Origin of wiring specificity in an olfactory map revealed by neuron type–specific, time-lapse imaging of dendrite targeting
- Department of Biology, Howard Hughes Medical Institute, Stanford University, United States
- Howard Hughes Medical Institute, Janelia Research Campus, United States
- Advanced Bioimaging Center, Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, United States
- Chan Zuckerberg Biohub, United States
- Departments of Molecular and Cell Biology and Physics, Howard Hughes Medical Institute, Helen Wills Neuroscience Institute, University of California, United States
Figures
Figure 1 with 2 supplements
Organization and development of the adult olfactory circuit in Drosophila.
(A, B) Timeline (A) and schematic illustration (B) of Drosophila olfactory circuit development. Green, red, and blue circles denote the birth of embryonic-born anterodorsal projection neuron (adPN), …
Figure 1—figure supplement 1
Visualization of larval- and adult-specific antennal lobes by co-staining of Ncad and nc82.
(A–C) N-Cadherin (Ncad) and nc82 staining of antennal lobes are shown in Figures 1D1—3. Adult-specific antennal lobes characteristic of strong Ncad and weak nc82 staining are outlined by a white …
Figure 1—figure supplement 2
Projection neuron (PN) axon development across pupal stages.
(A–F) Staining of fixed brains at indicated stages showing axon development of anterodorsal projection neurons (adPNs) (VT033006+ run+ ; labeled in yellow) and lPNs (VT033006+ run–; labeled in …
Figure 2 with 3 supplements
Expanded genetic toolkit for dual-color, type-specific labeling of projection neurons (PNs).
(A) tSNE plot of PN single-cell transcriptomes, color-coded according to CR45223 expression level in [log2(CPM +1)], where CPM stands for transcript counts per million reads. Zoom-in of boxes in the …
Figure 2—figure supplement 1
Expression of projection neuron (PN) marker genes across development.
(A–C) tSNE plots of PN transcriptomes, color-coded according to developmental stages (A), PN types (B), or the expression levels of the indicated gene [log2(CPM +1)] (C) using scRNA-seq data from Xie…
Figure 2—figure supplement 2
Generation of T2A-QF2/FLP transgenic flies by CRISPR/Cas9.
(A) Schematic of generation of transgenic driver lines by CRISPR/Cas9. acj6-T2A-QF2 is shown as an example. (B–E) Top: Transcripts of acj6 (B), run (C), CG14322 (D), and lov (E) visualized using …
Figure 2—figure supplement 3
Design of single- and dual-color FLP-out reporters.
Images, created with SnapGene, show four newly generated Q/UAS-based single- and dual-color FLP-out reporters.
Figure 3 with 4 supplements
Birth order–dependent spatial patterning of anterodorsal projection neuron (adPN) dendrites in the developing antennal lobe.
(A) Confocal images of fixed brains at indicated stages showing dendrite development of adPNs (acj6+; labeled in green) and DL1 adPNs (71B05+; labeled in yellow). Right column of A1 shows a zoom-in …
Figure 3—figure supplement 1
Dendrite development of early larval-born projection neurons (PNs).
(A) Dendritic extension of DL1 PNs (71B05+; labeled in yellow) across the developing antennal lobe at the wandering third instar larval stage (L3). Zoom-in of the dashed box shown on the right. N=3. …
Figure 3—figure supplement 2
MARCM-labeled single-cell projection neurons (PNs) of indicated lineages in adult brains.
(A) MARCM clone of DL1 PN (in yellow) generated by heat shock at 0–24 hr after larval hatching (ALH). (B) MARCM clones of early larval-born PNs (B1–3: adPNs in yellow; B4–10: lPNs in cyan) generated …
Figure 3—figure supplement 3
Dendrite development of DL1, middle larval-born, and late larval-born projection neurons (PNs) at early stages.
Adult-specific antennal lobes (revealed by high Ncad staining; not shown) are outlined by a white solid line. (A) MARCM clones of DL1 PNs (in yellow), generated by heat shock at 0–24 hr after larval …
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Figure 3—figure supplement 3—source data 1
Source data for Figure 3—figure supplement 3F and G.
- https://cdn.elifesciences.org/articles/85521/elife-85521-fig3-figsupp3-data1-v2.xlsx
Figure 3—video 1
3D rendering of z stacks of indicated projection neurons (PNs) in 12 hr APF antennal lobe.
This video shows a 3D rendering of z stacks with rotation along y-axis to visualize PN dendrites in the context of the antennal lobe in three dimensions. See Figure 3 for details.
Figure 4 with 3 supplements
Birth order–dependent spatial patterning of lPN dendrites in the developing antennal lobe.
(A) Confocal images of fixed brains at indicated stages showing dendrite development of DL1/DA3 adPNs (CG14322+; labeled in yellow) and DA1/DL3 lPNs (tsh+; labeled in cyan). Right column of A1 shows …
Figure 4—figure supplement 1
Dendrite development of DL1/DA3 and DA1/DL3 projection neurons (PNs).
(A) Dendritic extension of DL1/DA3 PNs (CG14322+; labeled in yellow) and DA1/DL3 PNs (tsh+; labeled in cyan) across the developing antenna lobe at the wandering third instar larval stage (L3). N=5. …
Figure 4—figure supplement 2
Expression patterns of tsh in the developing antennal lobe during mid-pupal stages.
(A) At 18 hr APF, tsh is only expressed in projection neurons (PNs) of the lateral linage (acj6–; in cyan). N=6. (B–D) From 24 hr APF onwards, tsh is expressed in some anterodorsal projection …
Figure 4—video 1
3D rendering of z stacks of indicated projection neurons (PNs) in 12 hr APF antennal lobe.
This video shows a 3D rendering of z stacks with rotation along y-axis to visualize PN dendrites in the context of antennal lobe in three dimensions. See Figure 4 for details.
Figure 5 with 3 supplements
Establishment of an explant system for time-lapse imaging of olfactory map formation.
(A) Schematic of the anatomical organization of the olfactory circuit in early pupal brain (0–3 hr APF). Green, red, and blue denote embryonic-born adPN, larval-born anterodorsal projection neuron …
Figure 5—figure supplement 1
Dendritic segregation of DC3/VA1d adPNs and DA1 lPNs targeting neighboring proto-glomeruli.
(A) Two-photon time-lapse imaging of DC3/VA1d adPN (Mz19+ acj6+; labeled in red) and DA1 lPN (Mz19+ acj6–; labeled in red) dendrites in pupal brain dissected at 24 hr APF and cultured for 8 hr ex …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1C and D.
- https://cdn.elifesciences.org/articles/85521/elife-85521-fig5-figsupp1-data1-v2.xlsx
Figure 5—video 1
Two-photon time-lapse imaging of projection neuron (PN) development.
See Figure 5E for details. Timestamp 00:00:00 refers to HH:mm:ss; H, hour; m, minute; s, second.
Figure 5—video 2
Two-photon time-lapse imaging of projection neuron (PN) dendritic segregation.
See Figure 5—figure supplement 1 for details. Timestamp 00:00:00 refers to HH:mm:ss; H, hour; m, minute; s, second.
Figure 6 with 2 supplements
Two-photon time-lapse imaging reveals active dendrite targeting.
(A) Two-photon time-lapse imaging of MARCM-labeled DL1 projection neuron (PN) (pseudo-colored in yellow) in a brain dissected at 3 hr APF and cultured for 21 hr ex vivo (A1–9). Arrowheads in A4–6 …
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Figure 6—source data 1
Source data for Figure 6C–G and Figure 6—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/85521/elife-85521-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Two-photon time-lapse imaging of DL1 projection neuron (PNs).
(A) Two-photon images of MARCM-labeled DL1 PNs (pseudo-colored in yellow) and other GH146 + cells (pseudo-colored in blue) in pupal brains dissected at 3 hr APF. Zoom-in time-lapse images of the …
Figure 6—video 1
Two-photon time-lapse imaging of DL1 projection neuron (PN) dendrites.
See Figure 6A for details. Timestamp 00:00:00 refers to HH:mm:ss; H, hour; m, minute; s, second.
Figure 7 with 4 supplements
AO-LLSM time-lapse imaging reveals cellular mechanisms of dendrite targeting specificity.
(A–C) AO-LLSM imaging of DL1 projection neurons (PNs) (71B05+; labeled in yellow) and anterodorsal projection neurons (adPNs) (acj6+; labeled in blue) in cultured brains dissected at 3 hr (A), 6 hr …
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Figure 7—source data 1
Source data for Figure 7E.
- https://cdn.elifesciences.org/articles/85521/elife-85521-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Analyses of DL1 projection neuron (PN) dendritic branches captured by AO-LLSM imaging.
(A) Categorization of branches into (1) stable, (2) transient, (3) emerging, and (4) retracting branches. Representative branches of each category are shown. Timestamp 00:00:00 refers to HH:mm:ss; …
Figure 7—video 1
AO-LLSM time-lapse imaging of 3 hr DL1 projection neuron (PN) dendrites.
See Figure 7A for details. Timestamp 00:00:00 refers to HH:mm:ss; H, hour; m, minute; s, second.
Figure 7—video 2
AO-LLSM time-lapse imaging of 6 hr DL1 projection neuron (PN) dendrites.
See Figure 7B for details. Timestamp 00:00:00 refers to HH:mm:ss; H, hour; m, minute; s, second.
Figure 7—video 3
AO-LLSM time-lapse imaging of 12 hr DL1 projection neuron (PN) dendrites.
See Figure 7C for details. Timestamp 00:00:00 refers to HH:mm:ss; H, hour; m, minute; s, second.
Figure 8 with 6 supplements
Embryonic-born projection neurons (PNs) timely participate in olfactory map formation via simultaneous pruning and re-extension.
(A) Confocal images of fixed brains at indicated stages showing dendrite development of lov+ PNs (embryonic-born; labeled in green) and 91G04+DC2 PNs (larval-born; labeled in magenta). As 91G04-GAL4 …
Figure 8—figure supplement 1
Dendrite development of lov+ embryonic-born projection neurons (PNs).
Figure 8—figure supplement 2
Dendrite re-extension of lov+ and Mz612+ embryonic-born projection neurons (PNs).
(A, B) Dendritic re-extension of lov+ (labeled in green) and Mz612+ (labeled in gray) PNs. (B) is the zoom-in of the dashed box in A. Magenta, cyan, and orange asterisks in A indicate cell bodies of …
Figure 8—figure supplement 3
Two-photon time-lapse imaging of Split7+ projection neuron (PN) dendrites.
(A) Confocal image of Split7+ PNs (labeled in yellow; Figure 8D) after 23 hr culture. N-Cadherin (Ncad; in blue) staining outlines the developing antennal lobe. Split7-GAL4 is expressed in more than …
Figure 8—figure supplement 4
Dual requirement of ecdysone signaling in pruning and re-extension of embryonic-born projection neuron (PN) dendrites.
(A) Normal dendrite development seen in control lov+ embryonic-born PNs (VT033006+ lov+ ; labeled in gray). Other PNs (referred to as most PNs; VT033006+ lov–) are labeled in green. Bottom row shows …
Figure 8—video 1
3D rendering of z stacks of indicated projection neurons (PNs) in 12 hr APF antennal lobe.
This video shows a 3D rendering of z stacks with rotation along y-axis to visualize PN dendrites in the context of the antennal lobe in three dimensions. See Figure 8 for details.
Figure 8—video 2
Two-photon time-lapse imaging of Split7+ projection neuron (PN) dendrites.
See Figure 8D for details. Timestamp 00:00:00 refers to HH:mm:ss; H, hour; m, minute; s, second.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | GH146-FLP | DOI: 10.1038/nn.2442 | ||
| Genetic reagent (D. melanogaster) | QUAS-FRT-stop-FRT-mCD8-GFP | DOI: 10.1016 /j.cell.2010.02.025 | ||
| Genetic reagent (D. melanogaster) | UAS-mCD8-GFP | DOI: 10.1016 /s0896-6273(00)80701–1 | ||
| Genetic reagent (D. melanogaster) | UAS-mCD8-FRT-GFP-FRT-RFP | DOI: 10.1016 /j.neuron.2014.06.026 | ||
| Genetic reagent (D. melanogaster) | VT033006-GAL4 | DOI: 10.1101/198648 | ||
| Genetic reagent (D. melanogaster) | Mz19-GAL4 | DOI: 10.1242/dev.00896 | ||
| Genetic reagent (D. melanogaster) | 91 G04-GAL4 | DOI: 10.1016 /j.celrep.2012.09.011 | ||
| Genetic reagent (D. melanogaster) | Mz612-GAL4 | DOI: 10.1242/dev.01614 | ||
| Genetic reagent (D. melanogaster) | 71B05-GAL4 | DOI: 10.1016 /j.celrep.2012.09.011 | ||
| Genetic reagent (D. melanogaster) | Split7-GAL4 | DOI: 10.7554/eLife.63450 | FlyLight:SS01867 | |
| Genetic reagent (D. melanogaster) | QUAS-FLP | DOI: 10.1016 /j.cell.2010.02.025 | ||
| Genetic reagent (D. melanogaster) | UAS-EcR.B1-ΔC655.F645A | DOI: 10.1242/dev.00205 | ||
| Genetic reagent (D. melanogaster) | tsh-GAL4 | Bloomington Drosophila Stock Center | BDSC:3040 | |
| Genetic reagent (D. melanogaster) | lov-GAL4 | Bloomington Drosophila Stock Center | BDSC:3737 | |
| Genetic reagent (D. melanogaster) | UAS-mCD8-GFP, hs-FLP; FRTG13, tub-GAL80;; GH146-GAL4 | DOI: 10.1016 /s0896-6273(00)80701–1 | ||
| Genetic reagent (D. melanogaster) | FRTG13, UAS-mCD8-GFP | DOI: 10.1016 /s0896-6273(00)80701–1 | ||
| Genetic reagent (D. melanogaster) | UAS-FRT10-stop-FRT10-3xHalo7-CAAX | this paper | on either II or III chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | UAS-FRT-myr-4xSNAPf-FRT-3xHalo7-CAAX | this paper | on III chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | UAS-FRT-myr-mGreenLantern-FRT-3xHalo7-CAAX | this paper | on II chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | QUAS-FRT-stop-FRT-myr-4xSNAPf | this paper | on III chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | run-T2A-FLP | this paper | on X chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | acj6-T2A-FLP | this paper | on X chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | acj6-T2A-QF2 | this paper | on X chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | CG14322-T2A-QF2 | this paper | on III chromosome; see Materials and methods | |
| Genetic reagent (D. melanogaster) | lov-T2A-QF2 | this paper | on II chromosome; see Materials and methods | |
| Antibody | chicken polyclonal anti-GFP | Aves Lab | RRID:AB_10000240; Aves Lab:GFP-1020 | (1:1000) |
| Antibody | rabbit polyclonal anti-DsRed | TaKaRa | RRID:AB_10013483; TaKaRa:632496 | (1:500) |
| Antibody | rat monoclonal anti-Cadherin DN | Developmental Studies Hybridoma Bank | RRID:AB_528121; DSHB:DN-Ex#8 | (1:30) |
| Antibody | mouse monoclonal anti-Bruchpilot | Developmental Studies Hybridoma Bank | RRID:AB_2314866; DSHB:nc82 supernatant | (1:30) |
| Recombinant DNA reagent | pBPGUw-HACK-QF2 | Addgene | RRID:Addgene_80276 | |
| Recombinant DNA reagent | pU6-BbsI-chiRNA | Addgene | RRID:Addgene_45946 | |
| Recombinant DNA reagent | pUAS-3xHalo7-CAAX | Addgene | RRID:Addgene_87646 | |
| Recombinant DNA reagent | pUAS-myr-4xSNAPf | Addgene | RRID:Addgene_87637 | |
| Recombinant DNA reagent | pcDNA3.1-mGreenLantern | Addgene | RRID:Addgene_161912 | |
| Recombinant DNA reagent | p5XQUAS | Addgene | RRID:Addgene_24349 | |
| Recombinant DNA reagent | p10xQUAS-CsChrimson | Addgene | RRID:Addgene_163629 | |
| Recombinant DNA reagent | pUAS-FRT10-stop-FRT10-3xHalo7-CAAX | this paper | backbone from pUAS-3xHalo7-CAAX; see Materials and methods | |
| Recombinant DNA reagent | pUAS-FRT-myr-4xSNAPf-FRT-3xHalo7-CAAX | this paper | backbone from pUAS-3xHalo7-CAAX; see Materials and methods | |
| Recombinant DNA reagent | pUAS-FRT-myr-mGreenLantern-FRT-3xHalo7-CAAX | this paper | backbone from pUAS-3xHalo7-CAAX; see Materials and methods | |
| Recombinant DNA reagent | pUAS-myr-mGreenLantern | this paper | backbone from pUAS-myr-4xSNAPf; see Materials and methods | |
| Recombinant DNA reagent | pQUAS-FRT-stop-FRT-myr-4xSNAPf | this paper | backbone from p5XQUAS; see Materials and methods | |
| Chemical compound, drug | SYLGARD 184 Silicone Elastomer Kit | DOW | DOW:2646340 | |
| Chemical compound, drug | Schneider’s Drosophila Medium | ThermoFisher Scientific | ThermoFisher Scientific:21720001 | |
| Chemical compound, drug | Fetal Bovine Serum | ThermoFisher Scientific | ThermoFisher Scientific:16140071 | used at 10% |
| Chemical compound, drug | Human recombinant insulin | ThermoFisher Scientific | ThermoFisher Scientific:12585014 | used at 10 µg/mL |
| Chemical compound, drug | Penicillin-Streptomycin | ThermoFisher Scientific | ThermoFisher Scientific:15140122 | (1:100) |
| Chemical compound, drug | Ascorbic acid | Sigma | Sigma:A4544 | used at 50 mg/mL in water |
| Chemical compound, drug | 20-hydroxyecdysone | Sigma | Sigma:H5142 | used at 20 µM and 2 µM |
| Chemical compound, drug | JF503-cpSNAP | DOI: 10.1038/nmeth.4403; DOI: 10.1021/jacsau.1c00006 | (1:1000); gift from Dr. Luke Lavis | |
| Chemical compound, drug | JF646-Halo | DOI: 10.1038/nmeth.4403; DOI: 10.1021/jacsau.1c00006 | (1:1000); gift from Dr. Luke Lavis | |
| Chemical compound, drug | JFX650-SNAP | DOI: 10.1038/nmeth.4403; DOI: 10.1021/jacsau.1c00006 | (1:1000); gift from Dr. Luke Lavis | |
| Chemical compound, drug | JFX554-Halo | DOI: 10.1038/nmeth.4403; DOI: 10.1021/jacsau.1c00006 | (1:10000); gift from Dr. Luke Lavis | |
| Chemical compound, drug | JF635-Halo | DOI: 10.1038/nmeth.4403; DOI: 10.1021/jacsau.1c00006 | (1:1000); gift from Dr. Luke Lavis | |
| Chemical compound, drug | JF570-Halo | DOI: 10.1038/nmeth.4403; DOI: 10.1021/jacsau.1c00006 | (1:5000); gift from Dr. Luke Lavis | |
| Chemical compound, drug | Sulforhodamine 101 | Sigma | Sigma:S7635 | used at 1 µM |
| Software, algorithm | ZEN | Carl Zeiss | RRID:SCR_013672 | |
| Software, algorithm | ImageJ | National Institutes of Health | RRID:SCR_003070 | |
| Software, algorithm | Python Programming Language | Python | RRID:SCR_008394 | http://www.python.org/ |
Additional files
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Supplementary file 1
Sample variability among individual brains.
A supplemental table describing the biological and technical variations we observed among individual brain samples, and measures we took to minimize them, if possible.
- https://cdn.elifesciences.org/articles/85521/elife-85521-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85521/elife-85521-mdarchecklist1-v2.pdf
