Cell type-specific driver lines targeting the Drosophila central complex and their use to investigate neuropeptide expression and sleep regulation
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Department of Psychology, College of Science, California State University, United States
Figures
Figure 1
Schematic diagram of CX neuropils.
(A) The brain areas included in the hemibrain connectome are shown with the CX and key connected brain areas highlighted. (B) The neuropils comprising the CX are shown: FB, fan-shaped body; PB, …
Figure 2 with 4 supplements
Maximum intensity projections (×20 confocal images) of the expression patterns driven by four stable split-GAL4 lines for the indicated cell types.
The brain and VNC are outlined in red. Original confocal stacks that include a neuropil reference channel can be downloaded from https://www.janelia.org/split-gal4. Similar images for 48 additional …
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Figure 2—source data 1
Table of split-GAL4 lines organized by CX structure and cell type.
- https://cdn.elifesciences.org/articles/104764/elife-104764-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Maximum intensity projections (×20 confocal images) of the expression patterns driven by stable split-GAL4 lines for the indicated 12 cell types.
The brain and VNC are outlined in red. Original confocal stacks that include a neuropil reference channel can be downloaded from https://www.janelia.org/split-gal4.
Figure 2—figure supplement 2
Maximum intensity projections (×20 confocal images) of the expression patterns driven by stable split-GAL4 lines for the indicated 12 cell types.
The brain and VNC are outlined in red. Original confocal stacks that include a neuropil reference channel can be downloaded from https://www.janelia.org/split-gal4.
Figure 2—figure supplement 3
Maximum intensity projections (×20 confocal images) of the expression patterns driven by stable split-GAL4 lines for the indicated 12 cell types.
The brain and VNC are outlined in red. Original confocal stacks that include a neuropil reference channel can be downloaded from https://www.janelia.org/split-gal4.
Figure 2—figure supplement 4
Maximum intensity projections (×20 confocal images) of the expression patterns driven by stable split-GAL4 lines for the indicated 12 cell types.
The brain and VNC are outlined in red. Original confocal stacks that include a neuropil reference channel can be downloaded from https://www.janelia.org/split-gal4.
Figure 3 with 1 supplement
Visualization of the entire expression pattern of a split-GAL4 line for the indicated five cell types revealed using UAS-myrGFP (A–E).
A subset of individual cells within those same cell types (A′–E′) are revealed by stochastic labeling using the MCFO method (Nern et al., 2015). The scale bar in E′ refers to all panels and = 50 μm. …
Figure 3—figure supplement 1
Individual cell morphologies revealed by stochastic labelling.
Individual cells within the indicated cell types revealed by stochastic labeling using the MCFO method (Nern et al., 2015) with the indicated split-GAL4 lines. Images are MIPs. Scale bars = 50 μm.
Figure 4
Using EASI-FISH to assess neurotransmitter expression.
(A–C) Expression of the transcripts encoding key enzymes required to synthesize tyramine (Tdc2) and octopamine (Tbh) has been examined as indicated. PFG neurons were marked by the split-GAL4 line …
Figure 5 with 1 supplement
Sparsely expressed neuropeptide genes.
(A-R) EASI-FISH was used to examine the expression of the indicated neuropeptide genes in the central brain (this brain area is shown as dashed box inS). Samples were counterstained with DAPI to …
Figure 5—figure supplement 1
Neuropeptides and neuropeptide receptor genes whose expression patterns were characterized by the EASI-FISH experiments shown in Figures 5—8.
Relevant figure panels are listed. Some predicted neuropeptides were not tested because of lack of evidence for their expression in adult brain and are shown in gray font and listed as ‘not tested’ …
Figure 6
More broadly expressed neuropeptide genes.
(A-P) EASI-FISH was used to examine the expression of the indicated neuropeptide genes in the central brain. Samples were counterstained with DAPI to visualize the outline of brain tissue. Images …
Figure 7
Neuropeptide receptor gene expression.
(A-T) EASI-FISH was used to examine the expression of the indicated neuropeptide genes in the central brain. Higher magnification views of regions of the brains shown in D, J, and M (indicated by …
Figure 8
Neuropeptide gene expression in specific cell types.
(A-R) EASI-FISH was used to examine the expression of the indicated neuropeptide genes in the brains also expressing myr-GFP driven by a cell type-specific split-GAL4 line. The line name and labeled …
Figure 9 with 2 supplements
Summary of neurotransmitter and neuropeptide expression as determined by EASI-FISH performed on adult brains in which selected cell types were marked by a split-GAL4 lines driving GFP expression.
About half of all CX cell types were examined using probes for the following 15 NPs: AstA, AstC, CCAP, CCHa1, CCHa2, Dh31, Dh44, FMRFa, Mip, Ms, NPF, Proc, SIFA, sNPF, and Tk. All NPs except CCAP, …
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Figure 9—source data 1
Table of split-GAL4 lines with EASI-FISH results for neurotrasmitter and neuropeptide expression.
- https://cdn.elifesciences.org/articles/104764/elife-104764-fig9-data1-v1.xlsx
Figure 9—figure supplement 1
RNA-seq data for genes related to neurotransmitter synthesis, transport, and receptors in the indicated cell types.
Mean log2 of TPM values for each gene are listed. See NCBI Gene Expression Omnibus (accession number GSE271123) for the raw data. The split-GAL4 drivers used for each cell type were as follows: ER5, …
Figure 9—figure supplement 2
RNA-seq data for genes related to neuropeptides and their receptors in the indicated cell types.
Figure 10 with 5 supplements
Activation of hDeltaF, comprised of eight intrinsic FB columnar neurons (see Hulse et al., 2021) decreases sleep.
(A) Thermogenetic activation and DAM-based monitoring using the split-GAL4 lines SS54903, SS53683, and SS63959 produced decreased sleep duration and increased P(Wake) in all three lines as compared …
Figure 10—figure supplement 1
Activation phenotype of PEN_b.
PEN_b is comprised of 22 columnar neurons linking the ellipsoid body (EB), protocerebral bridge (PB), and noduli (Wolff and Rubin, 2018; Turner-Evans and Jayaraman, 2016 ; Green et al., 2017; Hulse …
Figure 10—figure supplement 2
Activation phenotype of PFGs.
Thermogenetic (A) and optogenetic (B) activation of PFGs reveals sexually dimorphic sleep behavior. We tested four different split-GAL4 lines for this cell type. All four lines display expression in …
Figure 10—figure supplement 3
Activation phenotype of EL.
Thermogenetic activation (A) of EL neurons decreases sleep in one of the split-GAL4 lines (SS00026), while optogenetic activation (B) of both lines decreases sleep and increases P(Wake) on Day 2.
(C, D) MIP images of GFP-driven expression in the brain and VNC of the three spilt-GAL4 lines. The brain and VNC are outlined in red. (E) Higher resolution images of the relevant brain area of in SS00026. (F) Same as panel E, but with neuropil reference. (G) Morphology of individual neurons revealed by stochastic labeling. Original confocal stacks for panels C–G can be downloaded from https://www.janelia.org/split-gal4. The full genotypes of the driver lines are given there and in Figure 2—source data 1. Statistical comparisons were made by Kruskal–Wallis and Dunn’s post hoc test. Asterisk indicates significance from 0: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 10—figure supplement 4
Activation phenotype of hDeltaK.
This group of intrinsic columnar neurons is highly peptidergic and has been implicated in chronic social isolation-induced sleep loss (Li et al., 2021). We tested four split-GAL4 lines (SS59455, …
Figure 10—figure supplement 5
Activation phenotypes of combinations of dFB cell types.
The five split-GAL4 lines used here each utilize R84C10 as one of their hemidrivers. They represent different but largely overlapping subsets of layer 6 and 7 FB tangential cell types and have no …
Figure 11 with 1 supplement
Activation of SMP368 decreases sleep.
SMP368 connects central clock outputs to the CX (see Figure 12, Figure 12—figure supplement 1). Both split-GAL4 lines for SMP368 that we tested (SS74918 and SS74944) are strongly wake promoting in …
Figure 11—figure supplement 1
Activation phenotype of SMP531.
SMP531 is part of a potential pathway from clock outputs to the CX (see Figure 12—figure supplement 1) The SMP531 split-GAL4 line SS79089 suppresses sleep when activated thermogenetically (A) and …
Figure 12 with 1 supplement
Circuit diagram of CX cells implicated in regulating sleep.
Selected CX cell types, plus non-CX cell types SMP368, LNd, and OA-VPM3, are shown. The number within each circle denotes the number of cells in that cell type. The neurotransmitters used by each …
Figure 12—figure supplement 1
Circuit diagram showing some additional cell types potentially involved in regulating sleep.
Other studies have identified roles for some of these cell types as well as additional cells not shown in this diagram. Links between clock output DN1 neurons to the ExR1 have been described in Lamaz…
Videos
Video 1
Expression of genes encoding the neuropeptide receptors PDFR and Dh44R2 as well as the neuropeptide Dh31 in ER5 cells of the EB.
The ER5 cells were marked by membrane-bound GFP expression and expression of PDFR, Dh44R2, and Dh31 were assayed by EASI-FISH.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/104764/elife-104764-mdarchecklist1-v1.docx
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Supplementary file 1
Table of additional split-GAL4 lines organized by CX structure and cell type.
- https://cdn.elifesciences.org/articles/104764/elife-104764-supp1-v1.xlsx
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Supplementary file 2
Thermogenetic screen of selected stable-split lines in males.
- https://cdn.elifesciences.org/articles/104764/elife-104764-supp2-v1.xlsx
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Supplementary file 3
Thermogenetic screen of selected stable-split lines in females.
- https://cdn.elifesciences.org/articles/104764/elife-104764-supp3-v1.xlsx
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Supplementary file 4
Optogenetic screen of selected stable-split lines in males.
- https://cdn.elifesciences.org/articles/104764/elife-104764-supp4-v1.xlsx
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Supplementary file 5
Optogenetic screen of selected stable-split lines in females.
- https://cdn.elifesciences.org/articles/104764/elife-104764-supp5-v1.xlsx
