Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression

PLoS One. 2018 Oct 22;13(10):e0205883. doi: 10.1371/journal.pone.0205883. eCollection 2018.


The vertebrate pineal gland is dedicated to the production of the hormone melatonin, which increases at night to influence circadian and seasonal rhythms. This increase is associated with dramatic changes in the pineal transcriptome. Here, single-cell analysis of the rat pineal transcriptome was approached by sequencing mRNA from ~17,000 individual pineal cells, with the goals of profiling the cells that comprise the pineal gland and examining the proposal that there are two distinct populations of pinealocytes differentiated by the expression of Asmt, which encodes the enzyme that converts N-acetylserotonin to melatonin. In addition, this analysis provides evidence of cell-specific time-of-day dependent changes in gene expression. Nine transcriptomically distinct cell types were identified: ~90% were classified as melatonin-producing α- and β-pinealocytes (1:19 ratio). Non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular and leptomeningeal cells, and endothelial cells. α-Pinealocytes were distinguished from β-pinealocytes by ~3-fold higher levels of Asmt transcripts. In addition, α-pinealocytes have transcriptomic differences that likely enhance melatonin formation by increasing the availability of the Asmt cofactor S-adenosylmethionine, resulting from increased production of a precursor of S-adenosylmethionine, ATP. These transcriptomic differences include ~2-fold higher levels of the ATP-generating oxidative phosphorylation transcriptome and ~8-fold lower levels of the ribosome transcriptome, which is expected to reduce the consumption of ATP by protein synthesis. These findings suggest that α-pinealocytes have a specialized role in the pineal gland: efficiently O-methylating the N-acetylserotonin produced and released by β-pinealocytes, thereby improving the overall efficiency of melatonin synthesis. We have also identified transcriptomic changes that occur between night and day in seven cell types, the majority of which occur in β-pinealocytes and to a lesser degree in α-pinealocytes; many of these changes were mimicked by adrenergic stimulation with isoproterenol. The cellular heterogeneity of the pineal gland as revealed by this study provides a new framework for understanding pineal cell biology at single-cell resolution.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylserotonin O-Methyltransferase / metabolism
  • Adenosine Triphosphate / chemistry
  • Animals
  • Cluster Analysis
  • Female
  • Gene Expression Profiling*
  • Gene Expression Regulation, Enzymologic*
  • Male
  • Melatonin / metabolism
  • Pineal Gland / cytology*
  • Pineal Gland / metabolism
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Seasons
  • Sequence Analysis, RNA*
  • Serotonin / analogs & derivatives
  • Serotonin / metabolism
  • Transcriptome


  • RNA, Messenger
  • Serotonin
  • Adenosine Triphosphate
  • Acetylserotonin O-Methyltransferase
  • Melatonin
  • N-acetylserotonin

Grants and funding

This work was supported by the Intramural Research Programs of the National Institute of National Institute on Deafness and Other Communication Disorders (to J.C.M., M.C.K, and M.W.K) ( and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (to L.H., S.L.C., and D.C.K.) (, by Novo Nordisk Foundation (grants NNF15OC0015988 and NNF170C0026938, to M.F.R.)(, Lundbeck Foundation (grant R108-A10301, to M.F.R.)(, Carlsberg Foundation (grants CF15-0515 and CF17-0070, to M.F.R.)(, Independent Research Fund Denmark (grant 8020-000378, to M.F.R.)( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.