Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons

Abstract

Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.

Fig. 1

sNuc-Seq identifies cell types in adult mouse brain. (A) Representative images of isolated nuclei are more uniform than of dissociated neurons from adult brain. Scale = 10μm. sNuc-Seq method (right): nuclei are isolated, FACS sorted and profiled using modified Smart-Seq2 (21). (B) Major cell types identified from sNuc-Seq data reflected by clusters, shown as 2-D embedding of 1,188 nuclei from adult mouse hippocampus. (C) Cluster-specific genes across single nuclei. Color bar matches cluster color in B. (D) Identification of DG granule cell, CA1, CA2, and CA3 pyramidal cell clusters, by marker genes, shown as: 1, ISH image in hippocampus section (10) (arrowhead: high expression; Scale = 400μm.); 2, histogram quantifying expression in relevant cluster; and 3, 2-D embedding of nuclei (as in B) colored by relative expression.

Fig. 2

sNuc-Seq and biSNE distinguish cell subtypes and spatial expression patterns. (A) Pyramidal CA1 and CA3 biSNE sub-clusters. Shown is a 2-D embedding of the CA1 and CA3 pyramidal nuclei (colored by cluster). Inserts: the CA1 cluster (orange) and CA3 cluster (green) within all other clusters from Fig. 1B. (B) Mapping of CA1 and CA3 pyramidal sub-clusters to sub-regions. Sub-cluster assignments are numbered and color coded as in A. Top: hippocampus schematic. (C) Predictions by CA1 and CA3 sub-cluster spatial mapping match with Allen ISH data (10). Left illustrations: boxes: predicted differential expression regions; arrowhead: high expression; asterisk: low expression. (D) Mutually exclusive expression of Penk (facing up) and its receptor Oprd1 (facing down) across neuronal sub-clusters. Red line: median, box: 75% and 25% quantile. Single and double asterisks: GABergic clusters associated with Pvalb or Vip markers, respectively. (E) co-FISH of Penk or Oprd with markers of GABAergic sub-types (Pvalb and Vip as in D). Arrowheads: co-expression. Scale = 20μm.

Fig. 3

Transcriptional dynamics of adult neurogenesis by Div-Seq. (A) Div-Seq. EdU is injected into adult mice and incorporates into dividing cells (5), isolated EdU labeled nuclei are fluorescently tagged and captured by FACS for sNuc-Seq. (B) Adult neurogenesis in the DG (4). Tan box: timing of EdU labeling. Bottom panel: EdU labeling and tissue dissection (grey) time course. (C) A continuous trajectory of newborn cells in the DG. biSNE 2-D embedding of neuronal lineage nuclei (n = 269). Arrow: direction of trajectory determined by labeling time and marker expression. Top: Colored by labeling time (1–14 days). Bottom: Expression of markers, shown as: 1, 2-D embedding colored by the expression level; 2, average expression along the trajectory. Markers (clockwise from top left): Sox9 (NSC), Notch1 (proliferation/differentiation), Neurod1 (immature neurons), Eomes/Tbr2 (neuronal precursor). (D) Expression waves along the trajectory. Left: average expression of cluster genes along the trajectory. Middle: heatmap of average expression of each gene along the trajectory and neurogenic stages (labeled as in B). Right: representative enriched biological pathways.

Fig. 4. Dynamics of adult newborn GABAergic neurons in SC

(A) Div-Seq in SC captures OPCs and immature neurons. Distribution of cell types in non-EdU-labeled and 6–7 days EdU labeled nuclei. (B) Div-Seq captured nuclei expressing marker genes of immature (Sox11) and GABAergic (Gad1) neurons. Box plots for immature neurons, mature neurons and OPCs. Red: median, box: 75% and 25% quantiles. (C) Newborn cells in SC form a continuous trajectory. 2-D embedding of 1–7 days EdU labeled and non-labeled nuclei (n=184, neuronal lineage nuclei), colored by labeling time. Trajectory directionality is EdU labeling time and marker genes. (D) Dynamically expressed genes shared in SC and DG neurogenesis (347 genes from fig. S22B and Fig. 3D). (E) Gradual transition from a glia-like to neuronal state. Neuronal trajectories in the SC (as in C) and DG (as in Fig. 3C) colored by a glia-neuron RNA expression score. (F) Region specific gene expression in immature neurons (6–7 days post EdU). 236 genes differentially expressed between SC and DG (t-test FDR<0.05, log-ratio>1), in olfactory bulb (OB), SC and DG. Box: average expression of example genes up-regulated in OB and SC compared to DG.