularly distinct somatosensory glial and 41 distinct neuronal states across all time points. Analysis of differentiation trajectories revealed rare cells that coexpress two or more Trk receptors and overexpress stem cell markers, suggesting that these neurotrophic factor receptors play a role in cell fate specification. Comparison to previous RNA-based studies identified substantial differences between many protein/mRNA pairs, demonstrating the importance of protein-level measurements to identify functional cell states. Overall, this study demonstrates that mass cytometry is a high-throughput, scalable platform to rapidly phenotype somatosensory tissues.

neuroscience↗

bioRxiv2022

Isolation and Characterization of Sympathetic Extracellular Vesicles

Neuronal derived extracellular vesicles (EVs) have been well described in the central nervous system; however, studies in the peripheral nervous system have largely focused on EVs derived from supporting cell types such as endothelial cells or glia. Here we isolate EVs derived from sympathetic neurons and characterize them using immunoblot assays, nanoparticle tracking analysis and cryo-electron microscopy. Sizing of sympathetic EVs reveal a predominant peak between 45-75 nm as well as a range of larger sizes (90 nm to >350 nm), possibly due to multiple biogenic origins. We identified TrkA, a receptor for nerve growth factor (NGF), as a cargo for sympathetic EVs. Furthermore, TrkA on EVs was phosphorylated, indicating activated TrkA receptor. TrkA binds NGF at the axonal tip and is endocytosed and transported to the soma in signaling endosomes. We therefore examined if TrkA originating in the axon tip was subsequently able to be packaged into EVs and secreted by the somatodendritic domain of neurons. Using a compartmentalized culture system, we found that TrkA derived from endosomes originating in the distal axon can be detected on EVs secreted from the somatodendritic domain. In addition, inhibition of classic TrkA downstream pathways, specifically in somatodendritic compartments greatly decreases TrkA packaging into EVs. Our results suggest a novel trafficking route for TrkA: it can travel long distances to the cell body, be packaged into EVs and secreted. Secretion of TrkA via EVs appears to be regulated by its own downstream effector cascades, raising intriguing future questions about novel functionalities associated with TrkApositive EVs.

neuroscience↗