A library of lineage-specific driver lines connects developing neuronal circuits to behavior in the Drosophila ventral nerve cord
- School of Science and Engineering, Division Biological and Biomedical Systems, University of Missouri-Kansas City, United States
- Department of Genetics, Washington University School of Medicine, United States
- Department of Biology, New York University, United States
- Department of Biology, University of Toronto - Mississauga, Canada
- Department of Cell and Systems Biology, University of Toronto - Mississauga, Canada
- Department of Biology, University of Washington, United States
Figures
Figure 1 with 1 supplement
Intersecting the expression of acj6 and unc-4 genes with the split-GAL4 method faithfully marks hemilineage 23B.
(A–C) Projections of confocal stacks of the adult VNC. Blue: CadN; (A) acj6-GAL4 driven nls-tdTomato expression (displayed in green) marks Acj6 expressing neurons. (B) unc-4-GAL4 driven nls-tdTomato expression (displayed in green) marks Unc-4 expressing neurons. (C) The intersection of acj6 and unc-4 expression (displayed in green) (acj6-GAL4AD, unc-4-GAL4DBD> UAS-nls-tdTomato) marks lineage 23B neurons in the SEZ and VNC. (D) A partial confocal projection showing the complete overlap between membranous GFP (green) and Acj6 (magenta) immunostainings in acj6-GAL4AD, unc-4-GAL4DBD-marked 23B neurons in the adult VNC (T1 and T2 segments shown). (E) scRNAseq t-SNE plot shows Acj6 (Purple) and Unc-4 (Dark Blue) co-expression in a group of cell clusters.
Figure 1—figure supplement 1
acj6-GAL4AD, unc-4-GAL4DBD-driven myr-GFP marks 23B neurons throughout development.
(A, B) Acj6 (blue) and Unc-4 (magenta) co-expression shows robust overlap in GFP-marked embryonic progeny of NB7-4, 23B neurons, in a late-stage embryo. (C–D) Acj6 (blue) expression marks 23B neurons in an early stage larval VNC (C) and an early stage pupal VNC (D). The only lineages that express Acj6 are 23B, 8B, and 9B, and of these only the posterior-dorsal cells, corresponding to hemilineage 23B, co-stained for GFP and Acj6 in the larval and pupal VNC. (E) This driver combination marks a cluster of SEZ neurons (arrowhead) in the adult brain, presumably SEZ 23B neurons in addition to sensory neuron afferents (arrows). (F) Close-up of SEZ to highlight the corresponding cell bodies (arrowhead).
Figure 2
Matching the scRNAseq clusters to hemilineages.
(A–C) Confocal stack of larval VNC displaying the overlapping expressions between transcription factors identified from scRNAseq data (Fkh, Kn, and Sp1; green in (A), (B), and (C), respectively) and Hb9 (magenta) in three lineages: 4B, 10B, and 16B (dashed lines). Asterisk in A indicates the Fkh+Hb9- 0 A lineage neurons. (D) Sox21a-GAL4 driven UAS-GFP (green) marks lineage 2 A neurons. (E) HmxGFSTF reporter (green) marks lineage 17 A neurons. (F, G) Wild-type MARCM clones (green) immunostained for Tj (magenta). The insets show the clone location in the VNC counterstained with CadN (blue). (F) Tj marks subpopulations of neurons in lineage 0 A in the T2 segment. These neurons likely belong to cluster 88, the only Tj+ 0A cluster in scRNAseq data. (G) Tj marks nearly all neurons of lineage 21 A in the T1 segment. Lineage identification of MARCM clones was performed based on neuronal projections detailed in Truman et al., 2004; Kanca et al., 2019. scRNAseq clusters with the corresponding lineages shown under each panel. Only one thoracic segment is shown. Neuroglian-specific antibody BP104 labels axon bundles of all lineages (magenta in D-E).
Figure 3 with 58 supplements
The VNC expression of select driver lines from the split-GAL4 library targeting individual hemilineages.
Projections of confocal stacks showing the expression pattern of split-GAL4-driven membranous GFP (green) in the larval (A–O) and adult VNC (A’-O’). Only thoracic segments are shown in the larval images. (A, A’) Hemilineage 0 A, marked by inv-GAL4-DBD, tj-VP16.AD. (B, B’) Hemilineage 1 A marked by ets21c-GAL4-DBD, Dr-p65.AD. (C, C’) Hemilineage 2 A marked by sox21a GAL4-DBD, VGlut-p65.AD. (D, D’) Hemilineage 4B marked by ap-p65.AD, fkh-GAL4-DBD. (E, E’) Hemilineage 5B marked by vg-p65.AD, toy-GAL4-DBD. (F, F’) Hemilineage 6B marked by sens2-p65.AD, vg-GAL4-DBD. (G, G’) Hemilineage 7B marked by mab21-GAL4-DBD, unc-4-p65.AD. (H) Hemilineage 8 A marked by ems-GAL4-DBD, ey-p65.AD. (I, I’) Hemilineage 8B marked by lim3-GAL4-DBD, C15-p65.AD. (J, J’) Hemilineage 9 A marked by Dr-p65.AD, gad1-GAL4-DBD. (K, K’) Hemilineage 9B marked by acj6-p65.AD, VGlut-GAL4-DBD. (L, L’) Hemilineage 10B marked by knot-p65.AD, hb9-GAL4-DBD. (M, M’) Hemilineage 12 A marked by TfAP-2-GAL4-DBD, unc-4-p65.AD. (N, N’) Hemilineage 14 A marked by Dr-p65.AD, toy-GAL4-DBD. (O, O’) Hemilineage 17 A marked by unc-4-p.65AD, hmx-GAL4-DBD. The VNC was counterstained with CadN (magenta). The target lineage is indicated on the left bottom corner of each panel. Z-projections were made of selected regions of the VNC to highlight the cell-body clustering and axonal budling.
Figure 3—figure supplement 1
The rest of the driver lines from the Split-GAL4 library targeting individual hemilineages.
Projections of confocal stacks showing the expression pattern of Split-GAL4-driven membranous GFP (green) in the larval (A–O) and adult VNC (A’-O’). Only thoracic segments are shown in the larval images. (A) Hemilineage 1B marked by HLH4c-GAL4-DBD, H15-p65.AD. (B) Hemilineages 3 A, 7B, and 12 A are marked by H15-p65.AD, ChAT-GAL4-DBD. (C) Hemilineages 3B and 12B marked by fer3-GAL4-DBD, cg4328-AD. (D) Hemilineage 6 A marked by mab21-p65.AD, toy-GAL4-DBD. (E) Hemilineage 11 A marked by unc-4-GAL4-DBD, tey VP16.AD. (F) Hemilineage 11B marked by eve-p65.AD, gad1-GAL4-DBD. (G) Hemilineage 12B marked by HGTX-GAL4-DBD, gad1-p65.AD. (H) Hemilineage 13 A marked by dbx-GAL4-DBD, dmrt-p65.AD. (I) Hemilineage 13B marked by vg-GAL4-DBD, D-vp16.AD. (J) Hemilineage 15B marked by HGTX-GAL4-DBD, VGlut-p65.AD. (K) Hemilineage 16B marked by hb9-p.65AD, VGlut-GAL4-DBD. (L) Hemilineage 19 A marked by dbx-GAL4-DBD, scro-p65.AD. (M) Hemilineage 20/22 A marked by bi-GAL4-DBD, shaven-p65.AD. (N) Hemilineage 23B marked by unc-4-p65.AD, acj6-GAL4-DBD. (O) Hemilineage 24B marked by twit-p65.AD, ems-GAL4-DBD.
Figure 3—figure supplement 2
CRISPR-mediated insertion of Trojan Exons.
(A) Construction of CRISPR donor plasmids. For each gene of interest (GOI), a fragment is synthesized into the EcoRV restriction site of pU57_gw_OK2 as described before (Gratz et al., 2014). Briefly, this fragment contains a small sequence of the tRNA spacer, the gRNA against the gene of interest (GOI) (turquoise) and the Left HA and Right HA (turquoise) separated by a spacer containing SacI and KpnI restriction sites (black). A hemidriver cassette (gray, also see B) flanked by SacI and KpnI restriction sites is directionally cloned in between the HAs. (B) Six plasmids containing hemidriver cassettes (gray box) flanked by SacI and KpnI were made in the pBS-KS plasmid backbone. Each plasmid contains either a split-GAL4-DBD or p65.AD in phase 0, 1, and 2. Each hemidriver furthermore contains a 5’attP and FRT sequences, followed by a linker, splice acceptor (SA) and T2A proteolytic cleavage site. The linker length varies to keep the hemidriver in phase with the preceding exon (linker length: 24 nucleotides phase 0, 41 nucleotides phase 1 or 40 nucleotides phase2). An hsp70 termination sequence is introduced at the 3’end of the hemidriver followed by a splice donor (SD), FRT, and attP sequence. Note that the DBD cassettes do not contain a splice donor to keep them consistent with previously published split-GAL4 Trojan exon donors (Lacin et al., 2019). (C) The HAs promote HDR, and the entire hemidriver cassette is inserted at the site of the CRISPR/CAS9 cut, targeted by recognition sequence the gRNA-GOI. The attP sites allow for future cassette exchange with RMCE and genetic crosses.
Figure 3—figure supplement 3
Direct tagging with CRISPR.
Schematic representation of the direct tagging method that establishes split-GAL4DBD lines without any cloning. The gRNA against the gene of interest (GOI) cuts in the direct vicinity of the stop codon (+/-20 nt). The left HA 3’ end reaches up to, but does not include the stop codon, and the right HA 5’ end starts at the first nucleotide of the 3’ UTR. This ensures that the T2A-DBD fragment will be inserted at the 3’ end of the gene and is translated in frame with the GOI. (A) Construction of the CRISPR donor for direct tagging. A fragment that contains a small portion of the tRNA spacer, the gRNA-GOI, and the LHA, T2A-DBD, and RHA sequence is directly synthesized into the EcoRV site of pU57_gw_OK2. (B) Upon embryo injection, expression of gRNA1 linearizes the donor constructs, and the LHA-T2A-DBD-RHA fragment is used for CRISPR/Cas9 guided HDR. As a result, the T2A-DBD is inserted in frame at the 3’ end of the gene, and endogenous 3’ UTR posttranslational regulation mechanisms remain intact.
Figure 3—video 1
Hemilineage 1 A activation on a decapitated animal, 60 FPS.
Figure 3—video 2
Hemilineage 1 A activation on an intact animal, 50 FPS.
Figure 3—video 3
Hemilineage 1B activation on a decapitated animal, 40 FPS.
Figure 3—video 4
Hemilineage 1B activation on an intact animal, 40FPS.
Figure 3—video 5
Hemilineage 2 A activation on a decapitated animal, 60FPS.
Figure 3—video 6
Hemilineage 2 A activation on an intact animal, 40 FPS.
Figure 3—video 7
Hemilineage 4B activation on a decapitated animal, 72FPS.
Figure 3—video 8
Hemilineage 4B activation on an intact animal, 72FPS.
Figure 3—video 9
Hemilineage 5B activation on a decapitated animal, 60FPS.
Figure 3—video 10
Hemilineage 5B activation on an intact animal, 50 FPS.
Figure 3—video 11
Hemilineage 5B activation on an intact feeding animal, 25FPS.
Figure 3—video 12
Hemilineage 5B activation on an intact animal-tethered flight, 25FPS.
Figure 3—video 13
Hemilineage 5B activation on an intact animal walking, 25FPS.
Figure 3—video 14
Hemilineage 6B activation on a decapitated animal 40 FPSS.
Figure 3—video 15
Hemilineage 6B activation on an intact animal-tethered flight, 81FPS.
Figure 3—video 16
Hemilineage 7B activation on a decapitated animal, 40FPS.
Figure 3—video 17
Hemilineage 7B activation on a decapitated animal, 500FPS-5Xslower.
Figure 3—video 18
Hemilineage 7B activation on an intact animal, 40FPS.
Figure 3—video 19
Hemilineage 8 A activation on a decapitated animal, 40FPS.
Figure 3—video 20
Hemilineage 8 A activation on an intact animal, 40FPS.
Figure 3—video 21
Hemilineage 8B activation on a decapitated animal, 500FPS-10Xslower.
Figure 3—video 22
Hemilineage 8B activation on an intact animal, 500FPS-10Xslower.
Figure 3—video 23
Hemilineage 9 A activation on a tethered decapitated animal, 40FPS.
Figure 3—video 24
Hemilineage 9 A activation on a decapitated animal, 40FPS.
Figure 3—video 25
Hemilineage 9 A activation on an intact animal, 40FPS.
Figure 3—video 26
Hemilineage 9B activation on a decapitated animal, 40FPS.
Figure 3—video 27
Hemilineage 9B activation on an intact animal, 40FPS.
Figure 3—video 28
Hemilineage 10B activation on a decapitated animal, 60FPS.
Figure 3—video 29
Hemilineage 10B activation on an intact animal, 50FPS.
Figure 3—video 30
Hemilineage 11 A activation with a strong stimulation on a decapitated animal, 500FPS-10Xslower.
Figure 3—video 31
Hemilineage 11 A activation with a weak stimulation on a decapitated animal, 500FPS.
Figure 3—video 32
Hemilineage 11 A activation with a strong stimulation on an intact animal, 40FPS.
Figure 3—video 33
Hemilineage 11 A activation with a weak stimulation on an intact animal, 40FPS.
Figure 3—video 34
Hemilineage 11B activation on a decapitated animal, 40FPS.
Figure 3—video 35
Hemilineage 11B activation on an intact animal, 40FPS.
Figure 3—video 36
T1 clonal activation of hemilineage 12 A neurons on a decapitated animal, sample 1, 100FPS.
Figure 3—video 37
T1 clonal activation of hemilineage 12 A neurons on a decapitated animal, sample 2, 100FPS.
Figure 3—video 38
Hemilineage 13 A activation on a decapitated animal, 40FPS.
Figure 3—video 39
Hemilineage 13 A activation on two intact animals, 40FPS.
Figure 3—video 40
Hemilineage 13B activation on a decapitated animal, 40FPS.
Figure 3—video 41
hemilineage 13B activation on an intact animal, 40FPS.
Figure 3—video 42
Hemilineage 14 A activation on a decapitated animal, 60FPS.
Figure 3—video 43
Hemilineage 14 A activation on an intact animal, 40FPS.
Figure 3—video 44
hemilineage 15B activation on a decapitated animal, 50FPS.
Figure 3—video 45
hemilineage 15B activation on an intact animal, 40FPS.
Figure 3—video 46
hemilineage 16B activation on a decapitated animal, 60FPS.
Figure 3—video 47
hemilineage 16B activation on an intact animal, 40FPS.
Figure 3—video 48
hemilineage 17 A activation on a decapitated animal, 40FPS.
Figure 3—video 49
Hemilineage 17 A activation on an intact animal, 40FPS.
Figure 3—video 50
Hemilineage 19 A activation on a decapitated animal, 40FPS.
Figure 3—video 51
Hemilineage 19 A activation on an intact animal, 40FPS.
Figure 3—video 52
Hemilineage 21 A activation on a decapitated animal, 25FPS.
Figure 3—video 53
Hemilineage 21 A activation on an intact tethered animal, 200FPS.
Figure 3—video 54
Hemilineage 23B activation on a decapitated animal, 33FPS.
Figure 3—video 55
Hemilineage 23B activation on an intact animal, 47FPS.
Figure 4
Neurons of hemilineage 4B show profound morphological changes during development.
Projection of confocal stacks showing the morphology of 4B neurons (green) marked with the ap-GAL4AD and fkh-GAL4DBD driver combination across different developmental time points during metamorphosis: 0, 3, 12, 24, and 48 hr after puparium formation (APF). The VNC is counterstained with CadN (magenta). Cell bodies of 4B neurons in the T3 region are marked with asterisks. (A–F) Complete projections in T2-T3 segments. Anterior (A) up; posterior (P) down. (A’-F’) Transverse views of the entire T3 segments across the dorso-ventral (D–V) axis; Dorsal is up. Arrowheads in B’ mark growth cones. Arrowheads in C’ mark three new branches towards the medial (m), lateral (l) and dorsal (d) part of the leg neuropil. Scale bar is 20 micron.
Figure 5
Acj6-positive neurons in the VNC are glutamatergic or cholinergic.
(A–C) Split-GAL4 line reporting Acj6 expression intersected with a cognate split-GAL4 line reporting the expression of Gad1, ChAT or VGlut to visualize GABAergic, cholinergic, and glutamatergic populations of Acj6-positive neurons, respectively. The VNC is counterstained with CadN (magenta). (A) Split-GAL4 combination acj6-GAL4AD, gad1-GAL4DBD>UAS-GFP driven UAS-GFP shows that the optic lobes contain cholinergic Acj6-positive neurons in addition to a few clusters of neurons with prominent long projections. In the VNC, two cholinergic clusters per hemisegment corresponding to 8B (arrowheads) and 23B (arrows) hemilineages are labeled in addition to some sensory neurons (asterisks). (B) Split-GAL4 combination acj6-GAL4AD, VGlut-GAL4DBD> UAS-GFP marks a single glutamatergic lineage in the dorsal part of the brain and one 9 A glutamatergic cluster in the VNC. (C) Split-GAL4 combination acj6-GAL4AD, gad1-GAL4DBD>UAS-GFP marks two GABAergic lineages in the brain and nothing in the VNC.
Figure 6 with 1 supplement
Behavioral analysis with targeted lineage manipulation.
(A–D) Optogenetic activation of hemilineage 8 A in the VNC triggers jump behavior. lim3-GAL4DBD; c15-GAL4AD-driven CsChrimson::mVenus (green) targets 8B neurons in the VNC but also shows an unwanted broad brain expression (A), which can be suppressed via an additional layer of intersection using teashirt (tsh)-lexA-driven FLP strategy (B). (C, D) Overlay of video frames to capture the jump sequence induced by optogenetic activation of lineage 8B in the VNC. Intact flies (C) and decapitated flies (D) jump without raising their wings upon optogenetic activation, but decapitated flies were slower to initiate the jump. (E) Optogenetic activation of hemilineage 9 A induces forward walking in decapitated flies. (F, G) Clonal stimulation of hemilineage 12 A in the VNC in decapitated flies induces bilateral wing opening and single-step behavior. (F) Confocal stack displaying the lineage 12 A clone that extends from T2 into T1 and T3. (G) Overlay of movie frames. The fly folds both wings outward and swings its right front leg forward upon optogenetic activation. (H, L) Optogenetic activation of hemilineage 21 A in the VNC on a tethered, intact fly triggers flexion of the tibia-femur joint. (H) Without stimulus, all the legs move erratically in response to being tethered. (I) Upon optogenetic activation, all legs are pulled toward the body, the tibia-femur joints are flexed, and animals stay in this position until the end of stimulus. (J) Overlay of the movie shown in panel H and I, zoomed in on the left T1 leg. Note how the leg is pulled towards the body upon activation (520 ms) compared to its more lateral position without activation (315 ms). (K, L) Elimination of 21 A neurons makes hind leg femur-tibia joints protrude laterally (L) compared to control animals (K). For all overlays of movies, green display frames without optogenetic activation, magenta with optogenetic activation.
Figure 6—figure supplement 1
Giant fiber (GF) connectome.
Synaptic connectivity of the GF neuron extracted from the data generated by Marin et al., 2024. (A–C) Analysis of GF input connections. (D–F) Analysis of GF output connections. (A) Count of neurons per hemilineage that form synapses with GF dendrites. A total of ten hemilineages form synapses with GF dendrites. Five neurons originate from hemilineage 8B, six from hemilineage 7B, five from lineage 5B, and three from lineage 21 A. (B) Combined connectivity per hemilineage, cumulative count of synapses between GF dendrites and hemilineage neurons. The connectivity between hemilineage 8B and the GF is significant, spanning 339 synapses. Hemilineage 7B, 5B, and 21 A forms 45, 205, and 108 connections, respectively. (C) Weighted connectivity per hemilineage, calculated as the cumulative count of synapses between GF dendrites and hemilineage neurons, divided by the total number of GF output connections observed at a threshold of five synapses per neuron. Hemilineage 8B contributes heavily, making up 25% of GF input, followed by 15% from lineage 5B. Lineage 7B contributes 3.3% and lineage 21 A 8%. (D) Count of neurons per hemilineage that form synapses with GF axons. A total of 13 hemilineages are downstream synaptic partners of the GF. Of those, the synapses formed with lineage 8B are most divergent and span 12 neurons. (E) Combined connectivity per hemilineage, cumulative count of synapses between GF axons and hemilineage neurons. Hemilineage 8B makes 208 synaptic contacts. Hemilineages 18B and 6B also form strong connections, 206 and 121 connections, albeit with fewer neurons (5 and 6, respectively). (F) Weighted connectivity per hemilineage, calculated as the cumulative count of synapses between GF axons and hemilineage neurons, divided by the total number of GF output connections observed at a threshold of five synapses per neuron. 12.5% of output GF synaptic contacts are made with hemilineage 8B, followed by 12.4% with lineage 18B and 7.3% with lineage 6B.
Tables
Table 1
Overview of cluster annotation, lineage-specific marker genes, and tested split-GAL4 driver lines.
| Lineage | Clusters (Allen et al.) | Markers | Driver line combinations |
|---|---|---|---|
| 0A | 22, 88, 112 | En, Inv, Fkh, Tj, Lim1, grn, HLH3B, Mab-21, Gad1 | inv-GAL4-DBD, tj-p65.AD: * * * * fkh-GAL4-DBD, tj-p65.AD: * * * * mab21-p65.AD, fkhGAL4-DBD: * * * |
| 1A | 16 | Dr, Ets21C, Ptx1, ChAT | Dr-p65.AD, ets21C-GAL4-DBD: * * * |
| 1B | 12, 47 | HLH4C, H15, Mid, Gad1 | HLH4C-GAL4-DBD, H15-p65.AD: * * * |
| 2A | 15, 86 | HLH3B, Oc, Sox21a, Drgx, Lim1, grn, svp, VGlut | sox21a-GAL4-DBD, VGlut-p65.AD: * * * * sox21a-GAL4-DBD, lim1-VP16.AD: * * * |
| 3A | 7, 37, 85 | H15, HGTX, Grn, Lim1, ChAT | H15-p65.AD, ChaT-GAL4-DBD: * |
| 3B | 26 | Fer3, CG4328, Gad1 | fer3-GAL4-DBD, cg4328-p65.AD: * |
| 4B | 0, 100 | Exex, Ap, Fkh, Tey, HGTX, HLH4C, Oc, ChAT | ap-p65.AD, fkhGAL4-DBD: * * * ap-p65.AD, hgtx-GAL4-DBD: * * * * |
| 5B | 20, 87, 97 | Vg, Toy, Vsx2, Lim1, Gad1 | vg-p65.AD, toy-GAL4-DBD: * * * * |
| 6A | 9, 28 | Mab-21, Toy, Gad1 | mab21-p65.AD, toy-GAL4-DBD: * * |
| 6B | 3, 89 | Vg, Sens-2, En, CG4328, Vsx2, Gad1 | sens2-p65.AD, vg-GAL4-DBD sens2-GAL4-DBD, vg-p65.AD: * * CG4328-p65.AD, vg-GAL4-DBD: * * * |
| 7B | 2, 62 | Unc-4, Sv, Mab-21, ChAT | unc-4-p65.AD, mab21-GAL4-DBD: * * * unc-4-GAL4-DBD, sv-p65.AD: * * * |
| 8A | 6, 69, 110 | Ey, Ems, Toy, Ets65A, VGluT | ems-GAL4-DBD, eyAD: * * * * ems-GAL4-DBD, toy-p65.AD: * * ems-GAL4-DBD, vGluT-p65.AD: * * * |
| 8B | 8, 53, 76 | C15, Lim3, Acj6, ChAT | C15-p65.AD, lim3-GAL4-DBD: * * * |
| 9A | 31, 50, 56, 57 | Dr, Ets65A, grn, sox21a, Gad1 | Dr-p65.AD, gad1-GAL4-DBD: * * * * Dr-p65.AD, sox21a-GAL4-DBD: * * * * |
| 9B | 54, 76 | Lim3, Drgx, Sens-2, Acj6, Tup, HLH4C, VGluT | acj6-p65.AD, VGluT-GAL4-DBD: * * * |
| 10B | 39, 68, 91 | Exex, Kn, Sens-2, Lim3, ChAT | knot-p65.AD, hb9-GAL4-DBD: * * * * hb9-p65.AD, sens-2-GAL4-DBD: * * * * knot-p65.AD, nkx6-GAL4-DBD: * * * * knot-p65.AD, lim3-GAL4-DBD: * * * |
| 11 A | 21 | Unc-4, Tey, ChAT | unc-4-GAL4-DBD, tey-VP16: * * * unc-4-p65.AD, hgtx-GAL4-DBD: * * * |
| 11B | 38 | Eve, HLH4C, Gad1 | eve-p65.AD, gad1-GAL4-DBD: * * * * |
| 12 A | 40 | Unc-4, TfAP-2, Grn, ChAT | unc-4-GAL4-DBD, TfAP2-p65.AD: * * * |
| 12B | 30, 73, 81, 83, 94 | Fer3, HGTX, CG4328, H15, Tey, Gad1 | HGTX-GAL4-DBD, gad1-p65.AD: * * |
| 13 A | 48, 75, 79 | Dbx, Fer2, Dmrt99B, Gad1 | dbx-GAL4-DBD, dmrt99B-p65.AD: * * |
| 13B | 17, 25 | D, Vg, CG4328, tey, svp, Gad1 | vg-GAL4-DBD, D-VP16.AD: * * vg-GAL4-DBD, tey-VP16.AD: * * * |
| 14 A | 13, 41, 74 | Dr, Toy, Lim1, Ets65A, Grn, VGluT, | Dr-p65.AD, toy-GAL4-DBD: * * * |
| 15B | 36, 52, 80 | Tup, Lim3, HGTX, VGlut | HGTX-GAL4-DBD, VGlut-p65.AD: * * * nkx6- GAL4-DBD, twit-p65.AD: * * * |
| 16B | 5, 46 | Lim3, Exex, Bi, Sp1, VGlut, | hb9-p65.AD, bi-GAL4-DBD: * * * hb9-p65.AD, VGlut-GAL4-DBD: * * * |
| 17 A | 58, 77 | Unc-4, Hmx, Tup, ChAT | unc-p65.AD, hmx-GAL4-DBD: * * * * |
| 18B | N/A | Unc-4, ChAT | No line |
| 19 A | 19, 59, 82 | Dbx, Fer2, Scro, Gad1 | dbx-GAL4-DBD, scro-p65.AD: * * * |
| 19B | 27, 71 | Unc-4, Otp, ChAT | No line |
| 20/22 A | 14, 33, 34, 78, 108 | Bi, Ets65A, Sv, ChAT | sv-p65.AD, ets65-GAL4-DBD: * * * bi-GAL4-DBD, shaven-p65.AD: * * bi-p65.AD, ets65A-GAL4-DBD: * * |
| 21 A | 1 | Dr, Ey, Tj, VGluT | Dr-p65.AD, tj-GAL4-DBD: * * * * Dr-p65.AD, ey-GAL4-DBD: * * * |
| 23B | 35, 51, 67, 93 | Unc-4, Acj6, Slou, Otp, ChAT | unc-4-p65.AD, acj6-GAL4-DBD: * * * |
| 24B | A small subset of clusters 52 and 36 | Toy, Ems, Twit, VGlut | ems-GAL4-DBD, twit-p65.AD: * * * |
| **** Very specific for one hemilineage; *** Specific, some contamination from other neurons; ** Somewhat specific, significant contribution of e.g. motor neurons or sensory neurons; * More than one hemilineage marked | |||
Table 2
Overview of behavioral phenotypes upon optogenetic activation of specific hemilineages.
| Lineage | Genotype: Phenotype | Videos |
|---|---|---|
| 0A | tj-p65.AD, inv-GAL4-DBD: No apparent behavioral response observed in response to acute optogenetic activation. | N/A |
| 1A | Dr-p65.AD, Ets21C-GAL4-DBD: Activation in both intact and decapitated animals drove leg extension making fly taller. Our observation differed from previously observed phenotypes of erratic forward locomotion, occasionally interrupted by grooming in decapitated animals (Harris et al., 2015). | Figure 3—video 1; Figure 3—video 2 |
| 1B | H15-p65.AD, HLH4C-GAL4-DBD: Activation in both intact and decapitated flies drives leg rotational movement causing the joint between the femur and tibia to bend laterally, most pronounced by the hind legs. | Figure 3—video 3; Figure 3—video 4 |
| 2A | VGlut-p65.AD, Sox21a-GAL4-DBD: Activation in intact animals drove high-frequency wing flapping, consistent with the findings of Harris et al which showed the same phenotype with the decapitated flies. In our experiments with decapitated animals, no wing buzzing was observed, and only halteres moved ventrally upon stimulation. | Figure 3—video 5 ; Figure 3—video 6 |
| 4B | ap-p65.AD, HGTX-GAL4-DBD: Activation causes a full extension of all the legs in both decapitated and intact flies. | Figure 3—video 7; Figure 3—video 8 |
| 5B | vg-p65.AD, toy-GAL4-DBD: Activation of 5B neurons halts almost every movement in the animal, causing walking, grooming, flying (tethered flight assay), and feeding flies to halt these behaviors. Decapitated animals also halt their grooming activity in response to 5B activation. Active 5B neurons also halt the larval locomotion. | Figure 3—video 9; Figure 3—video 10; Figure 3—video 11; Figure 3—video 12 |
| 6B | CG4328-p65.AD, vg-GAL4-DBD: Activation in intact animals drove inhibition in wing buzzing and leg movements of the tethered flies. Activation in decapitated animals halted sporadic leg movements and drove a subtle change in the posture. | Figure 3—video 14; Figure 3—video 15 |
| 7B | sv-p65.AD, unc-4-GAL4-DBD: Upon 7B activation, both decapitated and intact animals raised their wings and attempted take-offs, but only a few showed modest take-off behavior. We also observed tibia levitation in response to activation. Harris et al. observed robust take-off behavior. | Figure 3—video 16; Figure 3—video 17; Figure 3—video 18 |
| 8A | ey-p65.AD, ems-GAL4-DBD: Activation brings the body of the fly closer to the ground likely flexing leg segments in both intact and decapitated animals. Harris et al. observed minimal effects after activation. | Figure 3—video 19; Figure 3—video 20 |
| 8B | C15-p65.AD, Lim3-GAL4-DBD: Activation drove intact animals lean backward and take-off. A few animals initiated wing flapping after the jump; others failed to initiate wing flapping and fell after the jump, then they jumped again under the continuous activation. Decapitated animals showed a similar response but never initiated the wing flapping after the take-off. | Figure 3—video 21; Figure 3—video 22-2 |
| 9A | Dr-p65.AD, Gad1-GAL4-DBD: Activation in intact animals drove erratic forward locomotion of the animal. Activation in tethered intact flies restricted the legs to stay in a specific posture. In decapitated animals, bodies were lowered toward the ground with legs becoming more splayed for approximately two seconds before occasional forward locomotion and leg grooming, consistent with previous research by Harris et al. | Figure 3—video 23; Figure 3—video 24; Figure 3—video 25 |
| 9B | acj6-p65.AD, VGlut-GAL4-DBD: Activation in intact animals did not lead to any robust behavior; occasionally animals changed their posture mildly. Decapitated animals halted their grooming in response to 9B activation. This halting behavior was less penetrant compared to the halting behavior observed with 5B activation. | Figure 3—video 26; Figure 3—video 27; |
| 10B | Hb9-p65-AD, sens-2-GAL4-DBD: Activation in intact animals drove erratic walking behavior. 10B activation in decapitated animals drove leg extension and body twisting. Our findings differed from Harris et al., 2015, which showed erratic leg movements causing backward locomotion with occasional wing flicking and buzzing. | Figure 3—video 28; Figure 3—video 29; |
| 11 A | tey-VP16.AD, unc-4-GAL-4-DBD: Low intensity light activation drove lateral wing waving with occasional jumping, while high intensity activation drove wing buzzing and jumping in intact and decapitated animals. | Figure 3—video 30; Figure 3—video 31; Figure 3—video 32; Figure 3—video 33 |
| 11B | eve-p65.AD, Gad1-GAL4-DBD: Harris et al. observed take-off behavior after activation of the 11B neurons. However, upon light activation, we observed wing movements without any take-off behavior. The wings moved from side to side in a buzzing behavior. | Figure 3—video 34; Figure 3—video 35 |
| 12 A | TfAP2-p65.AD, unc-4-GAL4-DBD: CsChrimson expression showed a lethal phenotype with no surviving adults. We generated lineage clones using TfAP-2-GAL4. Animals expressing CsChrimson in 12 A neurons in one side of the T1 segment showed a single swing movement of the leg that is located on the same side as the animal lineage clone. We also observed bilateral wing buzzing. | Figure 3—video 36; Figure 3—video 37 |
| 13 A | dmrt99B-p.65AD, dbx-GAL4-DBD: Upon 13 A activation, intact flies halt their walking and grooming behaviors and change the body posture, making flies slightly taller due to likely femur-coxa extension. Decapitated flies also halt the grooming behavior in response to 13 A activation. Both intact and decapitated flies buzz their wings in response to activation, a phenotype likely arising from contaminating neurons. | Figure 3—video 38; Figure 3—video 39 |
| 13B | D-VP16.AD, vg-GAL4-DBD: Intact flies lost control of their legs and fell on their back with uncoordinated leg movements upon activation of 13B neurons. Decapitated flies responded with a postural change and a weak leg extension phenotype. | Figure 3—video 40; Figure 3—video 41 |
| 14 A | Dr-p65.AD, toy-GAL4-DBD: Activation caused intact animals to fall on their back or side with uncoordinated leg movements; flies remained uncoordinated until the cessation of the stimulus. In decapitated animals, activation drove the femur-tibia joint to move anteriorly, most pronounced in the middle legs. We also observed flexion of the legs. | Figure 3—video 42; Figure 3—video 43 |
| 15B | VGlut-p65.AD, HGTX-GAL4-DBD: Upon light stimulation in both intact and decapitated flies, the legs showed a severe flexing phenotype. The legs flexed tightly against the body with the flies falling into a fetal position until after light stimulation ended. | Figure 3—video 44; Figure 3—video 45 |
| 16B | Hb9-p65.AD, Bi-GAL4-DBD: Activation in both intact and decapitated animals drove flexion at the femur-tibia joint and coxa-femur axis joint causing the animal to sink lower to the ground. | Figure 3—video 46; Figure 3—video 47 |
| 17 A | unc-4-p65.AD, Hmx-GAL4-DBD: Activation of 17 A neurons drove flexion of all the leg segments in both decapitated and intact animals. | Figure 3—video 48; Figure 3—video 49 |
| 19 A | scro-p65.AD, dbx-GAL4-DBD: Activation in decapitated animals drove flexion at the tibia-tarsus joint as well as anterior movement of the femur-tibia axis. In intact animals, we observed severe flexing of the legs against the body, making flies fall on their back. Harris et al. observed a leg-waving phenotype of the T2 legs in decapitated animals after stimulation. | Figure 3—video 50; Figure 3—video 51 |
| 21 A | Dr-p65.AD, tj-GAL4-DBD: Activation of 21 A neurons in decapitated animals drove flexion of the legs, bringing the body of the fly closer to the ground. We observed a similar phenotype in intact animals tethered to a pin. | Figure 3—video 52; Figure 3—video 53 |
| 23B | unc-4-p65.AD, acj6-GAL4-DBD: Activation caused intact animals to fall on their back due to uncoordinated leg movements and sustained flexion or extension of the leg segments; flies remained uncoordinated until the cessation of the stimulus. Flies also showed increased grooming activity. We also observed wing buzzing in response to activation. Decapitated animals showed similar responses. | Figure 3—video 54; Figure 3—video 55 |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | guinea pig anti-tj polyclonal | Gift from Dorothea Godt | 1:5000 dilution | |
| Antibody | Rabbit anti-tey polyclonal | Gift from Angelike Stathopoulos | 1:200 dilution | |
| Antibody | Rat anti-c15 polyclonal | Gift from Gerard Campbell | 1:1000 dilution | |
| Antibody | Chicken anti-GFP polyclonal | Life Technologies | A-10262 | 1:1000 dilution |
| Antibody | Rabbit anti-GFP polyclonal | Life Technologies | A-11122 | 1:1000 dilution |
| Antibody | Rabbit anti-Unc-4 polyclonal | Lacin et al., 2014 | A-10262 | 1:1000 dilution |
| Antibody | Mouse anti-Acj6 monoclonal | DSHB | Acj6 | 1:100 dilution |
| Antibody | Rat anti-CadN monoclonal | DSHB | DN-Ex #8 | 1:25 dilution |
| Antibody | Mouse anti-Neuroglian monoclonal | DSHB | BP104 | 1:25 dilution |
| Antibody | Goat anti-rabbit Alexa Fluor 488 | Life Technologies | A-11034 | 1:500 dilution |
| Antibody | Goat anti-rabbit Alexa Fluor 568 | Life Technologies | A-11011 | 1:500 dilution |
| Antibody | Goat anti-rabbit Alexa Fluor 633 | Life Technologies | A-21070 | 1:500 dilution |
| Antibody | Goat anti-rat Alexa Fluor 633 | Life Technologies | A-21094 | 1:500 dilution |
| Antibody | Goat anti-chicken Alexa Fluor 488 | Life Technologies | A-11039 | 1:500 dilution |
| Antibody | Goat anti-mouse Alexa Fluor 568 | Life Technologies | A-11001 | 1:500 dilution |
| Antibody | Goat anti-mouse Alexa Fluor 633 | Life Technologies | A-21050 | 1:500 dilution |
| Antibody | Goat anti-rat Alexa Fluor 568 | Life Technologies | A-21050 | 1:500 dilution |
| Genetic reagent (D. melanogaster) | unc-4DBD/FM7GFP; 20XUASCsChrimson_attp40/cyo | Lacin et al., 2020 | ||
| Genetic reagent (D. melanogaster) | unc-4AD/FM7; 20X-UASChrimson_attp40/cyo | Lacin et al., 2020 | ||
| Genetic reagent (D. melanogaster) | sens2-GAL4-DBD | Lacin et al., 2024 | ||
| Genetic reagent (D. melanogaster) | P{w[+mW.hs]=GawB}elav[C155]; P{w[+mW.hs]=FRT(w[hs])}G13 P{w[+mC]=tubP GAL80}LL2 | Tzumin Lee Lab | ||
| Genetic reagent (D. melanogaster) | 20XUAS-CsChrimson-mVenus_attp18 | V. Jayaraman lab | ||
| Genetic reagent (D. melanogaster) | 20XUAS >FRT-stop>CsChrimson-mVenus_attp18 | V. Jayaraman lab | ||
| Genetic reagent (D. melanogaster) | P{GawB}elav[C155], P{FRT(w[hs])}G13 P{UAS-mCD8::GFP.L}LL5 | Tzumin Lee Lab | ||
| Genetic reagent (D. melanogaster) | P{FRT(w[hs])}G13 P{tubP-GAL80}LL2 | Tzumin Lee Lab | ||
| Genetic reagent (D. melanogaster) | y[1] w1118; P{tubP-GAL80}LL9 P{FRT(w[hs])}2 A/TM3, Sb | Tzumin Lee Lab | ||
| Genetic reagent (D. melanogaster) | knot-p65.AD/CyO, weep; Dr/TM6 | Luo Lab, Hongjie Li | ||
| Genetic reagent (D. melanogaster) | pin/cyo; c15-p65.AD/TM6b | Luo Lab, Hongjie Li | ||
| Genetic reagent (D. melanogaster) | tj-vp16.AD | Desplan Lab- David Chen | ||
| Genetic reagent (D. melanogaster) | twit-p65.AD | Stephen Goodwin | ||
| Genetic reagent (D. melanogaster) | 13XLexAop2-IVS-myr::GFP in attP40 | BDSC | RRID:BDSC32210 | |
| Genetic reagent (D. melanogaster) | P{hsFLP}1; P{FRT(w[hs])}G13 P{tubP-GAL80}LL2/CyO | BDSC | RRID:BDSC5145 | |
| Genetic reagent (D. melanogaster) | P{tubP-GAL80}LL10 P{neoFRT}40 A/CyO | BDSC | RRID:BDSC5192 | |
| Genetic reagent (D. melanogaster) | w[*]; l(2)*[*]/CyO; Mi{Trojan-GAL4DBD.0}ChAT[MI04508-TG4DBD.0] CG7715[MI04508-TG4DBD.0-X]/TM3, Sb[1] | BDSC | RRID:BDSC60318 | |
| Genetic reagent (D. melanogaster) | w1118; PBac{RB}Fer2e03248 | BDSC | RRID:BDSC26028 | |
| Genetic reagent (D. melanogaster) | w1118; PBac{Sp1-EGFP.S}VK00033 | BDSC | RRID:BDSC38669 | |
| Genetic reagent (D. melanogaster) | w[1118]; PBac{y[+mDint2] w[+mC]=fkh GFP.FPTB}VK00037/SM5 | BDSC | RRID:BDSC43951 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]=MIC}twit[MI06552]/(SM6a) | BDSC | RRID:BDSC41449 | |
| Genetic reagent (D. melanogaster) | w[*]; Mi{Trojan-GAL4DBD.0}Dbx[MI05316-TG4DBD.0]/TM6B, Tb[1] | Lacin et al., 2019 | RRID:BDSC82989 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{Trojan-GAL4DBD.1}Lim3[MI03817-TG4DBD.1]/(CyO) | Lacin et al., 2019 | RRID:BDSC82990 | |
| Genetic reagent (D. melanogaster) | w1118; PBac{WH}Ets21Cf03639 | BDSC | RRID:BDSC18678 | |
| Genetic reagent (D. melanogaster) | w[*]; Mi{Trojan-p65AD.2}VGlut[MI04979-Tp65AD.2]/CyO | Lacin et al., 2019 | RRID:BDSC82986 | |
| Genetic reagent (D. melanogaster) | w[*]; betaTub60D[Pin-1]/CyO; Mi{Trojan-p65AD.1}Dr[MI14348-Tp65AD.1] | Lacin et al., 2019 | RRID:BDSC82991 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]=MIC}Dr[MI14348]/TM3, Sb[1] Ser[1] | BDSC | RRID:BDSC59504 | |
| Genetic reagent (D. melanogaster) | w[*]; betaTub60D[Pin-1]/CyO; TI{2 A-GAL4(DBD)::Zip-}HGTX[DBD]/TM6B, Tb[1] | Lacin et al., 2019 | RRID:BDSC82992 | |
| Genetic reagent (D. melanogaster) | ey-GAL4-DBD | Lacin et al., 2019 | RRID:BDSC6294 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]=MIC}Ets65A[MI05707] | BDSC | RRID:BDSC40235 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; TI{GFP[3xP3.cLa]=CRIMIC.TG4.2}sv[CR00370-TG4.2] | BDSC | RRID:BDSC78901 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; TI{GFP[3xP3.cLa]=CRIMIC.TG4.2}Sox21a[CR00451-TG4.2]/TM3 Sb[1] Ser[1] | BDSC | RRID:BDSC83174 | |
| Genetic reagent (D. melanogaster) | y[1] w[*] Mi{y[+mDint2]=MIC}bi[MI08152] lncRNA:CR32773[MI08152] | BDSC | RRID:BDSC51220 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]=MIC}ap[MI01996]/CyO | BDSC | RRID:BDSC42297 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]=MIC}inv[MI09433] | BDSC | RRID:BDSC52163 | |
| Genetic reagent (D. melanogaster) | y[1] w[*] Mi{y[+mDint2]=MIC}acj6[MI07818] | BDSC | RRID:BDSC51212 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{PT-GFSTF.2}Hmx[MI02025-GFSTF.2]/TM3, Sb[1] Ser[1] | BDSC | RRID:BDSC59785 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]=MIC}Hmx[MI02896] | BDSC | RRID:BDSC36161 | |
| Genetic reagent (D. melanogaster) | y[1] w[*]; Mi{y[+mDint2]=MIC}Ets65A[MI05707] | BDSC | RRID:BDSC40235 | |
| Genetic reagent (D. melanogaster) | y[1]; Mi{y[+mDint2]=MIC}toy[MI03240] | BDSC | RRID:BDSC61701 | |
| Genetic reagent (D. melanogaster) | P{Tub-dVP16AD.D} | BDSC | RRID:BDSC60295 | |
| Genetic reagent (D. melanogaster) | P{Tub-GAL4DBD.D} | BDSC | RRID:BDSC0298 | |
| Genetic reagent (D. melanogaster) | lim3-GAL4-DBD | Lacin et al., 2019 | RRID:BDSC82990 | |
| Genetic reagent (D. melanogaster) | ChAT-p65.AD | Lacin et al., 2019 | RMCE with RRID:BDSC37817 | |
| Genetic reagent (D. melanogaster) | y[*]w[*]/w[*];inv[MI09433.p65AD_1]/SM6a | this study | RMCE with RRID:BDSC52163, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | ap-GAL4-DBD | this study | RMCE with RRID:BDSC42297, request from Lacin lab BDSC52163 | |
| Genetic reagent (D. melanogaster) | ap-p65.AD | this study | RMCE with RRID:BDSC42297, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | mab-21-GAL4-DBD | this study | RMCE with RRID:BDSC59220, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | mab-21-p65.AD | this study | RMCE with RRID:BDSC59220, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | toy-GAL4-DBD | this study | RMCE with RRID:BDSC61701, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | toy-p65.AD | this study | RMCE with RRID:BDSC61701, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | shaven-p65.AD | this study | RMCE with RRID:BDSC78901, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | sox21a-GAL4-DBD | this study | RMCE with RRID:BDSC93174, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | bi-GAL4-DBD | this study | RMCE with RRID:BDSC51220, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | bi-p65.AD | this study | RMCE with RRID:BDSC51220, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | CG4328-p65.AD | this study | RMCE with RRID:BDSC42307, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | Ets65A-GAL4-DBD | this study | RMCE with RRID:BDSC56352, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | Hmx-GAL4-DBD | this study | RMCE with RRID:BDSC36161, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | dmrt99b-GAL4-DBD | this study | RMCE with RRID:BDSC92707, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | dmrt99b-p65.AD | this study | RMCE with RRID:BDSC92707, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | Dr-GAL4-DBD | this study | RMCE with RRID:BDSC59504, request from Lacin lab | |
| Genetic reagent (D. melanogaster) | exex-GAL4-DBD | this study | RMCE with exex-p65AD[attP2FRT2], request from Lacin lab | |
| Genetic reagent (D. melanogaster) | vg-GAL4-DBD | this study | RMCE with vg-p65AD[attP2FRT2], request from Lacin lab | |
| Genetic reagent (D. melanogaster) | sens2-p65.AD | this study | RMCE with sens2-GAL4-DBD[attP2FRT2], request from Lacin lab | |
| Genetic reagent (D. melanogaster) | Ets21C-GAL4-DBD | this study | RMCE with Ets21C-p65.AD[attP2FRT2], request from Lacin lab | |
| Genetic reagent (D. melanogaster) | eve-GAL4-DBD | this study | RMCE with eve-p65.AD[attP2FRT2], request from Lacin lab | |
| Genetic reagent (D. melanogaster) | exex-p65.AD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | exex-GAL4-DBD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | eve-p65.AD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | vg-p65.AD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | vg-GAL4-DBD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | H15-p65.AD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | scro-p65.AD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | scro-GAL4-DBD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | Ets21C-p65.AD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | Ets21C-GAL4-DBD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | eve-p65.AD[attP2FRT2] | this study | CRISPR /Trojan (CRIMIC), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | Fer3-GAL4-DBD | this study | CRISPR /In frame insertion (C terminus), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | ems-GAL4-DBD | this study | CRISPR /In frame insertion (C terminus), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | HLH4C-GAL4-DBD | this study | CRISPR /In frame insertion (2nd exon), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | w1118;; fkh-T2A-GAL4-DBD/TM6b | this study | CRISPR /In frame insertion (C terminus for RA isoform), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | w*;; D-VP16/TM6b | this study | CRISPR /In frame insertion (C terminus), request from Lacin lab | |
| Genetic reagent (D. melanogaster) | pJFRC29-10XUAS-IVS-myr::GFP-p10 in attP40 or attP2 | Rubin Lab | ||
| Genetic reagent (D. melanogaster) | pJFRC105-10XUAS-IVS-nlstdTomato in VK0003 | Rubin Lab | ||
| Genetic reagent (D. melanogater) | pJFRC12-10XUAS-IVS-myr::GFP attp40 or attP2 | Rubin Lab | ||
| Genetic reagent (D. melanogater) | pJFRC28-10XUAS-IVS-GFP-p10 in attP2 | Rubin Lab | ||
| Chemical compound, drug | Paraformaldehyde | EMS | 15713 | |
| Chemical compound, drug | Vectashield | Vectorlabs | H-1000 | |
| Chemical compound, drug | DPX | Electron Microscopy Sciences | 50980370 | |
| Chemical compound, drug | Gibson Assembly Master Mix | New England Biolabs | E2621S | |
| Recombinant DNA reagent | pCFD4-U6:1_U6:3tandemgRNAs | Addgene | 49411 | |
| Recombinant DNA reagent | pBS-KS-attB2-SA(1)-T2A-Gal4-Hsp70 | Addgene | 62897 | |
| Recombinant DNA reagent | pBS-KS-attB2-SA(1)-T2A-Gal4DBD-Hsp70 | Addgene | 62903 | |
| Recombinant DNA reagent | pBS-KS-attB2-SA(1)-T2A-p65AD-Hsp70 | Addgene | 62914 | |
| Recombinant DNA reagent | pBS-KS-attB2-SA(0)-T2A-Gal4-Hsp70 | Addgene | 62896 | |
| Recombinant DNA reagent | pBS-KS-attB2-SA(0)-T2A-Gal4DBD-Hsp70 | Addgene | 62902 | |
| Recombinant DNA reagent | pBS-KS-attB2-SA(0)-T2A-p65AD-Hsp70 | Addgene | 62912 | |
| Recombinant DNA reagent | pBS-KS-attP2FRT2-SA(0)-T2A-p65AD-Hsp70 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pBS-KS-attP2FRT2-SA(1)-T2A-p65AD-Hsp70 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pBS-KS-attP2FRT2-SA(2)-T2A-p65AD-Hsp70 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pBS-KS-attP2FRT2-SA(0)-T2A-gal4DBD-Hsp70 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pBS-KS-attP2FRT2-SA(1)-T2A-gal4DBD-Hsp70 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pBS-KS-attP2FRT2-SA(2)-T2A-gal4DBD-Hsp70 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pCFD4-exex | this study | request from Lacin lab | |
| Recombinant DNA reagent | pCFD4-vg | this study | request from Lacin lab | |
| Recombinant DNA reagent | pCFD4-H15 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_Hb9 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_vg | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_H15 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_gw_OK2_Scro | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_gw_OK2_Ets21C | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_gw_OK2_eve | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_gw_OK2_Fer3 | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_gw_OK2_ems | this study | request from Lacin lab | |
| Recombinant DNA reagent | pUC57_gw_OK2_HLH4C | this study | request from Lacin lab |
Additional files
-
Supplementary file 1
Detailed description of the expression patterns of the driver lines used in Figure 3, Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/106042/elife-106042-supp1-v1.xlsx
-
Supplementary file 2
Synaptic inputs of the Giant Fiber neuron related to Figure 6.
- https://cdn.elifesciences.org/articles/106042/elife-106042-supp2-v1.xlsx
-
Supplementary file 3
Synaptic outputs of the Giant Fiber neuron related to Figure 6.
- https://cdn.elifesciences.org/articles/106042/elife-106042-supp3-v1.xlsx
-
Supplementary file 4
Additional information on CRISPR genomic edits.
- https://cdn.elifesciences.org/articles/106042/elife-106042-supp4-v1.xlsx
-
Supplementary file 5
Genotypes of animals used for each figure and video.
- https://cdn.elifesciences.org/articles/106042/elife-106042-supp5-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/106042/elife-106042-mdarchecklist1-v1.docx
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A library of lineage-specific driver lines connects developing neuronal circuits to behavior in the Drosophila ventral nerve cord
eLife 14:RP106042.
https://doi.org/10.7554/eLife.106042.3
