Extended Data Figure 4: P-EN neuroanatomy: explanation for the numbering scheme, sytGFP localization, and multicolour single cell labelling.

a, Numbering used in the literature for the protocerebral bridge and ellipsoid body. b, Rearrangement of the left and right bridges and a linearized ellipsoid body that highlights the pattern of anatomical projections for E-PGs and P-ENs. Arrows indicate the expected direction of signalling (dendrite to axon) for each cell^21,22 (also see e, f). The dashed line in a shows where the ellipsoid body is opened to display it linearly. Tile 1 is repeated as a visual aid, as the ellipsoid body is circular. c, d, Same as a, b but using a modified numbering scheme. In d, the numbers are constant along each column (with the exception that glomerulus 9 from either side of the bridge matches up with ellipsoid body tile 1), highlighting the fact that E-PGs project within the same column, whereas left-bridge P-ENs project to the right (+1, or clockwise) and right-bridge P-ENs project to the left (−1, or anticlockwise). e, f, Sample images of synaptotagmin–GFP (sytGFP, labelling putative axonal terminals) and tdTomato (labelling the entire cell) expressed in P-EN1 (e) and P-EN2 (f) neurons. These data are consistent with P-ENs having extensive presynaptic terminals in the ellipsoid body and noduli but few in the protocerebral bridge. g–l, Sample multicolour flip-out images for P-EN neurons driven by VT032906–Gal4 (P-EN1, g, h), VT020739–Gal4 (P-EN2, i, j), and 12D09–Gal4 (P-EN2, k, l). The multicolour flip-out method⁴¹ allows one to visualize single randomly selected cells from a Gal4 driver line (which might label a dense thicket of cells) in their entirety, like a multicoloured Golgi stain. The neuropil is shown in grey. Single neurons are coloured. Glomerulus numbers, including L for left and R for right, are shown in the bridge. After tracing each neuron from the bridge to the ellipsoid body, we labelled the terminals in the ellipsoid body with the bridge glomerulus from which they originated, using our revised numbering scheme (c, d). VT032906–Gal4 stains a neuron type that passes near the bridge, but does not innervate the bridge, ellipsoid body or noduli (for example, the green neuron in g). VT020739–Gal4 stains a neuron type that innervates the noduli, but not the ellipsoid body or bridge (for example, the blue neurons innervating the noduli from the sides in j). Virtually all neurons labelled in the bridge and ellipsoid body were consistent with P-ENs (see Supplementary Information Table 1). 12D09–Gal4 very rarely revealed flip-outs of protocerebral bridge local neurons, not shown here (see Supplementary Information Table 1). eb, ellipsoid body; no, noduli; pb, protocerebral bridge.