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. 2017 Nov 16;171(5):1206-1220.e22.
doi: 10.1016/j.cell.2017.10.019.

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing

Hongjie Li  1 , Felix Horns  2 , Bing Wu  1 , Qijing Xie  3 , Jiefu Li  1 , Tongchao Li  1 , David J Luginbuhl  1 , Stephen R Quake  4 , Liqun Luo  5
Affiliations

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing

Hongjie Li et al. Cell. .

Abstract

The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes of closely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.

Keywords: Drosophila; Single-cell RNA-seq; cell type; combinatorial code; connectivity; development; neuronal identity; olfactory system; projection neuron; transcriptome.

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Figures

Figure 1.
Figure 1.. Single-cell RNA-seq Protocol for the Drosophila Pupal Brain
(A) Schematic of fly olfactory system organization. Olfactory receptor neurons (ORNs) expressing the same odorant receptor (same color) target their axons to the same glomerulus in the antennal lobe. Projection neuron (PN) dendrites also target single glomeruli, and their axons project to the mushroom body (MB) and lateral horn (LH). (B) Schematic of single-cell RNA-seq protocol. (C) Representative confocal images of Drosophila central brains labeled by UAS-mCD8GFP crossed with PN driver GH146-GAL4 (24h APF) or astrocyte driver alrm-GAL4 (72h APF). N-cadherin (Ncad, red) staining labels neuropil. Scale, 50 μm. (D) Heat map showing expression levels of genes that are specific for neurons or astrocytes. Each column is an individual cell. 67 alrm-GAL4+ and 946 GH146-GAL4+ cells are shown, with driver indicated by the color above. Cell type-specific genes are enriched in astrocytes (top 9) and PNs (bottom 5). Expression levels are indicated by the color bar (CPM, counts per million). Cells and genes were ordered using hierarchical clustering. (E) Visualization of astrocyte and PN populations using t-distributed Stochastic Neighbor Embedding (tSNE). Each dot is a cell.See also Figure S1.
Figure 2.
Figure 2.. Single-cell RNA-seq Analysis of GH146+ PNs
(A) Representative confocal projection and schematic of GH146+ PNs, which include (per antennal lobe) 50 adPNs (acj6+), 35 lPNs (vvl expression begins to decrease from 18h APF; Komiyama et al., 2003), and 6 vPNs (Lim1+). The cell bodies of adPNs, lPNs, and vPNs are located respectively anterodorsal, lateral, and ventral to the antennal lobe neuropil (circled; stained by Ncad). All GH146+ adPNs and lPNs send dendrites to a single glomerulus. The schematic shows the stereotyped locations of a large subset of glomeruli (named according to their locations; Laissue et al., 1999), color-coded according to adPNs or lPNs. Scale, 20 μm. D, dorsal; L, lateral. (B) Visualization of GH146+ PNs using dimensionality reduction by PCA followed by tSNE. Each dot is a cell. Cells are arranged according to transcriptome similarity. (C) Schematic of Iterative Clustering for Identifying Markers (ICIM), an unsupervised machine-learning algorithm for identifying genes that distinguish cell types. (D) Visualization of GH146+ PNs using tSNE based on 561 genes identified using ICIM. GH146+ adPNs and lPNs form 30 distinct clusters (differentially colored). Black dots are cells that could not be assigned to any cluster. (E) Visualization of GH146+ PNs as in Figure 2D, colored according to acj6 and vvl expression level. acj6 and vvl are expressed in GH146+ PNs in a mutually exclusive manner. See also Figure S2.
Figure 3.
Figure 3.. Mapping Clusters to PN Classes Using Known Markers
(A and B) Intersecting GH146-Flp with 91G04-GAL4 (A) or with Mz19-GAL4 (B) labels only one PN class (DC2) or 3 PN classes (acj6+ VA1d and DC3; acj6− DA1), respectively, at 24h APF and in adults. Driver schematics are shown on the right. Scale for (A), 20 μm; for (B), 50, 50, and 20 μm. (C) Visualization of GH146+, 91G04+, and Mz19+ PNs using tSNE as in Figure 2D. Cells are colored according to driver (left) or by expression level of acj6 (right). 91G04+ cells (green) map to a single cluster (#1) of GH146+ cells (orange); thus Cluster #1 corresponds to DC2 PNs. DA1 PNs (Mz19+/acj6−) map to two clusters, #2 and #2’. VA1d and DC3 PNs (Mz19+/acj6+) map to two clusters, #3 and #4.
Figure 4.
Figure 4.. Mapping Clusters to PN Classes Using Newly Identified Markers
(A) Visualization of GH146+ PN cells using tSNE as in Figure 2D, showing expression of trol enriched in one cluster (#5). Clusters #1–4 from Figure 3 are also indicated. (B) After intersecting with GH146-Flp, trol-GAL4 labels 2–3 PNs in each hemisphere at 24h and 72h APF, which project dendrites to the VM2 glomerulus. (C) Visualization of GH146+ and trol+ PNs using tSNE based on 561 genes previously identified using ICIM (Figure 2D). Cells are colored according to driver (left) or by expression level of trol (right; color bar in Figure 4A). trol-GAL4+ PNs map to one GH146+ PN cluster (left), which expresses high levels of trol (right). Thus, Cluster #5 corresponds to VM2 PNs. (D) Visualization of GH146+ PNs using tSNE as in Figure 4A showing expression of CG31676 (color bar in Figure 4A). Among two Mz19+/acj6+ clusters, CG31676 is highly expressed in Cluster #3, but not #4. Several acj6− (see Figure S3B) clusters also express CG31676, including #2 and #2’ (DA1) and #6 and #6’. (E) Schematic of CRISPR/Cas9 mediated insertion of T2A-GAL4 into the first intron of CG31676. (F) CG31676-GAL4 expression in PNs after intersecting with GH146-Flp. Similar numbers of PNs are labeled at 24h, 48h, and 72h APF. VA1d, but not DC3, is labeled, enabling us to map Cluster #3 to VA1d (CG31676+) and Cluster #4 to DC3 (CG31676−). In addition, DA1 and DL3 are labeled. Thus, the remaining acj6−/CG31676+ cells (Clusters #6 and #6’) correspond to DL3 PNs. (G) Summary of the mapping of 6 PN classes to corresponding transcriptome clusters. Markers used for unambiguous mapping (Figures 3, 4A–D, and S3B, C) are listed.Ncad is used as a neuropil marker. Scale, 20 μm.See also Figure S3.
Figure 5.
Figure 5.. Identification of New Lineage-specific Transcription Factors using Single-cell RNA-seq
(A) Visualization of GH146+ PNs using tSNE as in Figure 2E showing expression of acj6, C15, knot, and unpg. adPNs are outlined (based on acj6 expression) and remaining cells are lPNs. (B) Consistent with RNA-seq data in (A), 24h APF expression of C15 and Knot (antibody staining) in GH146+ PNs (green) is restricted to adPNs, while unpg (anti-β-gal staining) is restricted to lPNs. (C) Loss-of-function analysis of C15 using elav-GAL4 driven UAS-C15-RNAi (line #2; see Figure S4C). Wild type (WT) control: elav-GAL4 × w1118. When C15 is knocked down, the VA1d glomerulus (visualized by VA1d ORN axons labeled by Or88a-mtdT) displays a dorsal shift. In addition, GFP signal in VA1d PN dendrites (visualized by Mz19-QF driven QUAS-mCD8GFP) is undetectable. (D) Quantification of position shift of the VA1d glomerulus due to C15 knockdown in (C). θ is the angle between the dorsoventral axis and a line drawn through the centers of the VA1d and DC3 glomeruli. Error bars are SEM. ***, P < 0.001 (t test). (E) Gain-of-function analysis of C15 in Mz19-GAL4+ MARCM misexpression clones. In WT, dendrites of adPN neuroblast (adNB) clones target VA1d and DC3 and lPN neuroblast (lNB) clones target DA1. When C15 is misexpressed, dendrite targeting of adNB clones is not affected, while dendrite targeting of lNB clones is affected with 100% penetrance. Ncad is used as a neuropil marker. Scale, 20 μm.See also Figure S4.
Figure 6.
Figure 6.. Transcriptome Analysis of Mz19+ PNs across Developmental Stages
(A) Representative confocal projections of Mz19+ PNs from 5 stages. Schematic (right) shows cell body and glomerular targets of Mz19+ PNs. Ncad, red. D, dorsal; L, lateral. Scale, 20 μm. (B) Visualization of Mz19+ PNs from all developmental stages using tSNE based on 497 genes identified using ICIM. Color shows expression of acj6 and CG31676 (CPM, counts per million). acj6+ cells are adPNs (VA1d and DC3, outlined), and acj6− cells are lPNs (DA1). Within adPNs, CG31676+ cells are VA1d PNs, and CG31676− cells are DC3 PNs. CG31676 is turned off in all adult adPNs (see also C). (C) Visualization of Mz19+ PNs as in (B), with color indicating developmental stages. Expression patterns of acj6 and CG31676 (B) enabled unambiguous identification of three PN classes. Dashed lines indicate the developmental trajectories of these classes. VA1d and DC3 PNs are distinct at all pupal stages, but merge to form one cluster in the adult. The densely and sparsely dashed red lines represent the trajectories of Cluster 2 and 2’, respectively, which become indistinguishable by 72h APF and remain so in the adult. (D) Type identity score of VA1d and DC3 PNs from five developmental stages. Each dot represents a cell. Colors show developmental stages as in (C). The identity score is calculated as a scaled sum of the 22 most significantly differentially expressed genes between VA1d and DC3 PNs (STAR Methods). Scores range from −1 (high expression of the DC3 signature genes and no expression of the VA1d signature genes) to +1 (the opposite expression profile). (E) Violin plot showing the distribution of transcriptome similarity between all pairs of Mz19+ adPNs. Peaks are indicated by asterisks. The upper peak consists of pairs in which both cells are from the same class. The lower peak consists of pairs in which the two cells are from different classes. The adult distribution is unimodal, indicating a lack of transcriptome differences between the two classes. (F) Schematic summary. VA1d and DC3 PNs derive from a common neuroblast (NB) lineage. Their transcriptomes are distinct at pupal stages and become indistinguishable in the adult. (G) Differentially expressed genes between PN classes belonging either to the same lineage (VA1d and DC3; DL3 and DA1) or different lineages (VA1d and DA1). Adult data does not exist for VA1d vs DC3 PNs as they are indistinguishable. For DL3 PNs, we only have data for 24h APF.See also Figure S5.
Figure 7.
Figure 7.. Combinatorial Molecular Codes of PN Subtype Identity
(A) Minimal combinatorial code for subtype identity among Mz19+ PNs identified using an information theoretic approach. Left, mean expression level of each gene among cells belonging to each Mz19+ class. Right, binarized expression levels of the same genes [cutoff: log2(CPM+1) = 3]. Each Mz19+ PN class expresses a distinct combination of these two genes. (B) Information contained in minimal combinatorial codes for GH146+ subtype identity. X-axis is the number of genes included in the code. Y-axis is the amount of uncertainty (entropy) of cell type classification that is explained by the code. Colors denote codes constructed from different sets of genes. The genome-wide code (pink) is constructed from all genes, while the TF (green) or CSM (orange) codes use only 1045 TF or 955 CSM genes. Gray denotes codes constructed from 1,000 randomly sampled non-TF and non-CSM genes, with the line indicating the median and the shading indicating the standard deviation across 100 replicates, respectively. (C–E) Minimal combinatorial codes for GH146+ subtype identity constructed from (C) all genes, (D) TFs, or (E) CSMs. Heat map indicates the binary expression state of genes in each cluster, as in (A). Clusters and genes are arranged by hierarchical clustering. (F) Representation of TFs and CSMs among the top 30 differentially expressed (DE) genes between pairs of Mz19+ PN subtypes as indicated. Y-axis shows the fraction of the 30 most differentially expressed genes that are TFs (green), CSMs (orange), or TF + CSM (blue) at each developmental stage. Adult stage is absent from the VA1d vs DC3 comparison because their transcriptomes cannot be distinguished. (G) Enrichment of TFs and CSMs among the top differentially expressed genes between pairs of clusters of GH146+ cells (435 pairs). X-axis shows the number of top differentially expressed genes under consideration. Y-axis shows the distribution of enrichment of either TFs or CSMs within these genes. Enrichment is calculated relative to the genomic representation of TFs (6.7%) and CSMs (6.2%), indicated by the horizontal line.See also Figures S6 and S7.
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