FIGURE 3.

Bitbow2 enables broad neuron morphology labeling with a simple transgenic setup. (A) Schematic of Bitbow2 design. A self-regulating Flp (srFlp) module is added to ensure proper transient Flp activity without the need of an additional cross to the heat-shock Flp fly. Flp expression is driven by a neuron-specific n-Synaptobrevin (nSyb) promoter and terminated by self-excising between the flanking FRT sites, which have lower efficiency compared to those used in the Bitbow1 modules. This ensures proper Bitbow recombination before Flp self-excision to reach a stable genetic outcome. Compared to a (B) Bitbow1 labeling experiment, a (C) Bitbow2 labeling experiment requires only a direct cross to the TRH-Gal4 driver fly without the need of heat-shock. (B’,C’) indicate that mBitbow1.0 labeled fewer serotonergic neurons than Bitbow2.0 does. Inserted schematics indicate the somas of the labeled serotonergic neurons. (D) Quantification of the percentage of serotonergic neurons being labeled in different Bitbow2 flies, normalized to the labeling of a UAS-TagBFP fly. Each dot that overlays on the violin plots corresponds to the cell counting from one brain. (E) Adult neurons labeled in an offspring of the 2x mBitbow2.1 fly crossed to the R67A06-Gal4 fly. White and yellow insets show the somas of a group of neurons and their neurites projections, respectively. (F) Larva neurons labeled in an offspring of the 2x mBitbow2.1 fly crossed to the elav-Gal4 fly. Red and orange insets show one neuron cluster each in the central brain and in the VNC, labeled in distinct Bitbow colors, respectively. Dotted outline indicates the border of the neuron cluster. Scale bars: (B’,C’,F) 100 μm, (E) 50 μm, (E,F, inserts) 10 μm.