Tissue specific effects of βFTZ-F1 loss-of-function on the early gene E93 transcription during Drosophila melanogaster metamorphosis



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Steroid hormones, in conjunction with their receptors, regulate the transcription of target genes in higher organisms. During Drosophila melanogaster s metamorphosis, two pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) direct both morphogenesis and the destruction of tissues by programmed cell death. An increase in ecdysone titer at the end of the third larval instar triggers puparium formation, marking the onset of metamorphosis. As a result, the anterior larval muscles and larval midgut undergo apoptosis (Broadus, 1999). Roughly ten hours after puparium formation (APF), another ecdysone pulse induces the prepupal-pupal transition during which the larval salivary glands undergo programmed cell death and leg imaginal discs complete their extension. Induced by these ecdysone pulses are the early genes, namely Broad Complex (BR-C), E74A ,and E75A. These genes encode transcription factor for genes involved in tissue morphogenesis and programmed cell death. In addition to these genes, the gene E93 is expressed during the prepupal ecdysone pulse. The gene E93 induces programmed cell death of the salivary land and midgut (Lee, 2002). During the interval of low ecdysone titer in mid-prepupae, the gene βFTZ-F1 is expressed. This gene is believed to provide the early genes with the competence to respond to ecdysone (Woodard, 1994). This experiment examines RNA isolated from salivary glands, midgut, hindgut and fat from animals aged at 10 and 12 hours APF. The expression level of E93 in control (w1118) animals was compared to FTZ-F1 loss of function mutants to determine if FTZ-F1 is required for the proper expression of E93. Real Time PCR was used to quantify the amount of E93 mRNA in these tissues at specific metamorphic stages. The comparative CT (∆∆CT) analyses of relative real-time PCR results showed an underexpression of E93 at 10 hrs APF in hindgut and salivary gland tissues of FTZ-F1 mutants, suggesting that FTZ-F1 was necessary for the expression of E93. Both midgut and fat body tissues of FTZ-F1 mutants showed similar expression as control animals. This indicates that FTZ-F1 was not necessary for the expression of E93 in these tissues in late prepupae.