Volume 27, Issue 10, Pages 1521-1528.e4 (May 2017) Temporal Cohorts of Lineage-Related Neurons Perform Analogous Functions in Distinct Sensorimotor Circuits  Christopher C. Wreden, Julia L. Meng, Weidong Feng, Wanhao Chi, Zarion D. Marshall, Ellie S. Heckscher  Current Biology  Volume 27, Issue 10, Pages 1521-1528.e4 (May 2017) DOI: 10.1016/j.cub.2017.04.024 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 ELs Can Be Subdivided into Early-Born and Late-Born Temporal Cohorts (A) Even-skipped (Eve) and R11F02 expression (segments A2–A4). (A′) Higher magnification of NB3-3 (arrow), Eve and R11F02 co-expression (dashes), and cells expressing only R11F02 (∗). (B) A schematic of R11F02 and Eve expression in NB3-3 and ELs. (C–E) R11F02(+) neurons (circled) co-express Nab (late-born, C), but not Pdm2/Kruppel (early-born, D and E). A single hemisegment shown with number of hemisegments (n) co-expressing two markers. (F–G′) Expression patterns for early-born or late-born ELs in larval nerve cords. (F′ and G′) Higher magnification with co-expression of Eve. In all images anterior up; scale bar, 10 μm; midline at arrowheads or on left. Genotypes were as follows: (A–E) R11F02-GAL4/UAS-nls-GFP, (F) R11F02-GAL80/UAS-myr-GFP; EL-GAL4/+, and (G) EL-AD/UAS-myr-GFP; R11F02-DBD/+. See Figure S1. Current Biology 2017 27, 1521-1528.e4DOI: (10.1016/j.cub.2017.04.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Stimulation of Late-Born versus Early-Born ELs Yields Distinct Behavioral Responses (A) A behavioral rig uses infrared (IR) light to illuminate larvae, which is detected by the camera. An amber LED stimulates optogenetic effectors (Light ON, amber in B–D and H–K). (B–D) Average (± SEM) larval speeds normalized to pre-stimulus speed for late-born ELs (B), early-born ELs (C), or all ELs (D). Experimental larvae with all-trans-retinal (+ATR, black) allow activation of optogenetic effectors; controls lack ATR (–ATR, gray). (E–G″) Behavior before (E) or during (F and G) stimulation. Images shown with anterior up and box width of 500 μm. (E′ and G′) X-Y time projection shows total movement. (E″ and G″) Schematics with gray lines representing trachea. In G″, trachea disappear below the body during rolling. (H and I) Instances of rolling for late-born ELs (H) and early-born ELs (I). Each row represents response of a larva. Each black box indicates larval rolling. (J and K) Proportion of larvae rolling for late-born ELs (J) and early-born ELs (K). Genotypes were as follows: late-borns (B, E, F, H, and J), EL-AD/+; R11F02-DBD/UAS-CsChrimson.mVenus; early-borns (C, G, I, and K), R11F02-GAL80/+; EL-GAL4/UAS-CsChrimson.mVenus; and all ELs (D), EL-GAL4/UAS-CsChrimson.mVenus. See Figure S2 and Movie S1. Current Biology 2017 27, 1521-1528.e4DOI: (10.1016/j.cub.2017.04.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 A Majority of ELs Are Sensory Processing Interneurons (A and B) Examples of single-cell clones of early-born ELs (A and B) as visualized by light microscopy and their matching neurons in a TEM volume. Anterior up; midline (arrowhead). (C and D) Sensory neurons directly and indirectly synapse onto ELs: late-born (C) and early-born (D). Arrow thickness represents number of direct synaptic connections. (E and F) Schematic diagrams of late-born (E) and early-born EL (F) circuits, with neurons potentially related by lineage indicated in rounded boxes. ELs from NB3-3 shaded. Arrows represent direct connections. Current Biology 2017 27, 1521-1528.e4DOI: (10.1016/j.cub.2017.04.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Early-Born and Late-Born ELs Differentially Respond to Sound (A) Sound/vibration activates mechanosensitive chordotonal sensory neurons (Mechano CHOs) and causes larval hunching. (B) Behavioral arena used to record responses to stimulus (Sound ON, gray in C and D, and F and H). (C and D) Larvae hunch upon sound/vibration only when CHOs are present. Larvae before (left) or during (right) sound/vibration. Anterior up, head-to-tail larval length 3.5 mm (C and D). Average (± SEM) larval speeds normalized to pre-stimulus speed (C′ and D′) or larval perimeter normalized to pre-stimulus perimeter (C″ and D″). Genotypes were as follows: control (C; n = 14), UAS-RPR, UAS-HID/+; R11F02-DBD/+; and CHO ablation (D; n = 11), UAS-RPR, UAS-HID/+; IAV-GAL4/+. See Movie S2 and Audio S1. (E) The stimulus was played to a larva with the anterior portion, containing the CNS, immobilized and the posterior free. (F–H) Neuronal activity was monitored with GCaMP6s. Average (± SEM) change in fluorescence (ΔF/F) over time is shown, scale in H (F–H). Example responses to sound stimulation (F′ and H′). Fluorescence in pseudo color, red = maximum, blue = minimum. Anterior up; box width 72 μm. Genotypes were as follows: CHOs (F; n = 11), IAV-GAL4/UAS-syt-GCaMP6s; early-born ELs (G; n = 25), UAS-FLP, actin-FRT-stop-FRT-GAL4/+; R11F02-GAL80/UAS-syt-GCaMP6s; EL-GAL4/+; and late-born ELs (H; n = 17), UAS-FLP, actin-FRT-stop-FRT-GAL4/+; EL-AD/UAS-syt-GCaMP6s; R11F02-DBD/+. See Figure S3 and Movies S3 and S4. Current Biology 2017 27, 1521-1528.e4DOI: (10.1016/j.cub.2017.04.024) Copyright © 2017 Elsevier Ltd Terms and Conditions