LC4 and LPLC2 occupy distinct regions on GF dendrites

(A) GF (green), LPLC2 (magenta, one hemisphere), and LC4 (red, one hemisphere) maximum intensity projections superimposed over neuropil label Bruchpilot (Brp, gray) Scale bar, 50μm.

(B) Optic glomeruli as identified by Brp labeling with the LPLC2 (magenta) and LC4 (red) glomeruli highlighted. Maximum intensity projection of a substack located within the dashed box in (A). Scale bar, 20μm.

(C) Drosophila hemibrain EM reconstruction of GF (green) with colored dots indicating synapses from LC4 (red, top) and LPLC2 (magenta, bottom).

(D) (Left) Maximum intensity projections of dual labeled GF and VPNs. (Right) Colocalized pixels (orange) between GF and respective VPNs superimposed over GF maximum intensity projections. Scale bar, 20μm.

LC4 and LPLC2 territories on GF dendrites are established early in development

(A) Maximum intensity projections of GF (green) 36 hAPF (left), 60 hAPF (middle), and in adult (right) with the VPN dendritic region highlighted in yellow at distinct developmental stages. Scale bar, 20μm.

(B) Quantification GF lateral dendrite volume from (A). Unpaired Kruskal-Wallis test (p = 1.339 x 10−18), Tukey-Kramer multiple comparison test post hoc, * = p < .05 as compared to 24 hAPF, + = p < .05 as compared to 36 hAPF, # = p < .05 as compared to 48 hAPF, and % = p < .05 as compared to adult. N > 13 hemibrains from > 10 flies.

(C) Quantification of maximum dendrite extension length across the medial-lateral axis. Unpaired Kruskal-Wallis test (p = 2.072 x 10−12), Tukey-Kramer multiple comparison test post hoc, * = p < .05 compared to 24 hAPF, + = p < .05 compared to 36 hAPF, # = p < .05 compared to 48 hAPF.

(D,E) Left, maximum intensity projections of GF (green) with respect to LC4 (red, D), and LPLC2 (magenta, E) axonal membrane at distinct developmental stages. Right, maximum intensity projections of GF with VPN colocalized pixels (orange) superimposed along GF dendrites. Arow and arrowheads indicate divergent dorsal and ventral VPN axons, respectively. Scale bar, 20μm.

(F,G) Quantification of colocalization in (D,E) with colors corresponding to VPN type. Unpaired Kruskal-Wallis test (LC4, p = 2.088 x 10−12; LPLC2, p = 1.983 x 10−10), Tukey-Kramer multiple comparison test post hoc, * = p < .05 as compared to 36 hAPF, + = p < .05 as compared to 60 hAPF, # = p < .05 as compared to 72 hAPF. N > 6 hemibrains from > 3 flies.

(H,I) 3D renderings of GF lateral dendrites (green) with LPLC2 (H, magenta) or LC4 (I, red) colocalized pixels superimposed at distinct timepoints during development. Scale bar, 20μm. D - dorsal, V – ventral, M – medial, L – lateral.

(J) Histograms of the spatial distribution of LC4 and LPLC2 contacts along the normalized medial-lateral GF dendrite axis across development; colors are the same as in (H, I). N are as stated in (F,G).

Synaptogenesis and the emergence of stimulus-independent neural activity

(A) Schematic of GF and VPN developmental interactions

(B) Heatmap timecourse of average, normalized VPN mRNA expression of genes for electrical and chemical synapse function. Data are from the optic lobe transcriptional atlas22 and individual VPN expression patterns can be found in Supplemental Figure 9.

(C)Max intensity projections of a substack of DLG expression in GF (DLG1-V5, green) and Brp puncta in LC4 (Brp-Short, red) at selected timepoints. Scale bar, 10μm.

(D)Quantification of volume of Brp colocalized with DLG from (C). Unpaired Kruskal-Wallis test (p = 0.001), Dunn-Sidak comparison test post hoc, * = p < .05, N= 3-7 hemibrains from 2-5 flies.

(E)Schematic of ex-plant pupal electrophysiology preparation.

(F)The total number of identified depolarizing events increases exponentially (fit, dotted red line) over time. N = 5 flies.

(G)Representative traces of GF membrane potential recordings using the pupal electrophysiology preparation for two timepoints.

(H)Zoomed in recording showing features resolvable with electrophysiology. Arrow indicates hyperpolarization following large depolarizing events, arrowheads indicate different event amplitudes.

(I)Distribution of event frequencies from inter-event intervals.

(J)Timecourse of developmental stages as estimated from anatomical, scRNA-seq and electrophysiology data.

Developmental ablation of LC4 with Kir2.1 alters the morphology of the LPLC2 glomerulus and increases LPLC2 contacts and functional drive onto GF.

(A) Maximum intensity projections of LPLC2 expressing tdTomato (magenta) and LC4 (white) expressing GFP (left) or Kir2.1 (right) using LC4_4, a driver line that turns on early in development. Scale bar, 20μm.

(B) Maximum intensity projections of a substack of the LPLC2 glomerulus (magenta) in a fly where LC4 express GFP (top) or are ablated through Kir2.1expression (bottom). Scale bar, 20μm.

(C) Quantification of LPLC2 glomerulus volume in (B). Two-sample t-test, p = .0273, N = 16-24 hemibrains from 8-13 flies.

(D) Maximum intensity projections of Brp (NC82, gray) with the LPLC2 glomerulus highlighted (magenta) in a fly where GFP was expressed early in LC4 (left). Maximum intensity projections of GF dendrites (tdTomato, green) extending into the LPLC2 glomerulus (middle). Maximum intensity projections of colocalized pixels (magenta) between GF and the LPLC2 glomerulus superimposed onto the GF (right).

(E) Maximum intensity projections of Brp (gray) with the LPLC2 glomerulus highlighted (magenta) in a fly where Kir2.1 was expressed early to ablate LC4 (left). Maximum intensity projections of GF dendrites (tdTomato, green) extending into the LPLC2 glomerulus (middle). Maximum intensity projections of colocalized pixels (magenta) between GF and the LPLC2 glomerulus superimposed onto the GF (right). Scale bar, 20μm.

(F) Quantification of colocalization between GF and the LPLC2 glomerulus from (D,E). Unpaired Mann-Whitney U test, p = .0159, N > 4 hemibrains from > 4 flies.

(G) Maximum intensity projections of GF expressing smGFP (green) with colocalized pixels (white) between LC4 expressing GFP (left) or silenced by Kir2.1 (right) using an LC4-split-Gal4 driver that turns on late during development (LC4_1-split-GAL4). Scale bar, 20μm.

(H) Quantification of colocalization in (G). Unpaired Mann-Whitney U test, p = .1486, N > 11 hemibrains from > 6 flies.

(I) Schematic representing in-vivo electrophysiology setup for head-fixed adult flies. Visual stimuli (looms) were presented ipsilateral to the side of the recording via projection onto a screen positioned in front of the fly.

(J) Average GF responses to select looming stimuli presentations of different radius to speed ratios (r/v).

(K) Quantification of peak amplitude responses to looming stimuli presentations in (J). Unpaired Kruskal-Wallis test (r/v = 10ms, p = .1105; r/v = 20ms, p = .0443; r/v = 40ms, p = .0556; r/v = 80ms, p = .0385), Tukey-Kramer multiple comparison test post hoc, * = p < .05, N = 6-8 flies.

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