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A Steroid-Triggered Transcriptional Hierarchy Controls Salivary Gland Cell Death During Drosophila Metamorphosis

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A Steroid-Triggered Transcriptional Hierarchy Controls Salivary Gland Cell Death During Drosophila Metamorphosis Molecular Cell, Vol. 5, 445±455, March, 2000, Copyright 2000 by Cell Press A Steroid-Triggered Transcriptional Hierarchy Controls Salivary Gland Cell Death during Drosophila Metamorphosis Changan Jiang,* Anne-FrancË oise J. Lamblin,²§ critical to the survival of the animal, as misregulation of Hermann Steller,² and Carl S. Thummel*³ programmed cell death has been associated with a wide *Howard Hughes Medical Institute range of human diseases (Thompson, 1995; Rudin and Department of Human Genetics Thompson, 1997). University of Utah Hormones play a key role in controlling apoptosis. Salt Lake City, Utah 84112 The term ªprogrammed cell deathº was first used by ² Howard Hughes Medical Institute Lockshin and Williams (1965) in their study of steroid- Department of Biology regulated intersegmental muscle degeneration during Massachusetts Institute of Technology silkmoth metamorphosis. Similarly, thyroid hormone± Cambridge, Massachusetts 02139 induced RNA and protein synthesis are required for the destruction of the tadpole tail during Xenopus metamor- phosis (Tata, 1966). Steroid hormones also control pro- Summary grammed cell death in a wide range of mammalian tis- sues, including neurons, mammary glands, testes, the The steroid hormone ecdysone signals the stage-spe- prostate, ovaries, and the uterus (Kiess and Gallaher, cific programmed cell death of the larval salivary 1998). The best studied vertebrate model for hormone- glands during Drosophila metamorphosis. This re- regulated programmed cell death is the apoptosis of sponse is preceded by an ecdysone-triggered switch immature thymocytes and mature T cells in response in gene expression in which the diap2 death inhibitor to glucocorticoids. This response is dependent on the is repressed and the reaper (rpr) and head involution steroid, the glucocorticoid receptor, and de novo tran- defective (hid) death activators are induced. Here we scription, suggesting that the hormone is triggering a show that rpr is induced directly by the ecdysone± death-specific genetic program (Evans-Storms and Cid- receptor complex through an essential response ele- lowski, 1995). The genes that direct this death response, ment in the rpr promoter. The Broad-Complex (BR-C) however, remain unknown. is required for both rpr and hid transcription, while The programmed cell death of larval tissues during E74A is required for maximal levels of hid induction. Drosophila metamorphosis provides an ideal system to define the molecular mechanisms of steroid-regulated diap2 induction is dependent on ␤FTZ-F1, while E75A and E75B are each sufficient to repress diap2. This apoptosis. Metamorphosis in Drosophila is controlled study identifies transcriptional regulators of pro- by pulses of the steroid hormone 20-hydroxyecdysone (referred to here as ecdysone) (Riddiford, 1993). A pulse grammed cell death in Drosophila and provides a di- of ecdysone at the end of larval development triggers rect link between a steroid signal and a programmed puparium formation, signaling the onset of metamor- cell death response. phosis and initiating the prepupal stage in development. This is followed by another ecdysone pulse, approxi- Introduction mately 12 hr after puparium formation, that signals the prepupal±pupal transition. In response to these sequen- Programmed cell death provides a critical means by tial ecdysone pulses, obsolete larval tissues are de- which multicellular organisms remove obsolete or dam- stroyed in a stage-specific manner as adult tissues and aged cells. Unlike necrosis, which results from external structures develop from small clusters of progenitor injury, apoptosis is initiated inside the cell through an cells, resulting in the transformation of a larva into an evolutionarily conserved genetic regulatory pathway immature fly (Robertson, 1936). The larval midgut initi- and is characterized by distinct morphological and bio- ates cell death at puparium formation in response to chemical changes (Kerr et al., 1972; Jacobson et al., the late larval pulse of ecdysone, while the salivary 1997; Vaux and Korsmeyer, 1999). Over the past few glands are not destroyed until approximately 15 hr after years, significant advances have been made in our un- puparium formation, in response to the prepupal pulse derstanding of the cell death pathway and how it is of ecdysone. The destruction of these tissues is accom- regulated by intracellular and extracellular signals. panied by nuclear staining with acridine orange and Though the cell death machinery appears to be present DNA fragmentation, indicative of apoptosis (Jiang et al., in all cells, its activation is determined by a balance 1997). Furthermore, midgut and salivary gland cell death between death activators and death inhibitors. Multiple can be inhibited by ectopic expression of the p35 anti- signals, including growth factors, DNA-damaging agents, apoptotic protein, implicating a role for caspases in the and hormones, can influence the balance between these death response. This is consistent with ecdysone-induc- regulators and thereby induce or suppress cell death in tion of the DRONC caspase in both the larval midgut and a precisely regulated manner. This level of control is salivary glands before the onset of cell death (Dorstyn et al., 1999). The larval midgut and salivary glands thus appear to be destroyed by a steroid-triggered pro- ³ To whom correspondence should be addressed (e-mail: carl. grammed cell death pathway that has hallmark features [email protected]). § Present address: National Cancer Institute, National Institutes of of apoptosis. Health, Building 37, Room 4D11, 37 Convent Drive, Bethesda, Mary- As in vertebrate organisms, caspase activation in Dro- land 20892. sophila is controlled by both cell death activators and Molecular Cell 446 cell death inhibitors (Bergmann et al., 1998b; Abrams, switch in death gene expression suggests that ecdy- 1999). Three cell death activators have been identified sone triggers salivary gland cell death by simulaneously in DrosophilaÐreaper (rpr), head involution defective repressing a death inhibitor and inducing death activa- (hid), and grim (White et al., 1994; Grether et al., 1995; tors, shifting the balance between pro- and antiapo- Chen et al., 1996). Removal of these genes by use of ptotic activities. the Df(3L)H99 deletion effectively blocks programmed Ecdysone functions through a heterodimeric receptor cell death in Drosophila (White et al., 1994). In addition, comprised of two members of the nuclear receptor su- specific hid alleles lead to embryonic lethality with de- perfamily, EcR (NR1H1) and the RXR homolog, Ultraspi- fects in programmed cell death (Grether et al., 1995). racle (USP, NR2B4) (Koelle et al., 1991; Koelle, 1992; The expression patterns of rpr and grim foreshadow cell Yao et al., 1992; Thomas et al., 1993). The ecdysone± death during Drosophila development, whereas hid is EcR/USP complex directly regulates primary response also expressed in some cells that fail to die (White et genes, including three early genes that were originally al., 1994; Grether et al., 1995; Chen et al., 1996; Robinow identified as ecdysone-inducible puffs in the larval sali- et al., 1997). Ectopic expression of each of these genes vary gland polytene chromosomesÐthe Broad-Com- is sufficient to induce cell death by activating a caspase plex (BR-C), E74, and E75 (Russell and Ashburner, 1996). cascade. More recent studies, however, indicate that The BR-C encodes a family of zinc finger transcription rpr and hid,orrpr and grim, function together in a coop- factors (DiBello et al., 1991). E74 encodes two isoforms erative manner to direct death in some cell types (Zhou of an ETS domain transcription factor, designated E74A et al., 1997; Draizen et al., 1999). Cell death in Drosophila and E74B (Burtis et al., 1990), and E75 (NR1D3) encodes can also be triggered by UV or X irradiation, as in mam- three orphan members of the nuclear receptor super- malian cells, and this process is preceded by induction family, designated E75A, E75B, and E75C (Segraves and of rpr transcription (Nordstrom et al., 1996; A.-F. Lamblin Hogness, 1990). These early genes, in turn, regulate and H. Steller, submitted). Taken together, these obser- large batteries of secondary response late genes, defin- vations indicate that expression of the Drosophila death ing ecdysone-triggered regulatory hierarchies (Russell activators serves as a central life/death switch in re- and Ashburner, 1996; Thummel, 1996). Genetic studies sponse to different stimuli. It seems likely that these have demonstrated that the BR-C and E74 are required genes are controlled by cis-acting regulatory elements for appropriate developmental responses to ecdysone that function together to integrate the input from a num- during metamorphosis (Kiss et al., 1988; Restifo and ber of cell death signaling pathways, precisely coordi- White, 1992; Fletcher et al., 1995). Furthermore, BR-C nating the patterns of cell death during development. and E74A proteins have been shown to directly regulate Hence, understanding the regulation of rpr, hid, and late gene transcription (Urness and Thummel, 1995; grim transcription provides a critical step for defining Crossgrove et al., 1996). Ectopic expression studies the control of programmed cell death in Drosophila. have demonstrated that E75B can repress the ␤FTZ- Like vertebrate cells, the fate of a Drosophila cell is F1 orphan nuclear receptor (NR5A3), suggesting that also modulated by IAP homologs (Inhibitor of Apoptosis
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