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The Role of ßFTZ-F1 as a Tissue Specific
Regulator in Metamorphosis
‘‘Transformation…Transformation is a marvelous thing.
Though wonderful to watch, transformation from larva to
pupa is not a particularly pleasant process for the subject
involved. There comes for every caterpillar a difficult
moment when he begins to feel pervaded by an odd sense of
discomfort. It is a tight feeling-- here about the neck and
elsewhere, and then an unbearable itch. Of course he has
molted a few times before, but that is nothing in comparison
to the tickle and urge that he feels now.
He must shed that tight dry skin, or die.’’
(Nabokov, ‘On transformation’)
BACKGROUND
• The life cycle of Drosophila melanogaster has a duration of ten to
twelve days, during which the embryo develops into a larvae
to a stationary pupa and finally ecloses into the adult fly. This
transition from larvae to adult is known as metamorphosis and
is controlled by the steroid hormone, ecdysone.
•
The Life Cycle of Drosophila melanogaster
(Courtesy of The Official Web Site of The Nobel Foundation
BACKGROUND
•In metamorphosis, the developmental transitions are triggered by two
successive pulses of ecdysone.
•THE ONSET OF METAMORPHOSIS is marked by a high titer pulse in
late- third instar larvae which triggers puparium formation. The imaginal
discs begin to evaginate and the salivary glands secrete a polypeptide glue
in their lumen.
The prepupal-pupal transition takes place after 10-12 hrs in response to
a second pulse of ecdysone that causes the head to burst out of the thorax,
leg imaginal discs complete their elongation and the larval salivary
glands histolyze.
•The ßFTZ-F1 gene codes for a protein that acts as a ‘competence factor’
during the prepupal stage. In salivary glands, ßFTZ-F1 regulates the
expression of early genes such as E74A, E75A and BR-C and the
stage-specific E93 gene by enabling them to respond to ecdysone in
prepupae.
Ecdysone Timeline in Drosophila melanogaster
What is the molecular mechanism by which ßFTZ-F1 exerts its
function to regulate early gene expression?
OUR HYPOTHESIS
ßFTZ-F1 provides target genes with the competence to respond
to ecdysone by inducing expression of the ecdysone-receptor
complex (EcR) to facilitate the induction of the early genes.
EXPERIMENTAL DESIGN
• Transformant Flies called P[F-F1] were used that express a
high level of ßFTZ-F1 mRNA upon heat shock.
• Control w1118 and transformant w;P[F-F1] mid-third instar
larvae were heat shocked for 30 min and the tissues were
immediately dissected in oxygenated Robb’s saline.
• The organs were then cultured in the presence of oxygen at
25 C for 2 hr with or without ecdysone.
• Total RNA was extracted from the tissues and analyzed for
EcR mRNA by Northern blot hybridization. The Northern
blot was also probed with rp49 (gene encoding ribosomal
protein) as a control for loading and transfer.
Third Instar Larvae showing Blue Gut
Fat Bodies
Imaginal Disc
Central Nervous
System
Salivary Glands
RESULTS AND CONCLUSION
There is no EcR induction in any of the tissues!
This indicates that ßFTZ-F1 does not regulate the
genetic response to ecdysone by simply upregulating
the expression of the ecdysone-receptor complex.
EcR
rp49
Figure 2. Tissue-Specific Effects of Ectopic ßFTZ-F1 expression
Ecdysone Timeline in Drosophila melanogaster
IN WHICH OTHER TISSUES DOES
THE EXPRESSION OF ßFTZ-F1
AFFECT THE ECDYSONE INDUCTION
OF BR-C, E74A, E75A AND E93
TRANSCRIPTION?
RESULTS
The induction of E93 by ßFTZ-F1 expression differs from
tissue to tissue in mid-third instar larvae.
Table 1.Induction of E93 by ßFTZ-F1 in late-third instar larvae
CONDITION
w1118
[-Ecd]
w;P[F-F1]
[-Ecd]
W1118
[+Ecd]
w;P[F-F1]
[+Ecd]
SALIVARY
GLANDS
---+
GUT
---+
IMAGINAL
DISCS
-----
CNS
FAT
-+
-+
-+
-+
CONCLUSION
E93 displays different temporal expression patterns in
different tissues.Based on our results and previous studies,
we hypothesize that E93 may be regulated via several
different mechanisms depending on tissue and temporal
context
• independently of ßFTZ-F1 and ecdysone
• by ßFTZ-F1 and ecdysone
• by ecdsyone alone
• by ßFTZ-F1 alone
REMINDER:
ßFTZ-F1 does NOT regulate levels of EcR
FUTURE EXPERIMENTS
Determine in which tissues does ßFTZ-F1 enhance the
ecdysone response of the early genes: E74A, E75A and BR-C.
Determine the molecular mechanism by which ßFTZ-F1 exerts
its function by mapping the cis-regulatory sequences of E93.
ACKNOWLEDGEMENTS
Craig Woodard, The President of the Fly Den
Department of Biology, Mount Holyoke College
National Science Foundation
The Alicia Baruch Fellowship