Ecdysone receptor

Promoter

During the development of the adult central nervous system (CNS) of the fruitfly, the A-isoform of the Ecdysone receptor (EcR-A), a typical nuclear hormone receptor, is expressed at high levels in the Type II neurons, a set of neurons that die shortly after the emergence of the adult. To understand the role that transcriptional regulation of nuclear receptor genes plays in CNS development, the region controlling the transcription of EcR-A has been dissected by analyzing the ability of this region to drive the expression of reporter genes in transgenic animals. These analyses have demonstrated that the Type II neurons are a heterogeneous collection of neurons that utilize different regulatory elements to coordinate the expression of the same transcript (Sung, 2000).

Most neurons express low levels of EcR-A during metamorphosis and in the adult. However during the early stages of metamorphosis approximately 300 neurons, termed Type II, up-regulate the expression of EcR-A and maintain high levels throughout metamorphosis and into the adult. Shortly after emergence of the adult (eclosion), 100% of the Type II neurons undergo programmed cell death and die by apoptosis. The death of these neurons requires the withdrawal of ecdysteroids. Twelve of these 300 Type II neurons, designated n1, n2, n3, n4, and n5, are readily identifiable based on the expression of high levels of EcR-A protein and their unique position in the ventral nervous system. Of these twelve Type II neurons, all are recognized as pairs except the quartet of n4 neurons. The four n4 neurons are likely to represent two distinct pairs, but these pairs cannot be distinguished based on their position within the ventral nervous system. In addition to the doomed Type II neurons and the majority of neurons that survive and express low levels of EcR-A, four additional identifiable neurons, the n6 and n7 pairs, regulate EcR-A expression in a unique temporal manner. While the n6/n7 neurons express high levels of EcR-A protein in the newly eclosed adult as do the Type II neurons, the n6 and n7 neurons do not die. Developmentally, the n6 neurons up-regulate EcR-A at the outset of metamorphosis only to down-regulate expression during mid-metamorphosis. Approximately 24 h before eclosion EcR-A is again upregulated in these neurons. Expression of EcR-A remains high in the n6 neurons for the remainder of the life of the fly. In contrast, the n7 neurons up-regulate EcR-A only once, ~24 h prior to eclosion in concert with the n6 neurons. Curiously, the n7 neurons express high levels of EcR-A briefly. These neurons down-regulate EcR-A 24-48 h posteclosion but do not die. The differences in temporal patterns of expression between the Type II neurons and the n6/n7 neurons may be due to the utilization of different cis-acting EcR-A regulatory sequences, or may be due to the temporal modulation of transcription factors acting on the same cis-regulatory sequences in the different cell types (Sung, 2000).

The regulatory properties of a 13.75 kb genomic region are located immediately upstream of the EcR-A transcription unit were examined. The combined effects of interval AG result in high level expression of the reporter gene in all neurons n1-n7. Analysis of lines carrying various constructs has allowed the identification of the position of regulatory elements that confer robust reporter gene expression in specific sets of these neurons. Regulatory elements for neurons n1 and n3 map to interval B (the interval A is the most upstream, while G is the most proximate). Elements for the n2 neurons map to both intervals A and E. Interval E also appears to contain elements specifying expression in all four n4 neurons. Regulatory elements specifying expression in the n5 neurons could not be mapped to any individual interval. However there must be at least two such elements and they are not located in interval GH. Regulatory elements for neurons n6 and n7 map to the same two positions. One n6/n7 element maps to interval F. Mapping of the second n6/n7 element is based on the observations that intervals AB and CE do not drive expression in n6 or n7, yet interval BD does drive reporter gene expression in neurons n6 and n7. These results are contradictory if the proposed n6/n7 element is located within an interval. However, these results are consistent with a model in which an n6/n7 regulatory element spans the B/C border. Interval AG confers not only the correct spatial expression, but this interval also confers the correct temporal expression based on the observation that onset of EcR-A expression precedes the onset of reporter gene expression by as little as 4 h. The nature of this lag is unknown but may reflect constraints imposed by the chromosomal position of the transgene or delays in post-transcriptional events, and may be independent of the regulatory properties of interval AG. Thus, it is proposed that interval AG contains the essential elements required to drive the correct temporal expression of EcR-A in the n4 neurons and in all Type II neurons (Sung, 2000).

In addition to mapping elements of the EcR-A transcription unit that regulate gene expression in neurons n1-n7, an element was also mapped to interval B that activates high level expression of beta-galactosidase in the motorneurons that innervate the dorsal longitudinal flight muscles, MN1-5. This element was mapped to interval B based on the observations that intervals AG and BD drive reporter gene expression in MN1-5, but that interval CE does not. The identification of this regulatory element was surprising because these motorneurons express low levels of EcR-A. To reconcile these seemingly contradictory results, it is proposed that in addition to positive regulatory elements, there also exist negative regulatory elements that suppress EcR-A transcription in these flight motorneurons and that these various elements modulate the expression of EcR-A in these motorneurons. Such a negative regulatory element might reside in interval H or upstream of interval A. Further experimentation is necessary to thoroughly characterize the positive regulatory element and to assay for the presence of this proposed negative regulatory element (Sung, 2000).

Transcriptional Regulation

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).

EcR and E74B are potential targets of Crooked legs. In order to determine if crol functions in gene activation hierarchies during metamorphosis, the temporal patterns of transcription for a number of ecdysone primary- and secondary-response genes were examined in crol mutant animals. These include the EcR ecdysone receptor gene as well as the BR-C, E74A, E74B, E75A, E75B, DHR3 and betaFTZ-F1. E75A and E75B are two isoforms of the E75 early puff gene that encodes orphan members of the nuclear receptor superfamily. DHR3 and betaFTZ-F1 encode distinct orphan receptors, with DHR3 functioning as an inducer of ßFTZ-F1 expression in mid-prepupae. E75B inhibits this DHR3 activation function through direct heterodimerization. betaFTZ-F1, in turn, appears to function as a competence factor that facilitates the reinduction of the early genes by ecdysone in late prepupae. DHR3 is specifically expressed in early prepupae and is unaffected by crol mutations. In contrast, the other genes are all expressed at later stages and, interestingly, their transcription is selectively reduced in mid- and late crol 4418 mutant prepupae. EcR and E74B are both submaximally transcribed in crol 4418 mid-prepupae. Similarly, the peak of BR-C, E74A, E75A and E75B transcription in response to the prepupal ecdysone pulse is significantly reduced, while the earlier induction of these genes in response to the late larval ecdysone pulse is unaffected. Consistent with the stage-specificity of this mutant phenotype, a significant reduction in the transcription of the stage-specific early gene E93 is also seen. The timing of these transcriptional responses confirms that crol mutations have no effect on the duration of larval and prepupal development, but rather indicates that crol is required for the proper magnitude of ecdysone-induced gene expression in prepupae. The level of betaFTZ-F1 mRNA is also reduced in crol 4418 /Df mutants. However, crol 6470 homozygotes show only an approximate two-fold reduction in betaFTZ-F1 mRNA levels, yet the reduction in early gene transcription in these mutants is indistinguishable from that seen in crol 4418 mutants. This observation suggests that crol works independently of betaFTZ-F1 to regulate the prepupal genetic response to ecdysone (D'Avino, 1998).

Metamorphosis of the Drosophila brain involves pruning of many larval-specific dendrites and axons followed by outgrowth of adult-specific processes. From a genetic mosaic screen, two independent mutations were recovered that block neuronal remodeling in the mushroom bodies (MBs). These phenotypically indistinguishable mutations affect Baboon function, a Drosophila TGF-ß/activin type I receptor, and Smad on X (Smox, or dSmad2), its downstream transcriptional effector. Punt and Wit, two type II receptors, act redundantly in this process. In addition, knocking out Activin-beta (dActivin) around the mid-third instar stage interferes with remodeling. Binding of the insect steroid hormone ecdysone to distinct Ecdysone receptor isoforms induces different metamorphic responses in various larval tissues. Interestingly, expression of the Ecdysone receptor B1 isoform (EcR-B1) is reduced in activin pathway mutants, and restoring EcR-B1 expression significantly rescues remodeling defects. It is concluded that the Drosophila Activin signaling pathway mediates neuronal remodeling in part by regulating EcR-B1 expression (Zheng, 2003).

It was of interest to identify possible ligands that participate in the remodeling process. Seven TGF-β type ligands are present in the Drosophila genome. Three of these, dpp, scw, and gbb, are clearly of the BMP family. The remaining, maverick (mav), myoglianin (myo), dActivin (dAct), and activin-like-protein (alp), have not been assigned either genetically or biochemically to a particular family or signaling pathway. Phylogenetic considerations place dAct clearly within the Activin subfamily, while Myo is most similar to BMP-11 and GDF-8, and Mav and Alp are equidistant from both the BMP and TGF-β/Activin subgroups. Therefore, possible involvement of dAct in the Babo signaling was examined (Zheng, 2003).

First, in situ hybridization revealed that dAct is widely expressed in larval brains. Next, when conditioned media from cells expressing dAct was added to S2 cells transfected with Smox, it was found that this ligand is able to stimulate phosphorylation of Smox, while the prototypical BMP ligand Dpp is not. Finally, attempts were made to knock out dAct activity using two independent approaches and dAct, like Babo, was found to be essential for both optic lobe development and EcR-B1 expression in larval brains. Since dAct mutations are currently unavailable, attempts were made to produce a partial loss-of-function condition by overexpression of a dominant-negative form of the protein or RNAi. All TGF-β type ligands that have been examined dimerize and are processed prior to secretion. Previous studies have shown that overexpression of a cleavage-defective form of a particular ligand can interfere with processing and secretion of endogenous ligand. Therefore, a cleavage defective form of dAct (CMdAct) was expressed using either a general GAL4 driver (tubP-GAL4) or an MB-specific driver (GAL4-OK107). CNS development was observed to be retarded only when the CMdAct is ubiquitously expressed and not when it is expressed in MBs. This suggests that dAct does not function within MBs in an autocrine-like fashion. Poor development of the optic lobes is apparent in the tubP-GAL4>CMdAct larval brains, similar to that observed in babo and punt mutant larvae. More importantly, EcR-B1 expression is largely absent in γ neurons of animals that ubiquitously express CMdAct, similar to what is observed in babo mutants. Hs-GAL4-mediated transient expression of CMdAct around the mid-third instar stage also blocks both optic lobe development and EcR-B1 expression (53%). Consistent results are obtained after induction of RNAi using a hairpin-loop dAct construct (UAS-HLdAct). For instance, no EcR-B1 expression was detected in 65% of the late third instar larval brains that were heat shocked to express UAS-HLdAct transiently around the mid-third instar stage. Again, absence of EcR-B1 expression is tightly associated with poor optic lobe development. Similar treatments yield no detectable phenotypes when UAS-CMdAct/UAS-HLdAct is absent or replaced with other UAS-transgenes, such as UAS-mCD8-GFP and UAS-antisense dActivin. In addition, punt mutants, despite having small brains, continue to show EcR-B1 expression. Taken together, these results suggest that dAct, like Babo and dSmox, is indispensable for EcR-B1 expression in the CNS of wandering larvae (Zheng, 2003).

Therefore, from forward genetic mosaic screens, it was found that the Babo TGF-β/Activin type I receptor and a well-known TGF-β/Activin receptor downstream effector, Smox, are both cell autonomously required for remodeling of MB neurons during metamorphosis, providing definitive evidence for involvement of TGF-β/Activin signaling in neuronal plasticity. No evidence exists for any cell fate change in babo mutant MB neurons. For instance, expression of multiple cell type-specific markers remains normal, and mutant γ neurons, unlike wild-type α'/β' neurons, consistently acquire mature dendritic morphological features before metamorphosis. In addition, MB γ neurons that are born at various stages all commit to expressing EcR-B1 in response to TGF-β signaling at the same time and after they all develop into morphologically mature neurons. Therefore, TGF-β signaling probably plays a direct role in programming neuronal plasticity and is not required for cell specification (Zheng, 2003).

Changes in gene expression are believed to mediate most TGF-β-dependent biological processes. The observation that restoration of EcR-B1 expression significantly rescues remodeling defects in babo mutant neurons supports the model that the Babo/dSmox-mediated TGF-β signaling mediates neuronal remodeling via upregulation of the EcR-B1 expression. Interestingly, ecdysone has also been implicated in regulating synaptic efficacy at the Drosophila NMJ, as has BMP signaling. However, as yet no connection between TGF-β signaling and the ecdysone pathway has been established in this system. In C. elegans, the DAF-7 TGF-β ligand as well as the DAF-12 nuclear hormone receptor are involved in dauer formation. In response to hormonal signals, DAF-12 and EcR coordinate changes in diverse tissues during dauer formation and metamorphosis, respectively. Therefore it might be a common theme that TGF-β signaling patterns tissue-specific responses to steroid hormones in diverse organisms by regulating expression and/or activities of specific steroid hormone receptors (Zheng, 2003).

Several lines of evidence support the model that patterned EcR-B1 expression in the late third instar larval CNS is likely established as a consequence of stage-regulated, cell type-specific responses to TGF-β signaling: (1) dActivin is broadly expressed in the CNS, while expression of EcR-B1 is selectively restricted; (2) despite the persistent presence of dActivin expression during all developmental stages and the fact that γ neurons are born at different times, programming of EcR-B1 expression in γ neurons does not occur until the mid-third instar stage; (3) ubiquitous expression of activated Babo fails to activate EcR-B1 expression ectopically. Determining how TGF-β signaling induces such stage-specific, cell type-dependent responses will provide mechanistic cues for how EcR-B1 is differentially expressed to pattern metamorphosis of the CNS. Possible models might include differential expression of a Smox cofactor or the requirement for a second signal that cooperates with the dActivin signal. It will be important to determine how dActivin reaches its target MB neurons. As has been recently suggested for BMP signaling at the NMJ, this might involve retrograde signaling from the MB synapse or it may occur via a juxtacrine mechanism from nearby cells (Zheng, 2003).

No direct connection has been shown between TGF-β signaling and regulation of cytoskeleton dynamics. Consistent with vital roles of TGF-β pathways in regulating gene expression, the results suggest that Babo/Smox signaling might simply lead to activation of EcR-B1 expression to capacitate MB neuronal remodeling during metamorphosis. Although this study provides novel insights into how differential expression of EcR isoforms is achieved, the challenge now is how ecdysone-induced transcriptional hierarchies mediate complex cytoskeletal changes in remodeling neurons. Identifying mutations that block various aspects of the MB neuronal remodeling in mosaic organisms will continue to shed new light on the molecular mechanisms underlying neuronal plasticity (Zheng, 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 GAL4/UAS transgene system was used to examine Dlimk function in vivo. Overexpression of Dlimk in the imaginal wing disc via several different wing-specific GAL4 drivers causes notched wings, missing wing veins, vein fusion, and blistered wings. The notched wing phenotype appears to reflect an increase in apoptosis and is rescued by the p35 viral caspase inhibitor. In addition, wings exhibit enlarged cells (indicated by low wing hair density) and alterations in the number and polarity of wing hairs. Notably, similar defects in wing hair number and polarity are also seen in rho1 and Drok (the Rho effector kinase that activates LIMK) mutants, suggesting that Dlimk functions in the same pathway. Mammalian LIMKs promote actin assembly in cultured cells, and prominent F-actin accumulation and aberrant actin organization are observed in the wing discs of transgenic flies specifically overexpressing Dlimk. Thus, Dlimk can regulate actin assembly in developing tissues (Chen, 2004).

To verify that Dlimk normally regulates morphogenesis during the larval-pupal transition, a kinase-deficient form of Dlimk (Dlimk^D522A) was used as a dominant-negative protein. An analogous mutation in mammalian LIMK1 gives rise to a protein that specifically interferes with LIMK function in cultured cells. Use of a T80-GAL4/UAS-Dlimk^D522A transgenic line to express Dlimk^D522A during development results in viable and fertile animals, with approximately 85% of adults exhibiting malformed wings and legs, consistent with a normal requirement for Dlimk in proper disc morphogenesis. In wild-type adult legs, the femur and tibia are elongated and slender structures; however, in Dlimk^D522A mutant flies, the femur is bent and twisted, and the tibia is often shorter and twisted. In addition, wings are malformed and are approximately 40% smaller than those of wild-type flies. Coexpression of Dlimk^D522A and wild-type Dlimk results in flies whose wings appear normal, indicating that the effects of dominant-negative Dlimk result from specific inhibition of the endogenous wild-type Dlimk as opposed to nonspecific interference with an unrelated signaling pathway (Chen, 2004).

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

Defects in leg morphogenesis resembling those in Dlimk^D522A flies are seen in mutants of several ecdysone-inducible genes, including those encoding the transcription factor, Broad-complex (BR-C or br), and Stubble (Sb), a transmembrane serine protease (Appel, 1993). Both genes are required for disc morphogenesis during larval-pupal transition. Significantly, br mutants interact genetically with mutants of Sb, zipper, and blistered during imaginal-disc morphogenesis (Beaton, 1988: Gotwals, 1991), suggesting that the observed role for a Rho-Dlimk pathway in leg morphogenesis could reflect a requirement for this pathway in the response to ecdysone (Chen, 2004).

To determine if the Rho-Dlimk pathway interacts genetically with br or Sb, a heat-shock-inducible Dlimk^D522A transgene that exhibits a low-penetrance malformed leg phenotype was crossed with br and Sb mutants. Dlimk^D522A and mutants of several Rho signaling components strongly interact with Sb^63b and Sb⁷⁰, two dominant-negative alleles of Stubble, to produce malformed legs at a high frequency. However, mutants of the Rho-related GTPases, Rac1, Rac2, and Cdc42, do not enhance the frequency of leg defects. Similarly, several components of the Rho-Dlimk pathway, but not Rac and Cdc42, strongly interact with br¹ in an analogous genetic interaction test (Chen, 2004).

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

Expression of the ecdysone receptor (EcR) itself is similarly regulated by Rho1 and Dlimk. However, loss-of-function alleles of the EcR or Sb fail to rescue the effects of overexpressing Dlimk, suggesting that Rho-LIMK signaling controls additional aspects of metamorphosis independently of its effects on the ecdysone response. Thus, Rho-LIMK signaling may play a role in coordinating Rho-directed cell shape changes and movements with ecdysone-induced gene expression during tissue morphogenesis (Chen, 2004).

Many of the identified transcriptional targets of the ligand-activated ecdysone receptor are, themselves, transcription factors, which are not the actual effectors of tissue morphogenesis. However, the Stubble gene, which is highly sensitive to Rho-LIMK signaling, encodes a protein that participates directly in morphogenesis through its ability to promote remodeling of the extracellular matrix. The ability of Rho to direct both actin-mediated cell shape changes and the expression of a cell surface protease provides a potential mechanism for coordinately regulating these two major components of tissue morphogenesis during development (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).

It is concluded that Rho GTPase-mediated signal transduction to the actin cytoskeleton and ecdysone-induced gene expression are both critical regulatory components of tissue morphogenesis during Drosophila development. A direct relationship has been described between these two pathways in the context of metamorphosis. Specifically, these findings indicate that Rho, through its ability to activate LIMK and promote actin polymerization, regulates the expression of several ecdysone-responsive genes, including the ecdysone receptor itself. By modulating the expression of ecdysone-responsive genes, including a cell surface protease, the Rho-LIMK signaling pathway appears to play a critical role in regulating the proper morphogenesis of adult structures from the imaginal discs of larvae. This connection represents a previously unrecognized link between Rho GTPase signaling and nuclear hormone signaling that potentially plays a broader role in additional developmental contexts (Chen, 2004).

microRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in Drosophila

The insect steroid hormone Ecdysone and its receptor play important roles during development and metamorphosis and regulate adult physiology and life span. Ecdysone signaling, via the Ecdysone receptor (EcR), has been proposed to act in a positive autoregulatory loop to increase EcR levels and sensitize the animal to ecdysone pulses. Evidence is presented that this involves EcR-dependent transcription of the EcR gene, and that the microRNA miR-14 modulates this loop by limiting expression of its target EcR. Ecdysone signaling, via EcR, down-regulates miR-14. This alleviates miR-14-mediated repression of EcR and amplifies the response. Failure to limit EcR levels is responsible for the many of the defects observed in miR-14 mutants. miR-14 plays a key role in modulating the positive autoregulatory loop by which Ecdysone sensitizes its own signaling pathway (Varghese, 2007).

These experiments provide evidence that Ecdysone signaling reciprocally regulates transcription of the miR-14 and EcR genes. Thus ecdysone acts in two ways to induce EcR activity: first by promoting EcR transcriptional autoregulation and second by alleviating miR-14-mediated repression of EcR activity. Prior to an ecdysone pulse the balance between EcR autoinduction and the mutually repressive interaction between EcR and miR-14 will keep a stable low level of EcR activity. On ecdysone stimulation, the balance shifts to a higher level of EcR activity, but to do so it must overcome repression by miR-14. In the absence of miR-14 one means to limit EcR autoinduction loop is lost, and defects result due to excess EcR activity (Varghese, 2007).

This 'belt and suspenders' approach to EcR regulation may be needed because of the intrinsic lability of a positive autoregulatory mechanism based on a simple transcriptional loop. Such systems can permit a sharp switch-like response, but stochastic variation in EcR levels due to transcriptional 'noise' could trigger the autoregulatory loop at random. The requirement to overcome miR-14-mediated repression thus provides a buffer. Transient fluctuations in EcR levels will transiently repress miR-14 transcription, but the existing miR-14 would take some time to decay. Therefore a transient 'noise' in EcR levels is unlikely to overcome miR-14-mediated repression. In contrast, a sustained induction of EcR by ecdysone allows for decay of miR-14 and sustained autoregulation (Varghese, 2007).

Most miRNAs are predicted to have hundreds of potential target genes, and often the predicted target sites are conserved in evolution, providing some confidence that they are functional. Yet in several cases much of the biological output of the miRNA, as assessed by its mutant phenotype, has been linked to only one or a few of the predicted targets. miR-14 is no exception, with ~180 predicted targets. It is noted that EcR misregulation accounts for only some of the defects in the miR-14 mutants, indicating that other potential targets may play important roles mediating the effects of miR-14 in other contexts (Varghese, 2007).

The relationship between miR-14 and EcR is similar in some respects to that of miR-9a, and the positive feedback loop involved in sense organ specification and senseless expression in Drosophila. The proneural basic helix-loop-helix (bHLH) transcription factors form a positive regulatory loop with the zinc-finger transcription factor Senseless. miR-9a sets a threshold of activity that must be exceeded for the loop to activate by limiting Senseless expression. When coupled with a positive autoregulatory circuit, miRNAs can provide an effective means by which to set thresholds and limit noise-induced errors to ensure robustness in development (Varghese, 2007).

Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster

In most animals, steroid hormones are crucial regulators of physiology and developmental life transitions. Steroid synthesis depends on extrinsic parameters and autoregulatory processes to fine-tune the dynamics of hormone production. In Drosophila, transient increases of the steroid prohormone ecdysone, produced at each larval stage, are necessary to trigger moulting and metamorphosis. Binding of the active ecdysone (20-hydroxyecdysone) to its receptor (EcR) is followed by the sequential expression of the nuclear receptors E75, DHR3 and βFtz-f1, representing a model for steroid hormone signalling. This study has combined genetic and imaging approaches to investigate the precise role of this signalling cascade within the prothoracic gland (PG), where ecdysone synthesis takes place. These receptors operate through an apparent unconventional hierarchy in the PG to control ecdysone biosynthesis. At metamorphosis onset, DHR3 emerges as the downstream component that represses steroidogenic enzymes and requires an early effect of EcR for this repression. To avoid premature repression of steroidogenesis, E75 counteracts DHR3 activity, whereas EcR and βFtz-f1 act early in development through a forward process to moderate DHR3 levels. These findings suggest that within the steroidogenic tissue, a given 20-hydroxyecdysone peak induces autoregulatory processes to sharpen ecdysone production and to confer competence for ecdysteroid biosynthesis at the next developmental phase, providing novel insights into steroid hormone kinetics (Parvy, 2014).

During the past few years, the Drosophila steroidogenic tissue has been extensively used to investigate how extrinsic and intrinsic parameters integrate to coordinate growth with developmental progression. This study shows that EcR, E75, DHR3 and βFtz-f1, which mediate ecdysone signalling, are expressed in the steroidogenic cells and are required for ecdysone synthesis during Drosophila larval development. These findings are consistent with previous studies showing that E75A mutation and βFtz-f1 disruption in the PG induce developmental arrest as a consequence of steroid deficiency. During the past few years, the Drosophila steroidogenic tissue has been extensively used to investigate how extrinsic and intrinsic parameters integrate to coordinate growth with developmental progression. This study shows that EcR, E75, DHR3 and βFtz-f1, which mediate ecdysone signalling, are expressed in the steroidogenic cells and are required for ecdysone synthesis during Drosophila larval development. These findings are consistent with previous studies showing that E75A mutation and βFtz-f1 disruption in the PG induce developmental arrest as a consequence of steroid deficiency signal, while other cells do not respond to this signal. In the case of 20E responsiveness, the gap for acquisition of competence is associated with low ecdysone titres and high βFtz-f1 levels. These findings support the notion that βFtz-f1 also acts as a competent factor for ecdysone biogenesis through a step-forward moderation of DHR3 expression. In addition, as both EcR and DHR3 knockdown also act through an early induced event and affect βFtz-f1 expression, it is conceivable that they participate in the competence acquisition through βFtz-f1. Although the molecular mechanisms that link βFtz-f1 and EcR to DHR3 must still be elucidated, this study reveals that the response following the L2 ecdysone peak is necessary to confer competence for ecdysone biogenesis at the late L3 stage by delaying the DHR3-mediated repression of steroidogenic enzymes (Parvy, 2014).

In summary, this study unravels an autoregulatory mechanism in cyclic ecdysone production. This autoregulation is likely to be coordinated with the processes that adjust ecdysone biogenesis at the L3 stage in response to environmental cues. These include nutrition, insulin signalling and the circadian rhythm that integrates through the prothoracicotropic hormone (PTTH). Interestingly, a downstream effector of PTTH is the NR DHR4, also shown to modulate the ecdysone response at the onset of metamorphosis. However, the fact that DHR4 mutants are not arrested earlier than the prepupal stage, while each RNAi tested in this study provokes a significant arrest at larval stages, suggests that DHR4 acts through an independent mechanism. Moreover, as intermediates of the nutrient, insulin and PTTH signalling interact with NRs that respond to 20E, this study provides a framework to further investigate how environmental parameters integrate with the autoregulation of cyclic ecdysone production (Parvy, 2014).

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 ecd^ts 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 ecd^ts 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 ecd^ts 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).

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