Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism
- Brain Mind Institute, EPFL - Swiss Federal Institute of Technology, Switzerland
- Department of Microbiology and Cell Biology, Montana State University, United States
Figures
Figure 1
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 …
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Figure 1—source data 1
Source Data for Figure 1.
- https://cdn.elifesciences.org/articles/74968/elife-74968-fig1-data1-v1.xlsx
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…
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Figure 2—source data 1
Source Data for Figure 2.
- https://cdn.elifesciences.org/articles/74968/elife-74968-fig2-data1-v1.xlsx
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 …
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 …
Figure 3
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 …
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Figure 3—source data 1
Source Data for Figure 3.
- https://cdn.elifesciences.org/articles/74968/elife-74968-fig3-data1-v1.xlsx
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-G…
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Figure 4—source data 1
Source Data for Figure 4.
- https://cdn.elifesciences.org/articles/74968/elife-74968-fig4-data1-v1.xlsx
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 …
Scheme 1
Z-position colour code employed in 2D representations.
Scheme 2
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 resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (Escherichia Coli) | One shot top10 | Invitrogen | Cat#: C404010 | |
| genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]= MIC}Syt1[MI02197] | Bloomington Drosophila Stock CenterPMID: 21985007 | BDSC: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: 21985007 | BDSC: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: 21985007 | BDSC: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: 21985007 | BDSC:34115FLYB: FBal0249123 RRID:BDSC_34115 | FlyBase symbol: Mi{MIC}ShabMI00848 |
| genetic reagent (D. melanogaster) | nSyb-GAL4 | Bloomington Drosophila Stock CenterPMID: 18621688 | BDSC: 39171FBgn0013342 RRID:BDSC_39171 | Flybase symbol: P{GMR57C10-GAL4} |
| genetic reagent (D. melanogaster) | repo-GAL4 | Bloomington Drosophila Stock Center PMID: 7926782 | BDSC:7415FLYB: FBal0127275 RRID:BDSC_7415 | FlyBase symbol: P{GAL4}repo |
| genetic reagent (D. melanogaster) | repo-QF2 | Bloomington 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-GAL4 | Bloomington Drosophila Stock CenterPMID: 21985007 | BDSC:60325FLYB: FBal0304243 RRID:BDSC_60325 | FlyBase symbol: GAL4Shaw- MI01735-TG4.1 |
| genetic reagent (D. melanogaster) | Ddc-GAL4 | Bloomington 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-GAL4 | Bloomington Drosophila Stock CenterPMID: 12555273 | BDSC:8848FLYB: FBtp0114847 RRID:BDSC_8848 | FlyBase symbol: P{ple-GAL4.F}3 |
| genetic reagent (D. melanogaster) | Trh-GAL4 | Bloomington 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-GFP | Steve Stowers (Montana SU)doi:10.1371/journal. pgen.1008609 | FLYB: FBgn0001196 | |
| genetic reagent (D. melanogaster) | QUAS_H2A-mCherry | Steve Stowers(Montana SU)doi:10.1371/journal. pgen.1008609 | FLYB: FBgn0001196 | |
| genetic reagent (D. melanogaster) | brp-GAL4 | This paper, available upon request, https://www.epfl.ch/labs/mccabelab/resources/ | FLYB:FBgn0259246 | See Materials and Methods, Section 2 |
| genetic reagent (D. melanogaster) | Syt1-Gal4 | This paper, available upon request, https://www.epfl.ch/labs/mccabelab/resources/ | FLYB: FBal0314405 | See Materials and Methods, Section 4 |
| genetic reagent (D. melanogaster) | Sh-Gal4 | This paper, available upon request,https://www.epfl.ch/labs/mccabelab/resources/ | FLYB: FBal0297530 | See Materials and Methods, Section 4 |
| genetic reagent (D. melanogaster) | Shal-Gal4 | This paper, available upon request,https://www.epfl.ch/labs/mccabelab/resources/ | FLYB: FBal0249123 | See Materials and Methods, Section 4 |
| genetic reagent (D. melanogaster) | Shab-Gal4 | This paper, available upon request,https://www.epfl.ch/labs/mccabelab/resources/ | FLYB: FBal0249123 | See 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: FBgn0001196 | See Materials and Methods, Section 3 |
| antibody | anti-myc9EH10(Mouse monoclonal) | DSHB | DSHB Cat# 9E 10, RRID:AB_2266850 | IF(1:100) |
| antibody | anti-GFP(Chicken polyclonal) | Abcam | Cat#ab13970 | IF(1:500) |
| antibody | anti-Deadpan(Rat monoclonal) | Abcam | Cat#ab195173 | IF(1:50) |
| antibody | Goat Anti-Mouse IgG (H+L), Alexa Fluor 488 (Goat polyclonal Secondary Antibody) | Jackson ImmunoResearch | Cat#115-545-166 | IF(1:400) |
| antibody | Goat Anti-Horseradish Peroxidase,Alexa Fluor 647 (Goat polyclonal) | Jackson ImmunoResearch | Cat#123-605-021 | IF(1:200) |
| antibody | Goat anti-Chicken IgY (H+L), Alexa Fluor 488 (Goat polyclonal Secondary Antibody) | ThermoFisher | Cat#A-11039 | IF(1:500) |
| antibody | Goat anti-Rat IgG (H+L) Cross-Adsorbed, Alexa Fluor 594 (Goat polyclonal Secondary Antibody) | ThermoFisher | Cat#A-11007 | IF(1:500) |
| recombinant DNA reagent | pBID_DSCP-G-Gal4 (plasmid) | McCabe Lab,Available upon request.https://www.epfl.ch/labs/mccabelab/resources/ | Cat# #35,200 | |
| recombinant DNA reagent | pJFRC81-10XUAS-IVS- Syn21-GFP-p10 (plasmid) | Addgene | Cat#: 36,432 | |
| recombinant DNA reagent | pHD-sfGFP Scareless dsRed (plasmid) | Addgene | Cat#: 80,811 | |
| recombinant DNA reagent | pCFD3 (plasmid) | Addgene | Cat#: 49,410 | |
| recombinant DNA reagent | pCR4 brp-Gal4 | This paper, available upon request, https://www.epfl.ch/labs/mccabelab/resources/ | CRISPR construct inserted in D. Melanogaster | |
| recombinant DNA reagent | pBID LexO_H2A-mCherry | Gift from Steve Stowers (Montana SU), DOI: 10.1371/journal.pgen.1008609 | ||
| recombinant DNA reagent | pCS2+ChMermaid S188 | Addgene | Cat#: 53,617 | |
| recombinant DNA reagent | pBID-UAS_H2A::GFP-T2A-mKok::Caax | This paper, available upon request, https://www.epfl.ch/labs/mccabelab/resources/ | construct inserted in D. Melanogaster | |
| sequence-based reagent | pCFD3 gRNA brp-5’ | This paper | Guide RNA for Brp CRISPR knock in | GGTGAACCGA CCGGGACAAC |
| sequence-based reagent | pCFD3 gRNA brp-3’ | This paper | Guide RNA for Brp CRISPR knock in | GGGAGCCCCGC GACCGCTCC |
| sequence-based reagent | brp Ha1 Fo | This paper | PCR primer | GAGAGAGCATCT CGATTGTGCCGTGTG |
| sequence-based reagent | brp Pam7 Re | This paper | PCR primer | AATGTTGTCCCG GTCGGTTCACCG |
| sequence-based reagent | brp Pam7_In1 Re | This paper | PCR primer | TTCTAGCGTCCAA CGGCTCAGCTGTG GGCCATTTTCTAGT AATGTTGTCCCGG TCGGTTCACCG |
| sequence-based reagent | brp HA_In1 Fo | This paper | PCR primer | ACTAGAAAATGGCC CACAGCTGAGCC |
| sequence-based reagent | brp V5_In1 Re | This paper | PCR primer | TAGAATCGAGACCG AGGAGAGGGTTAGGG ATAGGCTTACCCATT GCTGAAATTCACACA CACACAGAATTCATGAG |
| sequence-based reagent | brp V5_ Fo | This paper | PCR primer | GGTAAGCCTATCCC TAACCCTCTCCTC |
| sequence-based reagent | brp PB5’ Re | This paper | PCR primer | TTAAGGGATCTTTCTA TTAGTATAACACTGCATGC |
| sequence-based reagent | brp Ex2 fo | This paper | PCR primer | AAATTGCATGCAGTGTT ATACTAATAGAAAGATCC CTTAATCGGCAGTCCAT ACTACCGCGACATGGATG |
| sequence-based reagent | brp Pam2_Re | This paper | PCR primer | TCTGGAGCGGT CGCGGGGC |
| sequence-based reagent | brp Pam2_Brp Ha2 Re | This paper | PCR primer | GCTCGTCCTCTAGGTAC AGGCCCCGTTCGAGGGA TCTGTCTCTGGAGC GGTCGCGGGG |
| sequence-based reagent | brp Ha2 Fo | This paper | PCR primer | GACAGATCCCTC GAACGGGGCC |
| sequence-based reagent | Syn21 H2A Fo | This paper | PCR primer | AACTTAAAAAAAAAA ATCAAAATGTCTGGA CGTGGAAAAGGTGGC |
| sequence-based reagent | H2A Re | This paper | PCR primer | CCCAAGAAGACC GAGAAGAAGGCC |
| sequence-based reagent | H2A-GFP Fo | This paper | PCR primer | ACAGGCTGTTCTGT TGCCCAAGAAGACC GAGAAGAAGGCCAT GGTGTCCAAGGG CGAGGAG |
| sequence-based reagent | GFP-T2A Re | This paper | PCR primer | GGGTTCTCCTCCAC ATCGCCGCAGGTCAG CAGGCTGCCGCGGC CCTCCTTGTACAGCT CATCCATGCCCAGG |
| sequence-based reagent | T2A-mKok Fo | This paper | PCR primer | GCGGCAGCCTGCT GACCTGCGGCGATG TGGAGGAGAACCCC GGGCCCATGGTGAGT GTGATTAAACCAG AGATGAAGATG |
| sequence-based reagent | mKok-Caax Re | This paper | PCR primer | TTACATAATTACACA CTTTGTCTTTGACTT CTTTTTCTTCTTTTTA CCATCTTTGCTCATGG AATGAGCTACTGCAT CTTCTACCTGC |
| chemical compound, drug | Formaldehyde 37% | Sigma | Cat#: 252,549 | |
| chemical compound, drug | Low melt agarose | Peq gold | Cat#: 35–2010 | |
| chemical compound, drug | VECTASHIELD Antifade Mounting Media | VECTOR Laboratories | Cat#: H-1000 | |
| chemical compound, drug | FluoSpheres, 0.2 µm, red fluorescent (580/605) | Thermo Fisher Scientific | Cat#: F8810 | |
| commercial assay or kit | Zero Blunt TOPO PCR Cloning Kit | Invitrogen | Cat#: 450,245 | |
| commercial assay or kit | pCR8/GW/TOPO TA Cloning Kit | Invitrogen | Cat#: K250020 | |
| commercial assay or kit | Gateway LR Clonase II Enzyme mix | Invitrogen | Cat#:11791020 | |
| commercial assay or kit | NEBuilder HiFi DNA Assembly Master Mix | New England Biolabs | Cat#: E2621S | |
| commercial assay or kit | KLD enzyme mix | New England Biolabs | Cat#:M0554S | |
| commercial assay or kit | Platinium Superfi polymerase | Invitrogen | Cat#:12359010 | |
| software, algorithm | CNS nuclei distance (code for Matlab) | https://doi.org/10.5281/zenodo | This manuscript | |
| software, algorithm | Fiji | https://fiji.sc/ | RRID:SCR_002285 | |
| software, algorithm | Arivis Vision4D 3.0.0 | Arivis | RRID:SCR_018000 | |
| software, algorithm | MATLAB (R2018a) | MathWorks | RRID:SCR_001622 | |
| software, algorithm | GraphPad Prism 9.0 | GraphPad | RRID:SCR_002798 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/74968/elife-74968-mdarchecklist1-v1.pdf
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Supplementary file 1
CNS nuclei center of geometry coordinates.
- https://cdn.elifesciences.org/articles/74968/elife-74968-supp1-v1.xlsx
