Transcriptional interpretation of the EGF receptor signaling gradient

Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During Drosophila oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. This study reports the regulatory elements of broad (br) and pipe (pip), two important targets of EGFR signaling in Drosophila oogenesis. br is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. This pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to pip, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. This similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo (Fuchs, 2012).

Current models of pattern formation in Drosophila oogenesis involve multiple components, signaling pathways, and network motifs. Critical tests of these models require direct analysis of the cis-regulatory sequences of genes comprising the network. As a first step in this direction, this study identified the regulatory elements of br, a gene that plays a key role in eggshell patterning and morphogenesis. The dynamic pattern of br was found to be generated by superposition of the activities of two distinct regulatory regions, which drive br expression in nonoverlapping regions of space and display differential sensitivity to three transcription factors that act downstream of EGFR (Fuchs, 2012).

It was shown that loss of Mirr induces ectopic br expression in the dorsal midline follicle cells, but leads to a complete loss of br in the lateral cells, which form dorsal appendages. This region-specific effect can be now explained, and is fully consistent with our finding that Mirr represses the brE and activates brL regions, respectively. Previous studies suggest that Mirr functions as a dedicated repressor. Based on this theory, it is speculated that the activating effect of Mirr on the expression of the brL region is indirect and involves intermediate factors. In contrast, these results strongly suggest that Mirr represses the brE region directly (Fuchs, 2012).

In contrast to the brL region, which generates br expression in a two-domain pattern that is necessary for the formation of two eggshell appendages, the function of the brE region is unclear. At the same time, this regulatory region was instrumental in identification of a critical cis-element that controls the expression of pip, a gene which must be repressed in the dorsal follicle cells for proper induction of the DV polarity of the embryo. The regulatory regions of both br and pip contain a sequence essential for their transcriptional restriction to the ventral follicle cells. Moreover, the data suggest that the identified sequence is a direct sensor of Mirr, which is derepressed by EGFR. Thus, thus this study has upheld an earlier proposal that Mirr connects the EGFR-mediated patterning of the follicle cells to the DV patterning of the embryo. In the emerging transcriptional cascade, EGFR signaling down-regulates CIC, which derepresses Mirr, which in turn represses pip (Fuchs, 2012).

Previous studies have demonstrated that Mirr can repress pip, but suggested that this effect requires a relay mechanism. The current results, based on marked mirr overexpression clones, demonstrate that the effect is cell-autonomous. Other studies argue against Mirr-dependent pip repression, based on the fact that mirr mutant clones did not induce ectopic expression of pip. These results may be because of the fact that the mirr allele that was used is not a complete null and has residual activity sufficient for pip repression. It is argued that the current data, demonstrating pip derepression by deletion of a sequence that binds Mirr, provide a strong support for Mirr-dependent repression of pip. Thus, these findings close a long-standing gap in the chain of events that convert EGFR signaling to pipe repression, a key step in transmitting the DV polarity from the egg to the embryo (Fuchs, 2012).

EGFR-dependent patterning of the follicle cells and the resulting effects for patterning of the embryo represent canonical examples of inductive effects in development. Indeed, genetic connection between EGFR signaling and pipe repression are found in essentially all textbooks of development. However, as discussed above, the identity of transcription factors involved in pipe regulation remained controversial and the cis-regulatory sequences responsible for pipe repression were unknown. The current results, which established Mirr as a direct repressor or pipe and identified the regulatory element responding to Mirr, clearly change this status. Thus, the results provide a significant addition to a very important model of inductive signaling. The regulatory element of pipe was discovered using an approach that harnesses both conventional and modern techniques of gene regulation research and can be extended to other transcriptional targets of EGFR pathway in the follicle cells. Finally, it is noted that most of the available information on the transcriptional effects of EGFR signaling is related to gene activation (mediated by Pnt) or derepression (mediated by Cic). The current work reveals a mechanism for EGFR-dependent gene repression, mediated by Mirr. Given the central role played by the EGFR signaling in development, the identified regulatory sequences can shed light on other EGFR-dependent pattern formation events (Fuchs, 2012).

Dynamic model for the coordination of two enhancers of broad by EGFR signaling

Although it is widely appreciated that a typical developmental control gene is regulated by multiple enhancers, coordination of enhancer activities remains poorly understood. This study proposes a mechanism for such coordination in Drosophila oogenesis>, when the expression of the transcription factor Broad (BR) evolves from a uniform to a two-domain pattern that prefigures the formation of two respiratory eggshell appendages. This change reflects sequential activities of two enhancers of the br gene, early and late, both of which are controlled by the epidermal growth factor receptor (EGFR) pathway. The late enhancer controls br in the appendage-producing cells, but the function of the early enhancer remained unclear. This study found that the early enhancer is essential for the activity of the late enhancer and induction of eggshell appendages. This requirement can be explained by a mechanism whereby the BR protein produced by the early enhancer protects the late enhancer from EGFR-dependent repression. This complex mechanism is illustrated using a computational model that correctly predicts the wild-type dynamics of BR expression and its response to genetic perturbations (Cheung, 2013).

Temporal control of transcription can be provided by changes in the levels of inductive signals, by cross-regulatory interactions between genes, and by dynamic use of different enhancers. For example, the dynamic expression of rho in the early Drosophila embryo results from sequential activities of two different rho enhancers, responding to two different inductive cues. In another control strategy, the early enhancer initiates expression, and the late enhancer maintains it through positive autoregulation. This mechanism controls Krox20 during the hindbrain segmentation in vertebrates. Both of these scenarios are different from the mechanism that coordinates br enhancers in Drosophila oogenesis. First, both the early and late enhancers respond to the same inductive signal. Second, the early enhancer is needed not to the initiate the expression of the late enhancer, but to protect it from ectopic and premature repression (Cheung, 2013).

In the wild-type egg chamber, brL, the late enhancer, is repressed only in cells exposed to the maximal levels of GRK. In the absence of brE, the early enhancer, signaling levels sufficient for repression are realized in the appendage primordia, due to amplification of EGFR activation resulting from ectopic expression of rho. This model is supported by eggshell defects induced by the RNAi-based disruption of BR expression by brE, and by ectopic expression of rho mRNA and PNT-dependent loss of brL activity in the absence of BR. The requirement for the rho-dependent amplification of EGFR signaling was tested computationally, by analyzing a simplified model in which BR and PNT repress each other directly, without feedback by rho. Extensive exploration of the parameter space in this model could not identify a set of parameters that would be consistent with both the wild-type expression of brL and its response to genetic perturbations. Based on this, it is argued that amplification of EGFR signaling by rho is essential for explaining the results (Cheung, 2013).

Going beyond br and rho, it is noted that dozens of genes regulated by GRK are expressed in dynamic patterns. Some of these patterns may be explained using the proposed computational model based on the interplay of multiple enhancers and dynamic signals. Although these models are more complex than existing models of developmental patterning, their analysis is essential for understanding temporal control of gene expression in development (Cheung, 2013).

A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila

The early genes are a key group of ecdysone targets that function at the top of the signaling hierarchy. In the presence of ecdysone, early genes exhibit a highly characteristic rapid and powerful induction that represents a primary response. Multiple isoforms encoded by early genes then coordinate the activation of a larger group of late genes. While the general mechanism of ecdysone-dependent transcription is well characterized, it is not known whether a distinct mechanism governs the hormonal response of early genes. Previous work has found that one of the Drosophila early genes, E75, harbors multiple functional ecdysone response elements (EcREs). This study extends the analysis to Broad and E74 and has found that EcRE multiplicity is a general feature of the early genes. Since most of the EcREs within early gene loci are situated distantly from promoters, the chromosome conformation capture method was used to determine whether higher order chromatin structure facilitates hormonal activation. For each early gene chromatin loops were detected that juxtapose their promoters and multiple distant EcREs prior to ecdysone activation. These findings suggest that higher order chromatin structure may serve as an important mechanism underlying the distinct response of early genes to ecdysone (Bernardo, 2014).

Transcriptional regulation

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 DlimkD522A 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 DlimkD522A. 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).

Pattern formation by a moving morphogen source.

During Drosophila melanogaster oogenesis, the follicular epithelium that envelops the germline cyst gives rise to an elaborate eggshell, which houses the future embryo and mediates its interaction with the environment. A prominent feature of the eggshell is a pair of dorsal appendages, which are needed for embryo respiration. Morphogenesis of this structure depends on broad, a zinc-finger transcription factor, regulated by the EGFR pathway. While much has been learned about the mechanisms of broad regulation by EGFR, current understanding of processes that shape the spatial pattern of broad expression is incomplete. It is proposed that this pattern is defined by two different phases of EGFR activation: an early, posterior-to-anterior gradient of EGFR signaling sets the posterior boundary of broad expression, while the anterior boundary is set by a later phase of EGFR signaling, distributed in a dorsoventral gradient. This model can explain the wild-type pattern of broad in D. melanogaster, predicts how this pattern responds to genetic perturbations, and provides insight into the mechanisms driving diversification of eggshell patterning. The proposed model of the broad expression pattern can be used as a starting point for the quantitative analysis of a large number of gene expression patterns in Drosophila oogenesis (Zartman, 2011).

The EGFR-mediated patterning of the follicular epithelium provides a striking example of how complex gene expression domains can be specified parsimoniously by a single pathway. A simple model is presented that explains the two-dimensional pattern of BR, a transcription factor that marks the cells contributing to the roof part of the dorsal appendages in Drosophila oogenesis. This model can be summarized as follows: the two BR domains are limited to the anterior domain by the earlier phase of EGFR signaling, which defines an anterior band of cells competent to express high levels of BR necessary for the formation of dorsal appendages. At a later stage of oogenesis, the level sets, i.e. lines of constant concentration, of the dorsoventral EGFR signaling gradient intersect with this competence zone, splitting the BR expression domain into two patches (Zartman, 2011).

In addition to rationalizing the wild-type pattern of BR, the model can be used to generate hypotheses regarding the patterning of eggshell morphologies in other Drosophila species. The number and size of dorsal appendages vary greatly across the phylogenetic spectrum, providing a mechanism of adaptation to the nature of the oviposition substrate. Since BR is a key regulator of dorsal appendage morphogenesis, one can expect that changes in the expression pattern of BR can provide a mechanism for the diversification of dorsal appendages. As a first step toward testing this hypothesis, the expression of BR was analyzed in mid-oogenesis, when the domains of elevated BR are first established (Zartman, 2011).

It was found that the BR pattern is dynamic and shows significant transitions during development; however, several stereotypic patterns at stage 10B of oogenesis emerge for species that have 2, 3 or 4 dorsal appendages. The number of contiguous BR patches is not equal to the number of dorsal appendages. At the same time, a clear pattern emerges regarding changes in the concavity of the BR domain. For species with two appendages, the BR patches are split along the dorsal midline (along the DV axis) and each of the two patches has a dorsal anterior boundary that is concave relative to the DV/AP coordinate system. Species with three dorsal appendages show a continuous BR patch that is convex. For four appendages, the curvature of the boundary appears to switch between concave and convex and then back to concave (Zartman, 2011).

Thus, the shape of the boundary of the BR expression domain diverges across species and may alternate between convex and concave (D. virilis) or may be simply convex (D. phalerata). With the change in concavity, the number of appendages also changes from 2 for Dm to 3 for Dp and 4 for Dv. A mathematical model can recapitulate qualitatively some aspects of the transition in BR expression, simply by varying the strength of the negative feedback and thresholds. One mechanism for converting a Dm pattern into a Dp pattern involves reducing the strength of inhibition (or the shape of the GRK source) and shifting the anterior/posterior pre-pattern in the posterior direction. A comprehensive comparison of the shape of the GRK source and the function of the feedback inhibitors across species will provide a further test of this model. At the same time, investigation of BR patterning in other species can establish the limits of the model (Zartman, 2011).

One of the most important questions for future work is how the quantitative changes in the expression pattern of BR give rise to discrete changes in the number of dorsal appendages. It is speculated that the local concavity of the BR pattern drives the temporal order of cell intercalations and specifies where the floor cells form a hinge that closes the forming tube. As such, changes in concavity could lead to mechanical 'instabilities' that further subdivide the BR cells into smaller domains to form extra tubular appendages. Interestingly, recent work by Boyle (2010) provides possible support for this model: that study found that genetic perturbations or laser ablation of the cells along the dorsal anterior boundary of the roof domain blocks tube formation. The next steps in increasing the scale of understanding regarding patterning and morphogenesis will require models that integrate geometry and mechanics with signaling dynamics, as well as quantitative approaches to validating model predictions (Zartman, 2011).

Wnt signaling cross-talks with JH signaling by suppressing Met and gce expression

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous juvenile hormone analogs (JHA). A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression are suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, it is concluded that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages (Abdou, 2011).

JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).

In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).

The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).

Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).

As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-armS10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-armS10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).

Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).

JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).

Drosophila Kdm4 demethylases in histone H3 lysine 9 demethylation and ecdysteroid signaling

The dynamic regulation of chromatin structure by histone post-translational modification is an essential regulatory mechanism that controls global gene transcription. The Kdm4 family of H3K9me2,3 and H3K36me2,3 dual specific histone )emethylases has been implicated in development and tumorigenesis. This study shows that Drosophila Kdm4A and Kdm4B, both members of the JHDM3 histone demethylase family are together essential for mediating ecdysteroid hormone signaling during larval development. Loss of Kdm4 genes leads to globally elevated levels of the heterochromatin marker H3K9me2,3 and impedes transcriptional activation of ecdysone response genes, resulting in developmental arrest. It was further shown that Kdm4A interacts with the Ecdysone Receptor (EcR) and colocalizes with EcR at its target gene promoter. These studies suggest that Kdm4A may function as a transcriptional co-activator by removing the repressive histone mark H3K9me2,3 from cognate promoters (Raueumi, 2013).

This study have discovered a role for Kdm4 in the transcriptional regulation of a subset of ecdysone pathway components. Furthermore, an interaction was demonstrated between Kdm4A and EcR in vivo, providing evidence that Kdm4 demethylases may act as co-activators of EcR. A genetic approach has allowed facilitated the detection of a previously uncharacterized, but essential, role of Kdm4 in development, and has identified a direct Kdm4 target gene in euchromatin. Interestingly, Human Kdm4 members interact with the nuclear hormone receptors, Androgen Receptor (AR) and Estrogen Receptor α (ERα), and has been proposed to serve as co-activators, suggesting a molecular mechanism by which Kdm4 can act as an oncogene in prostate and breast cancers. Kdm4B was shown to be a direct target gene of ERα, yielding a feed-forward loop for an augmented hormonal response. The results indicate that a similar epigenetic mechanism exists in Drosophila, where a nuclear hormone receptor requires the Kdm4 family of demethylases to remove H3K9 methylation at the promoter of a target gene. Taken together, the Kdm4 family of demethylases may function as transcriptional co-factors required for transcriptional activation by nuclear hormone receptors (Raueumi, 2013).

Previous studies have shown that the Trithorax-related (Trr) H3K4 methyltransferase, the Nurf nucleosome remodeling complex component, Nurf301, the Brahma (Brm)-containing chromatin remodeler, and the histone acetyltransferase CREB-binding protein (CBP) are also co-activators of EcR, indicating that activation of ecdysone pathway genes requires substantial regulation of the chromatin environment. Since H3K4 hyper-methylation at promoters is a marker of active transcription, and since H3K9 hypo-methylation also promotes upregulation of gene expression, it is feasible that synchronizing these two events would lead to more robust target gene activation. The mammalian Kdm4B (JMJD2B) forms a complex with the mixed-lineage leukemia (MLL) 2 H3K4 methyltransferase and serves as a co-activator of Estrogen Receptor. The complex couples H3K9 demethylation with H3K4 methylation in order to facilitate ERα target gene activation. Similar functional cross-talk between H3K9 demethylation and H3K4 methylation has been described in S. pombe, where the Lsd1 H3K9 demethylase and the Set1 H3K4 methyltransferase were found in a complex. Since, in Drosophila, the Nurf301 subunit, Brm and CBP were also found to interact with EcR, nucleosome remodeling may cooperate as well in the rapid and dynamic activation of ecdysone regulated genes (Raueumi, 2013).

These studies of the Kdm4A and Kdm4B homozygous double mutants demonstrate a requirement for these genes in the ecdysone pathway. This observation is similar to results obtained with mutant alleles of Nurf301 and trr, two seemingly ubiquitous chromatin regulators, where specific downregulation of ecdysone signaling genes has been detected. Additionally, this study is consistent with the reports that adult male Kdm4A mutants display abnormal courtship behavior and concomitant downregulated fru, a gene speculated to be a direct downstream target of EcR (Beckstead, 2005; Dalton, 2009). The specific defects in ecdysone signaling, rather than general transcription, exhibited by the double mutants indicate that either Kdm4 may not be essential for regulating all genes, or that the aberrant expression of ecdysone responsive genes is the earliest manifestation of loss of Kdm4. However, this study does not rule out the possibility that Kdm4 proteins regulate other crucial transcription factors that in turn regulate ecdysone pathway components by secondary effects. Further molecular and genomic studies are required to resolve this issue (Raueumi, 2013).

H3K9 demethylation-dependent transcriptional activation of BR-C was demonstrated. It is possible however, that H3K36 demethylation also contributes to ecdysone pathway component regulation. Previous studies have shown that HP1a is recruited to developmental puffs in polytene chromosomes and that it stimulates H3K36 demethylation by Kdm4A. Perhaps H3K36 demethylation in the gene body and subsequent displacement of the HDAC complex is important for transcriptional elongation or for the activation of downstream nested promoters of ecdysone pathway components. Moreover, H3K36 plays a role in exon splice choice and thus ecdysone pathway genes that produce multiple splice variants may require Kdm4 regulation. However, immunostaining experiments show that HP1a and Kdm4A signals are mostly non-overlapping. Thus, it seems that HP1a's involvement in the demethylase activities of Kdm4 toward H3K9 or H3K36 would have to be transient and dynamic (Raueumi, 2013).

In summary, this study has shown that double homozygous mutants of the two Kdm4 genes in Drosophila display developmental delays and lethality, with compromised activation of ecdysone related genes. Furthermore, it was found that BR-C may be a direct target of H3K9 demethylation, and that the interaction between Kdm4A and EcR may be important in transcriptional activation of BR-C. These results provide insight into the physiological functions and mechanistic roles of Kdm4 in vivo. The interaction between Kdm4 and EcR awaits further investigation. It is conceivable that EcR directs the recruitment of Kdm4A to the promoter of its target genes, or alternatively, that EcR allosterically regulates the demethylase activity of Kdm4A, allowing removal of H3K9m2,3 only upon hormone signaling (Raueumi, 2013).

The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. This study found that the COP9 signalosome (CSN; see CSN5) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. A model is proposed in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM (Huang, 2014: PubMed).

Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

During development cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. This study examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. A cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa. In the wing, ecdysone signaling at the larva to puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva to puparium pulse during early metamorphosis, Broad expression plummets allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing (Guo, 2016).

This study presents a model for how the pulse of ecdysone at the larval to pupal transition impacts the cell cycle dynamics in the wing during metamorphosis. Ecdysone signaling at the larva to puparium transition induces Broad, which in turn represses Stg to generate a temporary G2 arrest, which synchronizes the cell cycle in the wing epithelium. As ecdysone levels decline, Broad expression plummets, allowing Stg to be re-activated resulting in a pulse of cdc2 activity that promotes a rapid G2/M progression during the final cell cycle in the wing. This ultimately culminates in the relatively synchronized cell cycle exit at 24h APF, coinciding with the second large pulse of ecdysone. This second pulse in the pupa activates a different set of transcription factors (not Broad), promoting the acquisition of terminal differentiation characteristics in the wing. In this way, two pulses of ecdysone signaling can both synchronize the final cell cycle by a temporary G2 arrest and coordinate permanent cell cycle exit with the acquisition of terminal differentiation characteristics in the wing (Guo, 2016).

Over 30 years ago it was shown that 20-HE exposure in Drosophila tissue culture cells induces a cell cycle arrest in G2-phase. This response appears to be shared among 3 different cell lines, Cl-8, Kc and S2. This study shows that in Kc cells, pulsed 20-HE exposure also leads to a G2 arrest followed by rapid cell cycle re-entry after 20-HE removal and a subsequent prolonged G1. This cell cycle response to a pulse of 20-HE is reminiscent of the cell cycle changes that occur during early metamorphosis in the pupal wings and legs (Guo, 2016).

It is worth considering why Kc and S2 cells, which are thought to be derived from embryonic hemocytes would exhibit a similar cell cycle response to 20-HE to the imaginal discs. Relatively little is known about how ecdysone signaling impacts embryonic hemocytes, although recent work suggests that ecdysone signaling induces embryonic hemocyte cell death under sensitized conditions. More is known about larval hemocytes, which differentiate into phagocytic macrophages and disperse into the hemolymph during the first 8h of metamorphosis. Ecdysone is involved in this maturation process, as lymph glands of ecdysoneless (ecd) mutants fail to disperse mature hemocytes and become hypertrophic in the developmentally arrested mutants. This suggests that the high levels of systemic ecdysone signaling at the larval-puparium transition mediates a switch from proliferation to cell cycle arrest and terminal differentiation for lymph gland hemocytes during metamorphosis. Without ecdysone signaling, hemocytes may continue to proliferate and fail to undergo terminal differentiation leading to the hypertrophic lymph gland phenotype observed. Interestingly, while the loss of broad also prevents proper differentiation of hemocytes similar to loss of ecd, loss of broad does not lead to the hypertrophy observed in ecd mutants. Further studies will be needed to examine whether the ecdysone induced cell cycle arrest in larval hemocytes occurs in the G2 phase, or whether their cell cycle arrest proceeds via a similar pathway to that shown in this study for the wing (Guo, 2016).

Multiple lines of evidence suggest that the ecdysone receptor complex in the larval wing acts as a repressor for certain early pupa targets and that the binding of ecdysone to the receptor relieves this repression. For example loss of EcR by RNAi or loss of the EcR dimerization partner USP, de-represses ecdysone target genes that are high in the early pupal wing such as Broad-Z1 and βFtz-F1. The EcR/USP heterodimer also cooperates with the SMRTR co-repressor in the wing to prevent precocious expression of ecdysone target genes such as Broad-Z1. Consistent with the hypothesis that a repressive EcR/USP complex prevents precocious expression of Broad-Z1 and thereby a precocious G2 arrest, inhibition of SMRTR can also cause a G2 arrest. Thus, in the context of the early pupal wing, it is proposed that the significant pulse of ecdysone at the larval to puparium transition relieves the inhibition of a repressive receptor complex, leading to Broad-Z1 activation. Consistent with this model, high levels of Broad-Z1 in the larval wing lead to precocious neural differentiation at the margin and precocious inhibition of stg expression in the wing pouch. Interestingly, a switch in Broad isoform expression also occurs during the final cell cycle in the larval eye, such that Broad-Z1 becomes high in cells undergoing their final cell cycle and entering into terminal differentiation. However in this case, Broad-Z1 expression is not associated with a G2 arrest and occurs in an area of high Stg expression, suggesting the downstream Broad-Z1 targets in the eye may be distinct or regulated differently from those in the wing (Guo, 2016).

The ecdysone receptor has also been shown to down regulate Wingless expression via the transcription factor Crol at the wing margin, to indirectly promote CycB expression. While a loss of EcR at the margin decreased CycB protein levels, the effects of EcR loss on CycB levels in the wing blade outside of the margin area were not obvious. It is suggested that in the wing, the role for EcR outside of the margin acts on the cell cycle via a different mechanism through stg. Consistent with a distinct mechanism acting in the wing blade, over-expression of Cyclin B in the early prepupal wing could not promote increased G2 progression or bypass the prepupal G2 arrest. Instead the results on the prepupal G2 arrest are consistent with previous findings that Stg is the rate-limiting component for G2-M cell cycle progression in the fly wing pouch and blade (Guo, 2016).

In order to identify the gene expression changes in the wing that occur in response to the major peaks of ecdysone during metamorphosis, RNAseq was performed on a timecourse of pupal wings. Major changes were observed in gene expression in this tissue during metamorphosis. In addition, known ecdysone targets were identified that are affected differently in the wing during the first larval-to-pupal ecdysone pulse and the second, larger pulse at 24h APF. Ecdysone signaling induces different direct targets with distinct kinetics. Furthermore specific targets, for example Ftz-F1 can modulate the expression of other ecdysone targets, to shape the response to the hormone. Thus, it is expected that a pulse of ecdysone signaling leads to sustained effects on gene expression and the cell cycle, even after the ecdysone titer returns to its initial state. These factors together with the differences in the magnitude of the ecdysone pulse may contribute to the differences in the response to the early vs. later pulses in the wing (Guo, 2016).

Ecdysone signaling can also affect the cell cycle and cell cycle exit via indirect mechanisms such as altering cellular metabolism. This is used to promote cell cycle exit and terminal differentiation in neuroblasts, where a switch toward oxidative phosphorylation leads to progressive reductive divisions, (divisions in the absence of growth) leading to reduced neuroblast cell size and eventually terminal differentiation. Although reductive divisions do occur in the final cell cycle of the pupa wing, this type of mechanism does not provide a temporary arrest to synchronize the final cell cycle in neuroblasts as is see in wings. Importantly, a striking reduction is seen in the expression of genes involved in protein synthesis and ribosome biogenesis in the wing during metamorphosis, consistent with the lack of cellular growth. Instead the increased surface area of the pupal wing comes from a flattening, elongation and apical expansion of the cells due to interactions with the extracellular matrix creating tension and influencing cell shape changes. This is also consistent with the findings that a significant number of genes associated with protein targeting to the membrane are increased as the wing begins elongation in the early pupa. Further studies will be needed to determine whether the changes in expression of genes involved in ribosome biogenesis and protein targeting to the membrane are controlled by ecdysone signaling, or some other downstream event during early wing metamorphosis (Guo, 2016).

Perhaps the most interesting and least understood aspect of steroid hormone signaling is how a diversity of cell-type and tissue-specific responses are generated to an individual hormone. Cell cycle responses to ecdysone signaling are highly cell type specific. For example abdominal histoblasts, the progenitors of the adult abdominal epidermis, become specified during embryogenesis and remain quiescent in G2 phase during larval stages. During pupal development, the abdominal histoblasts must be triggered to proliferate rapidly by a pulse of ecdysone to quickly replace the dying larval abdominal epidermis. This is in contrast to the behavior of the wing imaginal disc, where epithelial cells undergo asynchronous rapid proliferation during larval stages, but during metamorphosis the cell cycle dynamics become restructured to include a G2 arrest followed by a final cell cycle and entry into a permanently postmitotic state, in a manner coordinated with tissue morphogenesis and terminal differentiation (Guo, 2016).

How does the same system-wide pulse of ecdysone at the larval to puparium transition lead to such divergent effects on the cell cycle in adult progenitors? Surprisingly it seems to be through divergent effects on tissue specific pathways that act on the same cell cycle targets. In the abdominal histoblasts the larval to puparium pulse of ecdysone triggers cell cycle re-entry and proliferation via indirect activation of Stg, by modulating the expression of a microRNA miR-965 that targets Stg. This addition of the microRNA essentially allows ecdysone signaling to act oppositely on the same cell cycle regulatory target as Broad-Z1 does in the wing. Thus, tissue specific programs of gene regulatory networks can create divergent outcomes from the same system- wide hormonal signal, even when they ultimately act on the same target (Guo, 2016).

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. This study shows that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors (Syed, 2017).

This study shows that the steroid hormone ecdysone is required to trigger a major gene expression transition at mid-larval stages: central brain neuroblasts transition from Chinmo/Imp to Broad/Syncrip/E93. Furthermore, it was shown that Svp activates expression of EcR-B1 in larval neuroblasts, which gives them competence to respond to ecdysone signaling, thereby triggering this gene expression transition. Although a global reduction of ecdysone levels is likely to have pleiotropic effects on larval development, multiple experiments were performed to show that the absence or delay in late temporal factor expression following reduced ecdysone signaling is not due to general developmental delay. First, the EcR gene itself is expressed at the normal time (~56 hr) in the whole organism ecdysoneless1 mutant, arguing strongly against a general developmental delay. Second, a type II neuroblast seven-up mutant clone shows a complete failure to express EcR and other late factors, in the background of an entirely wild type larvae; this is perhaps the strongest evidence that the phenotypes that are described are not due to a general developmental delay. Third, lineage-specific expression of EcR dominant negative leads to loss of Syncrip and E93 expression without affecting Broad expression; the normal Broad expression argues against a general developmental delay. Fourth, live imaging was used to directly measure cell cycle times, and it was found that lack of ecdysone did not slow neuroblast cell cycle times. Taken together, these data support the conclusion that ecdysone signaling acts directly on larval neuroblasts to promote an early-to-late gene expression transition (Syed, 2017).

The role of ecdysone in regulating developmental transitions during larval stages has been well studied; it can induce activation or repression of suites of genes in a concentration dependent manner. Ecdysone induces these changes through a heteromeric complex of EcR and the retinoid X receptor homolog Ultraspiracle. Ecdysone is required for termination of neuroblast proliferation at the larval/pupal transition, and is known to play a significant role in remodeling of mushroom body neurons and at neuromuscular junctions. This study adds to this list another function: to trigger a major gene expression transition in mid-larval brain neuroblasts (Syed, 2017).

Does ecdysone signaling provide an extrinsic cue that synchronizes larval neuroblast gene expression? Good coordination of late gene expression is not seen, arguing against synchronization. For example, Syncrip can be detected in many neuroblasts by 60 hr, whereas Broad appears slightly later at ~72 hr, and E93 is only detected much later at ~96 hr, by which time Broad is low. This staggered expression of ecdysone target genes is reminiscent of early and late ecdysone-inducible genes in other tissues. In addition, for any particular temporal factor there are always some neuroblasts expressing it prior to others, but not in an obvious pattern. It seems the exact time of expression can vary between neuroblasts. Whether the pattern of response is due to different neuroblast identities, or a stochastic process, remains to be determined (Syed, 2017).

It has been shown preiously that the Hunchback-Krüppel-Pdm-Castor temporal gene transitions within embryonic neuroblasts are regulated by neuroblast-intrinsic mechanisms: they can occur normally in neuroblasts isolated in culture, and the last three factors are sequentially expressed in G2-arrested neuroblasts. Similarly, optic lobe neuroblasts are likely to undergo neuroblast-intrinsic temporal transcription factor transitions, based on the observation that these neuroblasts form over many hours of development and undergo their temporal transitions asynchronously. In contrast, this study shows that ecdysone signaling triggers a mid-larval transition in gene expression in all central brain neuroblasts (both type I and type II). Although ecdysone is present at all larval stages, it triggers central brain gene expression changes only following Svp-dependent expression of EcR-B1 in neuroblasts. Interestingly, precocious expression of EcR-B1 (worniu-gal4 UAS-EcR-B1) did not result in premature activation of the late factor Broad, despite the forced expression of high EcR-B1 levels in young neuroblasts. Perhaps there is another required factor that is also temporally expressed at 56 hr. It is also noted that reduced ecdysone signaling in ecdts mutants or following EcRDN expression does not permanently block the Chinmo/Imp to Broad/Syncrip/E93 transition; it occurs with variable expressivity at 120-160 hr animals (pupariation is significantly delayed in these ecdts mutants), either due to a failure to completely eliminate ecdysone signaling or the presence of an ecdysone-independent mechanism (Syed, 2017).

A small but reproducible difference was found in the effect of reducing ecdysone levels using the biosynthetic pathway mutant ecdts versus expressing a dominant negative EcR in type II neuroblasts. The former genotype shows a highly penetrant failure to activate Broad in old neuroblasts, whereas the latter genotype has normal expression of Broad (despite failure to down-regulate Chinmo/Imp or activate E93). This may be due to failure of the dominant negative protein to properly repress the Broad gene. Differences between EcRDN and other methods of reducing ecdysone signaling have been noted before (Syed, 2017).

Drosophila Svp is an orphan nuclear hormone receptor with an evolutionarily conserved role in promoting a switch between temporal identity factors. In Drosophila, Svp it is required to switch off hunchback expression in embryonic neuroblasts, and in mammals the related COUP-TF1/2 factors are required to terminate early-born cortical neuron production, as well as for the neurogenic to gliogenic switch. This study showed that Svp is required for activating expression of EcR, which drives the mid-larval switch in gene expression from Chinmo/Imp to Syncrip/Broad/E93 in central brain neuroblasts. The results are supported by independent findings that svp mutant clones lack expression of Syncrip and Broad in old type II neuroblasts (Tsumin Lee, personal communication to Chris Doe). Interestingly, Svp is required for neuroblast cell cycle exit at pupal stages, but how the early larval expression of Svp leads to pupal cell cycle exit was a mystery. The current results provide a satisfying link between these findings: Svp was shown to activate expression of EcR-B1, which is required for the expression of multiple late temporal factors in larval neuroblasts. Any one of these factors could terminate neuroblast proliferation at pupal stages, thereby explaining how an early larval factor (Svp) can induce cell cycle exit five days later in pupae. It is interesting that one orphan nuclear hormone receptor (Svp) activates expression of a second nuclear hormone receptor (EcR) in neuroblasts. This motif of nuclear hormone receptors regulating each other is widely used in Drosophila, C. elegans, and vertebrates (Syed, 2017).

The position of the Svp+ neuroblasts varied among the type II neuroblast population from brain-to-brain, suggesting that Svp may be expressed in all type II neuroblasts but in a transient, asynchronous manner. This conclusion is supported by two findings: the svp-lacZ transgene, which encodes a long-lived β-galactosidase protein, can be detected in nearly all type II neuroblasts; and the finding that Svp is required for EcR expression in all type II neuroblasts, consistent with transient Svp expression in all type II neuroblasts. It is unknown what activates Svp in type II neuroblasts; its asynchronous expression is more consistent with a neuroblast-intrinsic cue, perhaps linked to the time of quiescent neuroblast re-activation, than with a lineage-extrinsic cue. It would be interesting to test whether Svp expression in type II neuroblasts can occur normally in isolated neuroblasts cultured in vitro, similar to the embryonic temporal transcription factor cascade (Syed, 2017).

Castor and its vertebrate homolog Cas-Z1 specify temporal identity in Drosophila embryonic neuroblast lineages and vertebrate retinal progenitor lineages, respectively (Mattar, 2015). Although this study shows that Cas is not required for the Chinmo/Imp to Syncrip/Broad/E93 transition, it has other functions. Cas expression in larval neuroblasts is required to establish a temporal Hedgehog gradient that ultimately triggers neuroblast cell cycle exit at pupal stages (Syed, 2017).

Drosophila embryonic neuroblasts change gene expression rapidly, often producing just one progeny in each temporal transcription factor window. In contrast, larval neuroblasts divide ~50 times over their 120 hr lineage. Mushroom body neuroblasts make just four different neuronal classes over time, whereas the AD (ALad1) neuroblast makes ~40 distinct projection neuron subtypes. These neuroblasts probably represent the extremes (one low diversity, suitable for producing Kenyon cells; one high diversity, suitable for generating distinct olfactory projection neurons). This study found that larval type II neuroblasts undergo at least seven molecularly distinct temporal windows. If it is assumed that the graded expression of Imp (high early) and Syncrip (high late) can specify fates in a concentration-dependent manner, many more temporal windows could exist (Syed, 2017).

This study illuminates how the major mid-larval gene expression transition from Chinmo/Imp to Broad/Syncrip/E93 is regulated; yet many new questions have been generated. What activates Svp expression in early larval neuroblasts - intrinsic or extrinsic factors? How do type II neuroblast temporal factors act together with Dichaete, Grainy head, and Eyeless INP temporal factors to specify neuronal identity? Do neuroblast or INP temporal factors activate the expression of a tier of 'morphogenesis transcription factors' similar to leg motor neuron lineages? What are the targets of each temporal factor described here? What types of neurons (or glia) are made during each of the seven distinct temporal factor windows, and are these neurons specified by the factors present at their birth? The identification of new candidate temporal factors in central brain neuroblasts opens up the door for addressing these and other open questions (Syed, 2017).

Hormone-dependent control of developmental timing through regulation of chromatin accessibility

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. This study shows that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, it was demonstrated that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. A temporal cascade of transcription factors was uncovered following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, the ecdysone-induced transcription factor E93 was shown to control temporal identity by directly regulating chromatin accessibility across the genome. Notably, it was found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility (Uyehara, 2017).

The importance of master transcription factors in specifying spatial identity during development suggests that they may control where other transcription factors bind in the genome. One prediction of this model is that tissues whose identities are determined by different master transcription factors would exhibit different genome-wide DNA-binding profiles. However, it was recently found that the Drosophila appendages (wings, legs, and halteres), which use different transcription factors to determine their identities, share nearly identical open chromatin profiles. Moreover, these shared open chromatin profiles change coordinately over developmental time. There are two possible explanations for these findings. Either (1) different transcription factors produce the same open chromatin profiles in different appendages or (2) transcription factors shared by each appendage control open chromatin profiles instead of the master transcription factors of appendage identity. The second model is favored for several reasons. Since the appendage master transcription factors possess different DNA-binding domains with distinct DNA-binding specificities, it is unlikely for them to bind the same sites in the genome. Supporting this expectation, ChIP for Scalloped and Homothorax, two transcription factors important for appendage identity, shows clear tissue-specific binding in both the wing and eye–antennal imaginal discs. The second model is also preferred because it provides a relatively straightforward mechanism for the observed temporal changes in open chromatin: By changing the expression of the shared temporal transcription factor over time, the open chromatin profiles that it controls would change as well. In contrast, expression of appendage master transcription factors is relatively stable over time, making it unlikely for them to be sufficient for temporal changes in open chromatin (Uyehara, 2017).

It is proposed that control of chromatin accessibility in the appendages is mediated at least in part by transcription factors downstream from ecdysone signaling. According to this model, a systemic pulse of ecdysone initiates a temporal cascade of hormone-induced transcription factor expression in each of the appendages. These are referred to as 'temporal' transcription factors. Temporal transcription factors can directly regulate the accessibility of transcriptional enhancers by opening or closing them, thereby conferring temporal specificity to their activity and driving development forward in time. Master transcription factors then bind accessible enhancers depending on their DNA-binding preferences (or other means of binding DNA) and differentially regulate the activity of these enhancers to control spatial patterns of gene expression, thus shaping the unique identities of individual appendages (Uyehara, 2017).

The experiments with E93 provide direct support for this model. In wild-type wings, thousands of changes in open chromatin occur after the large pulse of ecdysone that triggers the end of larval development. In E93 mutants, ~40% of these open chromatin changes fail to occur. Importantly, nearly three-quarters of sites that depend on E93 for accessibility correspond to temporally dynamic sites in wild-type wings. Thus, chromatin accessibility is not grossly defective across the genome; instead, defects occur specifically in sites that change in accessibility over time. This finding, combined with the large fraction of temporally dynamic sites that depend on E93 for accessibility, indicates that E93 controls a genome-wide shift in the availability of temporal-specific transcriptional enhancers. Supporting this hypothesis, temporal-specific enhancers depend on E93 for both accessibility and activity. Since it is proposed that the response to ecdysone is shared across the appendages, it is predicted that similar defects occur in appendages besides the wing. It remains to be seen whether other ecdysone-induced transcription factors besides E93 control accessibility of enhancers at different developmental times. It also remains to be seen how the temporal transcription factors work with the appendage master transcription factors to control appendage-specific enhancer activity (Uyehara, 2017).

The findings suggest that E93 controls temporal-specific gene expression through three different modalities that potentially rely on three distinct biochemical activities. The enrichment of E93 motifs and binding of E93 to temporally dynamic sites indicate that it contributes to this regulation directly. It is proposed that these combined activities drive development forward in time by turning off early-acting enhancers and simultaneously turning on late-acting enhancers (Uyehara, 2017).

First, as in the case of the tenectin tncblade enhancer, active most strongly in the interveins between the first and second and between the fourth and fifth longitudinal veins and in cells near the proximal posterior margin, E93 appears to function as a conventional activator. In the absence of E93, tncblade fails to express at high levels, but the accessibility of the enhancer does not measurably change. This suggests that binding of E93 to tncblade is required to recruit an essential coactivator. Importantly, this finding demonstrates that E93 is not solely a regulator of chromatin accessibility. E93 binds many open chromatin sites in the genome without regulating their accessibility and thus may regulate the temporal-specific activity of many other enhancers. In addition, since the tncblade enhancer opens between L3 and 24 h even in the absence of E93, there must be other factors that control its accessibility, perhaps, for example, transcription factors induced by ecdysone earlier in the temporal cascade (Uyehara, 2017).

Second, as in the case of the nubvein enhancer, E93 is required to promote chromatin accessibility. In this capacity, E93 may function as a pioneer transcription factor to open previously inaccessible chromatin. Alternatively, E93 may combine with other transcription factors, such as the wing master transcription factors, to compete nucleosomes off DNA. Testing the ability of E93 to bind nucleosomal DNA will help to discriminate between these two alternatives. In either case, it is proposed that this function of E93 is necessary to activate late-acting enhancers across the genome. Since only half of E93-dependent enhancers are directly bound by E93 at 24 h, it is also possible that E93 regulates the expression of other transcription factors that control chromatin accessibility. Alternatively, if E93 uses a “hit and run” mechanism to open these enhancers, the ChIP time point may have been too late to capture E93 binding at these sites (Uyehara, 2017).

Finally, as in the case of the broad brdisc enhancer, E93 is required to decrease chromatin accessibility. It is proposed that this function of E93 is necessary to inactivate early-acting enhancers across the genome. Current models of gene regulation do not adequately explain how sites of open chromatin are rendered inaccessible, but the ability to turn off early-acting enhancers is clearly an important requirement in developmental gene regulation. It may also be an important contributor to diseases such as cancer, which exhibits widespread changes in chromatin accessibility relative to matched normal cells. Thus, this role of E93 may represent a new functional class of transcription factor (“reverse pioneer”) or conventional transcriptional repressor activity. Additional work is required to decipher the underlying mechanisms. Notably, recent work on the temporal dynamics of iPS cell reprogramming suggest a similar role for Oct4, Sox2, and Klf4 in closing open chromatin to inactivate somatic enhancers (Chronis, 2017; Uyehara, 2017 and references therein).

broad: Biological Overview | Evolutionary Homologs | Targets of Activity | Developmental Biology | Effects of Mutation | References

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