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and nucleus of prothoracic gland cells. This behavior closely matched the
oscillations that were reported for
Drosophila
PTTH mRNA levels
(
McBrayer et al., 2007
), which suggested a functional link between PTTH
and DHR4. Indeed, when PTTH neurons were ablated, DHR4 remained
primarily in the nucleus, but when the PTTH pathway was hyperactivated,
the protein was predominantly cytoplasmic. Consistent with this observa-
tion, hyperactivation of the PTTH pathway via expressing constitutively
active
Ras
(
Ras
V12
) in the prothoracic gland (
Caldwell, Walkiewicz, &
Stern, 2005; Karim & Rubin, 1998
) also results in accelerated entry into
metamorphosis, suggesting that this is achieved precisely because active
Ras prevents DHR4 from entering the nucleus (
Fig. 2.4
B).
If loss-of-
DHR4
function causes shortened feeding times and accelerated
entry into metamorphosis, one would predict that this might arise from a
premature occurrence of ecdysone pulses. This appears to be the case, as de-
pleting DHR4 in the ring gland via RNAi causes a faster rise of ecdysone
levels that fail to regress when hormone levels normally drop. We suspect
that the animal interprets these higher ecdysone levels as an early ecdysone
pulse, resulting in developmental acceleration. Our model therefore pro-
poses that DHR4 acts as a repressor of ecdysone pulses by counteracting
the PTTH-stimulated rise of ecdysone levels. This idea is further corrobo-
rated by the finding that prothoracic gland-specific overexpression of
DHR4
blocks molting, a phenotype that can be partially rescued by feeding 20E. If
DHR4 is a target of the PTTH pathway, then one would expect that ERK
phosphorylates DHR4 to trigger its removal from the nucleus when the
PTTH signaling is active. While this has not been demonstrated directly,
it appears that there is an inverse correlation between the subcellular local-
ization of ERK and DHR4, suggesting that there is a functional link
(
Fig. 2.4
B). Indeed, DHR4 is predicted to have several clusters of ERK tar-
get sites, and future studies such as mutational analysis of putative ERK
phosphorylation sites of DHR4 will have to be carried out in order to de-
termine whether these sites affect the subcellular localization of the protein.
What are the downstream targets of DHR4? Does DHR4 regulate the
expression of the Halloween genes? In
Drosophila
, the expression levels of
phantom
,
disembodied
, and
shadow
are transcriptionally upregulated roughly
8 h prior to the onset of metamorphosis, consistent with the idea that PTTH
induces the Halloween genes at the transcriptional level (
Parvy et al., 2005
).
However, the expression levels of these three Halloween genes are relatively
constant (and fairly high as we pointed out earlier) in the last larval instar
before the major PTTH pulses, suggesting that the three minor ecdysone
