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CCAP neuronal differentiation, and that the production of CCAP and
bursicon peptides might be linked to the second pulse. Indeed, blocking
the ecdysone hierarchy specifically in CCAP neurons by expressing domi-
nant negative forms of EcR resulted in a loss of CCAP and bursicon expres-
sion in the late CCAP neuron population. Similarly, expressing RNAi
against EcR in early and late CCAP-EN neurons caused strongly reduced
expression of CCAP and Burs-
a
. On a whole animal level, these disruptions
of EcR function resulted in leg extension defects, but not head eversion phe-
notypes. This however, was consistent with the observation that manipulat-
ing EcR function in CCAPs did not significantly affect CCAP expression in
the six CCAP-PL neurons, which correspond to the head eversion pheno-
type, and the authors reason that the GAL4 driver used in this experiment
has much weaker expression in CCAP-PL neurons, which would result in
reduced RNA interference.
While these results nicely demonstrated the requirement of EcR for the
onset of late CCAP neuronal differentiation, it was still unclear why this
event did not occur in response to an earlier ecdysone pulse. A body of
work has examined the issue of stage-specific ecdysone responses. In short,
the expression of
b
FTZ-F1, a nuclear receptor isoform acting directly
downstream of EcR, provides temporal specificity to the prepupal stage,
ensuring that the responses to the late-larval ecdysone pulse are different
from the responses to the later pulse that triggers head eversion (
Broadus,
McCabe, Endrizzi, Thummel, & Woodard, 1999; Woodard, Baehrecke,
& Thummel, 1994
). This prompted the authors to ask whether ectopic
expression of
b
FTZ-F1
would be sufficient to trigger the differentiation
of late CCAP neurons. Indeed, when
b
FTZ-F1
was ubiquitously expressed
in first instar larvae, they observed precocious production of CCAP and
bursicon peptides in late CCAP-EN neurons only 4 h after induction of
the transgene. Thus, it appears that CCAP neurons are competent through-
out larval development to receive an inductive ecdysone signal to undergo
terminal differentiation into peptide producing CCAP neurons, however,
this inductive signal is dependent on the presence of
b
FTZ-F1, allowing
the cells to put the ecdysone pulse into temporal context (
Fig. 1.4
B). While
the data is compelling, its interpretation is complicated by the fact that
b
FTZ-
F1 is expressed in most tissues during earlier stages (
Yamada et al., 2000
),
giving rise to the possibility that additional distinguishing factors must be
involved in triggering the differentiation of CCAP neurons. Alternatively,
it is possible that these neurons simply do not express
b
FTZ-F1 at earlier
stages, which can only be resolved by a careful expression analysis.
