The GeneSwitch GAL4 system. In the absence of an activator (uninduced), the GeneSwitch GAL4 protein is expressed in target tissues but remains transcriptionally silent; no expression of downstream UAS-linked genes therefore occurs. However, after systemic application of RU486 (induced), the binding of the RU486 ligand causes the GeneSwitch GAL4 protein to become transcriptionally active, resulting in expression of UAS-linked genes (shown here as UAS-GFP).

GeneSwitch GAL4 lines with expression in the larval brain. Z-projections of confocal sections of the L3 brain. GeneSwitch GAL4 expression patterns were visualized using UAS-2xEGFP (green). (A) GSG3516 and (B) GSG6124 are broadly expressed in the central brain region and inner brain lobes. (C) GSG1802-2 is expressed in a few clusters of neurons, including a prominent lateral cluster (arrow). (D) GSG5961 is expressed in a small cluster of neurons surrounding the esophageal canal (OC) and in the optic lobe primordia (OLP). (E) GSG6098 is expressed in the mushroom bodies (MB) and in a few additional neurons (arrow) with processes that are associated with the mushroom bodies. (F) GSGB15-1 is expressed in the mushroom bodies (MB). VNC, ventral nerve cord. Bar, 100 μm.

GeneSwitch GAL4 lines with expression in motoneurons. Each GeneSwitch GAL4 expression pattern was visualized using UAS-2xEGFP (green). Columns 1 and 2 show Z-projections of confocal sections of L3 ventral nerve cord (VNC) expression. Column 2 shows anti-Eve expression (magenta), which recognizes three dorsal midline neurons: the motoneurons aCC/MN1-Ib and RP2/MNISN-Is and the pCC interneuron. Columns 3 and 4 show Z-projections of confocal sections of peripheral NMJ expression. Muscle fibers are numbered. Column 4 shows anti-FasII expression (magenta), which recognizes all motoneuron endings. (A–D) GSG3705 is expressed by a cluster of dorsal midline neurons in the VNC, including both Eve-positive motoneurons but not the Eve-positive interneuron (A and B), and at all NMJs (C and D), including type II endings (arrow). (E–H) GSG1802-2 is expressed by type Ib motoneurons in the dorsal VNC (E and F) and shows peripheral NMJ expression at type Ib endings (G and H). (I–L) GSGB15-1 is expressed in one Eve-positive dorsal midline neuron in the VNC, RP2/MNISN-Is (I and J), and exclusively at type Is NMJs in the periphery (K and L). (M–P) GSG2451-1 is expressed in lateral VNC cells (M and N) and shows peripheral NMJ expression at a subset of muscle fibers innervated by SNa, SNc (O and P), and the TN. Peripheral expression also occurs in the chordotonal sensory neuron vchA. (Q–T) GSGB13-7 is expressed in a single lateral VNC motoneuron cell body (Q and R) that innervates muscle fiber 25 via the TN (S and T). Anterior is up and dorsal is to the left in columns 3 and 4. Bar, 50 μm.

GeneSwitch GAL4 lines with expression in sensory neurons. Each GeneSwitch GAL4 line expression pattern was visualized using UAS-2xEGFP. Z-projections of confocal sections of peripheral sensory expression (left column) and sensory axons projections within the VNC (right column) in L3 larva are shown. (A and B) GSG2553 expression in thoracic es neurons. (A) Peripheral cell body expression in one of the two es neurons labeled in each hemisegment is shown. (C and D) GSG3938 expresses in all ch neurons. C shows the lateral compound chordotonal organ (lch5) and v′ch1. (E and F) GSG734 expression in all md-da neurons. E shows expression in the dorsal cluster of md-da neurons. (F) In the VNC there is also expression in a small number of lateral cells (arrowhead) that form longitudinal axon projections at a lateral position (arrow). (G and H) GSG3147-1 expression in class III and IV md-da neurons. G shows expression in class III and IV md-da neurons in the dorsal cluster. (I and J) GSG5961 expression in class I md-da neurons. I shows expression in ddaE, a class I md-da neuron in the dorsal cluster. Anterior is up and dorsal is left in all PNS images. Bar, 50 μm.

GeneSwitch GAL4 lines with expression in muscles. Each GeneSwitch GAL4 expression pattern was visualized using UAS-2xEGFP (green) and stained with rhodamine phalloidin (red), which labels all muscle fibers. (A and B) GSG329-1 is expressed in all muscle fibers. (C and D) GSG907 is expressed specifically in muscle fibers 17 and 29. Expression also occurs in peripheral nerve glia. (E and F) GSG3678 is strongly expressed in transverse muscles 18 and 24 in thoracic (T) segments, but not in abdominal (A) segments. Expression is also observed in tendon cells, the epidermal attachment cells at the end of all muscle fibers. Bar, 200 μm.

GeneSwitch GAL4 lines with expression in glia. Z-projections of confocal sections of GeneSwitch GAL4 lines visualized using UAS-2xEGFP (green) and labeled with anti-Repo (magenta). (A–E) GSG8302-2. (A and B) Expression is observed in repo-negative cells on the surface of the VNC (arrowheads) and in a number of repo-positive surface glia (arrows). (C–E) Peripheral perineurial cells (PPG) surrounding the outer edge of the peripheral nerves are labeled. There is no expression in ensheathing glia (PG). (F–J) GSG1201-2. (F and G) Expression occurs in repo-negative cells on the surface of the VNC (arrowhead), as with GSG8302-2, and in repo-positive cortex glia (CBG-cell body glia). (H–J) In peripheral nerves, expression is observed in the inner ensheathing glia, but not in the outer peripheral perineurial cells. Bars: A, B, F, and G, 50 μm; C–E and H–J, 25 μm.

Transgene expression level can be controlled by varying RU486 concentration. GSG734 drives expression in multidendritic sensory neurons. Z-projections of confocal sections of peripheral cell body expression (left) and sensory axon expression in the VNC (right) in L3 larvae are shown. Expression was visualized after a 24-hr exposure to varying concentrations of RU486 using a UAS-2xEGFP reporter gene. (A and B) After a 24-hr exposure to 10 μg/ml RU486 strong expression is visible in peripheral cell bodies (A) and in axon terminals in the VNC (B). (C and D) At 5 μg/ml, strong expression is observed in cell bodies (C), with weaker axonal expression (D). (E–H) Peripheral cell body expression is variable at 2 μg/ml (E) and 1 μg/ml (G), although some cells show strong expression. Axon expression is also variable (F and H). (I and J) At 0.5 μg/ml weak cell body expression is visible (I), and axon expression is barely detectable (J). (K and L) At 0.2 μg/ml, cell body expression is barely detectable (K, arrows) and no axon expression is observed (L). Bar, 50 μm.

GeneSwitch GAL4 lines with expression in VNC interneurons. Z-projection of confocal sections of the L3 VNC. (A and B) UAS-2xEGFP expression driven by GSG1142 (green) and double labeled with anti-FMRF (magenta). (C–F) UAS-2xEGFP expression driven by GSG3315-1 and double labeled with anti-FasII (magenta). A schematic view of the axon tracts labeled by FasII is shown at the bottom. Tracts referred to in this figure are highlighted in magenta. (A and B) GSG1142 is strongly expressed in single cells in thoracic (T) segments that extend processes toward the midline. Weak expression is also observed in chordotonal sensory neurons (ch). Double labeling with anti-FMRF shows that GSG1142 is expressed in the neuroendocrine Tv neurons. (C and D) GSG3315-1 expression in the dorsal VNC. Intersegmental processes extend along the DL FasII axon tract. (E and F) GSG3315-1 expression in the ventral VNC. Intersegmental interneuron processes extend (1) along the VM FasII axon tract and (2) lateral to the CI1-3 FasII axon tract. Anterior is up in this and all other figures, unless otherwise stated. Bar, 50 μm.