Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism

  1. Wei Jiao
  2. Gard Spreemann
  3. Evelyne Ruchti
  4. Soumya Banerjee
  5. Samuel Vernon
  6. Ying Shi
  7. R Steven Stowers
  8. Kathryn Hess
  9. Brian D McCabe  Is a corresponding author
  1. Brain Mind Institute, EPFL - Swiss Federal Institute of Technology, Switzerland
  2. Department of Microbiology and Cell Biology, Montana State University, United States
6 figures, 14 videos, 1 table and 2 additional files

Figures

Intact whole CNS quantitation pipeline schematic and validation.

(a–d) Illustration of an intact whole central nervous system (CNS) genetic, imaging and computational pipeline. (a) Genetic reagents: GAL4 is introduced into the exons of genes encoding synaptic proteins (e.g. bruchpilot [brp]) to capture their expression pattern with high fidelity. GAL4 expression regulates the production of UAS fluorescent-histone reporters, which target to the nucleus of cells, producing a punctate signal. (b) Imaging: the intact CNS is imaged at high resolution using light-sheet microscopy. Images are captured at five different angles at 72° intervals. (c) Assembly: multiview light sheet images are registered, fused, and deconvolved. (d) Quantitation: the volume is segmented, and the nucleus number and relative position are measured. Three-dimensional coordinates of the geometric centre of every nucleus can be calculated to produce a point cloud of nuclei positions.(e–h) Pipeline validation. Three-dimensional images before segmentation (above) and subsequent to segmentation (below) of (e) dopaminergic (TH-GAL4) neurons, (f) serotonergic neurons (Trh-GAL4) and (g) dopa decarboxylase expressing (Ddc-GAL4) neurons. (h) Manual or automated quantification of nuclei numbers in these volumes are similar. Scale squares in (e) and (g) are 100 μm and in (f) is 50 μm. (h) Bars indicate minimum and maximum values.

Figure 2 with 2 supplements
Quantitation of neurons and glia in the whole female larval CNS.

(a–d) Multiview deconvolved images (left) and z-stack projections (right) (colours represent z position) of the central nervous system (CNS) of (a) brp-GAL4, (b) Syt1-GAL4, (c)nSyb-GAL4, and (d) repo-GAL4. (e) Distribution of inter-nuclei distances for each line. (f) Quantification of the number of labelled nuclei in each line. (a–d) left; scale squares (a) and (c) = 50 μm, (b) and (d) = 100 μm; right images identical magnification, scale bar = 100 μm. (f) Bars indicate minimum and maximum values.

Figure 2—figure supplement 1
Larval CNS stem cells.

Central nervous system (CNS) projections labelled with (a) insc-GAL4 and (b) elav-GAL4. insc-GAL4 labels neuronal stem cells. elav-GAL4 also labels a fraction of stem cells in addition to potentially glial cells. Colours represent z position. Scale bar: 50 μm.

Figure 2—figure supplement 2
Developmental expression of neuronal GAL4 lines.

(a) Expression UAS_smFP(myc) (green) under control of brp-GAL4, Syt1-GAL4, and nSyb-GAL4 at embryonic stages 15–16 (upper) and stage 17 (lower). Neuronal membranes are labelled with anti Horseradish Peroxidase (HRP) (red) (b) Labelling of L3 larval central nervous system (CNS) with Deadpan (red) and UAS_histone-sfGFP (green) under control of brp-GAL4, Syt1-GAL4, or nSyb-GAL4 (c) histone-sfGFP expression in the adult brain under control of brp-GAL4, Syt1-GAL4, or nSyb-GAL4. Scale bar = 50 μm in (a), (b), and (c).

Quantitation of neurons and glia in the male larval CNS and topological comparison of sex differences.

(a–d) Example z-stack projections (colours represent z position) of male larval central nervous system (CNS) of (a) brp-GAL4, (b) Syt1-GAL4, (c) nSyb-GAL4, and (d) repo-GAL4. (e) Quantification of the number of labelled nuclei in each line. (f) The distribution of correlations between the ground truth and the prediction made by the support vector machine (SVM) using topological features is indicative of sexual dimorphism of the higher order structure of neuron point clouds (g) Simpler point cloud features such as properties of the distributions of inter-nuclei distances are not indicative of this. (a–d): identical magnification, scale bar = 100 μm. (e) Bars indicate minimum and maximum values.

Figure 4 with 1 supplement
Quantitation of the number of neurons expressing voltage-gated potassium channel genes.

(a–d) Multiview deconvolved images (left) and z-stack projections (right) (colours represent z position) of potassium channel family members: (a) Sh-GAL4, (b) Shal-GAL4, (c) Shab-GAL4, and (d) Shaw-GAL4. (e) Quantification of the number of labelled nuclei in each line. (a–d) left, scale squares = 50 μm, right, identical magnification, scale bar = 50 μm. (e) Bars indicate minimum and maximum values.

Figure 4—figure supplement 1
Expression of voltage gated K+ channel GAL4 lines in motor neurons.

Expression in example motor neurons and neuromuscular junction terminals of UAS_mKO-CAAX by (a) Sh-GAL4, (b) Shal-GAL4, (c) Shab-GAL4, and (d) Shaw-GAL4. All four lines express in motor neurons as predicted from mutant analysis. All images identical magnification, scale bar = 10 μm.

Z-position colour code employed in 2D representations.
Illustrative 2-dimensional synthetic point cloud.

Videos

Video 1
Larval CNS labelled with TH-GAL4.
Video 2
Larval CNS labelled with Trh-GAL4.
Video 3
Larval CNS labelled with Ddc-GAL4.
Video 4
Female CNS labelled with brp-GAL4.
Video 5
Female CNS labelled with Syt1-GAL4.
Video 6
Female CNS labelled with nSyb-GAL4.
Video 7
Larval CNS labelled with brp-GAL4 and repo-QF2.
Video 8
Larval CNS labelled with Syt1-GAL4 and repo-QF2.
Video 9
Larval CNS labelled with nSyb-GAL4 and repo-QF2.
Video 10
Female CNS labelled with repo-GAL4.
Video 11
Larval CNS labelled with Sh-GAL4.
Video 12
Larval CNS labelled with Shal-GAL4.
Video 13
Larval CNS labelled with Shab-GAL4.
Video 14
Larval CNS labelled with Shaw-GAL4.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
strain, strain background (Escherichia Coli)One shot top10InvitrogenCat#: C404010
genetic reagent (D. melanogaster)y[1] w[*]; Mi{y[+mDint2]=
MIC}Syt1[MI02197]
Bloomington Drosophila Stock CenterPMID: 21985007BDSC:35973FLYB: FBal0314405
RRID:BDSC_35973
FlyBase symbol: Mi{y[+mDint2]=
MIC}Syt1[MI02197]
genetic reagent (D. melanogaster)y[1] w[*] Mi{y[+mDint2]=
MIC}Sh[MI10885]
Bloomington Drosophila Stock CenterPMID: 21985007BDSC:56260FLYB:
FBal0297530
RRID:BDSC_56260
FlyBase symbol: Mi{MIC}ShMI10885
genetic reagent (D. melanogaster)y[1] w[*];Mi{y[+mDint2]=
MIC}Shal[MI10881]
Bloomington Drosophila Stock CenterPMID: 21985007BDSC:56089FLYB: FBal0295200
RRID:BDSC_56089
FlyBase symbol: Mi{MIC}ShalMI10881
genetic reagent (D. melanogaster)y[1] w[*]; Mi{y[+mDint2]=MIC} Shab[MI00848]Bloomington Drosophila Stock CenterPMID: 21985007BDSC:34115FLYB: FBal0249123
RRID:BDSC_34115
FlyBase symbol:
Mi{MIC}ShabMI00848
genetic reagent (D. melanogaster)nSyb-GAL4Bloomington Drosophila
Stock CenterPMID: 18621688
BDSC: 39171FBgn0013342
RRID:BDSC_39171
Flybase symbol:
P{GMR57C10-GAL4}
genetic reagent (D. melanogaster)repo-GAL4Bloomington Drosophila
Stock Center PMID: 7926782
BDSC:7415FLYB: FBal0127275
RRID:BDSC_7415
FlyBase symbol: P{GAL4}repo
genetic reagent (D. melanogaster)repo-QF2Bloomington Drosophila Stock Centerdoi:10.1534/
genetics.116.191783
BDSC:66477FLYB: FBal0322908
RRID:BDSC_66477
FlyBase symbol: P{ET-QF2.GU}repo
genetic reagent (D. melanogaster)Shaw-GAL4Bloomington Drosophila
Stock CenterPMID: 21985007
BDSC:60325FLYB: FBal0304243
RRID:BDSC_60325
FlyBase symbol:
GAL4Shaw-
MI01735-TG4.1
genetic reagent (D. melanogaster)Ddc-GAL4Bloomington Drosophila Stock Center
doi:10.1006/
dbio.1994.1261
BDSC:7009FLYB: FBtp0012451
RRID:BDSC_7009
FlyBase symbol:
P{Ddc-GAL4.L}
genetic reagent (D. melanogaster)TH-GAL4Bloomington Drosophila Stock CenterPMID: 12555273BDSC:8848FLYB: FBtp0114847
RRID:BDSC_8848
FlyBase symbol:
P{ple-GAL4.F}3
genetic reagent (D. melanogaster)Trh-GAL4Bloomington Drosophila Stock Centerdoi:10.1371/journal.
pone.0010806
BDSC:38389FLYB:
FBtp0055412
RRID:BDSC_38389
FlyBase symbol:
P{Trh-GAL4.long}
genetic reagent (D. melanogaster)UAS_H2A-GFPSteve Stowers (Montana SU)doi:10.1371/journal.
pgen.1008609
FLYB: FBgn0001196
genetic reagent (D. melanogaster)QUAS_H2A-mCherrySteve Stowers(Montana SU)doi:10.1371/journal.
pgen.1008609
FLYB: FBgn0001196
genetic reagent (D. melanogaster)brp-GAL4This paper, available
upon request, https://www.epfl.ch/labs/mccabelab/resources/
FLYB:FBgn0259246See Materials and
Methods, Section 2
genetic reagent (D. melanogaster)Syt1-Gal4This paper, available
upon request, https://www.epfl.ch/labs/mccabelab/resources/
FLYB: FBal0314405See Materials and
Methods, Section 4
genetic reagent (D. melanogaster)Sh-Gal4This paper, available
upon request,https://www.epfl.ch/labs/mccabelab/resources/
FLYB: FBal0297530See Materials and
Methods, Section 4
genetic reagent (D. melanogaster)Shal-Gal4This paper, available upon request,https://www.epfl.ch/labs/mccabelab/resources/FLYB: FBal0249123See Materials and
Methods, Section 4
genetic reagent (D. melanogaster)Shab-Gal4This paper, available
upon request,https://www.epfl.ch/labs/mccabelab/resources/
FLYB: FBal0249123See Materials and
Methods, Section 4
genetic reagent (D. melanogaster)UAS_H2A::GFP-
T2A-mKok::Caax
This paper, available
upon request,https://www.epfl.ch/labs/mccabelab/resources/
FLYB: FBgn0001196See Materials and
Methods, Section 3
antibodyanti-myc9EH10(Mouse monoclonal)DSHBDSHB Cat# 9E 10, RRID:AB_2266850IF(1:100)
antibodyanti-GFP(Chicken polyclonal)AbcamCat#ab13970IF(1:500)
antibodyanti-Deadpan(Rat monoclonal)AbcamCat#ab195173IF(1:50)
antibodyGoat Anti-Mouse IgG (H+L), Alexa Fluor 488 (Goat polyclonal Secondary Antibody)Jackson
ImmunoResearch
Cat#115-545-166IF(1:400)
antibodyGoat Anti-Horseradish Peroxidase,Alexa Fluor 647 (Goat polyclonal)Jackson
ImmunoResearch
Cat#123-605-021IF(1:200)
antibodyGoat anti-Chicken IgY (H+L), Alexa Fluor 488 (Goat polyclonal Secondary Antibody)ThermoFisherCat#A-11039IF(1:500)
antibodyGoat anti-Rat IgG (H+L) Cross-Adsorbed, Alexa Fluor 594 (Goat polyclonal Secondary Antibody)ThermoFisherCat#A-11007IF(1:500)
recombinant DNA reagentpBID_DSCP-G-Gal4 (plasmid)McCabe Lab,Available
upon request.https://www.epfl.ch/labs/mccabelab/resources/
Cat# #35,200
recombinant DNA reagentpJFRC81-10XUAS-IVS-
Syn21-GFP-p10 (plasmid)
AddgeneCat#: 36,432
recombinant DNA reagentpHD-sfGFP Scareless dsRed (plasmid)AddgeneCat#: 80,811
recombinant DNA reagentpCFD3 (plasmid)AddgeneCat#: 49,410
recombinant DNA reagentpCR4 brp-Gal4This paper, available
upon request, https://www.epfl.ch/labs/mccabelab/resources/
CRISPR construct inserted
in D. Melanogaster
recombinant DNA reagentpBID LexO_H2A-mCherryGift from Steve Stowers
(Montana SU),
DOI: 10.1371/journal.pgen.1008609
recombinant DNA reagentpCS2+ChMermaid S188AddgeneCat#: 53,617
recombinant DNA reagentpBID-UAS_H2A::GFP-T2A-mKok::CaaxThis paper, available
upon request, https://www.epfl.ch/labs/mccabelab/resources/
construct inserted
in D. Melanogaster
sequence-based reagentpCFD3 gRNA brp-5’This paperGuide RNA for Brp CRISPR knock inGGTGAACCGA
CCGGGACAAC
sequence-based reagentpCFD3 gRNA brp-3’This paperGuide RNA for Brp CRISPR knock inGGGAGCCCCGC
GACCGCTCC
sequence-based reagentbrp Ha1 FoThis paperPCR primerGAGAGAGCATCT
CGATTGTGCCGTGTG
sequence-based reagentbrp Pam7 ReThis paperPCR primerAATGTTGTCCCG
GTCGGTTCACCG
sequence-based reagentbrp Pam7_In1 ReThis paperPCR primerTTCTAGCGTCCAA
CGGCTCAGCTGTG
GGCCATTTTCTAGT
AATGTTGTCCCGG
TCGGTTCACCG
sequence-based reagentbrp HA_In1 FoThis paperPCR primerACTAGAAAATGGCC
CACAGCTGAGCC
sequence-based reagentbrp V5_In1 ReThis paperPCR primerTAGAATCGAGACCG
AGGAGAGGGTTAGGG
ATAGGCTTACCCATT
GCTGAAATTCACACA
CACACAGAATTCATGAG
sequence-based reagentbrp V5_ FoThis paperPCR primerGGTAAGCCTATCCC
TAACCCTCTCCTC
sequence-based reagentbrp PB5’ ReThis paperPCR primerTTAAGGGATCTTTCTA
TTAGTATAACACTGCATGC
sequence-based reagentbrp Ex2 foThis paperPCR primerAAATTGCATGCAGTGTT
ATACTAATAGAAAGATCC
CTTAATCGGCAGTCCAT
ACTACCGCGACATGGATG
sequence-based reagentbrp Pam2_ReThis paperPCR primerTCTGGAGCGGT
CGCGGGGC
sequence-based reagentbrp Pam2_Brp Ha2 ReThis paperPCR primerGCTCGTCCTCTAGGTAC
AGGCCCCGTTCGAGGGA
TCTGTCTCTGGAGC
GGTCGCGGGG
sequence-based reagentbrp Ha2 FoThis paperPCR primerGACAGATCCCTC
GAACGGGGCC
sequence-based reagentSyn21 H2A FoThis paperPCR primerAACTTAAAAAAAAAA
ATCAAAATGTCTGGA
CGTGGAAAAGGTGGC
sequence-based reagentH2A ReThis paperPCR primerCCCAAGAAGACC
GAGAAGAAGGCC
sequence-based reagentH2A-GFP FoThis paperPCR primerACAGGCTGTTCTGT
TGCCCAAGAAGACC
GAGAAGAAGGCCAT
GGTGTCCAAGGG
CGAGGAG
sequence-based reagentGFP-T2A ReThis paperPCR primerGGGTTCTCCTCCAC
ATCGCCGCAGGTCAG
CAGGCTGCCGCGGC
CCTCCTTGTACAGCT
CATCCATGCCCAGG
sequence-based reagentT2A-mKok FoThis paperPCR primerGCGGCAGCCTGCT
GACCTGCGGCGATG
TGGAGGAGAACCCC
GGGCCCATGGTGAGT
GTGATTAAACCAG
AGATGAAGATG
sequence-based reagentmKok-Caax ReThis paperPCR primerTTACATAATTACACA
CTTTGTCTTTGACTT
CTTTTTCTTCTTTTTA
CCATCTTTGCTCATGG
AATGAGCTACTGCAT
CTTCTACCTGC
chemical compound, drugFormaldehyde 37%SigmaCat#: 252,549
chemical compound, drugLow melt agarosePeq goldCat#: 35–2010
chemical compound, drugVECTASHIELD
Antifade Mounting Media
VECTOR LaboratoriesCat#: H-1000
chemical compound, drugFluoSpheres, 0.2 µm, red fluorescent (580/605)Thermo Fisher ScientificCat#: F8810
commercial assay or kitZero Blunt TOPO
PCR Cloning Kit
InvitrogenCat#: 450,245
commercial assay or kitpCR8/GW/TOPO
TA Cloning Kit
InvitrogenCat#: K250020
commercial assay or kitGateway LR Clonase
II Enzyme mix
InvitrogenCat#:11791020
commercial assay or kitNEBuilder HiFi
DNA Assembly Master Mix
New England BiolabsCat#: E2621S
commercial assay or kitKLD enzyme mixNew England BiolabsCat#:M0554S
commercial assay or kitPlatinium
Superfi polymerase
InvitrogenCat#:12359010
software, algorithmCNS nuclei distance
(code for Matlab)
https://doi.org/10.5281/zenodoThis manuscript
software, algorithmFijihttps://fiji.sc/RRID:SCR_002285
software, algorithmArivis Vision4D 3.0.0ArivisRRID:SCR_018000
software, algorithmMATLAB (R2018a)MathWorksRRID:SCR_001622
software, algorithmGraphPad Prism 9.0GraphPadRRID:SCR_002798

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  1. Wei Jiao
  2. Gard Spreemann
  3. Evelyne Ruchti
  4. Soumya Banerjee
  5. Samuel Vernon
  6. Ying Shi
  7. R Steven Stowers
  8. Kathryn Hess
  9. Brian D McCabe
(2022)
Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism
eLife 11:e74968.
https://doi.org/10.7554/eLife.74968