Figure 7.
Bursicon secretion from the BAG, but not the BSEG, is impaired in rk4 mutants. AM, Western blot showing the bursicon content of hemolymph extracted from either Canton S control flies or rk4 mutants within 1 h of eclosion. Samples containing the indicated volumes of pooled hemolymph, collected from at least 40 animals of each genotype, were loaded into each lane, and the blot was probed with anti-burs antibodies. The positions of selected molecular weight markers (in kDa) are shown. The bursicon α-subunit (arrow, Bursα) runs at ∼16 kDa. B, Confocal images of anti-bursicon immunostaining in whole mounts of adults filleted to reveal the ventral nervous system and the abdominal nerves. Nervous system preparations are from either Canton S (CS) control animals or from rk4 mutants, killed 5 min or 60 min after eclosion, as indicated. Dotted boxes in the 5 min, CS preparation indicate the size and location of the fields used to calculate the intensity of immunolabeling of central processes belonging to the BSEG (T2; arrows) or peripheral processes belonging to the BAG in the abdominal nerves (AN; arrowheads). The position of the BAG is also indicated. C, Higher resolution images of the abdominal nerve regions of the preparations shown in B. Arrowheads indicate bursicon immunoreactive varicosities abundant in the abdominal nerves of animals 5 min after eclosion and also frequently found in the nerves of rk4 animals 60 min after eclosion (bottom). Such varicosities are, however, rare in the nerves of control animals after 60 min (top), where bursicon is largely depleted. D, Graphs showing the bursicon immunoreactivities (Burs-IR) associated with the BSEG fibers (left) or BAG fibers (right) in 13–18 preparations of the type shown in B. Each cross represents the Burs-IR value determined for an individual preparation, measured as the mean pixel intensity (mpi, background-subtracted) of an area centered over the proximal abdominal nerves (for the BAG) or the central T2 thoracic region (for the BSEG), as depicted in the top left panel in B (dotted areas). Filled diamonds represent the mean Burs-IR values for preparations of each indicated type. In all cases, the difference in mean Burs-IR values at 5 min and 60 min for like samples (i.e., samples representing same fiber type in animals of the same genotype) was determined to be highly significant by t test, as noted in Results. In contrast, the difference in mean Burs-IR values measured for a given fiber type at a given time, but in animals of different genotype, was not statistically significant except for BAG measurements at 60 min, as indicated by double asterisks (p = 2 × 10−4). E, Bar graph depicts the change in mean Burs-IR measured for the BAG of Canton S (CS) control and rk4 mutant animals between 5 min and 60 min.