ftz-f1

Transcriptional Regulation

FTZ-F1 represses its own transcription and is repressed by ecdysone, explaining its brief expression in mid-prepupae. (Woodard, 1994).

In the early stages of Drosophila metamorphosis Hr46/DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. DHR is shown to interact directly with the Ecdysone receptor. The mechanism of DHR3 repression may involve an interaction between the DHR3 and Ecdysone receptor ligand binding domains. DHR also induces ßFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The DNA binding domain of DHR3, and perhaps sequences NH2-terminal to it, are necessary for the activating function of DHR3. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming a complex with DHR3 on the ßFTZF1 promoter, thereby providing a timing mechanism for ßFTZF1 induction that is dependent on the disappearance of E75B. DHR3 has two high-affinity binding sites approximately 300 base pairs apart, that lie downstream of the transcription start site of ßFTZF1. DHR3 appears to bind as a monomer to these sites, since sequencing and footprinting analysis have uncovered single consensus DHR3 sites at each of these DNA sites. E75B fails to bind DNA in the absence of DHR3. Thus E75B acts like a co-repressor with DHR3, rather than as a competitor with DHR3 for DNA binding; the restricted temporal expression of E75B apparently acts as a precise timer for the onset of ßFTZF1 expression (White, 1997).

The Hr46/DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. Hr46 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Hr46 directly contributes to both of these regulatory responses. Hr46 protein binds to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory proteins, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Hr46 represses E74A, and to a lesser extent E74B, and it also represses BR-C, E75A, and E78B. Hr46 activates betaFTZ-F1. Three Hr46 binding sites are present downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of Hr46 regulation. Ectopic expression of Hr46 reveals that the polytene chromosome binding pattern is of functional significance. Hr46 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. Hr46 thus appears to function as a switch that defines the larval-prepupal transition by arresting the early regulatory response to ecdysone at puparium formation and facilitating the induction of the betaFTZ-F1 competence factor in mid-prepupae. This study also provides evidence for direct cross-regulation among orphan members of the nuclear receptor superfamily and further implicates these genes as critical transducers of the hormonal signal during the onset of Drosophila metamorphosis (Lam, 1997).

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. A heat-inducible DHR3 rescue construct and clonal analysis were used to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, but no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. About half of the DHR3 mutants rescued to the third larval instar display defects in their tracheal system. The taenidial folds of the tracheal cuticle can be severely distorted. In more severe cases, this can lead to collapse of the tracheal cuticle and obstruction of the lumen, followed by necrosis. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-F1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. Clonal analysis provides support for a role for DHR3 in larval muscle development. The larval muscles of DHR3 mutants may provide a defective template for adult muscle formation, leading to a held out wing phenotype. DHR3 is inhibitory to late third instar larval gene expression, including BR-C, E74A and E75A, while DHR3 acts positively on later gene expression in mid-prepupae. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis (Lam, 1999).

Various ecdysteroid responsive genes play important roles in insect moulting and metamorphosis. Late FTZ-F1, a member of the nuclear receptor superfamily, is a unique transcription factor that is induced by a pulse exposure of 20-hydroxyecdysone. Elucidation of the regulation mechanism of this gene during the prepupal period will further understanding of metamorphosis at a molecular level. Using transgenic fly lines carrying various transcription regulatory regions of the FTZ-F1 gene fused to the LacZ gene, cis-regulatory elements in the late FTZ-F1 transcription unit were investigated. The region that governs the stage-specific expression during prepupal period was narrowed down to 1.2kb, from -0.7 to +0.5kb, relative to the transcription start site. Electrophoresis mobility shift assays using staged extracts and various probes within the stage-specific region allowed the identification of binding sites for DHR3, an early late gene product, around 170 and 450bp downstream of the transcription initiation start site. Electrophoresis mobility shift assays using staged extracts and various probes within the stage-specific region allowed the identification of binding sites for DHR3, an early late gene product, around 170 and 450bp downstream of the transcription initiation site. Mutations disrupting these binding sites reduce the reporter gene expression without affecting the stage specificity. These deletion and mutation studies of the cis-regulatory element of the FTZ-F1 gene suggest that the DHR3 binding sites located in the 5' non-coding region are involved in the prepupal expression of the gene. These DHR3 binding sites confer high level expression while other elements are also involved in stage-specific expression (Kageyama, 1997).

The reduced level of ßFTZ-F1 expression in E75A mutant second instar larvae provides a functional link to explain the molting defects in these animals. ßFTZ-F1 has been shown to be required for larval molting. Moreover, ßFTZ-F1 can directly regulate the Edg84A pupal cuticle gene, and ectopic overexpression of ßFTZ-F1 leads to an abnormal larval cuticle structure. Taken together, these observations suggest that ßFTZ-F1 plays a key role in controlling larval molts, directly regulating larval cuticle gene expression. The severe reduction in ßFTZ-F1 expression in E75A mutant larvae is thus consistent with the inability of these animals to molt. It is also concluded that E75A does not directly regulate ßFTZ-F1, since molting can be rescued by feeding ecdysteroids to E75A mutant larvae. This experiment places ßFTZ-F1 downstream from ecdysteroid signaling and E75A expression, indirectly dependent on E75A activity (Bialecki, 2002).

DHR4 plays an essential role in ßFTZ-F1 regulation

Studies in several insect species have suggested the orphan nuclear receptor encoded in Drosophila by DHR4 (CG16902) may contribute to the crossregulatory nuclear receptor network during the early stages of metamorphosis. A critical determinant of insect body size is the time at which the larva stops feeding and initiates wandering in preparation for metamorphosis. No genes have been identified that regulate growth by contributing to this key developmental decision to terminate feeding. Mutations in the DHR4 orphan nuclear receptor result in larvae that precociously leave the food to form premature prepupae, resulting in abbreviated larval development that translates directly into smaller and lighter animals. In addition, DHR4 plays a central role in the genetic cascades triggered by the steroid hormone ecdysone at the onset of metamorphosis, acting as both a repressor of the early ecdysone-induced regulatory genes and an inducer of the ßFTZ-F1 midprepupal competence factor. It is proposed that DHR4 coordinates growth and maturation in Drosophila by mediating endocrine responses to achieve critical weight during larval development (King-Jones, 2005).

The spatial and temporal patterns of DHR4 expression provide a framework for interpreting phenotypic studies, suggesting that the growth defects arise from neuroendocrine functions of DHR4 during larval stages and the metamorphic requirement for DHR4 arises from its ecdysone-induced expression at pupariation. These two regulatory functions of DHR4 have been examined, focusing first on its roles in regulating gene expression at the onset of metamorphosis and then moving on to the mechanisms by which DHR4 regulates growth during larval stages (King-Jones, 2005).

The temporal pattern of DHR4 expression and the prepupal lethality of DHR4¹ mutants suggest that this gene plays a critical role in ecdysone-regulated transcription at the onset of metamorphosis. To test this hypothesis, staged mutant and control L3 and prepupae were analyzed by Northern blot hybridization using probes to detect early (EcR, E74, E75), early-late (DHR3), or midprepupal (βFTZ-F1, Imp-L1) ecdysone-regulated gene expression. The EcR, E74A, E75A, and E75B early mRNAs are submaximally induced at puparium formation in DHR4¹ mutants, with EcR, E74, E75A, and E75B also failing to be repressed at the appropriate time. DHR3 induction appears normal in DHR4¹ mutants; however, the repression of this gene is significantly impaired. βFTZ-F1 expression is highly reduced in DHR4¹ mutant prepupae, with consequent defects in E74A and E75A induction in 10 hr prepupae, phenocopying a βFTZ-F1 mutant. Imp-L1 expression, in contrast, accumulates to wild-type levels in DHR4¹ mutant prepupae, with an ~2 hr delay, demonstrating that the DHR4¹ mutation does not have a general effect on midprepupal gene expression. Similar effects on ecdysone-regulated gene expression are seen when DHR4 function is disrupted by RNAi (King-Jones, 2005).

As a further test of a role for DHR4 in repressing early gene expression, DHR4 was ectopically expressed in late L3 at the time when EcR and the classic early puff transcripts, E74A and E75A, are initially being induced by ecdysone. These transcripts are significantly downregulated under these conditions, resulting in almost complete suppression of the early transcriptional response to the hormone. Consistent with this effect on gene expression, most hsDHR4 transformants subjected to an identical heat-treatment regime failed to initiate metamorphosis, dying as L3 (King-Jones, 2005).

Microarray analysis of DHR4¹ mutant prepupae and heat-treated hsDHR4 L3 were used to expand understanding of DHR4 function. Total RNA was isolated from P427 control and DHR4¹ mutant prepupae staged at 0, 4, and 8 hr after pupariation, spanning the peak of DHR4 expression. Only DHR4¹ mutant prepupae with a normal appearance were selected for this study. Also, a gain-of-function study was performed using w¹¹¹⁸ or w¹¹¹⁸; hsDHR4 L3 that were heat treated at ~10 hr prior to puparium formation and harvested 6 hr later. RNA was purified from each set of animals, labeled, and hybridized to Affymetrix Drosophila genome arrays. The resultant gene lists were compared with two data sets that are enriched for ecdysone-regulated genes: genes that significantly change their level of expression between 0 and 4 hr after pupariation in P427 control animals, a time when ecdysone is known to exert global effects on gene activity, and the only published microarray study of EcR mutants, using larval midguts. These comparisons revealed a robust correlation between genes that are normally downregulated in wild-type early prepupae, or EcR-dependent genes that are downregulated in the midgut, and genes that are upregulated in DHR4¹ mutants, suggesting a central role for DHR4 in the repression of ecdysone-regulated genes (King-Jones, 2005).

Therefore, metamorphic functions for DHR4 are not restricted to puparium formation but also extend to prepupal stages through its essential role in βFTZ-F1 regulation. Like DHR3, DHR4 is required for maximal expression of this midprepupal competence factor. The effects seen on EcR, E74, and E75 transcription in 10 hr DHR4 mutant prepupae and the lethal phenotypes associated with DHR4 RNAi are indistinguishable from those seen in βFTZ-F1 mutants, suggesting that most if not all of the effects of DHR4 are channeled through βFTZ-F1 at this stage in development. Thus, as originally proposed based on the timing of DHR4 expression (Charles, 1999; Hiruma, 2001; Sullivan, 2003), this factor contributes to the crossregulatory interactions among orphan nuclear receptors in prepupae. Together with DHR3 and through βFTZ-F1, DHR4 directs appropriate genetic and biological responses to the prepupal pulse of ecdysone, ensuring that this response will be distinct from that induced by the hormone several hours earlier at pupariation (King-Jones, 2005).

Targets of Activity

The seven-stripe pattern of Fushi tarazu during early embryogenesis is largely specified by the zebra element located immediately upstream of the FTZ transcriptional start site. The FTZ-F1 protein, one of multiple DNA binding factors that interacts with the zebra element, is implicated in the activation of FTZ transcription, especially in stripes 1, 2, 3, and 6. Because vertebrate hormone response elements show perfect or imperfect dyad symmetry separated by a short variable space, the sequences of the FTZ-F1 binding sites were examined in the ftz gene. A similar symmetry can be discerned for the FTZ-F1 binding sites in the ftz gene (Lavorgna, 1991).

A complex array of activator and repressor elements located within 669 bp proximal to the fushi tarazu transcriptional start site is sufficient to generate the 'zebra-stripe' expression pattern characteristic of the ftz gene. P-element-mediated transformation and ftz promoter/lacZ fusion genes were used to characterize, in detail, several of these transcriptional control elements. By reconstructing promoters with synthetic oligonucleotides containing cis-regulators of stripe expression, it has been shown that these regulatory sites can function as independent units to direct position-specific transcription in the Drosophila embryo. In particular, multiple copies of a positive regulatory site can mediate expression in both the odd- and even-numbered parasegments throughout most of the germ band. Specifically, the fAE3 site serves as an activator recognition site. A protein that binds to this motif is a transcriptional activator of Ultrabithorax and engrailed.. Negative regulatory sites can also transform a continuous pattern of gene expression into discrete stripes. Deletion of the fAE3 site causes ectopic expression of ftz in interstripe regions. This result suggests that fAE3 has a repression function. Four copies of the adjacent fDE site are able to convert a continuous, graded band of expression into a highly resolved pattern of seven stripes, indicating that multiple copies of a single repressor site can selectively repress transcription in this assay. Hairy is somehow required for repression of expression through the fDE1 element. FTZ-F1 can recognize fDE1 and fDE2 sites, both of which are known to serve dual activating and repressing functions. The reconstructed promoter system presented provides an effective means of studying molecular mechanisms governing spatially restricted transcription in the early embryo (Topol, 1991).

Two isoforms of FTZ-F1 regulate fushi tarazu expression found in the blastoderm of the Drosophila. A motif in the zebra element, the FTZ-F1 recognition element (F1RE), has been shown to bind a transcription factor, FTZ-F1 alpha. A second isoform, FTZ-F1 beta, has been identified: it also binds to this motif. To investigate the possibility that FTZ-F1 alpha and FTZ-F1 beta coregulate ftz transcription through the F1RE, the DNA binding properties of FTZ-F1 alpha and FTZ-F1 beta have been studied. FTZ-F1 alpha and FTZ-F1 beta proteins produce similar in vitro DNase I footprint patterns on a 14-nucleotide region of the zebra element and bind to this site with similar affinities and sequence specificities. FTZ-F1 alpha and FTZ-F1 beta both bind as monomers to the 9-bp F1RE in the zebra element, as well as to an imperfect inverted F1RE repeat present in the Drosophila alcohol dehydrogenase gene. A polyclonal antibody raised against FTZ-F1 beta identifies a predominant F1RE-binding component in embryonic nuclear extracts. Although FTZ-F1 alpha is also present in these extracts, FTZ-F1 alpha and FTZ-F1 beta do not appear to form heterodimers with one another. Cotransfection assays in mammalian cell culture indicate that both receptors contribute to the net transcriptional activity of a reporter gene through their direct interaction with the F1RE. These data suggest that FTZ-F1 alpha and FTZ-F1 beta likely coregulate common target genes by competition for binding to a 9-bp recognition element (Ohno, 1994).

The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls the ftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. Nine binding sites for multiple Drosophila nuclear proteins have been identifed in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. The nuclear receptor Ftz-F1 interacts with this repeated module. Additional proteins interacting with this module were purified from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Tramtrack and the transcription factor Adf-1 were obtained. While Ttk is thought to be a repressor of ftz stripes, both Adf-1 and Ftz-F1 have been shown to activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the time ftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1/Adf-1/Ttk binding sites are functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis has identified cis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks (Han, 1998).

It was proposed several years ago that Ttk acts as a repressor of ftz stripes, since the protein is present before and after ftz is expressed in stripes but is not detected during the time that ftz is expressed in stripes (Harrison, 1990).

The proximal enhancer used in the current studies contains multiple binding sites for Ttk. Therefore, an initial intention was to test the role of Ttk as a repressor of ftz stripes by simultaneously mutating multiple Ttk binding sites. It was expected that fusion gene expression would initiate earlier and/or persist later in the absence of repression by Ttk. Fusion genes 12 and 13 carry mutations in four Ttk sites, while all five sites are mutated in fusion gene 14. However, three of the five Ttk binding sites overlap with binding sites for activator proteins that are necessary to activate expression of fusion genes (fusion gene 11). Therefore, it was not possible to test whether Ttk represses through its proximal enhancer binding sites, since mutations result in loss of activation due to this overlap. Currently, the role of Ttk in regulating ftz is unclear. Mutation of Ttk binding sites in the zebra element results in premature activation of ftz gene expression, and ectopic expression of Ttk at later stages causes a decrease in ftz expression levels. However, given the observation that most Ttk binding sites also interact with other nuclear proteins, it is difficult to know whether these observations are a result of direct negative regulation of ftz by Ttk. Preliminary results suggest that Ttk can act as a transcriptional activator, raising the possibility either that Ttk interacts with a corepressor to decrease ftz expression levels or that observed effects of Ttk overexpression in embryos are indirect (Han, 1998).

The segmentation genes runt and hairy are required for the proper transcriptional regulation of the pair-rule gene fushi tarazu during the blastoderm stage of Drosophila embryogenesis. The expression of different fushi tarazu reporter genes was examined in runt and hairy mutant embryos, as well as in runt over-expressing embryos, in order to identify DNA elements responsible for mediating these regulatory effects. The results indicate that runt and hairy act through a common 32 base-pair element. This element, designated as fDE1, contains a binding site for a small family of orphan nuclear receptor proteins that are uniformly expressed in blastoderm embryos. The pair-rule expression of reporter gene constructs containing multimerized fDE1 elements depends on activation by runt and repression by hairy. Examination of reporter genes with mutated fDE1 elements provides further evidence that this element mediates both transcriptional activation and repression. Genetic experiments indicate that the opposing effects of runt and hairy are not due solely to cross-regulatory interactions between these two genes and that fDE1-dependent expression is regulated by factors in addition to runt and hairy (Tsai, 1995).

Within an engrailed enhancer, adjacent and conserved binding sites for the Fushi tarazu protein and a cofactor are each necessary, and together sufficient, for transcriptional activation. Footprinting shows that the cofactor site can be bound specifically by Ftz-F1, a member of the nuclear receptor superfamily. Ftz-F1 and the Fushi tarazu homeodomain bind the sites with 4- to 8-fold cooperativity, suggesting that direct contact between the two proteins may contribute to target recognition. Even parasegmental reporter expression is dependent on Fushi tarazu and maternal Ftz-F1, suggesting that these two proteins are indeed the factors that act upon the two sites in embryos. The two adjacent binding sites are also required for continued activity of the engrailed enhancer after Fushi tarazu protein is no longer detectable, including the period when both engrailed and the enhancer become dependent upon wingless. A separate negative regulatory element exists that apparently responds to odd-skipped (Florence, 1997).

Developmental and tissue-specific transcription from the Alcohol dehydrogenase distal promoter is regulated in part by the Adh adult enhancer, located 450 to 600 bp upstream from the distal RNA start site. Four proteins (DEP1 to DEP4) present in cell nuclear extracts bind to this enhancer. DEP1 and DEP2 bind to a positive cis-acting element (-492 to -481) and share nucleotide contacts. A small linker replacement deletion mutation, which disrupts the overlapping DEP1- and DEP2-binding sites, reduces Adh distal transcription in an alcohol dehydrogenase (ADH)-expressing cultured cell line in the adult fat body (the major tissue of ADH expression), as well as in some but not all adult tissues where ADH is normally expressed. This enhancer element contains an imperfect palindromic sequence similar to steroid hormone receptor superfamily response elements. FTZ-F1 binds to this site. Anti-FTZ-F1 antibodies have identified DEP1 as FTZ-F1. DEP2 also binds to the FTZ-F1 site from the fushi tarazu zebra element, suggesting that DEP2 may also be a steroid receptor superfamily member. These results raise the possibility that Adh regulation in certain adult tissues involves a hormone-mediated pathway. Because FTZ-F1 and DEP2 contact some of the same nucleotides within the positive cis element, it is unlikely that they can bind simultaneously. Such alternative binding may play a role in the tissue-specific and developmental transcription of Adh (Ayer, 1992).

FTZ-F1 is expressed as a product of the previously identified, midprepupal chromosome puff at 75CD. The 75CD puff occurs in the midst of a period of intense puffing activity, triggered in response to the steroid hormone ecdysone at the onset of metamorphosis. Indirect immunofluorescent staining for FTZ-F1 on Drosophila polytene chromosomes reveals binding to over 150 chromosomal targets, including 75CD itself and prominent late prepupal puffs, predicted to be regulated by midprepupal puff proteins. These results suggest a role for FTZ-F1 as a regulator of insect metamorphosis and underscore the repeated utilization of a regulatory protein for widely separate developmental pathways (Lavorgna, 1993).

A putatitive pupal cuticle gene, EDG84A, is expressed slightly following FTZ-F1 expression during the prepupal period and carries a strong FTZ-F1 binding site upstream of its transcription start site. EDG84A is prematurely expressed upon heat induction of FTZ-F1 in prepupae carrying a heat shock promoter-FTZ-F1 gene construct. The FTZ-F1 binding site is positioned between two differently acting regions of the EDG84A promoter. The region between bp -193 and -104 is responsible for expression in the anterior epidermis, and the region between bp -103 and +50 is responsible for ectopic expression in the posterior epidermis. A repressor in the posterior epidermis acts through sequences between bp -408 and -194. FTZ-F1 binds between pb -100 and -92 (Murata, 1996).

In Drosophila, peaks of the titer of the steroid hormone ecdysone act as molecular signals that trigger all the major developmental transitions occurring along the life cycle. The EcR/USP heterodimer, known to constitute the functional ecdysone receptor, binds with high affinity to specific target sequences. The target sequences, known as ecdysone response elements (EcREs) still remain to be fully characterized at both the molecular and functional levels. In order to investigate the properties of EcREs composed of directly repeated half-sites (DRs), an analysis was carried out of the binding properties of the ng-EcRE, a DR element located within the coding region of ng-1 and ng-2, two highly homologous genes mapping at the ecdysone-regulated 3C intermolt puff. The ng-EcRE contacts the Ecdysone receptor through its directly repeated half-sites spaced by 12 bp, and this element may interact efficiently with at least three Drosophila orphan receptors, namely DHR38, DHR39 and beta FTZ-F1. Interestingly, DHR38 is bound alone or in combination with USP, providing the first evidence that the EcR-USP and DHR38-USP may directly compete for binding to a common response element. These results suggest that EcREs composed of widely spaced DRs may contribute to the establishment of extensive cross-talk between nuclear receptors, thus modulating ng-1 and ng-2 intermolt expression (Crispi, 1998).

Premature expression of the late FTZ-F1 protein has an effect on early gene induction by ecdysone. The inability of Eip93F (E93) to be induced by ecdysone in late-third instar larval salivary glands can be overcome by ectopic expression of FTZ-F1. FTZ-F1 also represses its own transcription (Woodard, 1994).

Competence is a critical mechanism for restricting the developmental capacity of cells to specific pathways. Cells that are competent to respond to a signal can undergo a developmental response, while other cells are refractory to the signal or undergo a default developmental pathway. Competence is progressively regulated during development such that cells achieve a series of competent stages. In this manner, widespread signals are refined to direct spatially and temporally restricted biological responses. Thus, the acquisition of competence is a key mechanism for refining global signals to distinct spatial and temporal responses. The molecular basis of competence, however, remains poorly understood. In Drosophila, competence to respond to ecdysone is acquired during the mid-prepupal period for both reinduction of the Br-C, E74A, and the E75A early puffs as well as induction of stage-specific puffs, such as 93F. The prepupal puffing response cannot be achieved in early prepupal salivary glands cultured in the continuous presence of ecdysone. Rather, a preceding period of low ecdysone concentration and protein synthesis is required before the salivary glands become competent to respond to the hormone. The simplest interpretation of these observations is that one or more proteins that are repressed by ecdysone and expressed in mid-prepupae provide competence for the prepupal genetic response to ecdysone (Broadus, 1999)

The beta FTZ-F1 orphan nuclear receptor functions as a competence factor for stage-specific responses to the steroid hormone ecdysone during Drosophila metamorphosis. beta FTZ-F1 mutants pupariate normally in response to the late larval pulse of ecdysone but display defects in stage-specific responses, adult head eversion, leg elongation and salivary gland death, in response to the subsequent ecdysone pulse in prepupae. The ecdysone-triggered genetic hierarchy that directs these developmental responses is severely attenuated in beta FTZ-F1 mutants, although ecdysone receptor expression is unaffected. Both E74A and E75A, whose levels of expression are normally increased several orders of magnitude by ecdysone, are significantly affected in betaFTZ-F1 mutants. The severity of these effects correlates with the intensity of polytene chromosome staining by FTZ-F1 antibodies. The Br-C locus is only weakly stained, while E74 is strongly stained, and E75 is the most intensely stained site in the genome. It thus appears that betaFTZ-F1 exerts specificity to the degree to which it can enhance the ecdysone-induction of different promoters. The E93 early gene is also submaximally induced in betaFTZ-F1 mutants, consistent with the proposal that this stage-specific response is dependent on betaFTZ-F1 function. In contrast, the levels of Ecdysone receptor and Ultraspiracle mRNA are not significanty affected by betaFTZ-F1. EDG84A, a gene that encodes a pupal cuticle protein that is specifically expressed in the imaginal discs of mid-prepupae, contains a betaFTZ-F1 binding site upstream from the start site, and EDG84A transcription is delayed and reduced in betaFTZ-F1 mutants. Thus this study defines beta FTZ-F1 as an essential competence factor for stage-specific responses to a steroid signal and implicates interplay among nuclear receptors as a mechanism for achieving hormonal competence (Broadus, 1999).

The steroid hormone ecdysone signals the stage-specific programmed cell death of the larval salivary glands during Drosophila metamorphosis. This response is preceded by an ecdysone-triggered switch in gene expression in which the diap2 death inhibitor is repressed and the reaper (rpr) and head involution defective (hid) death activators are induced. rpr is induced directly by the ecdysone-receptor complex through an essential response element in the rpr promoter. The Broad-Complex (BR-C) is required for both rpr and hid transcription, while E74A is required for maximal levels of hid induction. diap2 induction is dependent on FTZ-F1, while E75A and E75B are each sufficient to repress diap2. This study identifies transcriptional regulators of programmed cell death in Drosophila and provides a direct link between a steroid signal and a programmed cell death response (Jiang, 2000).

FTZ-F1 expression immediately precedes that of diap2 in larval salivary glands and is required for ecdysone-induced gene expression in late prepupae. These observations have led to the hypothesis that betaFTZ-F1 may induce diap2 expression. To test this possibility, diap2 transcription was examined in the salivary glands of FTZ-F1¹⁷ mutant prepupae. FTZ-F1¹⁷ is a hypomorphic betaFTZ-F1 allele that leads to severe defects in both genetic and biological responses to the prepupal pulse of ecdysone. Salivary glands were dissected from staged FTZ-F1¹⁷/+ controls and FTZ-F1¹⁷/Df(3L)Cat^DH104 mutant prepupae, and diap2 transcription was examined by Northern blot hybridization. The levels of diap2 mRNA are significantly reduced in betaFTZ-F1 mutant salivary glands, indicating that diap2 expression is dependent on betaFTZ-F1 function (Jiang, 2000).

Therefore ecdysone-regulated transcription factors encoded by betaFTZ-F1, BR-C, E74, and E75 function together to direct a burst of the diap2 death inhibitor followed by induction of the rpr and hid death activators. It is proposed that cooperation between rpr and hid allows these genes to overcome the inhibitory effect of diap2, by precisely coordinating when the salivary glands are destroyed. Evidence that the ecdysone-receptor complex directly induces rpr transcription through an essential response element in the promoter, providing a direct link between the steroid signal and a programmed cell death response. The diap2 death inhibitor is expressed briefly in the salivary glands of late prepupae, foreshadowing the imminent destruction of this tissue. This transient expression is directed by at least three ecdysone-regulated transcription factors: betaFTZ-F1, E75A, and E75B. diap2 induction is dependent on the betaFTZ-F1 orphan nuclear receptor. This is consistent with the timing of betaFTZ-F1 expression, which immediately precedes that of diap2, as well as the known role of betaFTZ-F1 as an activator of gene expression in late prepupae (Jiang, 2000).

The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. When expressed under the control of a heat-inducible promoter, dsRNA interfers efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generates defects in larval molting and metamorphosis, resulting in animals that fail to pupariate or prepupae that die with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor has no effect on puparium formation, but leads to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that express either EcR or betaFTZ-F1 dsRNA show defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis. These results confirm and extend understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila (Lam, 2000).

To test the generality of this method, attempts were made to interfere with betaFTZ-F1 function during the onset of metamorphosis. There are two reasons why betaFTZ-F1 provides a valuable additional test of this method. (1) Unlike EcR, betaFTZ-F1 exerts a stage-specific function at the onset of metamorphosis, with no apparent function at puparium formation and an essential role in providing competence for the ecdysone-triggered prepupal-pupal transition. (2) Only hypomorphic betaFTZ-F1 mutants have been studied during the onset of metamorphosis because null mutants die during early stages of development. Thus, more severe phenotypes associated with betaFTZ-F1 RNAi might provide new insights into the function of this receptor. Expression of betaFTZ-F1 dsRNA ~18 and 12 hours before puparium formation, which is identical to the double heat-shock regime used with hs-EcRi-11, results in normal puparium formation, although 37% of these animals failed to evert one (usually) anterior spiracle. The ability of these animals to pupariate is consistent with the absence of betaFTZ-F1 expression in third instar larvae as well as the absence of any effects of betaFTZ-F1 mutations on puparium formation. The majority of animals expressing betaFTZ-F1 dsRNA, however, failed to progress through the early stages of metamorphosis and died as prepupae. Sequential heat induction of betaFTZ-F1 dsRNA at 0 and 6 hours after puparium formation leads to a similar phenotype, with all animals arresting development at the prepupal stage. Although these animals display normal gas bubble formation, they fail to translocate the bubble to the anterior end, and die after several days with a prominent bubble in the middle of the body. In addition, eversion of the adult head is completely blocked and the larval mouthhooks that are normally expelled at head eversion remain attached at the anterior end of the animal. Although betaFTZ-F1 hypomorphic mutants also show defects in adult head eversion, this phenotype is more severe and more penetrant in animals that express betaFTZ-F1 dsRNA. Most betaFTZ-F1 hypomorphic mutants die as pupae with defects in head eversion and leg elongation, with some animals surviving to adulthood. The fully penetrant prepupal lethality associated with betaFTZ-F1 RNAi is likely to be due to a severe reduction in betaFTZ-F1 function, and indicates that betaFTZ-F1 is absolutely required for progression through the mid-prepupal stage (Lam, 2000).

The effects of betaFTZ-F1 dsRNA on ecdysone-inducible gene expression were examined. Similar to the kinetics of EcR dsRNA, betaFTZ-F1 dsRNA is expressed at high levels in response to heat treatment and then turned over very rapidly. Furthermore, the levels of endogenous betaFTZ-F1 mRNA are significantly reduced in these animals, consistent with their selective degradation by RNAi. Ecdysone-induced E74A transcription is significantly reduced in animals expressing betaFTZ-F1 dsRNA: E74B is not repressed, E75A fails to be expressed, and E93 is only weakly induced. The levels of EcR mRNA are similar to those of control animals although there is a slight decrease at 10 hours after puparium formation. It is likely that this reduction reflects a requirement for betaFTZ-F1 in directing this prepupal peak in EcR activity. The levels of usp mRNA are unaffected by the expression of betaFTZ-F1 dsRNA. Importantly, all of these effects on ecdysone-regulated gene expression are virtually identical to those seen in betaFTZ-F1 mutant prepupae, indicating that betaFTZ-F1 dsRNA acts as an effective and specific block to the activity of this competence factor (Lam, 2000).

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