Figures and data in Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism

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Tables
Additional files

6 figures, 14 videos, 1 table and 2 additional files

Figures

Figure 1

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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 1—source data 1 Source Data for Figure 1.: https://cdn.elifesciences.org/articles/74968/elife-74968-fig1-data1-v1.xlsx
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Figure 2 with 2 supplements

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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—source data 1 Source Data for Figure 2.: https://cdn.elifesciences.org/articles/74968/elife-74968-fig2-data1-v1.xlsx
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Figure 2—figure supplement 1

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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

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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).

Figure 3

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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 3—source data 1 Source Data for Figure 3.: https://cdn.elifesciences.org/articles/74968/elife-74968-fig3-data1-v1.xlsx
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Figure 4 with 1 supplement

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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—source data 1 Source Data for Figure 4.: https://cdn.elifesciences.org/articles/74968/elife-74968-fig4-data1-v1.xlsx
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Figure 4—figure supplement 1

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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.

Scheme 1

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Z-position colour code employed in 2D representations.

Scheme 2

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Illustrative 2-dimensional synthetic point cloud.

Videos

Video 1

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posterframe for video — Larval CNS labelled with TH-GAL4.

Video 2

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Video 7

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Video 8

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Video 14

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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