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Fig. 4. dark82 salivary glands are defective for
histolysis. (A,B) Confocal micrographs of salivary glands
from wild-type (A) and dark82 (B) animals at 16 hours APF stained
for a cytoplasmic protein, p127 (green), and a nuclear protein, BR-C (red).
Head eversion, which marks the prepupal-pupal transition, has occurred in
these animals. In wild type, larval salivary glands are completely histolysed,
but in dark82 animals the glands persist and structural integrity
is maintained. (C,D) Confocal micrographs showing
immunohistochemical staining of salivary glands for anti-cleaved caspase 3
(blue), a marker for active DRICE (Yu et
al., 2002), together with anti-actin, (red) and OliGreen, a
nuclear stain (green). (C) Caspase activity (blue) in wild-type salivary
glands is shown here at 12 hours APF, 
4 hours before final histolysis.
(D) Caspase activity is starkly reduced in salivary glands of
dark82 animals, shown here at 16 hours APF. (E-G) Ecdysone
signaling and expression of death-related genes are unperturbed in
dark mutant salivary glands. Immunohistochemical staining (E,F) shows
nuclear accumulation of ecdysone-responsive transcription factors in
persisting dark salivary glands at 16 hours APF. The confocal image
in E shows coincident nuclear accumulation of Ecdysone Receptor (EcR, red) and
BFTZ-F1 (green), counterstained for actin (blue). Overlapping stains for EcR
and BFTZ-F1 produces a robust yellow signal in gland cell nuclei. In F,
nuclear accumulation of E74A (red) is shown, with counterstaining for actin
(blue) and the cytoplasmic protein Rab11 (green). (G) Pre-death expression
profiles for the genes indicated were determined using real-time quantitative
RT-PCR on RNA prepared from salivary glands dissected from wild-type (OreR)
and dark82 animals at 11 hours and 13 hours APF (normalized from
18°C). The gene set analyzed here is a surrogate for profiles of
pre-histolytic gene expression (Gorski et
al., 2003). Expression levels are represented by
Ct values, where Ct=Ct of no
template control (set at 38 PCR cycles) - Ct of sample.
Ct, or threshold cycle, is the PCR cycle at which a statistically
significant increase in fluorescent signal can be detected above background.
Drosophila rp49, used here as a control, showed no significant
differences in expression. dark transcripts were not detected in
mutant salivary glands, but, in all other respects, profiles between wild-type
and dark glands were highly comparable.
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