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Cellular diversity in the Drosophila midbrain revealed by single-cell transcriptomics

  1. Vincent Croset
  2. Christoph D Treiber  Is a corresponding author
  3. Scott Waddell  Is a corresponding author
  1. The University of Oxford, United Kingdom
Research Article
Cite this article as: eLife 2018;7:e34550 doi: 10.7554/eLife.34550
10 figures, 1 table, 1 data set and 1 additional file

Figures

Figure 1 with 4 supplements
Drop-seq reveals neuronal clusters in the Drosophila brain.

(A) Schematic of the experimental procedure. Drosophila brains were dissected and dissociated prior to Drop-seq. After sequencing and alignment, a digital expression matrix containing information about the number of UMIs found for each gene, in each cell, was generated and used for PCA and subsequent analyses. See Materials and methods section for details. (B) Two-dimensional representation (t-SNE) of 10,286 Drosophila brain cells, manually classified into 28 clusters. Based on the recovery of cell-types of known abundance in the brain, we estimate that there are 45,000 cells in the fly midbrain.

https://doi.org/10.7554/eLife.34550.002
Figure 1—source data 1

Digital Expression Matrix.

Zip-archive containing individual digital expression matrices of all 8 biological replicates.

https://doi.org/10.7554/eLife.34550.007
Figure 1—source data 2

List of marker genes for each cluster in Figure 1.

Table showing the average log-fold change values of marker genes for each incluster, including adjusted p-values.

https://doi.org/10.7554/eLife.34550.008
Figure 1—figure supplement 1
Preliminary validation of Drop-seq on insect cells.

(A) Schematic of a Drop-seq experiment that was run to analyze a mixture of Drosophila (S2; blue) and Spodoptera (Sf9; red) cells. (B) Scatter plot (Barnyard) indicating the number of transcripts from Drosophila or Spodoptera associated with each STAMP. Of 768 STAMPs analyzed, only 12 (1.56%) carried transcripts from both species (purple dots).

https://doi.org/10.7554/eLife.34550.003
Figure 1—figure supplement 2
– Comparison of different quality filters.

(A–C) Left: histograms representing the distribution of numbers of UMIs per cell selected with cutoffs of 200 (A), 800 (B) and 1800 (C) UMIs per cell. Right: t-SNE plots of the same data set as in Figure 1B. obtained with these cutoffs to compare the impact of number of cells and UMI coverage on t-SNE clusters.

https://doi.org/10.7554/eLife.34550.004
Figure 1—figure supplement 3
– t-SNE plot showing all eight replicates.

t-SNE plot as in Figure 1B, with cells from each replicate in a different color.

https://doi.org/10.7554/eLife.34550.005
Figure 1—figure supplement 4
– Sex-determination of individual neurons.

(A) Histogram showing the number of UMIs per neuron. The dashed line indicates the threshold that was chosen to separate roX1-positive and roX1-negative neurons. (B) t-SNE plot showing putative male and female cells, based on roX1 expression. Pie-chart shows the frequency of ‘male’ and ‘female’ neurons.

https://doi.org/10.7554/eLife.34550.006
Figure 2 with 1 supplement
Identification of Kenyon Cells and mushroom body-specific genes.

(A) mCherry labeling of MB008B neurons in the Drosophila brain. Neuropil, labeled by nc82 anti-Brp antibody, is shown in grey. (B) Expression of mCherry in the t-SNE-clustered brain cells shown in Figure 1B. mCherry-positive cells are labeled red and identify this cluster as αβ Kenyon Cells (KCs). Intensity of red (or other colors in the panels below) is proportional to the normalized expression level. (C) and (D) Expression of eyeless and Dop1R2 (damb), in t-SNE-clustered brain cells. The three numbered clusters containing indigo cells are KCs. (E) Expression of sNPF, Fas2 and trio in the three t-SNE clusters numbered in (C) and (D). Cells in light blue, orange and purple express each of these genes in αβ, γ, and α´β´ KCs, respectively. sNPF and Fas2 are mostly expressed in αβ and γ KCs, while trio is mostly detected in γ and α´β´ KCs. (F) Violin plots showing the main markers that distinguish KC subtypes from each other (pairwise comparisons for genes expressed in >50% of cells in either cluster; Log2 FC >1.5, Wilcoxon rank-sum test with Bonferroni-corrected p-value<0.01). The column on the right (grey) indicates the level of expression of these genes across all non-MB neurons in the brain.

https://doi.org/10.7554/eLife.34550.009
Figure 2—source data 1

Primer pairs used for qPCR.

List of primer pairs and UPL probe numbers used for qPCR show in Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.34550.011
Figure 2—figure supplement 1
– Confirmation of Kenyon Cell type specific gene expression using qPCR on FAC-sorted cells.

Relative difference of expression levels for some of the genes identified in Figure 2F. For each gene, in each replicate, expression values were normalized to the lowest ΔCt across the three lobes. n = 3, * Adjusted p-value<0.05 (Two-way ANOVA with Tukey's multiple comparisons test).

https://doi.org/10.7554/eLife.34550.010
Sub-populations of olfactory projection neurons.

(A) Re-clustering of the two Projection Neuron (PN) clusters from Figure 1B. Clusters are color-coded. (B) Dot plots showing the main markers distinguishing PN populations from each other (pairwise comparisons for genes expressed in >50% of cells in either cluster; Log2 FC >1.5, Wilcoxon rank-sum test with Bonferroni-corrected p-value<0.01). Dot diameter represents the fraction of cells expressing each gene in each cluster, as shown in scale. Color intensity represents the average normalized expression level. (C) t-SNE-plots for some known markers of PNs. ct labels all PN clusters, acj6 and vvl are mutually exclusive in Clusters 1, 2 and 4, versus Cluster 3, while Lim1 is specifically expressed in Cluster 4. Some of these cells may be antennal lobe local interneurons.

https://doi.org/10.7554/eLife.34550.012
Figure 4 with 1 supplement
Genetic markers of glial subtypes.

(A) Re-clustering of the glial and astrocyte populations identified in Figure 1B. The same three sub-clusters are identified. (B) Violin plots showing the main markers that distinguish glial subtypes from each other (pairwise comparisons for genes expressed in >75% of cells in either cluster; Log2 FC >2, Wilcoxon rank-sum test with Bonferroni-corrected p-value<0.01). The column on the right (grey) indicates the level of expression of each gene across all neurons in the brain (excluding glia, astrocytes and fat body).

https://doi.org/10.7554/eLife.34550.013
Figure 4—figure supplement 1
Expression levels of glia-specific genes and Ilp 6.

(A–B) tSNE plots showing the expression levels of the neuronal markers embryonic lethal abnormal vision (elav) and neuronal synaptobrevin (nSyb). (C–E) tSNE plots showing the expression levels of the glial markers reversed polarity (repo) and nervana 2 (nrv2), and the astrocyte marker astrocytic leucine-rich repeat molecule (alrm) (Doherty et al., 2009). (F) tSNE plots showing the expression levels of Insulin-like peptide 6 (Ilp6), confirming its expression in glia.

https://doi.org/10.7554/eLife.34550.014
Distribution of fast-acting neurotransmitters.

(A) t-SNE plot showing the distribution of cells expressing vesicular acetylcholine transporter (VAChT, cholinergic neurons), vesicular glutamate transporter (VGlut, glutamatergic neurons) and glutamic acid decarboxylase 1 (Gad1, GABA-ergic neurons). For graphical reasons only cells expressing each marker above a log normalized value of 2 are shown. (B) Quantification of cells expressing markers displayed in A. The difference to 100% are cells that did not express any of the three markers.

https://doi.org/10.7554/eLife.34550.015
Co-expression of neuropeptides with fast-acting neurotransmitters.

(A) Radar plots showing the co-expression of 16 neuropeptides with the three fast-acting neurotransmitters. Data represents the relative abundance of cells expressing a neuropeptide and either VAChT (ACh), VGlut (Glut), Gad1 (GABA), combinations of these three genes, or none of them. (B) Co-expression of four insulin-like peptides, including the non-neuronal Ilp6, with fast-acting neurotransmitters.

https://doi.org/10.7554/eLife.34550.016
Genetic markers and co-transmission in monoaminergic neurons.

(A) Expression of the Vesicular Monoamine Transporter (Vmat) labels three clusters from Figure 1B (cells in indigo, highlighted with arrowheads and circles. (B) Re-clustering of the three cell populations labeled in (A). Four sub-clusters are identified, representing dopaminergic, tyraminergic, octopaminergic, and serotonergic neurons. (C) Dot plots showing the main markers distinguishing monoamine populations from each other (pairwise comparisons for genes expressed in >50% of cells in either cluster; Log2 FC >1, Wilcoxon rank sum test with Bonferroni-corrected p-value<0.01). Dot diameter represents the fraction of cells expressing each gene in each cluster, as shown in scale. Color intensity represents the average normalized expression level. (D) Percentage of cells in each monoaminergic cluster that are co-expressing markers for fast neurotransmitter-releasing neurons (VGlut, Gad1, and VAChT). (E) Dot plots showing expression of genes encoding neuropeptides and neuropeptide amidating enzymes across monoaminergic populations. Dot diameter represents the fraction of cells expressing each gene in each cluster, as shown in scale. Color intensity represents the average normalized expression level. (F) Comparison of genes overexpressed in PAM dopaminergic neurons compared to the rest of the brain, measured with Drop-seq or with FACS and Smart-seq2. 9 of the 15 genes identified with Drop-seq (labeled in bold) were also found in the Smart-seq2 dataset.

https://doi.org/10.7554/eLife.34550.017
Localization of genes involved in dopamine metabolism and signaling.

(A) Schematic of a dopaminergic synapse, representing the major proteins involved in dopamine signaling and metabolism. Drawing inspired by Yamamoto and Seto (2014) (B) Dot plots showing the expression of these genes across all cell populations identified in the Drosophila brain. Dot diameter represents the fraction of cells expressing each gene in each cluster, as shown in scale. Color intensity represents the average normalized expression level. (C) Histogram showing the number of DA receptor types in Kenyon Cells (as labeled in Figure 2C & D). Most cells express a combination of several receptor types.

https://doi.org/10.7554/eLife.34550.018
Expression patterns of nicotinic acetylcholine receptor subunits.

(A) Prevalence of nicotinic receptor subunits. (B) Heatmap showing Pearson correlation coefficient Z-scores for each receptor subunit pair.

https://doi.org/10.7554/eLife.34550.019
Co-expression of neuronal activity markers.

Heatmap showing Pearson correlation coefficient Z-scores of activity-regulated genes, as reported by Chen et al., 2016 (histogram on top). Ten most highly upregulated genes following ChR2-XXL-induced activation of all neurons that are expressed in the brain, ranked by their correlation.

https://doi.org/10.7554/eLife.34550.020

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)MB008BBloomington Drosophila Stock CenterRRID:BDSC_68291
Genetic reagent (D. melanogaster)MB131BBloomington Drosophila Stock CenterRRID:BDSC_68265
Genetic reagent (D. melanogaster)MB461BBloomington Drosophila Stock CenterRRID:BDSC_68327
Genetic reagent (D. melanogaster)uas-mCherry (III)otheruas-mCherry(III)lab stock
Cell line (D. melanogaster)Drosophila S2 Cells in Schneider's MediumGibco, Waltham, MAR69007
Cell line (Spodoptera frugiperda)Sf9 cells in Sf-900 III SFMGibco12659–017
Sequence-based reagentTemplate switch oligoSigma, St. Louis, MOAAGCAGTGGTATCAACGCAGAGTGAATrGrGrG
Chemical compound, drugSchneider's mediumGibco21720–001
Chemical compound, drugFBSSigmaF0804
Chemical compound, drugpenicillin-streptomycinGibco15070–063
Chemical compound, drugSf-900 III SFMGibco12658019
Chemical compound, drugDPBS (calcium and magnesium free)Gibco14190–086
Chemical compound, drugPapainSigmaP4762
Chemical compound, drugCollagenaseSigmaC2674
Chemical compound, drugd(−)−2-amino-5-phosphonovaleric acidSigmaA8054
Chemical compound, drug6,7-dinitroquinoxaline-2,3-dioneSigmaD0540
Chemical compound, drugtetrodotoxinAbcam, UKab120054
Other10 µm CellTrix strainerSysmex, Japan04-0042-2314
OtherFuchs-Rosental hemocytometerVWR, Radnor, PA631–1096
Commercial assay or kitSingle Cell RNA-Seq systemDolomite Bio, UK3200537
Chemical compound, drugBarcoded Beads SeqBChemGenes Corp., Wilmington, MA
Chemical compound, drugFicoll PM-400VWR17-0300-10
Chemical compound, drugN-Lauroylsarcosine sodium salt solutionSigmaL7414
Chemical compound, drugQX200 Droplet generation oil for EvaGreenBiorad, Hercules, CA1864006
Chemical compound, drugDTTLife Technologies, Carlsbad, CAP2325
Chemical compound, drugMaxima H Minus Reverse TranscriptaseThermo Scientific, Waltham, MAEP0753
Chemical compound, drugExonuclease INEB, Ipswich, MAM0293L
Sequence-based reagentSMART PCR primerSigmaAAGCAGTGGTATCAACGCAGAGT
Chemical compound, drugHifi HotStart ReadymixKappa Biosystems, SwitzerlandKK2602, KK2611
Chemical compound, drugAgencourt AMPure XP beadsBeckman-Coulter, Brea, CAA63880
Commercial assay, kitBioanalyzer High-Sensitivity DNA kitAgilent, Santa Clara, CA5067–4626
Commercial assay, kitNextera XT DNA Sample Preparation KitIllumina, San Diego, CAFC-131–1024
Sequence-based reagentNew-P5-SMART PCR hybridSigmaAAT GAT ACG GCG ACC ACC GAG ATC TAC ACG CCT GTC CGC GGA AGC AGT GGT ATC AAC GCA GAG T*A*C
Commercial assay, kitPicoPure™ RNA Isolation KitApplied Biosystems, Foster City, CAKIT0204
Commercial assay, kitSuperScript III First-Strand Synthesis SuperMixInvitrogen, Carlsbad, CA18080400
Commercial assay, kitQIAquick PCR Purification KitQiagen, Germany28106
Commercial assay, kitUniversal Probe Library systemRoche, Switzerland04683633001,04869877001
Commercial assay, kitLightCycler® 480 Probes MasterRoche4887301001
Commercial assay, kitSMART-Seq v4 Ultra Low Input RNA Kit for SequencingTakara Clontech, Japan634890
Commercial assay, kitTruSeq RNA Library Prep Kit v2IlluminaRS-122–2001

Data availability

Sequencing data have been deposited in SRA under accession code SRP128516. Digital Expression Matrix is provided as supplementary file.

The following data sets were generated
  1. 1
    Data from: Cellular diversity in the Drosophila midbrain revealed by single-cell transcriptomics
    1. Croset V
    2. Treiber CD
    3. Waddell S
    (2018)
    Publicly available at the NCBI Sequence Read Archive (accession no: SRP128516).

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