Fig. 3: Identification of wake-Gal4 drivers expressed in insulin-producing cells (IPCs) of the adult male brain.

a Gal4 transgene insertion locations on the wake gene in the different enhancer trap NP lines (wake^NP3168, wake^NP1350, and wake^NP3624) and the wake¹⁵¹⁸⁵-Gal4 line generated using the CRISPR/Cas9 system are indicated using blue arrowheads. b–e Patterns of b wake^NP3168, c wake^NP3624, d wake^NP1350, and e wake¹⁵¹⁸⁵-Gal4 expression, revealed using UAS-mCD8::GFP, in the adult male brain, represented in green in b1–e1, which show the cell bodies of median neurosecretory cells (MNCs; indicated by the arrowheads). The neuropil was immunostained using an anti-DLG antibody (magenta in b2–e2); merged in b3–e3 (n = 6 for each). f–i The MNCs cell bodies exhibiting with four independent drivers (green in f1–i1) are IPCs, as evidenced by anti-insulin-like peptide 2 (anti-Ilp2) immunolabelling (magenta in f2–i2); merged in f3–i3 (n = 6 for each). j Diagrammatic representation of the gene targeting strategy for restricting myr-4xSNAP expression to IPCs specifically using a Gal4 driven Bxb1 recombinase and a specific IPC-expression LexA intersectional method. The blue and yellow circles represent the patterns of ilp2-LexA and wake-Gal4 expression, respectively. The resulting overlapping cell populations that express myr-4 × SNAPs are limited to the regions where Gal4 induced UAS-Bxb1 expression, as the transcriptional stop cassette is removed (LexAop-myr > STOP > 4 × SNAP → LexAop-myr::4 × SNAP) to allow LexA-induced expression, which represents the SNAP proteins that can be labelled specifically with molecular probes. k–e Representative images showing expression in IPCs with four independent drivers; green indicates the chemically labelled 4 × SNAPs expression pattern resulting from the intersectional ilp2-LexA and independent wake-Gal4 drivers: k wake^NP3168, l wake^NP3624, m wake^NP1350, and n wake-Gal4¹⁵¹⁸⁵. Cell bodies of IPCs are indicated by dotted squares (k1–n1, respectively) (magnified in k2–n2; n = 8 for each). Scale bars, 20 µm.