Figure 1. Single-cell RNA-seq identifies molecularly distinct types of Htr3a-GFP+ INs during development.

(a) Cortices and white matter from Htr3a-GFP+ mice were dissected at E18, P2 and P5. (b) Htr3a-GFP+ INs were isolated using fluorescence-activated cell sorting (FACS). (c) Individual Htr3a-GFP+ INs were captured in Fluidigm C1 chips and single cell RNA sequencing (RNA-seq) was performed. (n=89 cells at E18 in two chips; n=76 cells at P2 in one chip; n=78 cells at P5 in two chips). (d) Density clustering by Seurat-based⁴³ t-Distributed Stochastic Neighbor Embedding (t-SNE) identifies three distinct groups of Htr3a-GFP+ INs across postnatal time-points and an E18 group. (e) Cluster stability analysis using random sampling combined with principal component analysis (PCA) and hierarchical clustering reveals three robust types (colour-coded) of Htr3a-GFP+ INs at P2 and P5. (f) t-SNE analysis indicates that cells belonging to Htr3a-GFP+ interneuron types previously identified independently at P2 and P5 (colour-coded) cluster together. Cells are colour-coded according to cluster assignment obtained using cluster stability analysis. (g) Heat maps displaying top 50 type-enriched genes identified using single-cell differential display (SDCE)⁴⁴ at P2 and P5. (h) Violin plots reveal enriched expression of Meis2 in type 1 INs. (i) Time-course expression of interneuron-expressed transcription factors enriched in type 1 INs (Meis2, Etv1, Pbx1 and Sp8) and types 2 and 3 (Prox1, Maf, Npas3 and Nr2f2). SVZ: subventricular zone, IZ: intermediate zone, CTX: cortex, WM: white matter, mle: maximum likelihood estimates. Gene expression levels were obtained using the joint posterior estimation from the SCDE R package. Scale bars: (a) 150 μm at E18, 200 μm at P2 and P5.