Figure 2: TRH–GAL4 and R29H01–GAL4 share three pairs of neurons in the SE0 cluster.

(a,b) The Br–RG complex from the late third instar larva expressing R29H01>GFP was immunostained with anti–GFP (a and green in b) and anti-serotonin antibodies (magenta in b). GFP was detected in the PG-projecting neurites (yellow arrows), three pairs of SE0 cluster cells (blue arrows, insets), and other cells that were not serotonin immunoreactive (blue arrowheads). (c) The dorsal side of the third instar larva expressing R29H01>GFP. The PG and serotonergic neurons were visualized with anti-Shroud (Sro, green) and anti-serotonin (magenta) antibodies, respectively. The boxed area is magnified in the inset. R29H01–GAL4-driven GFP expression was detected in the PG or PV-projecting neurons (yellow arrows), but not in the PM-projecting neurons (green arrows in the inset). At the FJ, the neural tracts bifurcated to PM and PG (green and yellow arrows). (d,e) Only three pairs of SE0 neurons (blue arrows) are labelled with GFP (d and green in e) as well as serotonin (magenta in e). Any of these cells innervated the PG (yellow arrows). By using the Q system in conjunction with the GAL4 system, R29H01–GAL4-driven GFP was expressed only in the overlapped region where both TRH–QF and GAL4 are expressed. (f–i) In GRASP analysis, spGFP1-10::Nrx was expressed in SOG neurons with hugS3–GAL4, and spGFP11::CD4 was expressed in serotonergic neurons with TRH–lexA. GRASP signal was detected between SOG and SE0 neurons with anti–GFP body (green, arrows). In contrast, negative controls did not give signals (h,i). SOG neurons were labelled with anti-PBAN antibody (magenta). The oesophagus foramen is outlined with yellow-dotted lines. The scale bar depicted in i corresponds to 183.8 μm (a,b,d,e), 126.3 μm (c), 101.8 μm (c, inset) and 50.0 μm (f–i).