Figure 4: Inhibition of SE0PG neurons is coupled to delayed pupariation.
(a) The pupariation ratio after hatching. When newly hatched larvae were raised at 29 °C, control larvae pupariate at 72–96 hAH. In contrast, TeTxLC-expressing larvae delayed pupariation by 1–2 days, and the delay was rescued by feeding with 20E. The number of animals: R29H01>TeTxLC (n=201, EtOH; n=251, 20E), w>TeTxLC (n=142, EtOH; n=172, 20E). (b) The prolonged larval growth period resulted in an increased pupal size. The pupal size (double-headed arrow) was compared between control (n=46) and R29H01>TeTxLC (n=48). (c) The levels of ecdysteroids in w>TeTxLC and R29H01>TeTxLC animals at 96–144 hAH. At 120 hAH, prepupae were collected from the control. (d) The pupariation timing was delayed after TeTxLC was expressed in three pairs of SE0 neurons (black, n=193). mCD8::GFP was expressed in these neurons of control (grey, n=381). (e) The expression of ecdysteroidogenic genes decreased in the RG of R29H01>TeTxLC larvae at 96 hAH. The fold changes compared with control larvae (w>TeTxLC) were calculated. (1) nvd, (2) spok, (3) sro, (4) phm, (5) dib, (6) sad. (f–n) Three examples of the third instar Br–RG complex containing MARCM clones that express GFP and rpr in SE0PG neurons. Those larvae that exhibited a delayed pupariation were dissected at 120 hAH and immunostained for GFP (green) and serotonin (magenta). In our experimental condition, some cells were eliminated by rpr but others were not. The presence of the cell bodies and the PG projections are indicated with yellow arrows and circles, while the absence of them are indicated with blue ones. In the example 1 (f–h), the right side of SE0PG cell bodies and the PG projection were lost (blue arrow and circle). In the examples 2 and 3 (i–n), the PG projections remained (yellow circles) but serotonin signals significantly decreased in these PG projections (blue circles in k,n). For statistical analysis, Student’s t-test was performed in b,c,e. *P<0.05. The average values of triplicate data sets are shown with s.e.m.. Bars: (b) 1.0 mm; (f–n) 50 μm. Each experiment was conducted independently at least three times.
