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Project Description 1. Results from Prior NSF Support The PI was funded previously by a CAREER Award (MCB-9722205) from the NSF. The PI is currently funded by an RUI grant from the NSF (MCB-0110238), which expires August 1, 2004. This proposal is for a renewal of the RUI grant. Steroid hormones control a wide range of physiological and developmental processes in higher organisms, acting in conjunction with receptor proteins to regulate the stage- and tissuespecific transcription of target genes. Although we understand much about how steroid receptors control transcription in cultured mammalian cells, we understand little about how these effects on gene expression result in the dramatic stage- and tissue-specific developmental changes associated with steroid hormone function. We are using the fruit fly, Drosophila melanogaster, as a model organism with which to examine how steroid-induced changes in gene expression control specific developmental processes. Pulses of the steroid 20hydroxyecdysone (referred to in this proposal as ecdysone) trigger genetic regulatory hierarchies that direct both programmed cell death and morphogenesis. In this project, we are focusing on the role of orphan nuclear receptor ßFTZ-F1 in controlling the morphogenesis of the adult body structures. The research in my lab is done mainly by undergraduate students and a professional research associate (technician). I spend much of my time training undergraduate students in the lab. I have found that my research team is particularly successful at using sophisticated microscopy techniques and cleverly designed assays to examine developmental processes. Here I describe a plan to continue this line of research. These hypothesis-driven experiments are perfect for training undergraduates in the practice of science, and undergraduate students will be intimately involved in each part of this research plan. In addition, this proposal describes plans to travel with undergraduate students to the lab of a collaborator, where they will benefit from the resources and intellectual community at his institution. In carrying out these studies, we hope to both gain an understanding of the mechanisms by which steroid hormones elicit distinct biological responses, while providing a fertile training ground for scientists at the beginning of their careers. 1.1 The role of ßFTZ-F1 in Drosophila morphogenesis Our work has provided strong evidence supporting the hypothesis that the ßFTZ-F1 gene encodes a regulatory protein that plays a key role in directing genetic and developmental responses to the steroid hormone 20-hydroxyecdysone (ecdysone) during Drosophila metamorphosis. We (including undergraduate students Dana Cruz, Lynn L'Archeveque, Margaret Lobo, Jennifer McCabe, Emily McNutt, Tetyana Obukhanych, Petra Scamborova, and Layla Rouse) have performed several EMS and P-transposable-element mediated mutageneses of the FTZ-F1 locus, and have characterized the mutations at the molecular and phenotypic levels. As described in our 1999 Molecular Cell paper {Broadus, 1999 #93} (see below), a loss-of-function mutation in ßFTZ-F1 leads to defects in gene expression and developmental events that occur during metamorphosis. One of the events disrupted by loss of ßFTZ-F1 function is leg development. The legs of ßFTZ-F1 mutant pupae are shorter than those of wild-type. Priya Vasa, an undergraduate, did an extensive examination of the leg imaginal discs (the structures that give rise to the adult legs) in control and ßFTZ-F1 mutant prepupae and pupae. Tina Fortier, Research Associate (technician), has continued Priya’s work, and we recently published a paper on this work in the journal, Developmental Biology {Fortier, 2003 #545} (see below). Tina and Priya have done confocal microscopy to examine changes in cell shape during early leg development in control and ßFTZ-F1 mutant prepupae. Tina has also made "movies" of leg morphogenesis in wild-type animals and ßFTZ-F1 mutant animals expressing GFP in their legs, beginning at the start of metamorphosis, and ending more than 50 hours later. This has enabled us to establish a precise time-course of leg development in mutant and control animals for comparison. Leg morphogenesis in ßFTZ-F1 mutants occurs normally until shortly after the prepupal-pupal transition, when a major extension of the legs, thought to be driven by contraction of body wall muscles, occurs in the wild-type controls. This leg extension is delayed by several hours and significantly reduced in ßFTZ-F1 mutants. Tina has also made careful observations to further characterize pupation in control and ßFTZ-F1 mutants. She has made time-lapse video recordings of control and ßFTZ-F1 mutant development. Tina collected animals at the beginning of metamorphosis, which is 0 hours after puparium formation (APF) and placed them in a moist chamber. She used a video recording system to make time-lapse movies starting at 0 hours APF and ending at 1824 hours APF. ßFTZ-F1 mutants appear to develop normally until the prepupal-pupal transition. In the mutant, the anterior translocation and subsequent extension of the legs are delayed by several hours and are incomplete. All subsequent leg development in the mutant appears to occur normally, indicating that the abnormalities seen in these mutants are in fact due to stage-specific defects, rather than to general weakness or poor health. In wild-type, during the prepupal-pupal transition, contraction of larval muscles shortens the prepupal body, translocates a mid abdominal gas bubble to the posterior end of the pupal case, and then moves the gas to the anterior, providing a space into which the head can evert {Robertson, 1936 #76}. These contraction events have long been thought to generate enough hydrostatic pressure to elongate the legs and wings in the animal. Detailed observation of the ßFTZ-F1 mutants in Tina’s study revealed defects in each of these developmental processes. Furthermore, we have observed that, in ßFTZ-F1 mutants, the muscle contractions that drive these events are much less deliberate, vigorous, and consistent than in controls. We have shown that gas bubble translocation, leg and wing elongation, and head eversion can be rescued by exposing mutant prepupae to decreased external pressure. This indicates that these defects result from failure to generate sufficient internal pressure at the appropriate time. This also provides direct evidence that hydrostatic pressure does, in fact drive the major extensions of legs and wings at the prepupal-pupal transition. We have done experiments demonstrating that a drop in pressure can rescue leg elongation, wing elongation, and gas bubble translocation in some ßFTZ-F1 mutants. This indicates that ßFTZ-F1 is required for the muscle contractions that drive major morphogenetic events at the prepupal-pupal transition. These results are consistent with our model for the role of ßFTZF1 as a competence factor necessary for genetic and developmental response to ecdysone that occur at the prepupal-pupal transition. 1.2 Tissue-specific control of target gene transcription and programmed cell death by ßFTZ-F1 1.2.1 The larval salivary gland In collaboration with Dr. Eric Baehrecke’s group at the University of Maryland Biotechnology Institute, we have examined the roles of ßFTZ-F1 and its target genes in directing programmed cell death in the larval salivary gland. We have published the findings from these studies in Developmental Biology {Lee, 2002 #546} (see below). We have analyzed the function of the steroid-regulated genes ßFTZ-F1, BR-C, E74A, and E93 during salivary gland programmed cell death. While mutations in the ßFTZ-F1, BR-C, E74A, and E93 genes prevent destruction of salivary glands, only ßFTZ-F1 is required for DNA fragmentation. Analyses of BR-C, E74A and E93 loss-of-function mutants indicate that these genes regulate stage-specific transcription of the rpr, hid, ark, dronc, and crq cell death genes. Ectopic expression of ßFTZ-F1 is sufficient to trigger premature cell death of larval salivary glands, and ectopic transcription of the rpr, dronc and crq cell death genes that normally precede salivary gland cell death. The E93 gene is necessary for ectopic salivary gland cell destruction, and ectopic rpr, dronc and crq transcription that is induced by expression of ßFTZ-F1. Together, these observations indicate that ßFTZ-F1 regulates the timing of hormone-induced cell responses, while E93 functions to specify programmed cell death. 1.2.2 Effects of ßFTZ-F1 gain-of-function on target gene transcription in other tissues We are continuing our analysis of the tissue-specificity of ßFTZ-F1 function. Using transgenic 3rd instar larvae that contain ßFTZ-F1 coding sequences under the control of a heatindicible promoter (w; P[F-F1] )we have been examining the effects of ectopic expression of ßFTZ-F1 on transcription of early genes in four late third instar larval tissues - gut, fat, imaginal discs, and CNS (as a control, we are also repeating the salivary gland experiment). Undergraduates Shahana Baig, Gitanjali Chimalakonda and Zareen Gauhar have examined the effects of ectopic expression of ßFTZ-F1 on the transcription of the early genes E93, E74A and E75A in fat tissue. We have found that E93, E74A and E75A expression are greatly enhanced by ecdysone in the presence of ectopic ßFTZ-F1 in cultured mid third instar larval fat. ßFTZF1 appears to regulate the ecdysone responses of these early genes in both salivary gland and fat tissue. 1.2.3 Effects of ßFTZ-F1 loss-of-function on target gene transcription We are continuing to examine the transcription of ßFTZ-F1 target genes in specific tissues (salivary gland, CNS, fat, gut and imaginal discs) in ßFTZ-F1 mutant, and control 0-14hour prepupae. Undergraduate students Samara Brown, Kimberly Bucknor, Paejonette Jacobs, and Kathryn McMenimen have done northern blot hybridization analyses to examine expression of the early genes in specific tissues from staged mutant and control animals. We have determined that, in very early prepupae, levels of E75A mRNAs are normal in ßFTZ-F1 mutant gut tissue. In contrast, levels of E75A mRNAs are greatly reduced in ßFTZ-F1 mutant gut tissue from late prepupae. This supports our model, which predicts that ßFTZ-F1 functions to control early gene response to ecdysone only in late prepupae. In addition, levels of E75A mRNAs are greatly reduced in ßFTZ-F1 mutant salivary glands from late prepupae (E75A is not expressed in the salivary glands of early prepupae). Kathryn’s findings indicate that the response of E75A to ecdysone in late prepupal salivary gland and gut is dependent on ßFTZ-F1. This is in contrast to the situation with E93. We have found in late prepupae, the ecdysone reponse of E93 is dependent on ßFTZ-F1 in salivary gland, but not in gut. 1.3 Effects of EcR loss-of-function on target gene transcription We have obtained two EcR loss-of-function mutant alleles from Dr. Michael Bender at the University of Georgia. A necessary step in examining the effects of these mutations on metamorphosis is balancing them over 2nd chromosomes with a marker that can be detected in larvae. A small team of undergraduate students, including Vidyamargaret Anegundi, Myriah Deal, Paejonette Jacobs, and Rhiana Menen, has performed the necessary crosses to establish the stocks we need for this study. 1.4 What is the molecular mechanism underlying stage-specific regulation of E93 by ecdysone and ßFTZ-F1? Undergraduates Divya Mathur, Yashaswi Shrestha, and Beatrice Tapawan have been performing gel shift assays to identify the fragments of the E93 gene to which ßFTZ-F1 protein binds. In an effort to improve our yields of ßFTZ-F1 protein synthesized in vitro, Divya and Beatrice have cloned the ßFTZ-F1 coding sequences into a bacterial expression vector. Divya presented her findings in a poster at an international meeting in March, 2003. 1.5 Publications Resulting from Prior NSF Support Fortier, T.M., *Vasa, P.P., and Woodard, C.T. (2003). Orphan Nuclear Receptor ßFTZ-F1 is Required for Muscle-Driven Morphogenetic Events at the Prepupal-Pupal Transition in Drosophila melanogaster. Developmental Biology 257: 153-165 Lee C., Simon C.R., Woodard, C.T. and Baehrecke E.H. (2002). Genetic Mechanism for the Stage- and Tissue-Specific Regulation of Steroid Triggered Programmed Cell Death in Drosophila. Developmental Biology 252: 138-148 Broadus, J., *McCabe, J., Endrizzi, B., Thummel, C.S., and Woodard, C.T. (1999). The Drosophila ßFTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone. Molecular Cell 3: 143-149 *Mount Holyoke College undergraduate author 2. Specific Aims In Drosophila, fluctuations in 20-hydroxyecdysone (ecdysone) titer coordinate gene expression, cell death, and morphogenesis during metamorphosis. Our previous studies have supported the hypothesis that ßFTZ-F1 (an orphan nuclear receptor) provides specific genes with the competence to be induced by ecdysone at the appropriate time, thus directing key developmental events at the prepupal-pupal transition. We have made a detailed study of morphogenetic events during metamorphosis in control and ßFTZ-F1 mutant animals (see previous section). In this project, we will continue examining the role of ßFTZ-F1 in directing morphogenesis during metamorphosis in Drosophila, testing several hypotheses explaining the morphogenetic defects seen in ßFTZ-F1 mutants. ßFTZ-F1 appears to be a high-level regulator that acts to control the ecdysone responses of other genes that, in turn, must play more direct roles in driving morphogenesis. Several ßFTZ-F1 target genes have been identified, and we will examine the roles of these genes in the morphogenetic events that occur during the prepupal-pupal transition. Futhermore, we will examine other potential interacting genes, and attempt to identify all genes that are regulated by ßFTZ-F1 during the prepupal-pupal transition. 2.1 Specific Aim 1. Determine the spatial expression pattern of ßFTZ-F1 protein during metamophosis Yamada et al. (2000) {Yamada, 2000 #556} state that ßFTZ-F1 is widely expressed in mid-prepupae. More precise characterization of this expression pattern will be helpful in understanding the function of ßFTZ-F1 in morphogenesis. We therefore plan to perform a careful and comprehensive analysis of the spatial patterns of ßFTZ-F1 protein expression at precise times throughout the prepupal and early pupal periods. 2.2 Specific Aim 2. Test hypotheses explaining the morphogenetic defects seen in ßFTZF1 mutants We have several hypotheses that attempt to explain the morphogenetic defects seen in ßFTZ-F1 mutants during metamorphosis. These include the following: 1) The defects result from premature destruction of larval muscles that drive the morphogenetic processes of the prepupal-pupal transition. 2) The defects result from defects in the innervation of larval muscles that drive the morphogenetic processes of the prepupal-pupal transition. 3) The defects result from inappropriate pupal cuticle properties at key developmental times. 2.2.1 Examine the muscles in ßFTZ-F1 mutants and controls The observed ßFTZ-F1 mutant phenotypes could result from premature destruction of larval muscles that drive the morphogenetic processes of the prepupal-pupal transition. To test this hypothesis, we will do microscopic studies comparing stained and sectioned muscular tissue from control and ßFTZ-F1 mutant animals aged 12-24???? hours APF (After Puparium Formation). We will look for indications that the muscles in the ßFTZ-F1 mutants degenerate before they can to drive the morphogenetic events of the prepupal-pupal transition. 2.2.2 Examine innervation of the muscles in ßFTZ-F1 mutants and controls The observed ßFTZ-F1 mutant phenotypes could result from defects in the innervation of larval muscles that drive the morphogenetic processes of the prepupal-pupal transition. To test this hypothesis, we will do a careful examination of the neurons that innervate these muscles in ßFTZ-F1 mutants and controls. 2.2.3 Measure pupal cuticle stiffness in ßFTZ-F1 mutants and controls throughout metamorphosis We will assay the stiffness of the pupal cuticle throughout metamorphosis throughout metamorphosis in control and ßFTZ-F1 mutant animals. By doing this, we will be able to determine when, and to what degree, cuticle stiffness contributes to the developmental defects seen in ßFTZ-F1 mutants. 2.3.4 To determine if the ultrastructure of the pupal cuticle is abnormal in ßFTZ-F1 mutants, we will use TEM to examine pupal cuticle in staged ßFTZ-F1 mutant and control prepupae and pupae. 2.3 Specific Aim 3. Examine genes for interaction with ßFTZ-F1 in morphogenesis 2.3.1 Examine the roles of known ßFTZ-F1 target genes in morphogenesis ßFTZ-F1 has been shown to regulate the expression of several genes during the lateprepupal stage, including BR-C, E74A, E75A, and E93 {Broadus, 1999 #93}, and the defects seen during pupation in the ßFTZ-F1 mutant are possibly due to the reduced expression of one or more of these genes. We will examine the relationships between ßFTZ-F1 and specific target genes in controlling morphogenesis. We will: 2.3.1.1 test for genetic interactions between ßFTZ-F1, and E74 and BR-C. 2.3.1.2 attempt to rescue ßFTZ-F1 mutant phenotypes by ectopic expression of transgenes encoding E74A and E75A proteins. 2.3.2 determine the role of the gene, who, in morphogenesis and assay potential interaction between who and ßFTZ-F1 during the prepupal-pupal transition Recent observations made by our colleague, Dr. Eric Baehrecke of the University of Maryland Biotechnology Institute, have identified another potential target gene of ßFTZ-F1. In collaboration with Dr. Baehrecke, we are analyzing the role of the who gene in leg development. who loss-of-function mutants display defects in morphogenetic events that occur during metamorphosis. We will carry out experiments aimed at determining the role of who in morphogenetic events that occur during metamorphosis. We will: 2.3.2.1 examine cell shape changes in the leg imaginal discs of who mutant early prepupae. 2.3.2.2 analyze leg development in who mutants by making movies of these animals expressing GFP in their legs.. 2.3.2.3 examine Who protein expression patterns in staged animals (control, who mutant, and ßFTZ-F1 mutant. 2.3.3 Determine the entire set of genes that are regulated by ßFTZ-F1 during the prepupalpupal transition In collaboration with Dr. Kevin White at Yale University, we will continue our microarray analyses in an attempt to identify all genes regulated by ßFTZ-F1 during metamorphosis. 3. Background and Significance The development of animal body structures requires precise coordination of many complex processes. This coordination is often achieved by hormonal signaling. For example, the construction of a leg in the fruit fly, Drosophila melanogaster, involves the orchestration of numerous morphogenetic events by the single steroid hormone, 20-hydroxyecdysone (referred to here as ecdysone). Drosophila leg development thus provides an ideal model system in which to examine the control of specific developmental processes by steroid hormones. In directing leg formation and other developmental processes that occur during Drosophila metamorphosis, ecdysone acts in cooperation with a number of factors, including the orphan nuclear receptor ßFTZ-F1 {Woodard, 1994 #53;Broadus, 1999 #93}. We are examining the role of ßFTZ-F1 and its target genes in directing leg development and other morphogenetic processes that occur in response to ecdysone during metamorphosis in Drosophila. 3.1 Steroid hormones regulate numerous biological responses Steroid hormones regulate the metabolism, reproduction, and development of higher eukaryotes {Gorbman, 1983 #264}. The regulation of these diverse biological phenomena has been conserved in organisms that are as different as insects and humans, thus illustrating the importance of steroids within metazoa. Steroids are synthesized in neuroendocrine cells and secreted into the circulatory system, which then distributes these long distance signaling molecules to a wide variety of target tissues throughout the body. The small hydrophobic chemical structure of steroids enables them to pass through the plasma membrane of the cell. Once in the target cell, the steroid interacts with a receptor protein that binds to DNA, activating gene expression. Steroid-mediated maintenance of homeostasis and regulation of development is very complex, indicating a requirement for both cell- and tissue-specific responses to these hormones. Although steroid hormones play a key role in coordinating the growth and development of higher eukaryotes, we are still trying to elucidate the molecular mechanisms by which steroids direct specific biological responses. 3.2 Drosophila is an ideal system for studies of steroid hormone action Although steroid hormones play a key role in coordinating the growth and development of higher eukaryotes, little is known about the molecular mechanisms whereby they direct different developmental pathways. The fruit fly, Drosophila melanogaster provides an ideal model system for unraveling the molecular mechanisms of steroid hormone action in the context of an intact animal. Pulses of ecdysone trigger the major postembryonic developmental transitions during the Drosophila life cycle {Richards, 1981 #40; Riddiford, 1993 #74}. The most dramatic of these events is the metamorphosis from the larval form to the adult fly. A high titer pulse of ecdysone in late third instar larvae triggers puparium formation, defining the beginning of prepupal development. This is followed, 10-12 hours later, by a subsequent ecdysone pulse that triggers head eversion, marking the prepupal-pupal transition {Richards, 1981 #40; Handler, 1982 #7; Sliter, 1992 #55; Riddiford, 1993 #74}. The entire body plan is reformed during metamorphosis. Most larval tissues are destroyed and replaced by adult tissues that develop from clusters of pre-determined adult progenitor cells [Robertson, 1936 #76; Bodenstein, 1965 #75]. These complex developmental events are initiated by dynamic changes in ecdysone concentration and can, in many cases, be reproduced and studied in cultured organs treated with the proper hormone regime {Boyd, 1977 #77; Martin, 1978 #78; Fristrom, 1982 #47}. Insights into the molecular mechanisms by which ecdysone exerts its effects on development have come from observation of the puffing patterns of the salivary gland giant polytene chromosomes. The puffs thus provide a means of visualizing the effects of ecdysone on gene expression. Based studies of polytene chromosome puffs, Ashburner et al. [Ashburner, 1974 #27] proposed that ecdysone directly induces a small set of early regulatory proteins that subsequently repress their own expression and induce the large set of late genes. These late genes, in turn, are thought to play more direct roles in metamorphosis. The isolation and characterization of the BR-C, E74, and E75 early genes that reside within the 2B5, 74EF, and 75B early puffs, respectively, has provided significant support for the Ashburner model [Burtis, 1990 #20; Segraves, 1990 #19; DiBello, 1991 #21]. In addition, the ecdysone receptor has been characterized at the molecular level, and is known to be a heterodimer between the EcR and USP proteins [Koelle, 1991 #36; Koelle, 1992 #37; Yao, 1992 #45]. The co-crystal structure of the heterodimeric ecdysone receptor DNA-binding complex has been determined {Devarakonda, 2003 #569}. 3.3 Drosophila exhibits diverse stage- and tissue-specific responses to steroid hormones Changes in chromosomal puffing continue through the prepupal period until the larval salivary glands are destroyed by programmed cell death in early pupae [Becker, 1959 #5; Ashburner, 1967 #3]. The mid prepupal puffs, represented by a puff at 75CD, appear as the ecdysone titer drops 3-8 hours after pupariation [Richards, 1976 #13; Richards, 1976 #15]. Richards [Richards, 1976 #15] showed that formation of the 75CD puff in cultured larval salivary glands requires the addition of a high concentration of ecdysone followed by ecdysone withdrawal, mimicking the changes in hormone titer that occur at puparium formation. Addition of ecdysone to mid prepupal salivary glands results in the rapid regression of the 75CD puff, indicating that this puff is repressed by the hormone and suggesting that its activity during the mid prepupal interval is dictated by the drop in ecdysone titer at this time. As the ecdysone titer increases in late prepupae, the mid prepupal puffs regress and the early puffs (including 2B5, 74EF, and 75B) are reinduced. In addition, a few stagespecific early puffs, typified by 93F, which contains the prepupal stage-specific E93 gene [Baehrecke, 1995 #54], are induced directly by ecdysone in late prepupae but display no response to the hormone in late larvae. Interestingly, Richards showed that the early puffs (including the prepupal stage-specific 93F puff) cannot be induced by ecdysone in early prepupal salivary glands. In fact, a preceding period of low ecdysone concentration and protein synthesis is required before these puffs become competent to respond to the hormone [Richards, 1976 #13; Richards, 1976 #18]. The simplest interpretation of Richards' observations is that one or more proteins encoded by the mid prepupal puff genes provide the competence for the early puffs to be induced by the prepupal ecdysone pulse. 3.4 ßFTZ-F1 plays a key role in directing the morphogenetic responses of the pepupal-pupal transition ßFTZ-F1 is one of two protein isoforms encoded by FTZ-F1, which maps on to the 75CD mid prepupal puff locus. ßFTZ-F1 is expressed at the larval molts and briefly during the mid prepupal period {Lavorgna, 1993 #10;Woodard, 1994 #53;Yamada, 2000 #556}. A growing body of evidence strongly suggests that ßFTZ-F1 acts as a competence factor for ecdysone-induction of early genes. Ectopic expression of ßFTZ-F1 from a transgene enhances the ecdysone-induction of the early genes BR-C, E74A and E75A, and enables the premature induction of E93 by ecdysone in cultured third instar larval salivary glands {Woodard, 1994 #53}. A loss-of-function mutation in ßFTZ-F1 results in pupal lethality, with defects in the expression of BR-C, E74A, E75A and E93 transcripts in the late prepupal stage {Broadus, 1999 #93}. ßFTZ-F1 mutants also exhibit defects in destruction of the larval salivary glands by programmed cell death {Lee, 2001 #557;Lee, 2002 #563}, and defects in head eversion wing elongation, and leg elongation. These mutants possess legs that are properly segmented but shorter than normal {Broadus, 1999 #93}. ßFTZ-F1 mutant legs have a malformed appearance very similar to that seen in mutants such as Sb, BR-C, and sqh (Kiss et al., 1988; von Kalm et al., 1995; Edwards et al., 1996). The adult legs and wings develop from imaginal discs, which are epithelial sacs that are formed during embryogenesis and give rise to parts of the adult epidermis and cuticle. During larval development, the cells of the imaginal discs multiply and acquire their proper fates and positions {Cohen, 1993 #547}. Formation of the mature adult leg begins at the end of larval stages, when a high-titer pulse of ecdysone triggers pupariation (puparium formation), which marks the beginning of the prepupal period and the onset of metamorphosis. This late larval ecdysone pulse directs the process of leg disc evagination, which transports the leg from the inside to the outside of the body, and includes two different morphological events: elongation and shaping of the appendage, and eversion of the appendage to the outside of the animal. During elongation, from approximately 6 hours before pupariation until 6 hours after puparium formation (APF), morphogenesis of the leg disc begins with the telescoping of the flat disc into a long tubular limb. Increase in length is partly accomplished by the unfolding of the epithelium and a selective change in cell shape from columnar to cuboidal, which increases the surface area of the disc {Fristrom, 1993 #255}. Prior to metamorphosis, cells in the dorsal-lateral regions of the leg discs are compressed in the proximal-distal axis and are elongated circumferentially {Condic, 1991 #545}. During appendage elongation these highly asymmetric cells become isometric, resulting in leg elongation in the proximal-distal axis. This change in cell shape is thought to account for most of the elongation in the basitarsal and tibial leg segments between 0 and 6 hours APF {Condic, 1991 #545}. The elaborate changes in cell shape observed in imaginal epithelia during this six hour period occur in response to ecdysone. Elongation requires changes in cell-cell adhesions and transmission of signals from the cell surface to specific cytoskeletal elements. These cell shape changes result from myosin-based shaping of the actin cytoskeleton, and contraction of the cortical belt, in a response that involves the Rho signaling pathway {Edwards, 1996 #548;Halsell, 2000 #549}. At approximately 10-12 hours APF, the prepupal pulse of ecdysone occurs, inducing the transition from prepupa to pupa. According to Chadfield and Sparrow (1985), the first visible event in this prepupal-pupal transition is the expulsion of the gas bubble from inside the animal into the posterior end of the puparium, where a local separation of the pupal cuticle from the puparium occurs. The abdomen then contracts rhythmically, forcing the gas to the anterior of the puparium. The prepupa gradually moves posteriorly, making a space at the anterior end, beneath the operculum. Contraction of larval abdominal muscles is thought to then cause head eversion as well as the inflation and final elongation of the wings and legs and {Handler, 1982 #7;Chadfield, 1985 #546}. The hydrostatic pressure thought to be responsible for head eversion, leg extension, and wing extension also causes the highly folded apical epithelium of the leg imaginal discs to stretch and flatten, while segment boundaries are temporarily obliterated. 4. Preliminary Studies 4.1 Specific Aim 2. Determine the spatial expression pattern of ßFTZ-F1 protein during metamorphosis 4.1.1 Examine the muscles in ßFTZ-F1 mutants The failures exhibited by ßFTZ-F1 mutants in the morphogenetic events of the prepupal-pupal transition result, at least in part, from failure of muscles to contract adequately and at the proper time. This failure could be caused by premature destruction of the larval muscles that drive these processes. To test this hypothesis, we have done light and transmission electron microscopic (TEM) studies comparing stained (with rhodamine phalloidin) muscle tissue from control and ßFTZ-F1 mutant animals aged 12 hours APF (After Puparium Formation), which is when the prepupal-pupal transition normally occurs in control animals. In this study (and throughout this proposal unless indicated otherwise), “FTZ-F1 mutants” will be animals of the genotype FTZ-F117/Df(3L)CatDH104 (as described in Broadus et al., 1999 {Broadus, 1999 #93}) animals. Figure 1 shows a representative light micrograph of a ßFTZ-F1 mutant, aged 12 hours APF. These analyses have so far detected no gross differences in muscle between ßFTZ-F1 mutants and controls. 4.1.2 Measure pupal cuticle stiffness in ßFTZ-F1 mutants and controls throughout metamorphosis Cuticle is a non-living, layered structure that serves as an exoskeleton to protect and support the structures within, and can be divided into the epicuticle and procuticle. Formation of the pupal cuticle begins in the prepupa as early as 2 hours APF and is completed by 14 hours APF. The secretion of cuticulin starts at 3-hours APF in the form of disconnected plates over individual cells that fuse to cover the entire surface of the animal by 6-7 hours APF. This strategy allows the discs to continue to grow and change conformation before the rigidity of cuticle prevents further morphogenesis {Fristrom, 1993 #255}. Leg morphogenesis requires appendage elongation to occur while the epidermis is free from overlying cuticle. Leg elongation takes place at two stages: before pupal cuticle deposition and between the apolysis of this cuticle and deposition of the adult cuticle {Fristrom, 1993 #255}. Therefore, it is possible that in wild-type, the pupal cuticle is relatively elastic during the leg and wing extension events of the prepupal-pupal transition, but changes to become stiffer very soon thereafter. We have shown that in the mutants, contraction of the larval muscles is delayed by several hours compared to controls. Perhaps by the time the contraction occurs, the pupal cuticle has already changed, and is too stiff to allow complete leg and wing extension. This may explain why the mutant legs appear bent at their ends. Perhaps they try to extend, but come up against the stiff cuticle and cannot elongate any more in a straight line. A related but slightly different hypothesis is that there is something inherently wrong with the pupal cuticle in the mutants, such that it is too stiff throughout metamorphosis. Consistent with the second hypothesis, premature expression of ßFTZ-F1 results in disruption of the larval cuticle{Yamada, 2000 #556}. In addition, the expression of EDG78E and EDG84A, genes that encode pupal cuticle proteins {Fechtel, 1988 #236}, is reduced and delayed in ßFTZ-F1 mutants {Broadus, 1999 #93; Murata, 1996 #561}. If these proteins are not expressed properly, the cuticle may be abnormally stiff at the end of the prepupal stage. Thus, a greater force of contraction would be required to elongate the legs and wings to their normal length during the prepupal-pupal transition. Preliminary findings we have made as part of the proposed project also support this hypothesis. Our preliminary microarray analyses have indicated that several genes involved in cuticle synthesis are misregulated in ßFTZ-F1 mutant prepupae (See section 4.2.3). In order to test these hypotheses, we have begun to analyze the stiffness of the pupal cuticle in staged control and ßFTZ-F1 mutant prepupae. With the help of our Colleague, Diane Kelly, of the University of Massachusetts, we built a "minitensometer", a scaled-down version of a basic tensile testing machine (including force and displacement transducers). The core of the minitensometer is a dovetail slider; an aluminium plate bolted to one end for mounting the force transducer and the linear variable differential transducer (LVDT, which measures displacement) and a grip carriage bolted to the slider to hold one grip and the LVDT core rod. In these experiments, we remove the puparium from a weighed and measured test animal, and glue separate silk threads to the anterior and posterior ends of the pupal cuticle. After calibrating the force transducer and the LVDT, the specimen is inserted in the apparatus. Force is then applied to the pupal cuticle via the silk threads, and displacement (stretching) is measured until the pupal cuticle breaks. The stiffer the cuticle, the less displacement will occur before breakage. In pilot experiments, we have succeeded in collecting data to generate stress.strain curves for the pupal cuticle from control animals aged 12-16 hours APF. We are confident this technique will be an effective means of examining the role of the pupal cuticle stiffness in ßFTZ-F1 mutant phenotypes. 4.2 Specific Aim 3. Examine genes for interaction with ßFTZ-F1 in morphogenesis 4.2.1. Determine the role of the gene, who in morphogenesis and assay potential interaction between who and ßFTZ-F1 during the prepupal-pupal transition We are analyzing the role of the who (wings held out) gene, also named how, struthio, and qkr93F, in the morphogenetic events of metamorphosis. who encodes a KH RNA binding protein that functions in muscle and wing development in Drosophila (Baehrecke, 1997; Volk, 1999). Animals that lack who function have defects in muscle attachment during embryogenesis (Volk, 1999). who mutants also display defects in metamorphosis. We have begun to utilize combinations of hypomorphic alleles to analyze who function during metamorphosis of Drosophila. In order to determine if who has pleiotropic function during metamorphosis, we have been taking several approaches. We are carefully analyzing gross phenotypes of the mutant animals during metamorphosis. The legs of the transheterozygote whoe44/whor17 are short and often bent. This phenotype is similar to that seen in E74 mutants (Fletcher et al., 1995), and ßFTZ-F1 mutants {Broadus, 1999 #93; Fortier, 2003 #566}. Using northern blot hybridization, we have detected who transcript in imaginal discs, gut, fat bodies and salivary glands at the 0-hour prepupal stage and reduced levels in the gut and salivary glands at 12 hour prepupal stage. These findings suggest that who may interact with ßFTZ-F1 in the control of leg morphogenesis during metamorphosis. We therefore propose several lines of experimentation to test this hypothesis, and to elucidate the role of who in leg morphogenesis. Our Colleague, Laurie von Kalm, from the University of Central Florida, is currently doing experiments to test for genetic interaction between ßFTZ-F1 and who. 4.2.2 Analyze leg development in who mutants by making movies of these animals expressing GFP in their legs In order to determine exactly when the defect in leg development occurs in who mutants, we are taking an approach similar to that used to examine leg development in ßFTZF1 mutants {Fortier, 2003 #566}. We have constructed control genotype and who mutant animals expressing GFP in their developing leg imaginal discs and legs, as described {Ward, 2003 #565}. Figure 2 shows still images of a who mutant and a control, both at the 50-hour prepupal stage, imaged side-by-side using a BioRad MRC 600 laser scanning confocal microscope (Nikon inverted Diaphot base). 4.2.3 Determine the entire set of genes that are regulated by ßFTZ-F1 during the prepupalpupal transition In collaboration with Dr. Kevin White at Yale University, we are doing microarray analyses in an attempt to identify all genes regulated by ßFTZ-F1 during metamorphosis. More specifically, we are examining the effects of ßFTZ-F1 loss-of-function on genome-wide transcription in late prepupae. We are also examining the effects of premature, ectopic FTZ-F1 expression on genome-wide transcription in late 3rd instar larvae. For the loss-of-function studies, we have isolated total RNA from ßFTZ-F1 mutants (FTZ-F117/Df(3L)CatDH104) and control (+/ Df(3L)CatDH104), as well as reference (w1118) animals aged either 8, 10 or 12 hours APF. In Kevin White’s lab, the RNA is amplified and labeled. RNA from mutant and control genotypes is labeled differently from reference RNA. The RNA from each ßFTZ-F1 mutant group is hybridized to DNA micorarrays representing nearly every Drosophila melanogaster protein-coding gene (made in Kevin’s lab) together with RNA from the appropriate reference group. Likewise, RNA from each control group is hybridized to DNA micorarrays together with RNA from the appropriate reference group. Microarray measurements are made using GenePix Pro microarray analysis software (Axon Instruments). This analysis compares the levels of expression of each gene in the genome in ßFTZ-F1 mutant vs. reference, and in control vs. reference. Each experiment is done in triplicate. We are interested in genes that are either over- or under-expressed in ßFTZ-F1 mutants, but expressed at normal levels (levels equal to those in the reference group) in the control group. To examine the effects of premature, ectopic FTZ-F1 expression on genome-wide transcription in late 3rd instar larvae, we are using the animals of the genotype w; P[F-F1], which express ßFTZ-F1 under the control of a heat-inducible promoter {Woodard, 1994 #53}, as well as reference animals of the genotype w1118. We have isolated total RNA from late third instars given a heat shock at 35˚C for 30 minutes, then aged to 0 hours APF. The results of our very preliminary loss-of-function analyses indicate that genes involved in cuticle formation and metabolism are underexpressed in ßFTZ-F1 mutant late prepupae. These include ebony, structural protein of cuticle, chitinase, and structural protein of larval cuticle. These findings are intriguing, since they are consistent with our hypothesis that abnormalities in the pupal cuticle cause the morphogenetic defects seen in ßFTZ-F1 mutants (See section 2.2.3). 5. Research Methods 5.1 Specific Aim 1. Determine the spatial expression pattern of ßFTZ-F1 protein during metamophosis We will perform a careful and comprehensive analysis of ßFTZ-F1 protein spatial expression patterns at precise times throughout the prepupal and early pupal periods. Robert Beckstead, from Carl Thummel’s lab at the University of Utah, has generated high-quality preparations of antibodies specific to ßFTZ-F1. He has sent us several of these antibody preparations, and we will use them to localize ßFTZ-F1 protein within prepupae and pupae. We will collect wild-type control animals as 0-hour prepupae, and age them to 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 hours APF. We will open up the animals to release their internal organs, as described in {Patel, 1994 #400}. To visualize musculature, we will make ventral mid-line incision, and pin the animal open using a dissection chamber that we have constructed (modified from a chamber designed in Vivian Budnick’s lab at the University of Massachusetts). The dissected tissues will be fixed in paraformaldehyde and treated with anti-ßFTZ-F1 antibodies. These will be treated with HRP-tagged secondary antibodies. The preparations will be treated with hydrogen peroxide and DAB and visualized using a fluorescence compound microscope. A negative control treated with secondary antibody only will be prepared for each time stage. 5.2 Specific Aim 2. Test hypotheses explaining the morphogenetic defects seen in ßFTZF1 mutants 5.2.1 Examine the muscles in ßFTZ-F1 mutants We will continue doing microscopic studies comparing stained muscular tissue from control and ßFTZ-F1 mutant animals. As described in section 4.1.2, we have detected no differences between muscle tissues from control and ßFTZ-F1 mutant animals aged 12 hours APF, which is when the prepupal-pupal transition normally occurs in control animals. We have determined, however, that the morphogenetic events of the prepupal-pupal transition are delayed by several hours in ßFTZ-F1 mutants. It is therefore possible that the muscles of these mutants do degenerate before they can properly drive head eversion, and leg and wing elongation. To test this hypothesis, we will do a careful and comprehensive examination of larval muscles in staged ßFTZ-F1 mutants and controls, including animals older than 12 hours APF. We will collect ßFTZ-F1 mutant and control animals as 0-hour prepupae, and age them to 12, 14, 16, 18, 20, and 22 hours APF. We will visualize musculature as described in section 4.1.2. If we see anything intriguing in mutant muscles from prepupae/pupae of a certain age, we will do a closer examination using transmission electron microscopy (TEM). Control and mutant animals of the appropriate age will be fixed, sectioned, stained and imaged by TEM following standard methods {McDonald, 1994 #568}. As another approach to viewing muscles in ßFTZ-F1 mutants and controls, we will generate animals expressing GFP specifically in muscles. To do this, we have obtained the stock e22C/SM5, which expresses GAL-4 under the control of a muscle specific promoter. We will generate flies carrying Df(3L)CatDH104 on their 3rd chromosome, and e22C on their 2nd chromosome. We will cross these to our laboratory stock flies that carry, on their 2nd chromosome, the GFP gene under the control of a GAL-4 promoter. Crossing these two genotypes together will yield FTZ-F117/Df(3L)CatDH104 animals expressing GFP in their muscles. We will also generate control flies expressing GFP in their muscles. We will stage these animals at 12, 14, 16, 18, 20, and 22 hours APF, and view them with a confocal microscope. 5.2.2 Examine innervation of the muscles in ßFTZ-F1 mutants and controls The observed ßFTZ-F1 mutant phenotypes could result from defects in the innervation of larval muscles that drive the morphogenetic processes of the prepupal-pupal transition. To test this hypothesis, we will do a careful examination of the neurons that innervate these muscles in ßFTZ-F1 mutants and controls. We will collect ßFTZ-F1 mutant and control animals as 0-hour prepupae, and age them to 12, 14, 16, 18, 20, and 22 hours APF. To visualize motor neurons, we will use our dissection chamber to make a ventral midincision. The dissected tissues will be fixed in paraformaldehyde and treated with antiFasciclin II antibodies (obtained from the Developmental Studies Hybridoma Bank at the University of Iowa), which are specific to axons. These will then be treated with HRPtagged secondary antibodies. The preparations will be treated with hydrogen peroxide and DAB and visualized using a fluorescence compound microscope. A negative control treated with secondary antibody only will be prepared for each time stage. 5.2.3 Measure pupal cuticle stiffness in ßFTZ-F1 mutants and controls throughout metamorphosis We will continue our experiments testing the hypotheses regarding the role of pupal cuticle stiffness in the morphogenetic defects seen in ßFTZ-F1 mutants. We will use our minitensiometer to generate stress/strain curves of pupal cuticle from these animals. We will collect control and ßFTZ-F1 mutant 0 ours prepupae, and age them to 4, 6, 10, 12, 14, 16, 17, 18, and 20 hours APF, and assay the stiffness of their pupal cuticles. 5.2.4 Examine the ultrastructure of the pupal cuticle in ßFTZ-F1 mutants In order to determine if the ultrastructure of the pupal cuticle is abnormal in ßFTZ-F1 mutants, we will use TEM to examine pupal cuticle in staged ßFTZ-F1 mutant and control prepupae and pupae. We will collect control and ßFTZ-F1 mutant 0-hour prepupae, and age them to 4, 6, 10, 12, 14, 16, 17, 18, and 20 hours APF. These animals will be fixed, sectioned, stained and imaged by TEM following standard methods {McDonald, 1994 #568}. We will look for abnormalities in the pupal cuticle of ßFTZ-F1 mutants. 5.3 Specific Aim 3. Examine genes for interaction with ßFTZ-F1 in morphogenesis 5.2.1 Examine the roles of known ßFTZ-F1 target genes in morphogenesis ßFTZ-F1 has been shown to regulate the expression of several genes during the lateprepupal stage, including BR-C, E74A, E75A, and E93 {Broadus, 1999 #93}, and the defects seen during pupation in the ßFTZ-F1 mutant are possibly due to the reduced expression of one or more of these genes. We will examine the relationships between ßFTZ-F1 and these target genes in controlling morphogenesis. These studies will include: 5.2.1a Tests for genetic interactions between ßFTZ-F1, and E74 and BR-C. Certain E74 and BR-C mutant alleles result in prepupal or pupal lethality, and morphogenetic defects similar to those seen in ßFTZ-F1 mutants. These defects include failures in head eversion and leg elongation {Fletcher, 1995 #108; Fletcher, 1995 #107}. In addition, animals heterozygous for both recessive E74 and recessive BR-C mutant alleles (BR-C/+; E74/+) show similar metamorphic defects {Fletcher, 1995 #107}. To test the hypothesis that the morphogenetic defects seen in ßFTZ-F1 mutants result from improper regulation of E74 and/or BR-C, we will generate animals carrying various combinations of FTZ-F1 mutant alleles, and E74 or BR-C mutant alleles. FTZ-F119 is a complete loss-offunction allele that we generated in a screen for deletions in FTZ-F1 resulting from imprecise excision of a P-element {Broadus, 1999 #93}. FTZ-F119 is recessive, so FTZ-F119/+ animals develop normally. We will set up crosses to generate animals that are BR-C/+; FTZ-F119 /+, and animals that are E74 FTZ-F1+/E74+ FTZ-F1. The E74 mutant alleles that we will use include E74P[neo] and E74[Dl-1]. The BR-C alleles will include 2Bc2, rbp5, and br5 {Fletcher, 1995 #108; Fletcher, 1995 #107}. As controls we will use FTZ-F1/+, E74/+ and BR-C/+, as well as wild-type animals. We will do a careful analysis of each genotype in this study, determining lethal phases, and characterizing the defects seen in animals that die before eclosion. Lethality and morphological defects in the double mutants will indicate a genetic interaction between FTZ-F1 and E74 and/or BR-C, supporting the hypothesis that ßFTZ-F1 directs developmental events via its regulation of these target genes. 5.2.1b attempts to rescue ßFTZ-F1 mutant phenotypes by ectopic expression of transgenes encoding E74A and E75A proteins. If the morphogenetic defects in ßFTZ-F1 mutants result from failures in expression of the target genes E74A and/or E75A, then it may be possible to rescue the defects in ßFTZ-F1 mutants by ectopic expression of E74A or E75A from a transgene. We have obtained stocks carrying E74A or E75A coding sequences under the control of a heat-inducible promoter. The transgenes in these stocks are inserted in the second chromosome, and are homozygous viable. They are marked with w+, so they their presence can be detected in a w mutant background. In order to do this analysis, we will first need to generate flies of the genotypes, w; P[hs-E74]; Df(3L)CatDH104 /TM6B, Tb, and w; P[hs-E75]; Df(3L) CatDH104 /TM6B, Tb. These will then be crossed to flies of the genotype w; FTZ-F117/TM6B, Tb. We will collect the Tb+ progeny as O-hour prepupae, which will be of the genotype w; P[hs-E74]/+; FTZ-F117/ Df(3L)CatDH104, or w; P[hs-E75]/+; FTZ-F117/ Df(3L)CatDH104. These animals will be aged to 10 hours APF, then given a 30 minute heat shock at 37˚C to express the transgene (other than during this heat shock, the animals will be kept at 25˚C). As controls animals of identical genotype will be kept at 25˚C, with no heat shock. We will do a careful analysis of each group in this study, determining lethal phases, and characterizing the defects seen in animals that die before eclosion. Rescue of lethality and morphological defects by ectopic expression of E74 or E75 will support the hypothesis that If the morphogenetic defects in ßFTZ-F1 mutants result from failures in expression of these target genes. 5.2.2. Determine the role of the gene, who, in morphogenesis and assay potential interaction between who and ßFTZ-F1 during the prepupal-pupal transition 5.2.2a Examine cell shape changes in the leg imaginal discs of who mutant early prepupae. Our findings that who mutants display defects in leg development, and that who mRNA is expressed in the imaginal discs of 0 hour prepupae, suggests that who may function in the early stages of leg imaginal disc elongation. In wild-type, during initial elongation of the leg imaginal discs, from approximately 6 hours before pupariation until 6 hours APF, morphogenesis of the leg disc begins with the telescoping of the flat disc into a long tubular limb. Increase in length is partly accomplished by the unfolding of the epithelium and a selective change in cell shape {Fristrom, 1993 #255}. Ecdysone induces these changes in cell shape, which are thought to account for most of the elongation in the basitarsal and tibial leg segments between 0 hours and 6 hours APF {Condic, 1991 #545}. In order to test the hypothesis that the leg imaginal discs of who mutants fail to undergo these cell-shape changes, we will use fluorescein phalloidin (Alexa FluorTM 488 from Molecular Probes), an actin stain, to visualize leg imaginal disc cell outlines in studies that compared cell shape changes in 0 hour and 6 hour APF control and who mutant prepupae. We will do these experiments in the same way as those analyzing cell shape changes in ßFTZ-F1 mutants {Fortier, 2003 #566}. Briefly, we will collect 0 hour prepupae from who mutant (whoe44/whor17) and control (whoe4/+) genotypes and dissect them in sterile saline. We will select leg discs, then prepare and visualize them as described {Fortier, 2003 #566}. Using low magnification, we will examine overall morphology of control and who mutant 6 hour APF leg discs. At higher magnification, we will carefully examine each disc, paying particular attention to the shape of cells in the proximal segments. 5.2.2b Analyze leg development in who mutants by making movies of these animals expressing GFP in their legs In order to determine exactly when the defect in leg development occurs in the who mutants, we will examine leg development throughout the course of metamorphosis in living animals. We will continue the studies described in 4.2.2b. We will collect animals of the appropriate genotype as 0 hour prepupae and for a period of up to 51.5 hours at a constant temperature of 19˚C. Images will be captured at 6-minute intervals, and we ill convert these data into time-lapse movies. This approach should enable us to determine what goes wrong in leg development in who mutants, and when the problem occurs. 5.2.2c Examine Who protein expression patterns in staged animals (control, who mutant, and ßFTZ-F1 mutant. We will perform a careful and comprehensive analysis of Who protein spatial expression patterns at precise times throughout the prepupal and early pupal periods. Talila Volk, from, ,has kindly sent us anti-Who antibody preparations, and we will use them to localize Who protein within prepupae and pupae. We will collect wild-type control animals as 0-hour prepupae, and age them to 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 hours APF. We will prepare these specimens as described in section 5.1.1, using anti-Who primary antibodies in this case. 5.2.3 Determine the entire set of genes that are regulated by ßFTZ-F1 during the prepupalpupal transition In collaboration with Dr. Kevin White at Yale University, we will continue our microarray analyses in an attempt to identify all genes regulated by ßFTZ-F1 during metamorphosis. Students from our lab will travel to Kevin’s lab during the summer to amplify and label our RNAs, hybridize microarrays with the RNAs, and analyze the data. The strategy for the ßFTZ-F1 studies is described in section 4.2.3. For the ßFTZ-F1 premature expression studies, we will hybridize the RNA from each w; P[F-F1] group to DNA micorarrays together with differentially labeled RNA from the reference group. This analysis compares the levels of expression of each gene in the genome in 0-hour prepupae expressing ßFTZ-F1 prematurely vs. reference. We are interested in genes that are either over- or under-transcribed in animals expressing ßFTZ-F1 prematurely, but expressed at normal levels (levels equal to those in the reference group) in the control group.