Multi-Scale molecular deconstruction of the serotonin neuron system

Systematic analysis and integration of multiple cellular characteristics at single-cell resolution within each raphe nucleus is still lacking.

“Pioneering work has introduced the molecular diversity of serotonin neurons across the midbrain and hindbrain (Okaty et al., 2015; Spaethling et al., 2014; Wylie et al., 2010), yet systematic analysis and integration of multiple cellular characteristics at single-cell resolution within each raphe nucleus is still lacking.”

Okaty et al. used intersectional genetic fate mapping to produce population RNA-seq data (and single-cell RNA-seq data from a small number of cells) from DR, MR, and caudal raphe nuclei derived from different rhombomeres during development.

“Okaty et al. used intersectional genetic fate mapping to produce population RNA-seq data (and single-cell RNA-seq data from a small number of cells) from DR, MR, and caudal raphe nuclei derived from different rhombomeres during development.”

Our results are broadly consistent with previous findings that utilized developmental origin to differentiate raphe serotonin neurons (Okaty et al., 2015).

“Our results are broadly consistent with previous findings that utilized developmental origin to differentiate raphe serotonin neurons (Okaty et al., 2015).”

Because DR serotonin neurons derive exclusively from rhombomere 1 (R1), and the single-cell RNA-seq data was obtained from just 8 cells, the Okaty et al. dataset is limited in providing information about heterogeneity within DR serotonin neurons.

“Because DR serotonin neurons derive exclusively from rhombomere 1 (R1), and the single-cell RNA-seq data was obtained from just 8 cells, the Okaty et al. dataset is limited in providing information about heterogeneity within DR serotonin neurons.”

They confirmed the in situ hybridization data of Okaty et al. that Met is expressed in the cDR and MR.

“Nevertheless, we confirmed the in situ hybridization data of Okaty et al. that Met is expressed in the cDR and MR.”

Comparing marker genes enriched in R1-R3-derived serotonin neurons from Okaty et al. and MR-1-MR-4 transcriptomic clusters from current study suggests an approximate correspondence: R1-derived serotonin neurons correspond to MR-1 cluster, R2 to MR-4 cluster, and R3 to both MR-2 and MR-3 clusters.

“Comparing marker genes enriched in R1–R3-derived serotonin neurons from Okaty et al. and MR-1–MR-4 transcriptomic clusters from our current study (Figure 3—figure supplement 4) indeed suggests an approximate correspondence: R1-derived serotonin neurons correspond to our MR-1 cluster, R2 to MR-4 cluster, and R3 to both MR-2 and MR-3 clusters.”

MR serotonin neurons derive from three separate rhombomeres (R1-R3), and Okaty et al. sampled these three populations separately, allowing the question of whether developmental origins correspond to transcriptomic clusters in adult.

“The MR serotonin neurons derive from three separate rhombomeres (R1–R3), and Okaty et al. sampled these three populations separately, allowing us to ask the question of whether developmental origins correspond to transcriptomic clusters in adult.”

Inspection of rhombomere-specific marker gene expression in the 5-HT neuron cluster showed a lack of markers for R2 (Hoxa2) and R3/5 (Egr2) in contrast to expression of R1 markers (En1 and En2), indicating that the dissection excluded most of the median raphe nucleus.

“Inspection of rhombomere-specific marker gene expression in the 5-HT neuron cluster showed a lack of markers for R2 (Hoxa2) and R3/5 (Egr2) derived neurons in contrast to the expression of R1 markers (En1 and En2), indicating that our dissection, as intended, excluded most of the median raphe nucleus (Okaty et al., 2015).”

Some DRN neurons co-express genes traditionally used as markers for different neuronal cell types based on neurotransmitter usage, such as Gad1, Gad2, Slc17a6, and Slc17a8.

“Some DRN neurons co-express genes that are traditionally used as markers for different neuronal cell types based on their neurotransmitter usage (e.g. Gad1, Gad2, Slc17a6, and Slc17a8) (Fu et al., 2010; Okaty et al., 2015).”