Single‐cell RNA Sequencing reveals molecular heterogeneity of glia within mouse sympathetic ganglia
Authors: van Weperen Valerie Yeh-Shing Hsieh , Contreras Jaime , Littman Russell , Ajijola Olujimi A. 
Published: April 2020

Stellate ganglion neurons, important mediators of cardiopulmonary neurotransmission, are surrounded by satellite glial cells (SGCs), which are essential for the maintenance and development of neurons. However, little is known about the heterogeneity and physiological functions of glia in adult sympathetic ganglia, and their role in modulating neurotransmission. Therefore, we performed single‐cell RNA sequencing of dissociated mouse stellate ganglia (n=8 animals), specifically focusing on the SGCs. SGCs were identified by high expression of glial specific transcripts, S100b and Fabp7 (n = 13370 cells). Microglia and Schwann cells were identified by expression of C1qa/C1qb/C1qc and Ncmap/Drp2, respectively, and excluded from further analysis. Subclustering of the SGCs revealed six distinct transcriptomic profiles. Expression levels of different glial marker genes were compared between these six subgroups to describe and study their distinct molecular profiles. The first subgroup was classified as glia‐progenitors (n = 1689; principle markers: Ng2 and Sox2). Pathway analysis demonstrated activation of pathways involved in self‐renewal of neural precursor cells and maintenance of radial glia cells. The second subgroup was identified as quiescent ‘astrocyte‐like’ glia (n = 3710; principle markers: Id3 and Aldoc). Accordingly, pathway analyses demonstrated active pathways involved in regulation of quantity and morphology of astrocytes. A third cluster (n = 2930) showed many similarities to the second cluster, but had a more reactive transcriptomic profile. Activated pathways included ones that were associated with astrocyte hypertrophy and quantity of reactive astrocytes. Hence, this cluster is referred to as the more active 'astrocyte‐like' cells and characterized by increased expression of the early response markers Mt1 and Klf2. The fourth cluster had a very distinct transcriptomic profile and had high expression levels of genes involved in the inflammatory response (n = 808; principle markers: Ifit3b and Gm4951). Correspondingly, pathways involved in immune response of cells and astrocytosis in the brain were enriched. Therefore, these cells may represent highly activated 'astrocyte‐like' SGCs. The fifth and sixth cluster were characterized by the expression of genes involved in axon development or maintenance and therefore showed more similarities with oligodendrocytes. These clusters were referred to as the 'oligodendrocyte‐like cells' and could be separated in myelinating ‘oligodendrocyte‐like’ cells (n = 1507; markers: Egr2 and Cryab) and non‐myelinating ‘oligodendrocyte‐like’ cells (n = 951; markers: Gap43 and Kcna1). Accordingly, both clusters showed much overlap in their activated pathways, most of which were associated with the cell cycle of oligodendrocytes or Schwann cells. However, pathways associated with cholesterol syntheses were downregulated in the sixth cluster. Our findings indicate transcriptomic heterogeneity within SGCs in mouse stellate ganglia and suggest functional subgroups that may subserve various physiological roles in health and disease.tSNE plot depicting the different subclusters of satellite glial cells within mouse sympathetic ganglia.Figure 1Cladogram depicting the relations between the different satellite glial cell subclusters. The different subclusters were characterised by their principle genetic markers.Figure 2