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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 embryodevelops intoa 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 ofearly genes such as E74A, E75A and BR-C and the • stage-specific E93 gene by enabling them to respond to ecdysone in • prepupae.
What is the molecular mechanism by which ßFTZ-F1 exertsits 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.
Figure 2. Tissue-Specific Effects of Ectopic ßFTZ-F1 expression EcR rp49
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 byßFTZ-F1 in late-third instar larvae
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 doesßFTZ-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