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pulses are not simply a consequence of modulating mRNA levels of the
Halloween genes. In line with this, none of the Halloween genes appeared
to be affected in ring glands of DHR4 mutants or DHR4 RNAi animals.
Through ring gland-specific microarrays, a hitherto uncharacterized cyto-
chrome P450 gene, Cyp6t3 , was identified as a candidate downstream target
of DHR4 ( Fig. 2.4 B). Several lines of evidence link Cyp6t3 to DHR4 and
ecdysone synthesis: (1) decreasing Cyp6t3 expression in the prothoracic
gland results in molting defects and developmental delays, which can be res-
cued by 20E, suggesting that these animals are ecdysone deficient; (2)
Cyp6t3 activity can be provisionally placed into the “Black Box” of the
pathway: The ecdysone intermediate 5 b -ketodiol can partially rescue the
Cyp6t3 RNAi phenotype, however, precursors upstream of the “Black
Box” cannot; (3) Cyp6t3 expression is significantly increased in the ring
gland of both DHR4 mutant and DHR4 RNAi animals, indicating the gene
is normally repressed by DHR4; (4) knockdown of Cyp6t3 expression in a
DHR4 mutant background can reverse the precocious pupariation pheno-
type; (5) Cyp6t3 mRNA levels oscillate, where lower levels correlate with
times when DHR4 is nuclear. Taken together, these data demonstrate that
Cyp6t3 is negatively regulated by DHR4 to downregulate ecdysone pro-
duction, but whether the gene is a direct target of DHR4 needs to be de-
termined by further experiments.
10. DHR3/E75-MEDIATED NO SIGNALING IS A CRITICAL
PLAYER OF ECDYSTEROIDOGENESIS
The recent report by Caceres et al. (2011) that NO plays a key role in
the production of ecdysone comes undoubtedly as a surprise. In brief, it was
shown that NO signaling regulates the interaction of DHR3 and E75 to
control the transcriptional activation of b ftz-f1 , all well-characterized players
of the ecdysone hierarchy ( Fig. 2.4 B).
NO is produced by nitric oxide synthase (NOS) and is a short-lived
diatomic molecule that acts as an intra- and transcellular messenger that reg-
ulates many physiological functions in both vertebrates and invertebrates
( Bredt & Snyder, 1994 ). The Drosophila genome harbors only one nitric ox-
ide synthase gene called NOS , which is expressed throughout Drosophila de-
velopment ( Stasiv, Regulski, Kuzin, Tully, & Enikolopov, 2001 ). Previous
studies have shown that NO plays important roles in imaginal disk develop-
ment, synaptogenesis, formation of retinal projection patterns, response to
hypoxia, and behavioral responses ( Suman, Seth, & Chandna, 2008 ).
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