Extended Data Fig. 1: Blood cells (hemocytes) circulate in the fly head cavity.
From: Sleep-dependent clearance of brain lipids by peripheral blood cells
a. Localization of HmlΔ-LexA+ hemocytes (Hml Δ-LexA LexAop-mCherry, red) in the fly. Structure of the fly was visualized with a UV laser (Autofluorescence, white). b-f. Localization of the hemocytes near the brain. Visualizing of hemocyte with Srp-Gal4 (Top left, green), Srp-hemo-Gal4DBD/AD (Top right, green), Ppn-Gal4 (Bottom left, green), or eated-dsRed (Bottom right, red). Brain was visualized with brp (magenta), DAPI (Blue). BBB was visualized with 9-137-Gal4 (bottom right, green) (b). Hemocytes (Hml; green, NimC1; red) are not detected in the ventral nerve cord (c). Heatmap of average Hml+ cell number across the brain (mean with standard deviation, n = 22) (left). Representative image of Hml+ cells (green) across the brain (brp, magenta) used for analysis (right) (d). NimC1+ hemocytes (red) are located next to the Perineurial glia (PG, green, NP6293-Gal4 UAS-mCD8GFP, top) or Sub-perineurial glia (SPG, green, moody-Gal4 UAS-mCD8GFP, bottom) (e). 3D reconstruction of Fig. 1e shows that NimC1+ (magenta) hemocytes physically interact with perineurial glial cells (green) (f). Cortex glial cells were visualized with RFP (red) (R85G01-LexA LexAop-mCD8GFP NP2222-Gal4 UAS-mCD8RFP). g-h. Strong GRASP signals (green) were detected between Repo+ glial cells (magenta) and hemocytes, using Repo-LexA and HmlΔ-Gal4 to drive the two halves of GFP (top). In contrast, little green signal was observed in the Repo-LexA GRASP control samples (top right) or HmlΔ-Gal4 GRASP control samples (bottom right). Neuropil was visualized using brp (white) (g). With the use of SPG-LexA (R54C07-LexA) and HmlΔ-Gal4, strong GRASP signals (green; left) were detected but not in SPG-LexA GRASP control samples (right). Cell membrane was visualized using phalloidin (magenta) (h). To detect SPG-LexA and HmlΔ-Gal4 GRASP signals, GFP antibody was used. DAPI: blue, White scale bar, 100 μm unless it’s not indicated. All images above are representative images obtained from at least three different experiments.
