Interactive Fly, Drosophila

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 ßFTZ-F1 orphan receptor loci. Hr46 represses E74A, and to a lesser extent E74B, and it also represses BR-C, E75A, and E78B. Hr46 activates ßFTZ-F1. Three Hr46 binding sites are present downstream from the start site of ßFTZ-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 ßFTZ-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 ecdysteroids direct Drosophila through its life cycle by activating stage- and tissue-specific genetic regulatory hierarchies. An orphan nuclear receptor, Hormone-receptor-like in 78 (DHR78), functions at the top of the ecdysteroid regulatory hierarchies. DHR78 is expressed throughout development, with peaks of expression in third instar larvae and prepupae that correlate with the known ecdysteroid pulses. Consistent with this observation, DHR78 transcription can be induced by ecdysone in cultured larval organs. DHR78 protein binds to a subset of Ecdysone receptor/Ultraspiracle binding sites in vitro, suggesting that it may interact directly with the Ecdysone receptor. Cotransfection assays have supported this model by demonstrating that DHR78 can inhibit the ecdysone induction of a reporter gene. Null mutations in DHR78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. Consistent with these phenotypes, DHR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. The expression of usp is not affected in DHR78 mutants, consistent with its relatively modest transcriptional regulation by ecdysteroids. In contrast, all other ecdysteroid-regulated transcriptional responses examined are disrupted in DHR78 mutants. The coordinate induction of EcR, E74B, and the BR-C in mid-third instar larvae is significantly reduced, and E74A is not expressed. Fbp-1, which is induced directly by ecdysone in fat bodies, is not expressed either in these mutants. Mid-third instar larval development is characterized by an ecdysteroid-triggered switch in salivary gland gene expression, from the ng genes to the Sgs glue genes. This switch fails to occur in DHR78 mutants: ng-1 is not repressed and Sgs-4 is not induced. DHR78 protein is bound to many ecdysteroid-regulated puff loci, suggesting that DHR78 directly regulates puff gene expression. Ectopic expression of DHR78 has no effects on development, indicating that its activity is regulated post-translationally. It is proposed that DHR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis (Fisk, 1998).

Interestingly, the available evidence indicates that EcR and usp do not function in mid-third instar larvae, when DHR78 plays its critical role in gene regulation. Both EcR and Usp proteins are expressed at very low or undetectable levels in mid-third instar larvae. Recent genetic studies have led to the surprising conclusion that EcR-B1 and usp do not function at this stage of development. Studies of the puffing patterns of the polytene chromosomes in EcR-B1 mutant third instar larvae have revealed that the 2B5 and glue gene puffs are present, but the 74EF and 75B early puffs fail to form. This observation suggests that the BR-C and glue genes are induced normally in EcR-B1 mutants, but that the response to the high-titer late larval ecdysteroid pulse is selectively blocked. Mutations in usp show a similar stage-specific effect on ecdysteroid-regulated gene expression, where the mid-third instar regulatory hierarchy occurs normally but the late third instar hierarchy is blocked. It remains possible that EcR-A could perform a function in mid-third instar larvae, although its level in the salivary gland is very low, and it would have to exert this function independently of usp. On the contrary, these studies suggest that DHR78 is the critical regulator that triggers the mid-third instar regulatory hierarchy, preparing the animal for puparium formation in response to the high-titer late larval pulse of 20E (Fisk, 1998 and references).

The function of DHR78 most closely resembles EcR and usp, in that ectopic expression of either half of the ecdysone receptor has no adverse effects on development, most likely because their activity is controlled by a hormone. In this regard, it is interesting to note that DHR78 is transcribed throughout development with peaks of mRNA accumulation in response to ecdysteroid pulses. This broad expression pattern contrasts with that of other orphan receptor genes, which are only expressed for very brief temporal intervals during development. These observations indicate that DHR78 activity is regulated posttranslationally. This regulation could be imposed at a variety of different levels, including covalent modification (such as phosphorylation or glycosylation) or interaction with a cofactor. The simplest possibility, however, is that DHR78 is regulated by an as yet unidentified hormone. The identification of the putative DHR78 ligand is clearly the next critical step toward an understanding of the function of this receptor (Fisk, 1998).

The Drosophila eggshell, which has a pair of chorionic appendages (dorsal appendages) located asymmetrically along both the anterior/posterior and dorsal/ventral axes, provides a good model to study signal instructed morphogenesis. Broad-Complex, a gene encoding zinc-finger transcription factors, is essential for the morphogenesis of dorsal appendages and is expressed in a bilaterally symmetrical pattern in the lateral-dorsal-anterior follicle cells during late oogenesis. This pattern of expression is induced and specified along the dorsoventral axis by an epidermal growth factor receptor signaling pathway, which in the oocyte includes Gurken, a localized transforming growth factor alpha-like molecule. In the surrounding somatic follicle cells, Torpedo, the Drosophila EGF receptor homolog that functions as the target of Gurken specifies BR-C expression. Mutants that result in the mislocalization of Gurken, such as fs(1)K10, induce a BR-C late expression pattern that is expanded to the ventral follicle cells surrounding the oocyte. This expanded BR-C expression results in expansion of the dorsal appendages to the ventral region. Four extra copies of grk gene increase the gap between the two groups of BR-C expressing cells to about 8 cells wide, in comparison with the 4-cell-wide gap in wild type, resulting in a widened dorsal gap between the two dorsal appendages. A decrease in Grk-Egfr signaling in a topQY1 mutation in Egfr results in BR-C expression in the dorsal most follicle cells, leading to a fusion of the dorsal appendages in the dorsal-most region. The Egfr target gene pointed regulates the number of BR-C expressing cells. Ectopic expression of pnt decreases the number of BR-C expressing cells, suggesting that Pnt regulates BR-C expression (Deng, 1997).

The precisely localized expression of BR-C along the AP axis requires a separate signaling pathway, initiated by a transforming growth factor-beta homolog, Decapentaplegic, in nearby follicle cells. The fact that BR-C expression is missing in the anterior most columnar follicle cells suggests that there is probably another repressor produced in these cells. If Dpp signaling acts as a repressor of BR-C expression in oogenesis, a decrease in Dpp levels should result in the expression of BR-C in the anterior most collumnar cells -- cells where it is not found in wild-type eggshells. This was confirmed using a temperature sensitive dpp mutant. When dpp is ectopically expressed in most of the follicle cells, BR-C expression is found over the middle part of the oocyte at a more posterior postion than in wild-type egg chambers, resulting in a more posterior localization of the dorsal appendages. These two signaling pathways (Gurken functioning from the oocyte and Dpp functioning from the follicle cells) co-ordinately specify patches of follicle cells to express the Broad-Complex in a unique position with respect to both DV and AP axes respectively, and which, in turn direct the differentiation of the dorsal appendages in the correct position on the eggshell (Deng, 1997).

During Drosophila oogenesis Gurken, a TGF-alpha like protein localized close to the oocyte nucleus, activates the MAPK cascade via the Drosophila EGF receptor (Egfr). Activation of this pathway induces different cell fates in the overlying follicular epithelium, specifying the two dorsolaterally positioned respiratory appendages and the dorsalmost cells separating them. Signal-associated internalization of Gurken protein into follicle cells demonstrates that the Gurken signal is spatially restricted and of constant intensity during mid-oogenesis. Gurken internalization can first be observed in all posterior follicle cells, abutting the oocyte from stage 4 to 6 of oogenesis. At the same time MAPK activation evolves in a spatially and temporally dynamic way and resolves into a complex pattern that presages the position of the appendages. Therefore, different dorsal follicle cell fates are not determined by a Gurken morphogen gradient. Instead they are specified by secondary signal amplification and refinement processes that integrate the Gurken signal with positive and negative feedback mechanisms generated by target genes of the Egfr pathway (Peri, 1999).

How does MAPK activation influence the morphogenesis of the developing egg shell? The gene Broad-complex is expressed and required in the anlagen of the dorsal respiratory appendages. It has been suggested, therefore, that Br-C acts as a marker specifying appendage fate. Gurken signaling leads to a repression of Br-C in the dorsalmost cells of the follicular epithelim. Br-C expression disappears in a sharply delineated dorsal-anterior patch coincident with the first observable MAPK activation. Later, when the MAPK activation refines to its distinct 'spectacle shape', Br-C expression is confined to two lateral domains showing weak MAPK activation surrounded by rings strongly staining for activated MAPK. Based on the striking complementarity of these patterns, it is proposed that at this stage Br-C is repressed by high levels and activated by low levels of MAPK activation. In addition, Br-C expression does not by itself specify dorsal appendage fate, as it is visible in duplicated anterior ringlike domains in completely ventralized ovaries that do not possess appendages. This adds a caveat to using Br-C expression as a fate marker for dorsoventral positions. It is proposed that Br-C instead is a general marker expressed in cells that undergo morphogenetic changes, consistent with its expression in the embryo and during earlier stages of oogenesis. It is proposed that Gurken initiates secondary processses in the follicular epithelium that modulate and amplify the initial activation of the Egfr pathway (Peri, 1999).

During Drosophila oogenesis, Gurken, a protein associated with the oocyte nucleus, activates the Drosophila EGF receptor in the follicular epithelium. Gurken first specifies posterior follicle cells, which in turn signal back to the oocyte to induce the migration of the oocyte nucleus from a posterior to an anterior-dorsal position. From this location Gurken signals again to specify dorsal follicle cells, which give rise to dorsal chorion structures, including the dorsal appendages. If Gurken signaling is delayed and starts after stage 6 of oogenesis, the nucleus remains at the posterior pole of the oocyte. Eggs develop with a posterior ring of dorsal appendage material that is produced by main-body follicle cells expressing the gene Broad-Complex. They encircle terminal follicle cells expressing variable amounts of the TGFbeta homolog, decapentaplegic. By ectopically expressing decapentaplegic and using clonal analysis with Mothers against dpp, it has been shown that Decapentaplegic signaling is required for Broad-Complex expression. Thus, the specification and positioning of dorsal appendages along the anterior-posterior axis depends on the intersection of both Gurken and Decapentaplegic signaling. This intersection also induces rhomboid expression and thereby initiates the positive feedback loop of EGF receptor activation, which positions the dorsal appendages along the dorsal-ventral egg axis (Peri, 2000).

The ecdysone response hierarchy mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates (Buszczak, 1999).

In order to investigate the role of ecdysone-responsive gene expression in the ovary, expression of three classical early ecdysone-responsive genes, E75, E74 and BR-C were examined. In situ hybridization revealed that the E75 and E74 genes are transcribed in remarkably similar patterns during oogenesis. Both E75 and E74 transcripts are first detected in region 2b of the germarium. Expression decreases during stages 2-4 and low levels of E75 and E74 mRNA are again detected in stage 5-7 egg chambers. Transcription of E75 and E74 appears to be upregulated during stage 8 in both the germline and soma. This expression continues to increase until stage 10B when transcription of both genes peaks in the follicle cells and the nurse cells. Immunofluorescent staining reveals the presence of BR-C protein in the follicle cell nuclei beginning between stages 5 and 6 of oogenesis. In most of the egg chambers examined, BR-C appears to be completely absent from the germline. However, in rare cases, low levels of expression could be detected in the nurse cell nuclei. These observations are consistent with a recent report that describes follicle cell expression of BR-C mRNA (Buszczak, 1999).

The expression of E75, E74 and BR-C in egg chambers suggests that these genes are co-regulated by a common signal. If these early response genes are being regulated by ecdysone, one would expect a dependence on the ecdysone receptor. To determine whether the ecdysone receptor is present in the ovary, egg chambers from Canton-S females were stained using anti-EcR antibodies. Antibody staining reveals that germline and somatic cells express EcR protein in their nuclei. This expression is first detected in the germarium, appears to be slightly upregulated during stage 4 and persists until the late stages of oogenesis. Additionally, border cells strongly express EcR during their migration through the nurse cell cluster. Uso has also been detected in all cells within the ovary. Thus, both components of the functional ecdysone receptor are present in the germline and soma during all stages of oogenesis (Buszczak, 1999).

To test the dependence of early response gene expression on ecdysone, a study was made of the effects in ovaries of the ecdysoneless¹ (l(3)ecd¹) mutation, in which low levels of ecdysone are generated. Females homozygous for the temperature-sensitive mutation l(3)ecd¹ lose the ability to lay eggs after just 2 days at the restrictive temperature and to have 13% of the wild-type ovarian ecdysone titer when shifted to the restrictive temperature for 4 days. E75 transcript levels were compared in wild-type and l(3)ecd¹ females shifted to the restrictive temperature for different lengths of time. Using in situ hybridization, no difference in E75 mRNA levels could be detected between ovaries taken from wild-type and l(3)ecd¹ females maintained at 25�C. However, there is a reproducible reduction of E75 mRNA in l(3)ecd¹ ovaries relative to wild-type controls shifted to the restrictive temperature for 2 days. An RNAse protection assay was used to quantitate the difference in E75 transcription in l(3)ecd¹ and wild-type ovaries. This analysis reveals that l(3)ecd¹ ovaries contain approximately half the E75 mRNA of wild-type ovaries when subjected to restrictive conditions. BR-C expression in wild-type and l(3)ecd¹ ovaries was also assayed. Immunofluorescent staining showed that BR-C protein levels appear to be reduced in l(3)ecd¹ ovaries relative to wild-type controls. A reduction of BR-C expression in ovaries from mutants shifted to 29�C was also detected on Western blots (Buszczak, 1999).

The steroid hormone 20-hydroxyecdysone (20E) initiates metamorphosis in insects by signaling through the ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP). Analysis of usp mutant clones in the wing disc of Drosophila shows that in the absence of USP, early hormone responsive genes such as EcR, DHR3 and E75B fail to up-regulate in response to 20E, but other genes that are normally expressed later, such as beta-Ftz-F1 and the Z1 isoform of the Broad-Complex (BRC-Z1), are expressed precociously. Sensory neuron formation and axonal outgrowth, two early metamorphic events, also occur prematurely. In vitro experiments with cultured wing discs show that BRC-Z1 expression and early metamorphic development are rendered steroid-independent in the usp mutant clones. These results are consistent with a model in which these latter processes are induced by a signal arising during the middle of the last larval stage but suppressed by the unliganded EcR/USP complex. These observations suggest that silencing by the unliganded EcR/USP receptor and the subsequent release of silencing by moderate steroid levels may play an important role in coordinating early phases of steroid driven development (Schubiger, 2000).

In clones lacking USP, BRC-Z1 is still developmentally regulated but it first appears about 103 hours AED rather than at its expected time of expression at mid-wandering. This precocious appearance of BRC-Z1 corresponds to the so called 'mid-third instar transition', which is a time when changes occur in preparation for metamorphosis. It has been proposed that the gene DHR78 may play a role in this transition and that these changes may occur independently of the 20E titers. It appears that events at this time serve to activate BRC-Z1 expression but a USP mediated mechanism suppresses expression until rising ecdysteroid titers remove the inhibition late in wandering (Schubiger, 2000).

The development of sensory precursor cells in the wing shows a similar response to the loss of USP. As sensory neurons differentiate and project their axons toward the CNS, the timing of these events is crucial. In order for axons to navigate centripetally, guidance cues must be present at the correct place and time. The synchrony of these events, as has been shown for the developing neurons along the presumptive wing margin, is disrupted in usp mutant clones. Differentiation and axon outgrowth occur precociously, and, as a consequence, the axons originating from these neurons are misrouted. Premature differentiation and faulty axon projection for the presumptive campaniform sensilla on the radius of the wing has been seen, and precocious differentiation of photoreceptors in the eye disc. Thus, USP appears to play a general role of suppressing early sensory neuron differentiation in the imaginal tissue (Schubiger, 2000).

Both the expression of BRC-Z1 and the differentiation of sensory neurons are dependent on exposure to ecdysteroids. Surprisingly, though, the presumed disruption of the ecdysone receptor complex by removal of USP allows these processes to proceed in a hormone-independent fashion. Observations on the developing eye disc show that 20E is required for the correct progression of the morphogenetic furrow, but that loss of USP leads to advancement of the morphogenetic furrow and precocious differentiation of the photoreceptors. It is assumed that in the case of the eye, the events in the morphogenetic furrow are also rendered hormone-independent by the removal of USP. The data suggest that USP is involved in two distinct types of ecdysteroid controlled responses and that these responses have distinct developmental roles. In some instances USP serves as a hormone-inhibited silencer whereas in others it is a hormone-dependent activator. Early metamorphic events in the wing, including neurogenesis and axonal outgrowth, clearly require ecdysteroids in order to occur, but this requirement is carried out through an ecdysteroid-dependent release of USP-mediated suppression. Thus, in the absence of USP these events occur in a steroid-independent fashion. Importantly, the rate of development in usp mutant clones is at least as fast or faster than in wild-type tissue exposed to 20E, suggesting that for these developmental processes the effects of 20E are at best permissive (Schubiger, 2000).

A molecular parallel to what is seen for early sensory neuron development is illustrated for the expression of BRC-Z1. Even though BRC-Z1 is expressed in the neurogenic regions of the disc, studies with BRC-Z1 mutants show that altered expression of BRC-Z1 does not interfere with the differentiation of the wing or its sensory neurons. Nevertheless the precocious expression of BRC-Z1 in cells lacking USP function provides insight into what would be expected for the genes directly involved in neuronal birth and differentiation. BRC-Z1 expression appears to be activated by events during the mid-third instar, but it is suppressed via USP until the titer of 20E is high enough to remove this USP-mediated silencing. In this context the presence of the hormone is permissive in that it allows other factors (induced by the mid-third instar transition?) to take control of gene expression. This de-repression contrasts with the other class of ecdysteroid-dependent events, such as the up-regulation of the early response genes (EcR, DHR3, E75B). EcRA, for example, is not up-regulated in the absence of USP, with or without hormone (Schubiger, 2000).

In Drosophila, pulses of the steroid hormone ecdysone trigger larval molting and metamorphosis and coordinate aspects of embryonic development and adult reproduction. At each of these developmental stages, the ecdysone signal is thought to act through a heteromeric receptor composed of the EcR and USP nuclear receptor proteins. Mutations that inactivate all EcR protein isoforms (EcR-A, EcR-B1, and EcR-B2) are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heat shock promoter drives expression of an EcR cDNA, temperature-dependent rescue of EcR null mutants has been employed to determine EcR requirements at later stages of development. EcR is required for hatching, at each larval molt, and for the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone-responsive genes is blocked and normal ecdysone responses of both imaginal and larval tissues are blocked at an early stage. These results show that EcR mediates ecdysone signaling at multiple developmental stages and implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis (Li, 2000).

Western analysis of extracts from rescued EcR mutants using antibodies directed against products of five ecdysone-responsive genes shows that expression of three early response genes are largely (BR-C) or completely (E74A, E75B) abolished. Expression of the early-late gene DHR3 and the mid-prepupal response gene betaFTZ-F1 are also severely affected. These results show that expression of ecdysone-responsive genes early in metamorphosis is dependent on EcR. The results confirm and extend to the whole animal earlier findings from analysis of larval salivary gland polytene chromosomes that transcriptional puffing of ecdysone-responsive genes is blocked in EcR-B1 mutants. The retention of low levels of BR-C 91 and 81 kDa products in EcR mutants is consistent with the incomplete block to BR-C puffing previously seen in EcR-B1 mutants (Li, 2000).

The Drosophila homolog of the retinoid X receptor, ultraspiracle (Usp), heterodimerizes with the ecdysone receptor (EcR) to form a functional complex that mediates the effects of the steroid molting hormone ecdysone by activating and repressing expression of ecdysone response genes. As with other retinoid X receptor heterodimers, EcR/USP affects gene transcription in a ligand-modulated manner. The functions were analyzed of two usp alleles, usp³ and usp⁴, which encode stable proteins with defective DNA-binding domains. Usp is able to activate as well as repress the Z1 isoform (BrC-Z1) of the ecdysone-responsive Broad Complex gene. Activation of BrC-Z1 as well as EcR, itself an ecdysone response gene, can be mediated by both the USP3 and USP4 mutant proteins. USP3 and USP4 also activate an ecdysone-responsive element, hsp27EcRE, in cultured cells. These results differ from the protein null allele, usp², which is unable to mediate activation. BrC-Z1 repression is compromised in all three usp alleles, suggesting that repression involves the association of Usp with DNA. These results distinguish two mechanisms by which Usp modulates the properties of EcR: one that involves the Usp DNA-binding domain and one that can be achieved solely through the ligand-binding domain. These newly revealed properties of Usp might implicate similar properties for retinoid X receptor (Ghbeish, 2001).

These data suggest a separation between the repressive functions of Usp and some of its activating functions, since the Usp DBD is dispensable for the activation of some ecdysone targets. usp³ and usp⁴ are capable of heterodimerizing with EcR, although they are defective in DNA binding. On some EcREs, USP3 or 4/EcR heterodimers mediate activation. In culture, the Usp LBD alone seems sufficient for the formation of an activating complex with EcR. Thus, for some genes, models explaining USP/EcR gene activation must accommodate the fact that the Usp DNA-binding domain is not necessary, whereas the LBD is. One such model posits that EcR monomers, homodimers, or alternative EcR complexes can bind some EcREs but only activate these response elements if the Usp LBD is present to promote formation of the EcR complex, ligand-binding, and/or interaction with coactivators. This model suggests that USP3, USP4, USPL, and possibly USP+ can activate through a multimeric complex in which the LBDs heterodimerize and DNA binding occurs largely via one or more EcR DBDs. In support of this model, ligand-induced EcR homodimers are able to form on DNA (Ghbeish, 2001).

In contrast to activation, repression of BrC-Z1 clearly requires functional Usp DNA-binding abilities, whereas its post-furrow activation in the eye imaginal disc does not. The apparent differential requirement for DNA binding in repression and activation suggests that, in some situations, the switch between repression and activation regulated by the EcR/Usp heterodimer may involve more than just changes in the LBD in response to ligand. It is also possible that normally the switch from repression to activation occurs without a change in the DNA binding of either EcR or Usp but that on some target sites in the absence of the wild-type complex, an alternative complex can form and allow activation (Ghbeish, 2001).

The ability of added wild-type Usp to restore BrC-Z1 repression in the eye imaginal disc suggests that the Z1 isoform of BrC may be a direct target of Usp regulation. Since it has been shown that Usp has the ability to homodimerize on a DNA element able to mediate repression in cultured Drosophila cells, it is possible that an alternative Usp complex other than EcR/Usp represses BrC-Z1. If Usp is able to repress target genes through a homodimer but requires heterodimerization with EcR to mediate activation, a situation could arise in which gene repression absolutely requires the DNA-binding activity of Usp while this function can be abolished for gene activation (Ghbeish, 2001).

In this study a dual role has been uncovered for Usp in the ecdysone response. Depending on the particular target gene, activation and repression may be more complicated than just a simple ligand-activated switch. This adds potential complexity to the roles that ecdysone, Usp, and EcR play in metamorphosis. This work separates aspects of the Usp component of the ecdysone response into repressive and activating functions, with unique and separable effects attributable to the DNA-binding and ligand-binding domains (Ghbeish, 2001).

Response to the insect hormone ecdysone is mediated by a nuclear receptor complex containing Ultraspiracle (Usp) and the Ecdysone Receptor (EcR). Among other phenotypes, loss of functional Usp in Drosophila eye development results in an accelerated morphogenetic furrow, although loss of ecdysone arrests the furrow. Usp both represses and activates a gene affecting furrow movement, the ecdysone-responsive Z1 isoform of Broad-Complex. Using targeted replacement of Usp to rescue usp mutant clones in the eye, various USP functions have been mapped and whether the USP nuclear receptor has an activating as well as a repressive effect on furrow movement has been tested. Furrow movement and related phenotypes are rescued by the presence of Usp in a limited domain near the furrow, while other phenotypes are rescued by Usp expression posterior to the furrow. These data indicate roles for Usp activity at multiple developmental stages and help explain why loss of functional Usp leads to furrow advancement while loss of ecdysone stops furrow movement (Ghbeish, 2002).

These data demonstrate that the usp gene regulates the expression of multiple genes involved in differentiation in developing imaginal discs. Most, but not all, usp mutant phenotypes in developing discs appear to involve premature gene expression in otherwise normal target cells. In the eye disc, normal morphogenetic furrow movement, photoreceptor differentiation and ommatidial organization depend on Usp activity in a limited domain near the furrow while other functions require Usp posterior to the furrow. The usp gene likely affects furrow movement and photoreceptor differentiation in part by regulating the BrC-Z1. Loss of the function of the Usp protein as a repressor of BrC-Z1, and perhaps other genes, in the furrow region is sufficient to account for many of the usp^- furrow-associated phenotypes. Conversely, stoppage of the furrow in the absence of ecdysone probably results from continued BrC-Z1 repression by the EcR/Usp complex (Ghbeish, 2002).

Examination of the role of the usp gene in the regulation of BrC-Z1 may be particularly informative. BrC-Z1, a known target for regulation by Usp/EcR protein complexes, is not normally expressed anterior to the furrow, but is upregulated just posterior to the furrow. Loss of Usp nuclear receptor repressive function leads to high level expression of BrC-Z1 protein both anterior and posterior to the furrow. A null allele of usp shows no further post-furrow activation, but the partially functional usp³ allele allows increased posterior expression consistent with Usp-mediated activation as well as repression. Readdition of usp⁺ in a position specific manner shows that wild-type BrC-Z1 levels return once Usp protein is expressed (Ghbeish et al., 2001).

The expression of BrC-Z1 protein in the presence and absence of usp gene activity allows the analysis of the relative magnitude of the Usp nuclear receptor repression and activation functions in an in vivo context. Surprisingly, the level of post-furrow BrC-Z1 protein in wild-type regions is substantially lower than the level of expression resulting from the complete loss of usp gene activity. Thus, for at least this particular situation, the repression of intrinsic gene expression activity by an RXR-containing complex is far greater than the level of gene activation. This may explain why the eye phenotypes of all three usp alleles are similar, since both null and missense alleles have levels of BrC-Z1 protein well above those seen in wild-type discs (Ghbeish, 2002).

The observations concerning the effects of loss of usp gene function on BrC-Z1 protein expression may resolve a superficial paradox of eye development. usp mutations increase the rate of furrow movement while loss of ecdysone stops the furrow. Additionally, loss of ecdysone decreases the level of BrC-Z1 protein posterior to the furrow and loss of BrC-Z1 results in disruption of neural differentiation and the furrow. This suggests that ecdysone regulation in the eye is mediated in part by the BrC-Z1 gene. It is possible that additional ecdysone-responsive genes are regulated in this dual manner by the EcR/Usp complex during furrow movement. These results are completely consistent with the observations made in this study. In the absence of hormone activity, BrC-Z1 gene activation is repressed. Posterior to the furrow, the consequences of such repression help explain the furrow stoppage phenotype, especially as loss of BrC-Z1 gene function leads to a loss of the critical furrow activator, Hedgehog. By contrast, loss of the fully functional Usp nuclear receptor, far from leading to a loss of BrC-Z1 protein expression, leads to high level expression, both anterior and posterior to the furrow. This high level of BrC-Z1 protein in usp mutant regions may help explain the furrow advancement phenotypes. In the region in or very near the furrow, other regulatory genes (such as hairy, extramacrochaete, atonal, hedgehog, patched and decapentaplegic) function to initiate eye differentiation. High levels of BrC-Z1 protein in this region may induce slightly premature differentiation of photoreceptor cells. This would lead to slightly premature Hedgehog protein expression and possibly incrementally faster furrow movement. Summed across a large usp mutant clone, this would appear as notable furrow advancement (Ghbeish, 2002).

In summary, these results show a role for the usp gene in controlling the timing of gene expression and differentiation in developing imaginal discs. In the eye disc some functions are very closely linked to events at the morphogenetic furrow, while others are clearly posterior to the furrow. It is suggested that the Usp nuclear receptor mediates the effects of ecdysone on furrow movement and neuronal differentiation in the eye imaginal disc in part by regulating the BrC-Z1. The specifics of gene repression and gene induction in this interaction account for the differences between loss of the Usp protein and loss of ecdysone on furrow movement. This analysis shows that in vivo the magnitude of repression of the BrC-Z1 gene is far greater than the magnitude of activation (Ghbeish, 2002).

Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers molting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homolog Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. TRR is a histone methyltransferase capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. It is proposed that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters (Sedkov, 2003).

Rho-LIM kinase signaling regulates ecdysone-induced gene expression and morphogenesis during Drosophila metamorphosis

The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. A link has been established between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development (Chen, 2004).

Metamorphosis in Drosophila is stringently controlled by pulses of the steroid hormone ecdysone at discrete developmental stages. During larval-pupal transition, ecdysone triggers coordinated changes in tissue morphology that involve histolysis of larval tissues and the initiation of adult structures. Rho GTPase-mediated signaling pathways have been implicated in several aspects of morphogenesis during Drosophila embryo formation. However, a role for Rho signaling in metamorphosis has not yet been reported. Among the downstream mediators of Rho signaling are the LIM kinases, and a closely related Drosophila ortholog of mammalian LIM kinases (designated Dlimk) is specifically expressed at relatively high levels in late larval and pupal stages, suggesting a potential role in Rho-LIMK signaling during this transition. In adult flies, Dlimk is expressed at substantially higher levels in males than in females, consistent with a potential evolutionarily conserved role in spermatogenesis, a process in which mammalian LIMK2 has been implicated. Dlimk mRNA is uniformly expressed throughout eye, wing, and leg imaginal discs (Chen, 2004).

The malformed legs in Dlimk^D522A flies closely resemble leg defects in flies in which Rho signaling is perturbed through genetic disruption of Rho1, DrhoGEF2 (a guanine nucleotide exchange factor for Rho1), sqh (myosin light chain), and zipper (nonmuscle myosin heavy chain). Sqh and zipper are downstream targets of Drok and regulate actomyosin contractility. Loss-of-function mutants of Rho1 or DrhoGEF2 strongly suppress the severity of wing defects associated with Dlimk expression. Reducing Rho activity by overexpressing the potent Rho inhibitor, p190 RhoGAP, also efficiently suppresses Dlimk-induced wing defects. Moreover, reducing levels of Diaphanous or Drok, two Rho targets that promote actin assembly, also substantially reduces the severity of Dlimk-induced wing defects. A loss-of-function allele of blistered, the Drosophila SRF ortholog, also suppresses the Dlimk-induced wing defects, suggesting that regulation of SRF-dependent transcription by Rho-LIMK signaling plays a role in wing morphogenesis. Significantly, in mammalian cells, LIMK and Diaphanous cooperate to regulate SRF activity (Geneste, 2002). Reducing levels of the Rho-related GTPases, Rac1, Rac2, and Cdc42, or the Rac activator, Myoblast city (Mbc), or the Rac/Cdc42 effector target, PAK, has very little effect on the Dlimk-induced wing phenotype. Thus, it appears that in the developing leg and wing, Dlimk specifically mediates a Rho-actin signaling pathway required for imaginal-disc morphogenesis (Chen, 2004).

The observed interactions among Rho1, Dlimk, br, and Sb support a role for Rho signaling in ecdysone-regulated metamorphosis. However, neither Rho1 expression nor activation is ecdysone inducible. In light of studies linking Rho-LIMK signaling to effects on gene expression (Sotiropoulos, 1999), BR-C and Sb expression were examined in flies overexpressing Rho1, Dlimk, or p190 RhoGAP during early puparium stages, when disc morphogenesis is underway. Expression of BR-C and Sb mRNA normally peaks approximately 2-4 hr after puparium formation. However, in flies overexpressing Rho1 or Dlimk, expression of these genes persists well beyond the normal peak of expression seen in 'driver-only' control flies (approximately 8–10 hr after puparium formation. Moreover, expression of these genes is greatly reduced at all stages of pupation in flies expressing p190 RhoGAP. Significantly, although most of the transgenic flies that overexpress p190 RhoGAP die at a late pupal stage, the few 'escapers' that eclose exhibit malformed wings and twisted and bent leg phenotypes that are very similar to those seen in flies expressing Dlimk^D522A. In addition, the pupal lethality that is frequently observed with overexpression of p190 RhoGAP is efficiently rescued by coexpressing Dlimk, indicating that the late developmental defects that arise as a consequence of Rho inactivation largely reflect defects in Rho-LIMK signaling (Chen, 2004).

To examine more directly a requirement for a Rho-actin-SRF pathway in the transcriptional response to ecdysone, Drosophila SL2 cells were used. In SL2 cells, as in developing discs, ecdysone induces the expression of EcR mRNA. Transfection of cells with the Rho-inhibitory C3 toxin or pretreatment with the actin polymerization inhibitor, latrunculin B, substantially reduces the ecdysone-induced increase in EcR mRNA but does not affect transcription of the ecdysone-insensitive gene rp49 or the Rho1 gene. As expected, latrunculin B completely inhibits morphogenesis of leg appendages, indicating a requirement for F-actin assembly. To examine the role of SRF in ecdysone-induced EcR expression, SL2 cells were treated with RNAi corresponding to the blistered gene. RNAi-treated cells exhibit reduced SRF expression and an absence of ecdysone-induced EcR mRNA expression. Together, these results suggest that the ability of Rho and Dlimk to promote F-actin assembly and SRF activation is responsible for their effects on ecdysone-responsive gene expression and tissue morphogenesis. In addition, the findings in SL2 cells indicate that the observed effects of Rho-SRF signaling on the ecdysone response are cell-autonomous effects. Interestingly, genetic interactions have been observed between zipper and sb and between zipper and br, suggesting that Rho-regulated actomyosin contractility, in addition to F-actin assembly, may also influence the ecdysone response. In this regard, it is interesting to note that mechanical stretching of cells reportedly promotes SRF activity. Alternatively, actomyosin contractility may play a parallel role in disc morphogenesis that is independent of any direct regulation of the ecdysone response (Chen, 2004).

No motif has been identified within the 5′ and 3′ regulatory sequences (2 kb each) of the EcR gene has been identified that matches the reported SRF binding consensus site. Hence, it remains possible that an SRF-regulated coactivator of ecdysone receptor gene expression is a primary target of Rho-Dlimk signaling. It is interesting to note that the Drosophila transcription factor, Crooked legs, regulates expression of ecdysone receptor mRNA and is encoded by an ecdysone-inducible gene that is also required for wing and leg morphogenesis. Such findings highlight the complexity of the gene expression hierarchy involved in the morphogenetic response to ecdysone and indicate a likely role for transcriptional feedback mechanisms (Chen, 2004).

Krüppel homolog 1 mediates juvenile hormone action upstream of broad during metamorphosis

Juvenile hormone (JH) given at pupariation inhibits bristle formation and causes pupal cuticle formation in the abdomen of Drosophila due to its prolongation of expression of the transcription factor Broad (BR). In a microarray analysis of JH-induced gene expression in abdominal integument, it was found that Krüppel homolog 1 (Kr-h1) was up-regulated during most of adult development. Quantitative real-time PCR analyses showed that Kr-h1 up-regulation begins at 10 h after puparium formation (APF), and Kr-h1 up-regulation occurs in imaginal epidermal cells, persisting larval muscles, and larval oenocytes. Ectopic expression of Kr-h1 in abdominal epidermis using T155-Gal4 to drive UAS-Kr-h1 results in missing or short bristles in the dorsal midline. This phenotype was similar to that seen after a low dose of JH or after misexpression of br between 21 and 30 h APF. Ectopic expression of Kr-h1 prolonges the expression of BR protein in the pleura and the dorsal tergite. No Kr-h1 was seen after misexpression of br. Thus, Kr-h1 mediates some of the JH signaling in the adult abdominal epidermis and is upstream of br in this pathway. It was also show that the JH-mediated maintenance of br expression in this epidermis is patterned and that JH delays the fusion of the imaginal cells and the disappearance of Dpp in the dorsal midline (Minakuchi, 2008).

In the Coleoptera and Lepidoptera, the epidermal cells make larval, pupal, and adult cuticles sequentially. By contrast, in higher Diptera such as Drosophila melanogaster, the adult epidermis on the head and the thorax is derived from imaginal discs, and the adult epidermis on the abdomen is formed by imaginal cells derived from the histoblast nests that make larval cuticle, but do not divide during larval life. After puparium formation, the histoblasts begin to proliferate rapidly, displacing the larval epidermal cells that subsequently die after making the pupal cuticle. This process is complete by 40 h after puparium formation (APF). Juvenile hormone (JH) application to D. melanogaster during the final larval instar or during the prepupal stage has little effect on the adult differentiation of the head and the thoracic epidermis, but it prevents the normal adult differentiation of the abdominal epidermis that is derived from the histoblasts. After JH treatment, the histoblasts continue to divide to form the imaginal epidermis, but the normal outgrowth of abdominal bristles is prevented, and a second pupal, rather than adult, cuticle is formed (Minakuchi, 2008).

In insects ecdysteroids trigger molting, while JH determines the nature of the molt. When JH is present, the ecdysteroid-induced molt is to another like stage; whereas in its absence, metamorphosis ensues. Little is known about how the JH signal is mediated in preventing insect metamorphosis. The broad (br) gene, an ecdysone-induced transcription factor in the Broad-Tramtrack-Bric-a-brac (BTB) family, is the key regulator of the onset of metamorphosis, since amorphic D. melanogaster mutants of br (npr) can develop normally until the final larval instar but fail to begin metamorphosis. In the silkworm Bombyx mori, RNAi knock-down of br in imaginal discs and primordia resulted in their failure to undergo metamorphosis properly. It has been shown that the treatment of either D. melanogaster or M. sexta with a JH mimic (JHM) at the onset of adult development induces the re-expression of the br gene in the abdominal epidermis and that misexpression of br during the adult development of D. melanogaster results in the truncation of bristles and the formation of pupal cuticle by the imaginal cells, both of the abdomen and of the disc-derived head and thorax. In hemimetabolous insects such as the milkweed bug Oncopeltus fasciatus, br is expressed during embryonic development and each nymphal molt, then disappears at the molt to the adult. In this animal, JH is necessary to maintain br expression during the nymphal stages. Clearly, br can be regulated by JH. Yet little is known about the genes that are either upstream or downstream of br in the JH signaling pathway (Minakuchi, 2008).

Therefore a genome-wide analysis of JH-regulated genes was performed in the abdominal integument of D. melanogaster to which pyriproxyfen, a JHM, had been applied at the time of pupariation to suppress the adult differentiation of abdominal histoblasts. One of the up-regulated genes was Krüppel-homolog 1 (Kr-h1, CG9167). This study shows that the misexpression of Kr-h1 in the epidermal cells results in missing or short bristles in the dorsal midline of the adult fly, a phenotype similar to that seen after treating wild-type animals with a low dose of JH and to that seen after br was misexpressed early in adult development. This action of KR-H1 was found to be accompanied by the prolongation of BR expression in the abdominal epidermis, indicating that Kr-h1 is a key regulator functioning upstream of br in the JH signaling pathway. It was also found that JHM treatment delayed the development of the abdominal epidermis, thus altering the timing of Dpp expression in the dorsal midline (Minakuchi, 2008).

This study has identified Kr-h1 as one of the genes up-regulated by JHM treatment of Drosophila at pupariation that then persists during the entire pupal-adult developmental period. Moreover, the presence of KR-H1 during early adult development can induce the abnormal re-expression of br in the abdomen that results in the formation of a second pupal cuticle (Zhou, 2002; Minakuchi, 2008).

KR-H1 is a zinc-finger type transcription factor with three putative isoforms with different N-terminal sequences (Pecasse, 2000). There are two main isoforms with the β isoform being expressed mainly during nervous system development in the embryo (Beck, 2004). Normally Kr-h1α is expressed at low levels in midembryogenesis, at high levels during larval life, then declines rapidly after pupariation (Pecasse, 2000) and is not expressed again until just before adult eclosion (Beck and Richards, personal communication to Minakuchi, 2008). KR-H1α appears to be necessary for metamorphosis since most of the mutants lacking Kr-h1α function die at the time of head eversion to the pupa (Pecasse, 2000) or shortly thereafter (Minakuchi, 2008).

In insects ecdysteroids cause the molt and JH is present during larval life to ensure that the molt is to another larval stage by preventing the developmental program-switching action of ecdysteroids necessary for metamorphosis. In most holometabolous insects where the epidermal cells are polymorphic so that they produce sequentially larval, then pupal, then adult cuticles, this switching occurs in the final larval instar when the JH titer declines and ecdysone appears in the absence of JH. By contrast, in the highly derived Drosophila, the onset of metamorphosis triggered by ecdysone in the absence of JH results in the death of most of the larval tissues and the development of the pupa and subsequent adult from the imaginal discs. One exception is the larval abdominal epidermis which switches from production of larval cuticle to that of pupal cuticle. The subsequent adult cuticle is then made by imaginal cells derived from the abdominal histoblasts that begin proliferation shortly after pupariation. Importantly, in Drosophila JH cannot prevent the metamorphosis of the imaginal discs or the proliferation of the histoblasts but can delay the onset of metamorphosis; it also causes the formation of a pupal rather than an adult cuticle by the new imaginal cells of the abdomen (Minakuchi, 2008).

Kr-h1α is regulated at least in part by 20-hydroxyecdysone (20E) and in turn regulates the ecdysone-regulated processes (Pecasse, 2000; Beckstead, 2005). It shows a dynamic pattern of binding to certain ecdysteroid-regulated chromosomal sites during the 20E-induced cell death of the salivary glands at metamorphosis (Beck, 2005). In mutants that lack Kr-h1α function, the normal ecdysteroid cascade of transcription factors at pupariation is disrupted with some appearing precociously and others being delayed or reduced in amount (Pecasse, 2000). The result of this misregulation is retention of the salivary glands and death around the time of head eversion that signals completion of pupal development. Interestingly, although overexpression of Kr-h1 suppressed the initial morphogenesis of mushroom body neurons, its loss caused no detectable defects in neuronal morphogenesis, but rather affected the patterning of EcR-B1 expression in the central nervous system at the onset of metamorphosis (Shi, 2007). Thus, KR-H1 is clearly necessary for the proper coordination of the ecdysone response (Minakuchi, 2008).

Interestingly, the few escapers among the Kr-h1α mutants formed cryptocephalic pupae that developed into adults with pigmented eyes and wings but no adult abdominal differentiation beyond the proximal segments (Pecasse, 2000). Thus, they resemble pharate adults formed after treatment with JH at the time of pupariation. In these JH-treated animals, it was found in this study that Kr-h1 is up-regulated in imaginal abdominal epidermal cells, derivatives of imaginal discs (wing, leg, and eye), persisting larval muscles necessary for eclosion and wing-spreading behavior, and in larval oenocytes during the ecdysteroid rises for pupal head eversion and adult development. Yet the adult head and thorax appeared grossly normal after JH treatment. Likewise, there was no significant difference in the number of persisting larval muscles in JH-treated pupae, but the normal differentiation and attachment of adult muscles and the outgrowth of abdominal bristles were either inhibited or delayed at 45 h APF has been seen for the thoracic muscles and the abdominal bristles. The larval oenocytes are involved in lipid metabolism during growth and larval development, then persist through much of adult development where they appear critical for normal utilization of the stored lipid. In other insects such as Tenebrio, the oenocytes have been shown to produce ecdysteroids. Whether the presence of KR-H1 in these oenocytes during this latter period is responsible for any of the defects seen in JH-treated animals is unknown (Minakuchi, 2008).

The role of JH in the normal developmental expression of Kr-h1α in the Drosophila larva is not known. In the red flour beetle, Tribolium castaneum, the transcript level of Kr-h1 is high during larval life, decreases at the end of the final larval stage and disappears just before pupation, then remains very low during the pupal stage and the ensuing adult development just as in Drosophila (Pecasse, 2000). Importantly, RNAi-mediated knockdown of Kr-h1 in young (pre-final instar) Tribolium larvae resulted in precocious metamorphosis, indicating that Kr-h1 is necessary for mediating JH signals in normal larvae. The finding that Kr-h1α reappears abnormally in the abdomen of pupae that were treated with JH at pupariation suggests that its appearance is the normal larval response to ecdysone in the presence of JH. Clearly further work is necessary to work out the details of the normal hormonal regulation of Kr-h1 in the Drosophila larva (Minakuchi, 2008).

In the brain of the honeybee, Apis mellifera, a homolog of Kr-h1 was identified as one of the genes down-regulated by queen mandibular pheromone (Grozinger, 2003) and up-regulated during the transition to foraging behavior of the adult, which is initiated by JH (Grozinger, 2006). Whether JH directly or indirectly controls the transcription of this gene has not been determined (Minakuchi, 2008).

In Drosophila at the onset of metamorphosis, 20E induces a cascade of transcription factors including the different isoforms of br, E74, and E75 that serve to regulate tissue-specific genes involved in metamorphosis. For most tissues, this is the first appearance of Broad (BR), a BTB-domain containing transcription factor, which is necessary for metamorphosis. At this time, br is apparently regulated by KR-H1 since this protein has been localized to the 2B5 br gene site on the salivary gland chromosomes (Beck, 2005). In the abdominal epidermis, br specifies pupal cuticle formation, whether the cells are initially larval or whether the cells are the imaginal cells derived from the histoblasts. This study found that misexpression of Kr-h1α during adult development caused the re-expression of br in the imaginal epidermis of both the pleura and the dorsal abdominal tergites, but that misexpression of br during normal adult development did not lead to Kr-h1 misexpression. These data strongly suggest that the JH-induced KR-H1α is acting somehow to prevent the permanent cessation of br expression in at least some of the imaginal abdominal epidermal cells during the onset of adult development. The nature of this action is not understood including whether or not it acts directly or indirectly on br transcription (Minakuchi, 2008).

Importantly, this interaction of Kr-h1α and br is not essential for the normal expression of BR during the late third larval instar since in Kr-h1α mutants, BR is present at the time of wandering as in wild-type larva. Whether the normal time course of br activation and expression occurs in these mutants has yet to be studied (Minakuchi, 2008).

The ectopic expression of Kr-h1α in the abdominal epidermis resulted in missing or short bristles in the dorsal midline. A similar phenotype was observed after low JHM was applied at pupariation, or after misexpression of br during early adult development between 21 and 30 h APF. The abdominal epidermis around the dorsal midline is the most sensitive to JHM treatment. As the dose of JHM is increased, more bristles on the tergite are affected. In wild-type Canton S, 100 ng of pyriproxyfen prevents the outgrowth of abdominal bristles, resulting in a bald abdomen with few or no bristles. When JHM was applied to JH-resistant lines such as the Methoprene-tolerant mutant, the outgrowth of the majority of abdominal bristles was not completely blocked except for the bristles in the dorsal midline (Minakuchi, 2008).

By misexpressing Kr-h1 with the Gal4 driver T155 that expresses in the abdominal histoblasts and the derivative imaginal epidermal cells, it was not possible to mimic the complete loss of bristles as seen with the higher dose of JHM. Whether this is due to the strength of the T155 driver or whether this indicates the involvement of other pathways in the action of JH is unknown. The fact that BR persists longer in the dorsal midline cells of the tergite under control of KR-H1 driven by T155 than it does in the dorsolateral cells suggests that patterning elements may also be affected by JH. Zhou (2002) has showed that misexpression of the various isoforms of br between 30 and 39 h caused a truncated bristle phenotype, at early times on the head and thorax and at later times on the abdomen. Misexpression of BR-Z1 between 44 and 60 h has little effect on bristle formation but causes the formation of pupal cuticle. The present study has shown that the misexpression of br at even earlier times in developing adults (between 21 and 30 h APF) caused the loss of bristles and hairs in the dorsal midline of the tergite. This effect is mimicked by the ectopic expression of Kr-h1α in the larval and imaginal epidermis that in turn causes the prolongation of BR protein in the dorsal midline until at least 70 h (at 29°C). Presumably this BR misexpression prevented hair formation, bristle outgrowth and normal adult cuticle formation. Whether this effect of Kr-h1α on br expression is constrained to the dorsal midline by Dpp signaling or is modulated by other patterning genes is unclear (Minakuchi, 2008).

Interestingly, the pattern of BR expression in Kr-h1α-containing cells is different from the JH-induced BR pattern. In the Kr-h1α-directed BR expression, there appear to be more BR-expressing cells near the mid-dorsal line and fewer such cells laterally. Also, in the a5 region, more of the Kr-h1α-directed cells express BR than in the JH-treated animals, whereas fewer of those in a3 and a4 express BR. Thus, the KR-H1α-directed BR pattern looks like a triangle with most of the cells at the dorsal midline and very few cells in the lateral area expressing BR. Possibly this patchy BR staining in the anterior tergite directed by Kr-h1α is dependent on other local patterning elements that are not disturbed by JH. Further study of this difference is warranted (Minakuchi, 2008).

The histoblast nests start rapid mitosis after puparium formation, and the cell division continues until the imaginal cells from several nests migrate and fuse. After the imaginal cells replace the larval epidermal cells, they start the formation of abdominal bristles followed by the secretion of adult cuticle. Using the enhancer trap line (dpp-lacZ), it has been shown that Dpp expression is confined to the pleura and the dorsal midline in the posterior edge of the anterior compartment at 45 h APF. This study also observed a similar pattern of Dpp expression. Importantly, the spatial patterns of Dpp expression were very similar between control and JHM-treated animals, but the expression of Dpp and the differentiation of the abdominal epidermis were delayed in JHM-treated animals. Zhou (2002) has reported that the proliferation of imaginal epidermal cells on the abdomen occurs normally after JHM treatment at puparium formation such that there os no apparent difference in the size of the imaginal nest between JHM-treated and control animals at 18 and 30 h APF. This study investigated the migration of imaginal cells between 29 and 45 h APF in detail; it was found that JHM treatment at puparium formation delayed the migration of imaginal epidermal cells and/or the death of the larval epidermal cells during this period. Since death of the larval cells is tightly linked to the migration of the adult cells, one may not be able to separate the effects on the two. Whether JH delays the earlier rapid proliferative events needs to be restudied. These results indicate that JHM treatment disrupts the coordination of events in development of the adult abdominal epidermis, including the timing of Dpp expression, which is likely a consequence of the delayed development. Whether the JH-induced re-expression of br in the abdominal epidermis mediates this effect on dpp expression or whether there is a direct effect of JH on dpp expression is unknown (Minakuchi, 2008).

These studies have shown for the first time that the JH-mediated maintenance of br expression is patterned in the developing adult dorsal abdominal epidermis (the ventral epidermal cells were not investigated). The cells in the a1 region, the anterior-most of the segment, do not express BR in response to JHM. These cells, under normal conditions, show high levels of hedgehog (hh), and do not receive wingless (wg) and optomotor-blind (omb) signals. The lack of BR expression in these cells may be due to the lack of Wg signal. Wg signaling has been shown to be required for BR expression in the follicle cells of the dorsal appendage primordia of egg chambers. However, the loss of br expression in the cells in the p2 region is not likely to be caused by lack of Wg signal, because Wg is present at higher levels in the p2 region than in the p1 region where BR is expressed. Thus, various patterning elements apparently are also involved in the regulation of the JHM-induced br re-expression during adult development (Minakuchi, 2008).

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