A transcriptomic taxonomy of Drosophila circadian neurons around the clock
- Howard Hughes Medical Institute, Brandeis University, United States
- Howard Hughes Medical Institute, Janelia Research Campus, United States
Figures
Figure 1 with 1 supplement
Single-cell RNA sequencing of Drosophila clock neurons around the clock.
(A) Confocal stack images of immunostained brains from Clk856-GAL4 > UAS-Stinger-GFP flies at ZT18. Anti-GFP (left), anti-TIM (middle) and a merge of these two images (right). The arrow indicates …
Figure 1—figure supplement 1
Methods and verification of single-cell RNA sequencing of Drosophila clock neurons.
(A) Schematic of single-cell RNA-seq protocol. The method is based on CEL-Seq2 with some modifications. EGFP-labeled clock neurons were sorted into 96- or 384-well plates by fluorescence-activated …
Figure 2 with 1 supplement
Cycling timeless (tim) expression and Clock (Clk) abundance defines key circadian neuron clusters.
(A–B) The mean tim expression (A) and Clk (B) throughout the day in light: dark (LD) and constant darkness (DD) conditions is graphed for each cluster. Seventeen clusters were chosen as high …
Figure 2—figure supplement 1
Cycling vrille (vri) expression helps to define key circadian neuron clusters.
The mean vri expression throughout the day in light:dark (LD) and constant darkness (DD) conditions is graphed for each cluster. vri expression in high confidence clusters are labeled with purple …
Figure 3 with 1 supplement
Identification of the lateral neuron clusters: LNvs and LNds.
(A) Seventeen Drosophila clock neuron clusters. t-SNE visualization of 2615 clock neurons from both light:dark (LD) and constant darkness (DD). Each cluster retains its original identifying number …
Figure 3—figure supplement 1
Identification of two Trissin-expressiong LNd neurons.
Confocal stack of images showing antibody staining for GFP and TIM in Trissin-LexA > LexAop-FLP; Clk856-GAL4 > UAS-FRT-STOP-FRT-Cschrimson.venus fly brains. GFP (left), TIM (middle), and a merge of …
Figure 4 with 1 supplement
Mapping the dorsal and lateral posterior neuron (LPN) clock neurons.
(A) t-SNE plots showing CCHa1 expression. Red indicates higher expression (color bar, TP10K). Light:dark (LD) and constant darkness (DD) data are plotted together. CCHa1 expression is specific to …
Figure 4—figure supplement 1
Mapping the dorsal clock neurons.
(A) Confocal stack of images showing antibody staining for GFP (top) and TIM (middle) and the merge (bottom) in CCHa1-LexA > LexAop-FLP; Clk856-GAL4 > UAS-FRT-STOP-FRT-Cschrimson.venus fly brains. …
Figure 5 with 1 supplement
Enriched transcripts in identified clock neuron clusters.
(A) Heatmap showing the enriched transcription factors expression in each cluster. (B–E) t-SNE plot showing ham (B), toy (C), Hr51 (D), and opa (E) expression in clock neurons. Red indicates higher …
Figure 5—figure supplement 1
Enriched transcripts in identified clock neuron clusters.
(A) Heatmap showing the enriched marker genes expression in each cluster. (B) Heatmap showing the enriched secretory gene expression in each cluster. (C) Violin plot showing the expression of DN1p- …
Figure 6 with 1 supplement
Neuron-specific cycling gene expression.
(A) Gene ontology (GO) analysis of the all the cycling genes found in all the clock neuron clusters. GO terms that were also identified in cluster-specific GO term analysis are indicated by …
Figure 6—figure supplement 1
Oscillating transcripts in Drosophila clock neurons.
(A) Pie chart of cycling transcripts in clock neurons in light:dark (LD) and constant darkness (DD) conditions. (B) Most cycling transcripts are specific to one or two clock neuron clusters. Bar …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | UAS-Stinger | BDSC | RRID:BDSC_84277 | |
| Genetic reagent (D. melanogaster) | R14F03-p65.AD | BDSC | RRID:BDSC_70551 | |
| Genetic reagent (D. melanogaster) | VT029514-GAL4.DBD | BDSC | RRID:BDSC_75062 | |
| Genetic reagent (D. melanogaster) | R18H11-p65.AD | BDSC | RRID:BDSC_68852 | |
| Genetic reagent (D. melanogaster) | R51H05-GAL4.DBD | BDSC | RRID:BDSC_69036 | |
| Genetic reagent (D. melanogaster) | P{10XUAS-IVS-myr::GFP}attP2 | BDSC | RRID:BDSC_32197 | |
| Genetic reagent (D. melanogaster) | Clk856-GAL4 | Gummadova et al., 2009 | Flybase: FBtp0069616 | |
| Genetic reagent (D. melanogaster) | Clk4.1M-LexA | Guo et al., 2016 | Flybase: FBtp0093698 | |
| Genetic reagent (D. melanogaster) | UAS-EGFP | BDSC | RRID:BDSC_5428 | |
| Genetic reagent (D. melanogaster) | UAS-FRT-STOP-FRT-CsChrimson.mVenus and LexAop-FLP | Guo et al., 2018 | ||
| Genetic reagent (D. melanogaster) | Trissin-LexA | Deng et al., 2019 | ||
| Genetic reagent (D. melanogaster) | Trissin-GAL4 | Deng et al., 2019 | ||
| Genetic reagent (D. melanogaster) | CCHa1-GAL4 | Deng et al., 2019 | ||
| Genetic reagent (D. melanogaster) | CNMa-GAL4 | Deng et al., 2019 | ||
| Antibody | Anti-PER Rabbit polyclonal | Laboratory of Michael Rosbash | 1:1000 | |
| Antibody | Anti-TIM Rat monoclonal | Laboratory of Michael Rosbash | RRID: AB_2753140 | 1:200 |
| Antibody | Anti-PDF Mouse monoclonal | DSHB | RRID: AB_760350 | 1:500 |
| Antibody | Anti-GFP Mouse monoclonal | Sigma-Aldrich | RRID: AB_390913 | 1:1000 |
| Antibody | Chicken anti-GFP | Abcam | RRID: AB_300798 | 1:1000 |
| Antibody | Rabbit anti-DsRed | Takara Bio USA | RRID: AB_10013483 | 1:200 |
| Antibody | Goat anti-mouse polyclonal | ThermoFisher | RRID: AB_2536185 | 1:200 |
| Antibody | Goat anti-rabbit polyclonal | ThermoFisher | RRID: AB_2576217 | 1:200 |
| Antibody | Goat anti-rabbit polyclonal | ThermoFisher | RRID: AB_2633281 | 1:200 |
| Antibody | Anti-PDF Mouse monoclonal | DSHB | RRID: AB_760350 | 1:500 |
| Software, algorithm | FIJI | https://fiji.sc/ | RRID: SCR_002285 | |
| Software, algorithm | Microsoft Office Excel | |||
| Software, algorithm | RStudio | https://rstudio.com | RRID: SCR_000432 | Version 1.2.5033 |
| Software, algorithm | Adobe Photoshop CC | RRID: SCR_014199 | ||
| Software, algorithm | Code for clustering and rhythmic gene expression analysis | This paper | R code Ma and Przybylski, 2020) |
Additional files
-
Source data 1
List of identified marker genes in each cluster.
Table displaying the average log-fold change values, percentage of expression and p-value for the list of differentially expressed genes among clusters.
- https://cdn.elifesciences.org/articles/63056/elife-63056-data1-v2.xlsx
-
Supplementary file 1
List of identified rhythmic genes in each cluster.
Table displaying the minimum and maximum gene expression level, phase, and p-value for the list of rhythmically expressed genes among clusters in light:dark (LD) and constant darkness (DD) conditions.
- https://cdn.elifesciences.org/articles/63056/elife-63056-supp1-v2.xlsx
-
Supplementary file 2
Gene ontology (GO) term analysis of the cyclers in each cluster.
Table displaying p-value for the GO term analysis of the rhythmically expressed genes among clusters in light:dark (LD) condition.
- https://cdn.elifesciences.org/articles/63056/elife-63056-supp2-v2.xlsx
-
Supplementary file 3
Gene ontology (GO) term analysis for all cyclers.
Table displaying the GO terms and their p-values of the pooled rhythmically expressed genes.
- https://cdn.elifesciences.org/articles/63056/elife-63056-supp3-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/63056/elife-63056-transrepform-v2.docx
