Targets of Activity

The different Broad isoforms have different functions in regulation of genes activated late in the molting hierarchy. Transgenic animals expressing individual BR-C isoforms have been tested for their ability to provide the BR-C rbp+ genetic function by monitoring the transcriptional activation of the L71 genes, a cluster of genes arranged as five sets of divergently transcribed gene pairs. BR-C Z1 isoforms can complement the transcriptional defects seen in rbp mutants but the Z2, Z3 and Z4 isoforms can not. It is concluded that the BR-C rbp+ function is provided by the BR-C Z1 isoform in prepupal salivary glands. L71 gene rescue is restricted to the prepupal salivary gland, suggesting the involvement of additional factors in L71 gene expression. One candidate gene that might encode the non-BR-C temporal specifier is the E74A protein. E74A is an ETS-domain DNA-binding protein that binds in vitro to sequences in the L71-5/6 intergenic region, including a site that overlaps with the BR-C Z1 isoform binding sites. In addition, the E74A and the BR-C function interact genetically in keeping with the idea that ETS-domain proteins often require a protein partner to facilitate DNA binding. Overexpression of Z3 or Z4 isoforms in salivary glands represses L71 expression, indicating that BR-C proteins might also function as transcriptional repressors. There are several Z1 isoform binding sites in the L71 cluster. Mutagenesis of these sites results in the failure to activate late gene expression in vivo. It is concluded that the BR-C prepupal protein directly activates late gene transcription by interacting with late gene cis-acting regulatory elements and that this interaction is responsible for the temporal linkage of early and late ecdysone-induced gene expression (Crossgrove, 1996).

Expression of Ecdysone-induced protein 74EF (E74A) is sufficient to prematurely induce the L71-6 late gene. There are four strong E74A binding sites within the 5' region of L71-6 and these sites are essential for proper L71-6 induction at puparium formation (Urness, 1995). However, ectopic expression of both Broad and E74A activators in an E74B mutant background is not sufficient to prematurely induce all late genes, indicating that other factors contribute to late gene induction. One candidate for this essential inducer is encoded by the Broad Z1 isoform, corresponding to the rbp+ function of Broad. The rbp+ function is essential for late gene expression and exerts its effects through the direct interaction of the Z1 protein with late promoters. In addition, Broad Z1 expression overlaps that of E74A, suggesting Z1 might be available to coregulate late gene function. Coexpression of Z1 and E74A reveals that both together are still insufficient for L71-6 transcription. Another candidate gene for late gene regulation is crooked legs. crol mutants have no effect on E74A transcription in late third instar larvae, but show a delay in L71 induction in prepupae that is similar to that seen in E74A mutants. The Ecdysone receptor itself exerts negative control on the late puffs, preventing their premature induction by ecdysone (Fletcher, 1997).

Analysis of the Broad-Complexgene suggests that it regulates myosin II (zipper) function during imaginal disc morphogenesis. Molecular genetic analysis shows that zinc-finger transcription factors encoded by Br-C are critical for imaginal disc morphogenesis. A screen for enhancers of a Br-C family member, broad1, has identified several loci that function during leg imaginal disc morphogenesis. Ebr, an enhancer of broad1, is a mutation in the myosin heavy chain locus. Defects in leg morphogenesis produce the malformed phenotype. The malformed phenotype reflects aberrations in cell shape changes during morphogenesis in pupal leg imaginal discs. The malformation ranges in severity from a small deformation in the femur to extreme twisting and gnarling of the femur and tibia. The genetic behavior of myosin, and the observation that myosin is subcellularly localized during leg elongation and during additional morphogenetic events, strongly support the hypothesis that myosin-based contraction drives these cell shape changes. Transcription of zip is not under ecdysone control in the imaginal discs; therefore, the gene expression directed by Br-C must affect other aspects of leg disc morphogenesis, rather than merely inducing zip expression. Genetic analysis reveals that genes other than E74 are involved with zipper as SNCCs. These studies promise to extend current understanding of the spatial and temporal control of myosin-based contractility in the cell shape changes required for metazoan development (Halsell, 1998 and references).

BR-C function is required for the regulation of the small heat shock protein genes, which are clustered in the 67B puff and are known to be activated by heat shock and by ecdysone during development. The genes of the 67B cluster are expressed differentially in the salivary glands. While hsp23 and hsp27 transcripts accumulate at relatively high levels, those of hsp22 and hsp26 are present at low and intermediate levels, respectively. The complete BR-C deficiency as well as mutations of the npr class reduce the expression of genes hsp23 and hsp27 by 95 to 99%. Analysis of mutants representing two subfunctions of the BR-C-l(1)2Bab and l(1)2Bc showns that the latter is principally required for complete hsp induction. As sites of DNase I hypersensitivity in chromatin are believed to correspond to gene regulatory sequences, the changes of chromatin structure have been studied in the 67B region at different states of hsp gene activity. Upon hormonal induction, at the onset of metamorphosis, additional DNase I hypersensitive sites (DHS) appear in the 5' regions, four DHSs are associated with hsp23 and two with hsp27. It is suggested that they are due to the binding of the hormone-receptor complex and/or transcription factors, related to ecdysone action. Finally, two DHSs (at -1400 of hsp23 and at -1200 of hsp27) are absent in the mutant nuclei, and thus may correspond to the target sequences for the BR-C-dependent regulatory protein(s) (Dubrovsky, 1994).

Broad is a tissue-specific modulator of the ecdysone response of the Drosophila hsp23 gene. hsp23 is one of seven closely related heat shock genes clustered at the 67B locus. Hormonal regulation of hsp23 and hsp27 is under the genetic control of the BR-C locus, since BR-C deficiency reduces expression by 95 to 99%. The downregulation of the hsp23gene in BR-C correlates with the defective absence of a DNase I-hypersensitive (DHS) site at position -1400 in its promoter region. Thus the DHS-1400 is a target sequence for BR-C regulatory action. The ecdysone response element at DHS-1400 is necessary but not sufficient for full developmental expression of hsp23 in the late third instar, and there is, indeed, another regulatory element in the vicinity. hsp23 developmental expression is not tissue specific. A construct lacking the ecdysone response element is unable to direct normal hsp23 expression in all tissues except the brain. Similarly, brain-specific expression is BR-C independent, although in the other tissues there are varying requirements for BR-C genetic functions. The effect of br mutation is restricted to wing imaginal discs and midgut tissue, while that of 2Bc is restricted to the fat body and Malpighian tubules: mutations in the rbp group have no effect in any of the tissues studied. Thus, BR-C regulatory action is mediated through different genetic functions in a tissue-specific manner (Dubrovsky, 1996).

The steroid hormone 20-hydroxyecdysone operating through BRC controls both induction and repression of the Drosophila "intermolt gene," Sgs-4. The ecdysone receptor binds to two sites, element I and element II, in the regulatory region of Sgs-4. Element II appears to be of no importance for Sgs-4 expression, while element I is an ecdysone response element that is necessary, but not sufficient, for induction of Sgs-4 expression. These results provide no evidence that repression of Sgs-4 expression is mediated by one of the two receptor binding sites. In the close vicinity of elements I and II are two binding sites of secretion enhancer binding protein 3 (SEBP 3). Like receptor element I, one of these sites also proved to be necessary, but not sufficient, for expression of Sgs-4. Therefore, induction of Sgs-4 requires binding of both ecdysone receptor and SEBP 3 to a complex hormone response unit that also contains binding sites for a third factor, SEBP 2. The SEBP 2 sites coincide with binding sites of products of the broad locus, which has been implicated in transduction of the hormonal signal. Thus, the available data suggest that induction of Sgs-4, and possibly other intermolt genes, is a combination of a primary and a secondary response to the hormone (Lehmann, 1995).

Broad mRNAs accumulate in mid third instar larval salivary glands prior to Sgs-4 induction, as is expected for the products of a gene that regulates the timing of Sgs-4 activation. There are a number of binding sites for these protein products, in sequences known to regulate the timing of Sgs-4 induction. Some of these binding sites are required in vivo for Sgs-4 activity. In addition, rbp+, a genetically defined broad function that is required for Sgs-4 induction, acts through these Broad binding sites. Thus, Broad directly mediates a temporal and tissue-specific response to ecdysone as larvae become committed to metamorphosis (van Kalm, 1994).

During the third larval instar, the steroid molting hormone ecdysone activates three temporally distinct puff sets on the Drosophila salivary gland polytene chromosome: the so-called intermolt as well as early and late puffs. Hormonal regulation of intermolt puffs is quite complex and, so far, largely not understood. The effects of mutations of the broad locus have been examined on the expression of genes mapping at the 3C intermolt puff. These genes can be subdivided into two groups. Each group is characterised by a different temporal expression profile, so that at the beginning of the wandering stage, the transcription of the first group declines as group II transcription is induced. Interestingly, the BRC locus appears to play a regulatory role in establishing this transcriptional switch. By using mutants of each of the three lethal complementation groups, the role of BRC in this developmental transition has been defined. This locus also plays an essential role in the early pre-metamorphic hormonal response (D'Avino, 1995).

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

The BR-C is defined by three genetic functions: broad (br), reduced bristles on palpus (rbp), and l(1)2Bc. Earlier studies have shown that the rbp function of the BR-C is required for salivary gland cell death during metamorphosis. This result has been confirmed by finding that larval salivary glands are not destroyed by 22 hr after puparium formation in pupae that carry the rbp5 null allele. The high penetrance of this mutant phenotype suggests that rpr and hid may not be properly expressed in rbp5 mutant salivary glands (Jiang, 2000).

To test this hypothesis, salivary glands were dissected from staged rbp5 mutants, and rpr and hid expression was examined by Northern blot hybridization. Both rpr and hid transcription is significantly reduced in rbp5 mutant salivary glands, indicating that the failure of salivary gland cell death in this mutant can be attributed to its inability to express these death activators. Both betaFTZ-F1 and the ecdysone-inducible E93 early gene are expressed in rbp5 mutant salivary glands, indicating that the block in rpr and hid transcription is not simply due to developmental arrest of the mutant animals. BR-C is expressed in midprepupal salivary glands and thus would be present in the late prepupal glands used for the cycloheximide experiment described above. This explains why the reduced level of rpr transcription observed in the absence of protein synthesis is not as severe as the rbp5 mutant phenotype (Jiang, 2000).

Metamorphosis in Drosophila is orchestrated by the steroid hormone ecdysone, which triggers a cascade of primary-response transcriptional regulators and secondary effector genes during the third larval instar and prepupal periods of development. The early ecdysone-response Broad-Complex (Br-C) gene, a key regulator of this cascade, is defined by three complementing functions (rbp, br, and 2Bc) and encodes several distinct zinc-finger-containing isoforms (Z1 to Z4). Using isoform-specific polyclonal antibodies a switch is observed in Br-C isoform expression in the fat body from the Z2 to the other three isoforms during the third instar. The 2Bc+ function that corresponds presumably to the Z3 isoform is required for the larval fat body-specific expression of a transgenic construct (AE) in which the lacZ gene is under the control of the ecdysone-regulated enhancer and minimal promoter of the fat body protein 1 (Fbp1) gene. Using hs(Br-C) transgenes, it has been demonstrated that overexpression of Z1, Z3, or Z4, but not Z2, is able to rescue AE activity with faithful tissue specificity in a Br-C null (npr1) genetic context, demonstrating a partial functional redundancy between Z1, Z3, and Z4 isoforms. Continuous overexpression of Z2 during the third instar represses AE, while conversely, expression of Z3 earlier than its normal onset induces precocious expression of the construct. This finding establishes a tight correlation between the dynamic pattern of expression of the Br-C isoforms and their individual repressive or inductive roles in AE regulation. Altogether these results demonstrate that the balance between Br-C protein isoforms in the fat body mediates, in part, the precise timing of the ecdysone activation of the AE construct but does not modulate AE tissue specificity (Mugat, 2000).

The finding that overexpressed Z2 is unable to rescue AE activity in a npr16 genetic context indicates that either Z2 is not normally required for induction of AE or it is not active on this construct. However, the significant elevation of AE expression in the absence of br+ function indicates that br+, i.e., Z2, has a repressor effect on the expression of this construct. The strong decrease observed in the level of AE expression resulting from the continuous overexpression of the Z2 isoform during the second half of the third instar and the facilitation by the br28 genetic context of the precocious expression of the AE construct by premature induction of hsZ3 strongly support a repressor function for Z2. However, no precocious expression of the AE construct is observed in the absence of functional Z2 in the br28 context or in the br28/Y; hsZ3/+; AE/+ context prior to heat shock. These results indicate that the absence of the Z2 repressor is not sufficient by itself to allow the expression of the AE construct and support the essential requirement for Z3, Z1, and Z4 as activators in this process. Interestingly, repression of target genes by Br-C has been observed; the 2Bc+ function is required for the repression of the 71E gene VII and Sgs-3, Sgs-4, and Sgs-5 salivary gland-specific genes at the prepupal stage. The Sgs-4 gene is repressed by overexpression of Z3 and L71 genes by Z3 and Z4. The component of the Ddc gene expressed in the epidermis in late third-instar larvae and white prepupae is repressed by induction of hsZ1 and hsZ4 in wild-type animals. Expression of the Brg-P9 gene in imaginal discs at puparium formation is repressed by br+/Z2 function. The observation that Z2 is expressed in larval tissues that survive metamorphosis (e.g., the ring of diploid imaginal cells at the base of the salivary glands and the small islands of diploid imaginal cells in the larval midgut) suggests that this isoform could also be involved in the repression of the genes that direct the death response (Mugat, 2000 and references therein).

Small hydrophobic hormones like steroids control many tissue-specific physiological responses in higher organisms. Hormone response is characterized by changes in gene expression, but the molecular details connecting target-gene transcription to the physiology of responding cells remain elusive. The salivary glands of Drosophila provide an ideal model system to investigate gaps in knowledge, because exposure to the steroid 20-hydroxyecdysone (20E) leads to a robust regulated secretion of glue granules after a stereotypical pattern of puffs (activated 20E-regulated genes) forms on the polytene chromosomes. A convenient bioassay for glue secretion is described in this study, and this bioassay is used to analyze mutants in components of the puffing hierarchy. 20E mediates secretion through the EcR/USP receptor, and two early-gene products, the rbp+ function of BR-C and the Ca2+-binding protein E63-1, are involved. Furthermore, 20E treatment of salivary glands leads to Ca2+ elevations by a genomic mechanism, and elevated Ca2+ levels are required for ectopically produced E63-1 to drive secretion. The results presented establish a connection between 20E exposure and changes in Ca+ levels that are mediated by Ca2+-effector proteins, and thus establish a mechanistic framework for future studies (Biyasheva, 2001).

Although the majority of data presented here focuses on the secretion of glue granules by the premetamorphic pulse of ecdysteroids, there are reports that speculate that a small ecdysteroid pulse induces a series of developmental events, including glue gene induction during the mid-third instar (90-100 h AEL). However, the idea that 20E acts through the EcR/USP heterodimer to mediate these changes has recently been questioned, and the results presented here substantiate the challenge. l(3)ecd1 mutants, severely compromised but probably not devoid of circulating ecdysteroids, synthesize SgsDelta3-GFP. In addition, animals depleted of Ultraspiracle (Usp), EcR-B1, or EcR-B2 also express the fusion gene. However, since neither the EcR nor the usp mutants tested remove all protein (EcR-A remains intact; Usp is truncated in usp2 mutants, and some leaky expression might occur from the hs-usp construct), the caveat that glue gene induction requires only a small amount of ecdysteroid-EcR/USP signaling must be considered. It is also noted that the studies reporting that BR-C null mutants do not produce glue, are consistent with the observation that an SgsDelta3-GFP transgene is not expressed in npr1 animals (Biyasheva, 2001).

Salivary glands cultured with 20E secrete glue, whereas glands cultured in the presence of both 20E and cycloheximide fail to secrete. This suggests that protein synthesis is required for glue secretion; however, no single early or early-late product, thus far identified, has been found to be necessary for this secretion. The rbp+ function of the BR-C early locus, however, is required for secretion to occur in a timely manner. These mutants secrete SgsDelta3-GFP, but the process is delayed by at least 4 h. Although the rbp5 lesion is expected to be an amorph (all BRC-Z1 proteins are truncated before the DNA-binding domain), it does produce a small protein with some reported function (Biyasheva, 2001).

Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, these results suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators (Renault, 2001).

DNaseI footprinting experiments with bacterially produced recombinant protein show that BR-C proteins can bind to the control regions of the BR-C-dependent genes Ddc, L71-6 and Sgs4. Consensus DNA-binding sequences have been defined in these studies for each of the four isoform-specific zinc-finger domains. However, the high variability of these A/T-rich sequences suggests that additional determinants are important for specific DNA recognition. One such determinant could be the BTB/POZ domain that is present in all BR-C isoforms. Nuclear proteins with this domain, like the GAGA or Mod(mdg4) proteins, are thought to act by remodeling chromatin structure. The BR-C dependence of a DNase I-hypersensitive site in the hsp23 gene suggests that BR-C proteins might act in a similar manner. The BTB/POZ domain of the GAGA factor mediates strong cooperative DNA binding to multiple sites but, like other BTB/POZ domains, inhibits binding to single sites. BTB/POZ domains are thus likely to play a critical role in targeting proteins to specific chromosomal loci. The data support this concept, as they show that DNA binding sites that are bound by BR-C proteins in vitro are not sufficient to bind BR-C in a chromosomal context. Such sites may, therefore, turn out to be irrelevant for the control of the gene with which they are connected. Mutation of in vitro BR-C binding sites in the promoter of the death gene reaper (rpr), for instance, has no effect on the expression of a reporter gene. It was therefore concluded that the BR-C might only indirectly regulate rpr transcription in the larval salivary glands, which foreshadows the BR-C-dependent destruction of this tissue during metamorphosis (Renault, 2001).

Mutations that eliminate BR-C binding to Sgs4 in vitro do lead to reduced reporter gene expression in vivo. However, these mutations also affect Fkh binding sites that are required for Sgs4 expression, and this effect may therefore be unrelated to the ability of these sites to be bound by BR-C. This interpretation is favored, not only because BR-C proteins cannot be detected at Sgs4 in situ, but also because BR-C proteins extracted from salivary gland nuclei or produced by in vitro translation are not able to recognize these sites in mobility shift DNA-binding assay. It is important to note that BR-C protein could not be detected at the Sgs4 locus 3C11, but also could not be detected at loci of other Sgs genes like 68C, which harbors Sgs3, Sgs7 and Sgs8. Interestingly, although expression of these three genes depends on BR-C function to a much larger extent than that of Sgs4, Fkh binding sites, but no BR-C binding sites, could be detected in the transcriptional control region of Sgs3. Fbp1 is another example of a gene that is clearly under BR-C control, although it does not seem to be bound directly by BR-C proteins. Fbp1 is activated in response to 20E in the larval fat body at about the time when the Sgs genes are activated in the salivary glands. Expression of an Fbp1 transgene depends on different BR-C isoforms, but attempts to demonstrate direct binding of these isoforms to elements within the transgene were not successful (Renault, 2001).

In this study, evidence has been presented not only for an indirect control of Sgs genes by the BR-C, but also for a possible mechanism that explains how this indirect control is achieved. Based on these results, a new model is proposed for the regulatory interactions between BR-C, Fkh and the 20E receptor EcR/Usp in Sgs gene regulation. The 2Bc function of the BR-C is required at puparium formation for downregulation of the Fkh transcription factor, which is involved in tissue-specific activation of the Sgs genes. Ectopic expression of fkh, which overrides repression of the endogenous gene, mimics the effects of 2Bc mutations on Sgs4 expression. The high level of Sgs4 mRNA that is maintained in the presence of ectopic Fkh clearly shows that the downregulation of fkh is necessary for Sgs repression. Together with downregulation of the Sgs activator SEBP3, it may even be sufficient for repression, leading to the surprising conclusion that direct binding of a repressor may not be required for this process. This study not only provides strong evidence for an indirect mode of action of the BR-C in Sgs gene repression but also suggests that activation might be indirect. BR-C proteins cannot be detected at Sgs loci when the genes are actively transcribed, however, they are present at 98D, the cytogenetic region that includes fkh and other genes. The presence of BR-C protein at 98D in mid- and late-third instar larvae is consistent with a model of direct repression of fkh by the BR-C. However, since fkh seems to be normally expressed in mid-third instar larvae of BR-C null mutants, BR-C activation of Sgs genes is likely to be mediated by a factor other than Fkh. Alternatively, BR-C protein might transiently bind to the Sgs genes at an earlier time when the polytene chromosomes are not yet accessible for immunolocalization studies (Renault, 2001).

A central player in this model is the Fkh protein, which plays an important role in development of the salivary glands as well as, later on, in tissue-specific gene control in this organ. Since fkh mutants show homeotic transformations, it has been suggested that Fkh is required for the establishment of tissue identity. Downregulation of fkh by the BR-C 2Bc function at puparium formation may therefore have a more global effect on the salivary glands than just repression of glue genes, altering the determined state of the glands. Downregulation of fkh could be the first step towards destruction of the larval salivary glands by programmed cell death that occurs about 14 hours later, at pupation. Interestingly, fkh mutations result in removal of the embryonic salivary gland placode by apoptosis, suggesting that Fkh might function as a survival factor that prevents the salivary glands from being eliminated by programmed cell death. Unfortunately, little is known about the mechanisms through which Fkh and other winged helix proteins exert their functions. Studies on the mouse serum albumin enhancer have implicated HNF3/Fkh proteins in chromatin organization. However, efforts to identify interacting proteins, including attempts to demonstrate a direct interaction between Fkh and BR-C proteins, have failed so far (Renault, 2001).

The study presented here integrates fkh in the 20E-controlled regulatory hierarchies that are active at the onset of metamorphosis and assigns BR-C to a new position within these hierarchies. Future studies will show if similar regulatory connections between the BR-C and tissue-specific factors exist in other responses to 20E signaling during development (Renault, 2001).

Ecdysone triggers the expression of golgi genes in Drosophila imaginal discs via Broad-Complex

One of the most significant morphogenic events in the development of Drosophila melanogaster is the elongation of imaginal discs during puparium formation. This macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. The fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks. Using mRNA in situ hybridization, it has been shown here that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, dsed5, and drab1) and downregulates the expression of others (such as dergic53, dß'COP, and drab6). The transcription factor Broad-complex, itself an 'early' ecdysone target, mediates this regulation. The ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing current understanding of the relationship between gene expression and organelle biogenesis (Dunne, 2002).

Two types of small transport vesicles, defined by their cytoplasmic coat proteins, mediate protein transport between the ER and the Golgi complex. A sequential function of these COPII- and COPI-coats/vesicles has been proposed based on biochemical, morphological and functional studies. It is generally considered that COPII-coated vesicles function during protein exit from the ER. After their budding from the ER and disassembly of the coats, COPII-vesicles are though to generate the more pleiomorphic intermediate compartment (IC) structures by homotypic fusion. Subsequently, the IC elements bind coatomers (subunits of COPI coats) in a process that is regulated by the small GTP-binding protein ARF1. The suggested functions of COPI coats/vesicles include molecular sorting and anterograde transport between the ER and Golgi, bidirectional transport within the Golgi, and retrograde transport of selected components back to the ER (Dunne, 2002).

How does golgi gene expression drive golgi stack biogenesis? At the onset of disc elongation, small larval Golgi clusters comprising vesicles and tubules grow in size to finally be converted to Golgi stacks in white pupae. During this period, dsar1, dsec23, drab1, dnsf1, dsnap, dsed5, and dgos28 genes and dSec23 protein are upregulated by ecdysone. These results suggest a possible model involving the activation of three pathways leading to the formation of large Golgi stacks from small larval clusters: a COPII vesicle budding pathway (dSec23/ dSar1) leading to increased ER-derived membrane; a docking mechanism involving dRab1 leading to increased vesicle tethering prior to fusion; and a fusion pathway (dNSF1 and dSNAP, dSed5 and dGos28) mediating fusion of vesicles and tubules to form Golgi cisternae (Dunne, 2002).

Larval clusters grow in size between mid- and late third instar larvae (their surface density increases ~3-fold), and ~60% of the larval clusters in the third instar larvae (TIL) are populated with COPII-derived vesicles. These findings could be explained by the present results. dsec23 and dsar1 are transcriptionally activated between mid- and late TIL (and dSec23 protein is being synthesised). This activation could be expected to activate the COPII budding pathway leading to more vesicles generated at ER exit sites. Interestingly, concomitant to the COPII budding mechanism being stimulated, observations of mRNA and protein expression dynamics of dß'COP suggest that the COPI budding mechanism is downregulated from mid- to late TIL (Dunne, 2002).

Together, these results suggest that, during late TIL, the COPII budding mechanism dominates over the COPI. Conversely, the dß'COP downregulation suggests that the COPI budding mechanism dominates during mid-third instar. This model requires that the Golgi larval clusters present in the mid-TIL would comprise predominantly COPI-derived vesicles, and the enlargement of larval clusters observed in late TIL imaginal discs could be accounted for by COPII vesicles. Immunofluorescence studies provide circumstantial evidence that this is the case, but further work needs to be undertaken to clarify the process (Dunne, 2002).

Rab1 is part of the Ras superfamily of small GTPases and has been shown to have a role in vesicular transport between the ER and the cis face of the Golgi stack. Between mid- and late-TIL, drab1 expression increases about 3.7-fold. This result represents the activation of a possible docking mechanism of the newly budded COPII vesicles. Recently, mammalian Rab1 has been shown to recruit p115 and GM130, two proteins known to be involved in vesicle tethering and docking (Dunne, 2002).

Although in Drosophila a similar interaction has yet to be characterized, the role for dRab1 could be as follows. The expression of dp115 and dGM130 was found to be elevated throughout the third larval instar. During mid-TIL, their gene products could serve a tethering mechanism (as they do in mammalian systems) for the COPI vesicles present in the larval clusters. During late third instar, dRab1 would switch dp115 and dGM130 function from tethering the COPI vesicles to docking and priming the newly formed COPII vesicles prior to their fusion to form Golgi cisternae (Dunne, 2002).

The upregulation of drab1 is specific. Indeed, drab6 (involved in retrograde transport in mammalian systems is downregulated ~4-fold. The fusion machinery including the SNAREs dSed5 and dGos28, and the fusion ATPase dNSF1 and cofactor dSNAP is also upregulated ~4-fold at the mRNA level. This up-regulation would provide the machinery necessary for COPII vesicle fusion and cisternae formation. This correlates well with results showing that dNSF1 is required for the conversion in Golgi morphology. A similar activity has also been shown in vitro by using a Golgi stack reassembly assay from mitotic Golgi fragments (Dunne, 2002).

A second homolog of mammalian NSF was identified in Drosophila that is expressed from the end of embryogenesis to adulthood. Throughout this study, dnsf2 was not detected in TIL imaginal discs, contrary to previous reports (Dunne, 2002).

What is the possible role of TER94 and cofactor dp47? They are not substantially regulated but present at all stages of disc elongation. This second fusion machinery has also been shown in vitro to be involved in Golgi stack reassembly. Nevertheless, because of the constant level of expression, the hypothesis is favored that this complex is not involved in Golgi stack biogenesis at puparium formation. Golgi clusters are not converted to Golgi stacks in the absence of dNSF1, using a comatose mutant maintained at restrictive temperature. Taken together, these results suggest that the dNSF1 fusion pathway is the major and possible sole contributor to the formation of Golgi stacks in disc cells. Only a hypomorphic ter94 mutant will solve the puzzle. p97, the mammalian homolog of TER94 has been shown to bind at least two other cofactors, ufd1 (required for ubiquitin degradation) and nlp4 (implicated in nuclear transport). Its presence in imaginal disc cells might reflect the activity of these two other pathways (Dunne, 2002).

Finally, it has been shown that the overexpression of either dNSF1 or dSNAP leads to the precocious formation of Golgi stacks from larval clusters that are comparable to pupal profiles. That the overexpression of either proteins is sufficient to stimulate Golgi stack biogenesis encouraged an investigation of whether dsec23 expression is influenced by the overexpression of dNSF1 or dSNAP. dsec23 expression was 1.8-fold increased, but resulted from the heat shock and not from the overexpression of either protein. This result argues against a direct cross talk between dNSF1 and dSNAP (protein or mRNA), and dsec23. Other genes will be investigated. The direct involvement of dNSF1 and dSNAP in the formation of active COP II vesicles could account for this biogenesis, especially the 2.7-fold increase in volume density. This has been shown before in mammalian systems for the COPI vesicles and for clathrin-coated vesicles in the endocytic pathway. The data presented here support the hypothesis that transcriptional regulation of a variety of Golgi genes is a significant factor contributing to Golgi stack biogenesis. The direct transcriptional activation of the budding, docking, and fusion pathways are likely to lead to the construction of Golgi stacks (Dunne, 2002).

Until present, experimental evidence has indicated that modulation of organelle architecture is mostly influenced and/or regulated by modifications at the translational and posttranslational level. This study shows, for the first time, that changes in the expression of genes known to participate in the building of the Golgi apparatus exert a direct effect on Golgi biogenesis. Furthermore, for the first time, it has been demonstrated that this gene expression regulation is developmentally controlled by the steroid hormone ecdysone. This novel mechanism is likely to be common to other organisms (Dunne, 2002).

Broad targets Dopa decarboxylase

The amount of epidermal Ddc transcript is much lower at pupariation in several broad mutants, which is one of the primary response loci of the molting hormone, ecdysone. The mutant effects are allele specific and the molecular basis of one of these alleles is known. This implicates a particular family of the zinc finger proteins encoded by the BRC locus in the hormone dependent induction of Ddc expression (O'Keefe, 1995).

Mutation in broad substantially reduces the levels of Dopa decarboxylase (DDC) in the epidermis of mature third instar larvae but not in mature second instar organisms. Enzyme levels appear to be normal in the central nervous system. The specificity of these effects suggests that a product of the BR-C locus mediates the rapid appearance of DDC in mature third instar larvae experiencing an elevated titer of ecdysone. Both the transcript and a protein carrying Broad DNA-binding domain are present in the epidermis and a BR-C recombinant protein carrying the Z2 finger binds to the first intron of the Ddc gene. Five binding sites have been identified within the intron by DNAase I footprinting. A core consensus sequence has been derived that shares some identity with the consensus binding site of the Z2 protein to the Sgs-4 regulatory region. This demonstration that Ddc is a target of BRC in the epidermis is the first direct evidence of a role for this early gene in a tissue other than the salivary glands. The data reinforce the idea that BRC, which clearly mediates a salivary gland-specific response to ecdysone, may play a widespread role in the hormone's activation of gene cascades in other target tissues (Hodgetts, 1995).

The induction of the Dopa decarboxylase gene (Ddc) in the epidermis of Drosophila at pupariation is a receptor-mediated response to the steroid molting hormone, ecdysone. Activity is also dependent on the Broad-Complex (BR-C), an early ecdysone response gene that functions during metamorphosis. BR-C encodes a family of zinc-finger protein isoforms, BR-CZ1-Z4. Genetic experiments have shown that the Z2 isoform is required for epidermal Ddc to reach maximum expression at pupariation. BR-C is shown to regulate Ddc expression at two different developmental stages through two different cis-acting regions. At pupariation, BR-C acts synergistically with the ecdysone receptor to up-regulate Ddc. DNase I foot printing has identified four binding sites of the predominant Z2 isoform within a distal regulatory element that is required for maximal Ddc activity. The sites share a conserved core sequence with a set of BR-C sites that had been mapped previously to within the first Ddc intron. Using variously deleted Ddc genomic regions to drive reporter gene expression in transgenic organisms, it has been shown that the intronic binding sites are required for Ddc expression at eclosion. At both pupariation and eclosion, BR-C releases Ddc from an active silencing mechanism, operating through two distinct cis-acting regions of the Ddc genomic domain at these stages. Transgenes, bearing a Ddc fragment from which one of the cis-acting silencers has been deleted, exhibit ß-galactosidase reporter activity in the epidermal cells prior to the appearance of endogenous DDC. The finding that BR-C is required for Ddc activation at eclosion is the first evidence to suggest that this important regulator of the early metamorphic events, also regulates target gene expression at the end of metamorphosis (Chen, 2002).

A mutation in BR-C br function substantially reduces the level of DDC in the epidermis of mature third instar larvae. Since the br28 allele is caused by a transposon insertion into the Z2 DNA binding domain, it is concluded that a product of the BR-C locus carrying this motif mediates the rapid appearance of DDC in mature larvae in response to an elevated titer of ecdysone. Both the transcript and a protein carrying the Z2 DNA-binding domain are present in the epidermis and a BR-C recombinant protein carrying the Z2 finger binds to five sites within the first intron of the Ddc gene. This paper examines whether these BR-C binding sites mediate Z2 action in vivo; surprisingly, it was found that all five sites are dispensable for Ddc induction at pupariation. Instead, deletion of these sites results in loss of induction at eclosion, suggesting that these sites play an important role in mediating BR-C function at this stage (Chen, 2002).

The cis-acting element through which BR-C regulates Ddc expression at pupariation was mapped between -743 and -382 bp. The four Z2 recombinant protein binding sites, located in this region, shared a perfect conserved triplet core sequence among themselves and with those present at other BR-C regulated genes. Consensus DNA-binding sequences have been defined for each of the four isoform-specific zinc-finger domains. Despite the many possible binding sites that exhibit the consensus core sequences within the 360 bp region between -743 and -382 bp, no binding of the BR-C protein isoforms other than Z2 was detected. The variability of these A/T-rich sequences suggests that along with the zinc-finger domain, additional determinants on the protein are important for specific DNA recognition. One such determinant could be the BTB/POZ domain that is present in all BR-C isoforms. The crystal structure of the BTB domain of PLZF (promyelocytic leukemia zinc finger) has been determined and has been shown to form a homodimer. The domain mediates a functionally relevant dimerization in vivo, promoting strong co-operative DNA binding to multiple sites. BTB/POZ domains are thus likely to play a critical role in targeting proteins to specific sites (Chen, 2002).

Eclosion resembles the first three molts during development in that the peak of DDC activity occurs after the ecdysone titer has dropped from a maximum attained earlier. However, unlike the early molts where the independence of Ddc induction from BR-C can be inferred (since none of the BR-C mutants die at these molts), DDC activity at eclosion is dependent on BR-C binding sites in the first intron. The low titer of ecdysone at eclosion makes it very unlikely that BR-C participates in the kind of co-operative interaction with the ecdysone receptor that is envisioned at pupariation. In fact, BR-C transcription itself can occur in an ecdysone-independent fashion at eclosion. The developmental cues that control eclosion have not been studied in much detail. However, it appears that an entirely different hormonal regime from that which directs the onset of metamorphosis operates. It is based on the eclosion hormone (EH) whose presence is required for eclosion in Drosophila. In pharate adults, the final release of EH occurs approximately 40 min before ecdysis. How it might control the BR-C- dependent epidermal gene cascade in which Ddc is a target remains a subject for further investigation (Chen, 2002).

All four BR-C isoform transcripts are detected at eclosion and the regulatory domain in the first Ddc intron, which has been defined in this study and contains binding sites for all four of the BR-C recombinant proteins. Thus, it is possible that a BR-C isoform other than Z2 is responsible for the Ddc activation at eclosion. Switches in isoform expression do occur during Drosophila development. In cultured imaginal discs, there is an isoform switch from accumulations of Z2, Z3 and Z4 to Z1, 6 h after incubating discs with ecdysone. A switch is also seen at puparium formation, where Z1 replaces Z2. These observations suggest that different isoforms could be involved in the temporal control of a downstream target gene and Z2, which up-regulates Ddc activity during pupariation, may be replaced by one of the other isoforms at eclosion (Chen, 2002).

The finding that the Ddc gene is actively repressed in pharate adults by what is called the downstream silencer, was an unexpected finding in this work. It is however, quite reminiscent of the discovery of an upstream cis-acting sequence that silences Ddc prior to pupariation. The rise in titer of ecdysone, which begins 18-19 h after puparium formation (APF), results in the apolysis of the pupal cuticle. In the absence of the downstream silencer, Ddc activity would appear prematurely in the epidermal tissue, as evidenced from transformants carrying P[Ddc-lacZ]BT, which start to show lacZ staining at this time. Formation of the adult cuticle commences well after the apolysis of the pupal cuticle and after the cellular morphogenesis that produces bristles and hairs. The onset of adult cuticulin deposition occurs 15-25 h after the peak in hormone titer. Moreover, even when cuticulin deposition begins, it is protracted over a 10-15 h period. This deliberate pace of cuticulin deposition provides additional time for morphogenesis. Deposition of most procuticle components occurs between 55 and 80 h APF when the titer of ecdysone is falling or is low. Thus, repression of Ddc in the epidermis ensures that tanning and hardening of the adult cuticle does not occur until after this extended period of adult cuticle deposition and maturation. Release of Ddc from the repressed state might be triggered by the falling ecdysone titer, since DDC activity starts to rise 80 h APF (Chen, 2002).

The identity of the repressor that acts through the downstream silencer remains to be worked out. Genetic and transgenic analysis of BR-C regulation of downstream genes shows that different BR-C isoforms can have opposite regulatory effects on the same target gene. In one case, the Z3 isoform activates the larval fat body-specific expression of the Fbp-1 gene and the Z2 protein represses it. In another case, Z3 acts as a repressive isoform for the expression of a salivary gland specific gene Sgs4, while Z1 activates it. Thus, the switch from one predominant isoform to another may mediate the precise temporal control of target genes. Although the sensitivity of the Northern analysis used to detect BR-C expression needs to be addressed, it is felt that the absence of BR-C mRNA during the pharate adult stage, makes it unlikely that an isoform of BR-C functions as a repressor during this stage (Chen, 2002).

Broad targets let-7

lin-4 and let-7 are founding members of an extensive family of genes that produce small transcripts, termed microRNAs (miRNAs). In Caenorhabditis elegans, lin-4 and let-7 control the timing of postembryonic events by translational repression of target genes, permitting progression from early to late developmental programs. To identify Drosophila melanogaster miRNAs that could play similar roles in the control of developmental timing, the developmental expression profile of 24 Drosophila miRNAs were characterized; seven miRNAs are either upregulated or downregulated in conjunction with metamorphosis. The upregulation of three of these miRNAs (mir-100, mir-125, and let-7), and the downregulation of a fourth (mir-34) requires the hormone ecdysone (Ecd) and the activity of the Ecd-inducible gene Broad-Complex. Interestingly, mir-125 is a putative homolog of lin-4. mir-100, -125, and let-7 are clustered within an 800-bp region on chromosome 2L, suggesting that these three miRNAs may be coordinately regulated via common cis-acting elements during metamorphosis. In S2 cells, Ecd and the juvenile hormone analog methoprene exert opposite effects on the expression of these four miRNAs, indicating the participation of both these hormones in the temporal regulation of mir-34, -100, -125, and let-7 expression in vivo (Sempere, 2003).

The 24-h lag between the addition of Ecd to cultured S2 cells and the expression of mir-100 and mir-125 suggest that the initial Ecd signal activates mir-100 and mir-125 expression indirectly via intermediate regulators. One such intermediate could be BR-C, which is required for mir-100 and mir-125 expression in animals. To test whether BR-C activity is required for the Ecd-induced expression of mir-100 and mir-125 in S2 cells, BR-C activity was inhibited by RNAi using a 700-nucleotide dsRNA corresponding to a common region of all BR-C isoforms. S2 cells were incubated for 30 min with BR-C dsRNA or mock dsRNA, corresponding to unrelated C. elegans sequence. Then, the transfected and nontransfected cultures were treated with Ecd and harvested 32, 40, and 48 h later. The levels of mir-100 and mir-125 RNAs were considerably lower in BR-C RNAi cells as compared with nontransfected or mock RNAi cells. This result further supports the conclusion that BR-C is required to mediate the activation of mir-100 and mir-125 by an Ecd signal in vivo. This result also argues against the possibility that mir-100, mir-125, and let-7 RNAs were detected at very low levels in nonpupariating ecd1 and npr6 mutants simply because these mutant animals were arrested at a stage before mir-100, mir-125, and let-7 are normally upregulated (Sempere, 2003).

It should be noted that BR-C RNAi does not result in complete loss of mir-100 and mir-125 expression, suggesting that RNAi treatment is not fully effective. Consistent with an incomplete knockdown of BR-C by RNAi, miR-34 levels are unaffected by BR-C RNAi in Ecd-treated cells. Based on results with npr6 mutant animals, one would have expected that mir-34 expression would be derepressed by BR-C RNAi in Ecd-treated cells. Since BR-C activity may not have been completely eliminated by RNAi, the requirement for BR-C activity in the repression of mir-34 could not be assesssed in S2 cells (Sempere, 2003).

Distinct promoter regions regulate spatial and temporal expression of the Drosophila caspase dronc: Broad targets dronc

Dronc is an apical Drosophila caspase essential for programmed cell death during fly development. During metamorphosis, dronc gene expression is regulated by the steroid hormone ecdysone, which also regulates the levels of a number of other critical cell death proteins. As dronc protein levels are important in determining caspase activation and initiation of cell death, the regulation of the dronc promoter was analyzed using transgenic flies expressing a LacZ reporter gene under the control of the dronc promoter. These results indicate that dronc expression is highly dynamic during Drosophila development, and is controlled both spatially and temporally. While a 2.3 kb dronc promoter region contains most of the information required for correct gene expression, a 1.1 kb promoter region is expressed in some tissues and not others. During larval-pupal metamorphosis, two ecdysone-induced transcription factors, Broad-Complex and E93, are required for correct dronc expression. These data suggest that the dronc promoter is regulated in a highly complex manner, and provides an ideal system to explore the temporal and spatial regulation of gene expression driven by nuclear hormone receptors (Daish, 2003).

Experiments outlined in this paper demonstrate that 2.3 kb of the dronc promoter is largely sufficient for temporal expression (compared to endogenous dronc) throughout development. Previous experiments have shown that dronc is predominantly expressed in the larval and prepupal salivary glands and midgut, and larval brain lobes. 2.3 kb of the dronc promoter contains all necessary elements for correct spatial regulation of dronc expression in these tissues (Daish, 2003).

In order to identify transcription factors responsible for both temporal and spatial regulation of dronc and ecdysone-mediated PCD, it is of vital importance to elucidate the regions of the promoter essential for dronc expression in different tissues. In addition, it would be of interest to determine if there is a single promoter region controlling the spatial expression profile of dronc, or if different promoter regions are required in different tissues. LacZ transgenic reporter experiments reveal that the 2.3 kb promoter is the minimal requirement for correct expression in brain lobes and salivary glands. Furthermore, the region between 1.1 and 2.3 kb contains transcription factor-binding sites essential for expression in these tissues. This region also seems to harbor a repressor element important to keep dronc levels low during periods when ecdysone titers are low. Surprisingly, regulation of dronc transcription is markedly different in the midgut. The region between 1.1 and 2.3 kb is not important for transcription in this tissue, because 1.1 kb of the promoter is sufficient for expression. These results clearly demonstrate that distinct regions of the promoter are required for expression in different tissues, and implies that different transcription factors regulate dronc expression in a tissue-dependent manner (Daish, 2003).

The two ecdysone-induced transcription factors BR-C and E93 are essential for dronc expression in salivary glands. In the midgut, however, only E93 seems to be important. The results of dronc promoter-LacZ transgenic expression in flies deficient in BR-C and E93 are consistent with recent findings. LacZ expression driven by the 2.8 kb promoter is severely impaired in salivary glands of BR-C (rbp5 and npr) or E93 mutants, whereas expression is impaired only in the midgut of E93 mutant background animals. This further supports the idea that the mechanisms governing dronc regulation are tissue specific. The key questions arising from these experiments are: why does the BR-C Z1 isoform (rbp5 mutant) regulate dronc in the salivary glands and not in the midgut? What factors are binding to the 1.1-2.3 kb region of the promoter in salivary glands, and why are they not as important in the midgut? Previous results show that either BR-C Z1- or BR-C Z1-regulated proteins bind to the dronc proximal promoter and control its expression. Transactivation of the 2.8 kb promoter by BR-C Z1, however, was only seen in specific cell types. Given that BR-C Z1 is also expressed in the midgut, this implies that it may be acting through cofactors which are not expressed in the midgut, yet are specifically recruited to the dronc promoter. Alternatively, BR-C Z1 induces the expression of another factor which binds to the promoter, and this factor is absent in the midgut (Daish, 2003).

Since the proximal promoter alone (0.54 kb) is not sufficient for expression in the salivary gland, it is believed that BR-C Z1 (or a Z1-regulated protein) is cooperating with other transcription factors binding upstream (1.1-2.3 kb), that are essential for salivary gland expression. It has been shown that E93 acts through the first 600 bp of the dronc promoter by transactivation studies; however, no direct binding of E93 to the dronc (or any other) promoter has been shown so far. Additionally, a preliminary analysis indicates the presence of an EcR/Usp-binding site between 1.1 and 2.3 kb of the dronc promoter, and in vitro experiments show that this element may be important in regulating dronc expression. Since the proximal promoter (0.54 kb) alone is not sufficient for expression, cooperation of BR-C and E93 with EcR/Usp and other unknown factors may be important for temporal and spatial regulation of dronc expression during development. Identification of these factors will be important for fully understanding dronc transcription during development (Daish, 2003).

Overall, this study has established the minimal dronc promoter requirement for spatial and temporal expression to be within the 2.3 kb region upstream of the dronc gene. This region is important for both BR-C- and E93-mediated transcription in salivary glands and E93 transcription in the midgut. Importantly, the 1.1-2.3 kb promoter region harbors elements important for salivary gland expression and a putative repressor element. The 0.54-1.1 kb promoter region is important for expression in the midgut. These regions will form the basis of future experiments designed to identify factors necessary for the regulation of dronc expression during PCD (Daish, 2003).

Ligand-dependent de-repression via EcR/USP acts as a gate to coordinate the differentiation of sensory neurons in the Drosophila wing: broad is required for the activation of senseless

Loss of function of either the ecdysone receptor (EcR) or Ultraspiracle (USP), the two components of the ecdysone receptor, causes precocious differentiation of the sensory neurons on the wing of Drosophila. It is proposed that the unliganded receptor complex is repressive and that this repression is relieved as the hormone titers increase at the onset of metamorphosis. The point in development where the receptor complex exerts this repression varies for different groups of sensilla. For the chemosensory organ precursors along the wing margin, the block is at the level of senseless expression and is indirect, via the repressive control of broad expression. Misexpressing broad or senseless can circumvent the repression by the unliganded receptor and leads to precocious differentiation of the sensory neurons. This precocious differentiation results in the misguidance of their axons. The sensory precursors of some of the campaniform sensilla on the third longitudinal vein are born prior to the rise in ecdysone. Their differentiation is also repressed by the unliganded EcR/USP complex but the block occurs after senseless expression but before the precursors undertake their first division. It is suggested that in imaginal discs the unliganded EcR/USP complex acts as a ligand-sensitive 'gate' that can be imposed at various points in a developmental pathway, depending on the nature of the cells involved. In this way, the ecdysone signal can function as a developmental timer coordinating development within the imaginal disc (Schubiger, 2005).

The ecdysone signal is transmitted via the ecdysone receptor to activate a number of direct target genes. It has generally been assume that the hormone and its receptor activate a hierarchy by activating early genes that then activate the many late genes. A large body of work based primarily on larval tissues has supported this model. Thus when the ecdysone receptor is non functional, the first step in the cascade fails, the early genes are not activated, and the tissues are unable to undergo a metamorphic response. In imaginal discs it has been shown that loss of function of USP leads to the inability to activate early genes, such as DHR3, EcR and E75B, but also results in precocious differentiation, rather than in a failure to initiate a particular metamorphic response. It has now been demonstrated that loss of EcR function in the wing discs gives similar results (precocious BR-Z1 expression and sensory neuron differentiation) to the ones reported for loss of USP function, and it is concluded that the unliganded EcR/USP heterodimer is the functional repressor. Thus at least some processes at the onset of metamorphosis are not controlled by the ecdysone-induced hierarchy, but rather through the relief of the repressive function of the unliganded EcR/USP complex once the ecdysone titers rise. The importance of this repressive function of the unliganded receptor is further demonstrated by experiments using a dominant negative EcR, which does not bind the hormone, and as a consequence repression cannot be relieved and target genes are not expressed. The repressive role proposed for the unliganded ecdysone receptor complex would also explain why loss-of-function USP clones, in general, result in the differentiation of normal adult bristle organs since loss of receptor function would only control the timing of differentiation. Such an interpretation is supported by early pigmentation of abdominal bristles in usp3 clones that has been observed on occasions. In vivo studies of activation by EcR/USP suggest that activation plays a major role in the metamorphic response of larval tissues but has only a minor role in the development of the imaginal discs. It remains to be seen which processes are activated by the ecdysone hierarchy and which by loss of the repressive actions of the unliganded receptor (Schubiger, 2005).

Adult chemosensory neurons on the wing margin undergo precocious differentiation in loss-of-USP clones. To understand which step is repressed by the EcR/USP complex the early expression patterns of a set of genes involved in neuron differentiation was examined in such mutant clones. In the absence of USP function the early pattern of Achaete (AC) expression in the margin is unaffected. In contrast, both Neur (as visualized by A101) and Sens are expressed in usp mutant cells before they are detected in the surrounding wild-type tissue. In vitro experiments revealed that A101 expression that is already on at the time cultures were set up, remains on through the culture period, but that there is a block by EcR/USP at the level of sens expression that prevents the maturation of the SOPs of the chemosensory neurons in the wing margin. The block is released once the hormone titers rise (Schubiger, 2005).

The repressive function of the unliganded receptor does not act directly on the genes tested. The block of SOP differentiation is controlled through BR-Z1, and br function is required for the activation of sens, a gene necessary and sufficient for sensory organ differentiation. Thus expressing BR-Z1 or Sens early in the margin allows the inhibition from the unliganded ecdysone receptor to be by-passed and the sensory neurons in the margin to differentiate precociously. When Sens is misexpressed it was found that clusters of extra neurons differentiate in the region of high driver expression. This is in agreement with reports that high levels of Sens activate the proneural genes and promote the formation of SOPs. By contrast, when BR-Z1 is misexpressed in the margin, a more normal pattern of sensory neuron arrangement is observed, that is very similar to what is observed in loss-of-function USP or EcR cells. This indicates that BR-Z1 does not induce the formation of SOPs but rather causes the up-regulation of Sens in cells that have already committed to the SOP fate. Occasionally expressing BR-Z1 in the margin leads to the differentiation of a sensory neuron in the posterior margin, normally devoid of neurons. It is possible that in such a situation BR-Z1 misexpression can at times lead to sufficiently high expression of Sens to cause SOP differentiation (Schubiger, 2005).

Loss-of-function of BR demonstrates the requirement for BR to activate the high levels of Sens in the SOPs, as well as the low levels in the posterior margin, but without molecular data it is not known if br is directly activating sens. Since BR-Z1 normally appears later than the initial low expression of Sens, it is proposed that early Sens expression is most probably controlled by BR-Z2. BR-Z2 is expressed shortly after the molt to the third instar, and ectopic BR-Z2 expression induces low levels of Sens. BR-Z3, which also induces Sens when ectopically expressed may induce low levels of Sens as well, but since BR-Z3 is normally expressed at very low levels in the wing disc, it is thought that Br-Z3 plays a minor role. BR-Z1 then is needed for the accumulation of Sens in the mature SOPs (Schubiger, 2005).

In summary the above genetic interactions suggest that unliganded EcR/USP represses BR expression that is required for sens activation and the formation of the mature SOPs in the margin (Schubiger, 2005).

The SOPs are born in a specific temporal sequence in the wing disc. The first SOPs arise in the third instar, 20-30 hours before pupariation; they include GSR, ACV and L3-2 along the third vein. The SOPs of the margin arise later, at 10-12 hours before pupariation, so they are at a very different stage from that of the early born SOPs at the time metamorphosis begins. Since the unliganded receptor is acting as a repressor it is postulated that the block must be occurring at different times during the progression of sensory organ differentiation for these two groups of sensilla. Based on genetic studies, the ecdysone-sensitive arrest for the chemosensory sensilla of the margin occurs in the up-regulation of Sens, since the SOP is undergoing maturation. For the early born sensilla, however, Sens levels are already elevated before the rise in 20E and are not dependent on BR function. For these early born sensilla the ecdysone-sensitive arrest occurs after high Sens expression but prior to the division of the SOP. It is thought that for different sets of sensilla the imposition of an ecdysone-sensitive arrest at different points in development is important to coordinate the differentiation of the sensilla. Such a mechanism would ensure that the outgrowing axons begin to elongate in a choreographed manner leading to the correct axon pathways and to their finding of the correct targets in the CNS according to their physiological function. This idea is supported by the observation that the axons of sensilla forced to differentiate precociously by the absence of a functional ecdysone receptor or by early expression of BR-Z1 or Sens often take abnormal routes (Schubiger, 2005).

Ecdysone is also acting as a timer for the formation of the chordotonal and Johnston's organs as well as for the initiation of the morphogenetic furrow. These structures arise early in the third instar (80 hours after egg laying) and appear to be under the control of the small ecdysone peak at that time. In the case of the leg chordotonal organ, ecdysone appears to be controlling the proneural gene atonal (ato). It is not known yet if this control also occurs via de-repression as is seen for the wing (Schubiger, 2005).

The subsequent progression of the morphogenetic furrow is also dependent on ecdysone. This action of ecdysone has been proposed not to occur via EcR. However, loss of USP leads to an advancement of the furrow and precocious differentiation of the photoreceptors. It has been reported that the progression of the morphogenetic furrow, as well as the timing of differentiation of the chordotonal organs in the leg, are controlled by the insulin receptor (InR)/Tor pathway, with increased InR signaling leading to precocious differentiation. In the wing margin, by contrast, increasing or decreasing InR signaling does not affect the timing of differentiation of the chemosensory neurons. Thus there must be multiple temporal control mechanisms for sensory structures. The current results have demonstrated repression of sensory organs by the unliganded ecdysone receptor at the end of the third instar, but do not rule out additional steps controlled by ecdysone or other factors. It remains to be elucidated which timer(s) is used when, and for which sensory structures (Schubiger, 2005).

In holometabolous insects functional larval tissues are replaced by the differentiating imaginal ones. The endocrine system is acting on larval tissues composed of differentiated cells that are thus in an equivalent state to initiate programs such as cell death and neuronal remodeling. Here EcR/USP's role is activational. For the differentiation of the imaginal tissues the endocrine system faces a varied cellular landscape where some cells may still be dividing while other have begun to differentiate. In these tissues the unliganded receptor acts as a repressor to interrupt the sequence of differentiation at different points in order to coordinate the response to the rising 20E titers. Release of repression by 20E may therefore function as a 'gate' at the onset of metamorphosis and thus would enable development of imaginal tissues to be coordinated and tightly controlled by the rising ecdysone titers. In metamorphosing amphibians a similar situation is seen with functional larval tissues such as the tail and the gills dying and adult limbs and lungs developing in response to thyroid hormone. It would not be surprising to find that the thyroid hormone receptor is activational in the larval tissues but that the forming adult tissues are controlled through de-repression (Schubiger, 2005).

Transcription of Myocyte enhancer factor-2 in adult Drosophila myoblasts is induced by the steroid hormone ecdysone: Broadis required for full activation of the myogenic program in these cells

The steroid hormone 20-hydroxyecdysone (ecdysone) activates a relatively small number of immediate-early genes during Drosophila pupal development, yet is able to orchestrate distinct differentiation events in a wide variety of tissues. This study demonstrates that expression of the muscle differentiation gene Myocyte enhancer factor-2 (Mef2) is normally delayed in twist-expressing adult myoblasts until the end of the third larval instar. The late up-regulation of Mef2 transcription in larval myoblasts is an ecdysone-dependent event that acts upon an identified Mef2 enhancer, and enhancer sequences have been identified required for up-regulation. Evidence is presented that the ecdysone-induced Broad Complex of zinc finger transcription factor genes is required for full activation of the myogenic program in these cells. Since forced early expression of Mef2 in adult myoblasts leads to premature muscle differentiation, these results explain how and why the adult muscle differentiation program is attenuated prior to pupal development. A mechanism is proposed for the initiation of adult myogenesis, whereby twist expression in myoblasts provides a cellular context upon which an extrinsic signal builds to control muscle-specific differentiation events, and the general relevance of this model for gene regulation in animals is discussed (Lovato, 2005).

Since a 175-bp Mef2 enhancer recapitulates the pattern of gene expression seen for Mef2 during larval development, attempts were made to identify the factors that might be interacting with this sequence. No consensus binding sites for the ecdysone receptor were found, nor for the transcription factors encoded by the immediate-early gene E74. Therefore, to identify cis-acting elements involved in enhancer activation, deletion analysis of the 175-bp enhancer was performed. Deletion of 20 bp from the 5′ end of the enhancer to generate a 156-bp Mef2 enhancer has a dramatic impact upon transgene expression in adult myoblasts. Lines carrying the wild-type enhancer showed strong β-galactosidase activity in the adult myoblasts, whereas the 156-bp enhancer was barely active in all lines tested. This result indicates that the 5′ portion of the enhancer is essential for its activity in adult myoblasts and identifies this region as a possible target for ecdysone-dependent gene regulation (Lovato, 2005).

The 175-bp enhancer is also active at the embryonic stage in skeletal muscle precursors; therefore, the embryonic activities of the 175-bp and 156-bp enhancers were compared. Interestingly, both enhancers are strongly active in embryos, although there was a slightly reduced activity of the 5′ deleted enhancer compared to the full-length. This observation suggested that the 20 bp deleted from the 175-bp enhancer to generate the 156-bp construct contains specific response elements for activation of Mef2 in adult myoblasts, rather than a general factor necessary for enhancer activation in all contexts (Lovato, 2005).

Within the 20 bp of DNA deleted in the above experiment were sequences that weakly resembled the binding sites for the zinc finger transcription factors of the BR-C. Since the BR-C has been shown to mediate gene activation in response to ecdysone, the BR-C gene products were considered to be excellent candidates for direct regulation of Mef2. Therefore, the accumulation of MEF2 was studied in the imaginal discs of control wild-type siblings and brnpr-3 mutants which lack BR-C function. There was a significant, but not complete, reduction in the level of MEF2 in mutants. However, there were no effects upon Mef2 expression of mutations affecting any of the Z1, Z2, or Z3 BR-C isoforms individually, perhaps due to the documented functional redundancy between many of these products (Lovato, 2005).

These results indicate that although the 5′ enhancer sequence is required for enhancer activity in myoblasts, it is not likely to be a direct target of proteins of the BR-C. This is because removal of the 5′ sequence ablates enhancer activity almost completely, whereas removal of BR-C function has an incomplete effect upon Mef2 expression. In further support of this conclusion, although antibody stains have indicated that isoforms Z1, Z2, and Z4 are detected in myoblasts of larvae and young pupae, it has not been possible to demonstrate binding of any of these three factors to the Mef2 enhancer sequence using in vitro DNA binding assays. It is therefore concluded that while the BR-C influences ecdysone-dependent Mef2 expression, it does so indirectly and not through direct binding to the 175-bp enhancer (Lovato, 2005).

The steroid hormone ecdysone functions broadly in Drosophila to control molting and metamorphosis during the life cycle, and much research has concentrated upon the mechanisms of its action. Ecdysone is known to induce a number of immediate-early genes, which mediate transcriptional responses to hormone levels. However, since ecdysone is a systemic signal, and since many of the immediate-early genes are expressed in multiple tissues, a central challenge in the field has been to understand how widespread activation of immediate-early genes can have specific effects in different target tissues. This study shows that temporal expression of Mef2 in adult myoblasts occurs as a result of the ecdysone pathway. The finding that Mef2 expression in adult myoblasts is low prior to the onset of pupariation is consistent with the known role of Mef2 in muscle differentiation. Since the adult myoblasts do not initiate differentiation until after puparium formation, this might account for why Mef2 expression is absent in young myoblasts. In fact, early expression of Mef2 in adult myoblasts causes premature differentiation of these cells. While the possibility cannot be excluded that this premature differentiation results from the demonstrated ability of high levels of Drosophila Mef2 to inappropriately induce myogenesis, this seems unlikely given the profound myogenesis which was observed in the imaginal discs (Lovato, 2005).

Sustained expression of twist in the adult myoblasts prevents normal muscle differentiation, and Twist levels must decline during the pupal stage for normal adult muscle development to occur. Forced expression of Mef2 in the discs can induce muscle differentiation; however, it is not known if such Mef2 expression attenuates Twist levels, or if the myogenesis observed occurs concurrently with twist expression (Lovato, 2005).

Since the BR-C is expressed in many tissues late during larval development, the myoblast-specific activation of Mef2 must also depend upon an additional factor(s). This factor is likely to be the myoblast marker Twist, which is expressed in the adult myoblasts throughout the larval stage and which has been shown to be an essential activator of Mef2 transcription. Furthermore, the temporal and tissue-specific signals are integrated at the genome level via the 175-bp Mef2 enhancer (Lovato, 2005).

It is therefore proposed that systemic signals such as ecdysone and immediate-early gene activation have specific effects in distinct tissues via interpretation of the systemic signals by a tissue-specific factor: Twist in the case of the adult myoblasts. These findings are analogous to the activation of Fbp1 in fat body cells at the late third larval instar. Fbp1 activation results from the combined effects of ecdysone/EcR complex and the tissue-specific factor dGATAb. The current findings support this model of the specificity of hormone action and extend them to apply to the formation of the adult musculature. The interpretation of systemic hormone signals by cell-autonomous factors is a powerful mechanism to control gene expression. It has recently been shown that gut epithelial cell-specific response to the nuclear hormone receptor PPAR-gamma requires the tissue-specific co-activator Hic-1 (Lovato, 2005).

The data also provide a novel mechanism for regulating Mef2 expression in the animal. This is the first demonstration that Mef2 levels in vivo can be regulated by hormone action, and this may be a broadly relevant paradigm. Indeed, it has been shown that in cultured mammalian myotubes, mef2 mRNAs are induced by treatment with nonapeptide Arg-8 vasopressin. This mechanism is also similar to that demonstrated in vertebrates where Mef2 and thyroid hormone receptor interact with each other and synergistically activate the alpha cardiac myosin heavy-chain gene. These results, and the the results presented in this study, support an important role for hormones in impacting muscle development and underline the utility of the Drosophila system for defining these important mechanisms (Lovato, 2005).

To date, it has not been possible to identify how the effects of ecdysone are directly mediated on the Mef2 gene. There is a requirement for the function of the BR-C, and involvement of this gene complex is attractive given both the expression of BR-C isoforms in the developing adult muscles and a demonstrated role of the Z1 and Z4 isoforms in controlling indirect flight muscle development. Several studies have identified complex cross-regulatory interactions among ecdysone immediate-early genes. This complexity may explain the partial requirement of the BR-C for Mef2 activation and suggests that the direct regulation of Mef2 in adult myoblasts might be complex. Nevertheless, these studies define how the onset of adult myogenesis is orchestrated and also define the Mef2 enhancer as an ecdysone-responsive element. Identification of the factors that interact with the Mef2 enhancer will ultimately provide important insight into the mechanisms of hormone-induced gene regulation and differentiation (Lovato, 2005).

Border of Notch activity establishes a boundary between the two dorsal appendage tube cell types; Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression

Boundaries establish and maintain separate populations of cells critical for organ formation. Notch signaling establishes the boundary between two types of post-mitotic epithelial cells, the Rhomboid- and the Broad-positive cells. These cells will undergo morphogenetic movements to generate the two sides of a simple organ, the dorsal appendage tube of the Drosophila egg chamber. The boundary forms due to a difference in Notch levels in adjacent cells. The Notch expression pattern mimics the boundary; Notch levels are high in Rhomboid cells and low in Broad cells. Notch mutant clones generate an ectopic boundary: ectopic Rhomboid cells arise in Notch+ cells adjacent to the Notch mutant cells but not further away from the clonal border. Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression. It is further shown that Broad represses rhomboid cell autonomously. These data provide a foundation for understanding how a single row of Rhomboid cells arises adjacent to the Broad cells in the dorsal appendage primordia. Generating a boundary by the Notch pathway might constitute an evolutionarily conserved first step during organ formation in many tissues (Ward, 2006).

At the boundary, cells with high Notch express rhomboid, whereas cells with lower Notch express Broad. A new boundary is established at Notch mutant clone borders, where Notch+ cells adjacent to Notch cells ectopically express rhomboid and do not express Broad. Thus, in the dorsal anterior, when two cells with different Notch levels are adjacent to one another, the cell with higher Notch levels simultaneously represses Broad and promotes rhomboid expression. broad cells ectopically express rhomboid, indicating that Broad normally represses rhomboid expression. It is inferred that cells with higher Notch levels repress Broad, thereby allowing rhomboid expression. It is now proposed that when cells with different levels of Notch are located next to each other, the cells with high Notch repress Broad, allowing rhomboid expression. In contrast, cells with low Notch express Broad and therefore repress rhomboid expression (Ward, 2006).

Notch, an important modulator of boundary function in other tissues, establishes the boundary that defines the Rhomboid and the Broad dorsal appendage cell types. When Notch is removed from cells that should span the boundary, rhomboid is not expressed, and Broad is ectopically expressed. Thus, at the boundary, Notch regulates the patterning of both Rhomboid and Broad cell types. When Notch activity is removed from Region 1, ectopic Rhomboid cells (Notch+) arise adjacent to Notch (Broad) cells, thus resembling the normal Notch border. It is proposed that these Notch mutant clones produce ectopic borders of differential Notch activity, which in turn generate ectopic boundaries between Rhomboid and Broad domains (Ward, 2006).

Normally, Rhomboid cells arise all along the high–low Notch boundary in each dorsal appendage primordium. Based upon this observation, one might expect that Rhomboid cells would surround the Notch clones. In the current studies, however, it was found that only those cells close to the normal boundary turned on ectopic rhomboid. Two factors probably contribute to this result. First, other signaling pathways, most notably EGFR and DPP, are involved in specifying and positioning the Rhomboid and Broad cell populations within the follicular epithelium. Presumably, these other signaling pathways influence Broad/rhomboid expression in cells adjacent to Notch clones. Second, the ectopic Notch borders generated by Notch clones arise within the Broad domain, which normally has low levels of Notch. Therefore, many cells at the ectopic border may not have sufficient Notch activity to repress Broad and activate rhomboid (Ward, 2006).

Within the domain that would normally express Broad, loss of Notch causes the loss of Broad non-cell autonomously in adjacent cells and the appearance of ectopic rhomboid in these same cells. Furthermore, Notch clones spanning the boundary ectopically express Broad and do not express rhomboid. These findings are consistent with previous results demonstrating that dorsal appendage cells express either rhomboid or Broad, but never both markers. This work shows that broad cells ectopically express rhomboid, suggesting that one function of Broad in the follicular epithelium is to directly or indirectly repress rhomboid expression. Such regulation must occur (at least in part) in the 2.2-kb fragment that drives lacZ expression in a reporter construct. CONSITE software detects twenty Broad binding sites clustered together in this region; all four zinc-finger isoforms have the potential to bind. Thus, high levels of Broad could directly regulate rhomboid in Region 1. Additional work is needed to test this hypothesis (Ward, 2006).

Other factors must also regulate rhomboid expression in Region 2. Within clones spanning the boundary, ectopic expression of Broad prevents rhomboid expression. In cells adjacent to Notch clones, loss of Broad expression allows ectopic rhomboid expression. Nevertheless, the simple absence of Broad is insufficient to induce rhomboid expression, since the majority of cells in Region 2 lack Broad expression and do not express rhomboid. Presumably, high levels of EGFR and DPP signaling prevent rhomboid expression in these cells (Ward, 2006).

The Notch loss- and gain-of-function data, as well as the Notch expression pattern, all suggest that juxtaposition of two cells with different Notch levels is critical for establishing the boundary between Rhomboid and Broad cell types. How, then, is Notch protein level regulated? The restricted pattern of Notch in the dorsal anterior follicle cells suggests that Notch expression is determined by a combination of patterning instructions from DPP along the anterior/posterior axis and EGFR signaling along the dorsal/ventral axis (Ward, 2006).

The importance of regulating Notch protein levels is underscored by data showing that overexpression of full-length Notch represses Broad expression throughout the follicular epithelium. Since the full-length Notch receptor must be bound by ligand to initiate Notch signaling, a Notch ligand is either present throughout the follicular epithelium or is presented to the follicle cells by the underlying germ line. The Drosophila genome encodes two known Notch ligands, Delta and Serrate, and several potential ligands, such as CG9138. The absence of both Delta and Serrate in the follicular layer did not affect Broad or rhomboid expression. The function of other potential ligands in follicle cells is not currently known. It is also possible that the ligand for this process is present in the germ line. Delta is expressed in the germ line at the appropriate time and functions in the germ line to regulate follicle cell processes, such as the pinching-off of egg chambers in the germarium and the mitotic-to-endocycle transition at stage 7. Additionally, previous work demonstrates that egghead and brainiac, which encode modulators of Notch function, act in the germ line to pattern the dorsal anterior follicle cells. Regardless of the tissue distribution of the ligand, however, the ability to uniformly activate the Notch pathway throughout the follicle cell layer is note-worthy. This observation suggests that Notch levels, rather than spatial location of a ligand (or ligand modulator), determines where or how Notch signals in follicle cells of late stage egg chambers (Ward, 2006).

One of the most surprising aspects of the work presented here is that Notch clones act in a non-cell-autonomous manner to regulate Broad and rhomboid expression in adjacent cells. While surprising, non-cell-autonomous Notch activity occurs in the embryo, and most notably, at the D/V boundary in the wing disc. In the third-instar wing disc, Wingless is expressed in a 3- to 6-cell wide stripe spanning the D/V boundary, which separates the dorsal and ventral portions of the future wing blade. In this system, wingless-lacZ is repressed both within and adjacent to Notch clones. Thus, Notch clones act non-cell autonomously in two different tissues where boundaries act to distinguish different cell types (Ward, 2006).

What is the nature of the non-autonomous signal from the Notch clones? It is proposed two potential mechanisms to explain this process. First, Notch itself measures Notch levels in adjacent cells, either directly through homophilic adhesion or indirectly through interaction with Notch-binding proteins. When a Notch clone occurs in the dorsal anterior, adjacent cells sense the absence of Notch and respond as wild-type cells do when high-Notch cells neighbor low-Notch cells; they either repress Broad directly, or they repress Broad indirectly by affecting Pangolin (or some other component of the Wingless signaling pathway). Pangolin is needed to express Broad and therefore down-regulate rhomboid throughout the follicle cell layer. A second possibility is that when cells have little or no Notch activity, they might secrete an inhibitor of the Pangolin pathway that only affects cells with high Notch. The first mechanism is favored for its simplicity in accounting for rhomboid expression only at the border between high- and low-Notch-expressing cells (Ward, 2006).

The establishment of a border between Rhomboid and Broad cells is important for preventing intermingling of these cell types during tube formation (Ward, 2005). It is not clear, however, what mechanism separates the Broad and Rhomboid cells from each other at the border. In some situations, the non-transcriptional branch of the Notch pathway regulates F-actin (Major, 2005), which creates a “fence” that could help separate the two cell types from each other in the border. In dorsal anterior follicle cells, however, the canonical Notch pathway acts through the transcription factor Su(H). It is possible that in this cell type, the Notch pathway transcriptionally regulates a cell adhesion molecule or other component of an actin-binding protein complex, which in turn coordinates the cytoskeleton, thereby maintaining a separation between the Rhomboid cells and the Broad cells. Unlike cells at other boundaries in which an actin fence is evident, the Rhomboid and Broad cells undergo dramatic morphological changes and reorganize their actin networks to produce these effects. A fence that could maintain the separation of these cells during apical constriction, directed elongation, and convergent extension would be critical during these processes. One such Notch-interacting candidate gene that links to actin filaments is Echinoid. Future experiments will define whether Echinoid plays a role during border formation between Rhomboid and Broad cells (Ward, 2006).

Animals have a wide variety of organs containing different cell types arranged in a stereotypical manner. While the general morphogenesis of most organs has been described, little is known about the molecular mechanisms required to specify boundaries between diverse cell types and direct their subsequent reorganization to produce a functional structure. This study has shown that canonical Notch signaling is necessary to establish a boundary between the Broad and Rhomboid cells, which will form the dorsal and ventral portions of the dorsal appendage tube. Notch is also required in the vertebrate hindbrain for rhombomere boundary formation. Thus, in simple and more complex organs, Notch specifies boundaries between distinct cell populations needed for organ formation. Generating a boundary through Notch signaling could be an evolutionarily conserved first step during organ formation in many tissues. The next challenge is to define the molecular nature of the physical power that keeps the two different cell types separated from each other in the border (Ward, 2006).

Drosophila eggshell is patterned by sequential action of feedforward and feedback loops

During Drosophila oogenesis, patterning activities of the EGFR and Dpp pathways specify several subpopulations of the follicle cells that give rise to dorsal eggshell structures. The roof of dorsal eggshell appendages is formed by the follicle cells that express Broad (Br), a zinc-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp signaling. The resulting changes in the Br pattern affect the expression of Dpp receptor thickveins (tkv), which is essential for Dpp signaling. By controlling tkv, Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its own repression in a negative-feedback loop. These observations have been synthesized into a model, whereby the dynamics of Br expression are driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of Br expression, while the feedback loop modulates this pattern in time. This mechanism demonstrates how complex patterns of gene expression can emerge from simple inputs, through the interaction of regulatory network motifs (Yakoby, 2008a).

These results provide new insights into the dynamics and function of the Dpp pathway in oogenesis. First, it was demonstrated that, contrary to the current model of Drosophila eggshell patterning, the pattern of Dpp signaling in oogenesis is not static, and undergoes a clear transition from purely AP to DV pattern in late stages of eggshell patterning. This transition reflects the change in the expression of the type I Dpp receptor and is conserved in fly species separated by more than 40 million years of evolution. Second, it was shown that the early and late patterns of Dpp signaling control the dynamic pattern of br, a transcription factor expressed in the roof of future dorsal appendages. While the AP phase of Dpp signaling represses br in the anterior region of the egg chamber, the DV phase of Dpp signaling limits the duration of br expression in the roof cells. Third, it was established that, in addition to being regulated by Dpp, Br actively controls Dpp signaling, thus regulating its own repression via a negative-feedback loop (Yakoby, 2008a).

The results lead to a new model for the dynamics of Br expression in the roof cells. Within the framework of this model, the rising phase of Br expression is due to an incoherent feedforward loop, a network in which the input activates both the target and its repressor. In this case, the feedforward loop, formed by EGFR, Pointed, and Br, determines the spatial pattern of Br. This pattern is then modulated in time by a negative-feedback, which depends on the Br-mediated increase of tkv expression and Dpp signaling. The feedforward part of the model is supported by the previously published gain- and loss-of-function experiments with Pointed and EGFR signaling, and by the current analysis of Br expression in ras- mosaics. The negative-feedback loop is supported by the correlation of patterns of Br, Tkv, and P-Mad, by published experiments with manipulation of the levels of Dpp, by analysis of Br protein and br transcript in the Dpp pathway loss-of-function experiments, and by the effects of br- clones and Br overexpression on tkv and Dpp signaling (Yakoby, 2008a).

Four phases in the dynamics of the Br pattern are distinguished. (1) Low levels of Br before stage 9 of oogenesis are independent of EGFR signaling and insensitive to repression by Dpp. (2) Following the formation of the DV gradient of EGFR activation, Br is repressed in the midline and in the dorsoanterior cells. The midline repression is due to Pointed, a transcription factor induced by high levels of EGFR activation in the dorsal midline. The dorsoanterior repression is due to the early phase of Dpp signaling, which reflects the anterior secretion of Dpp and uniform expression of Tkv. (3) Levels of Br begin to rise in the roof cells. Changes in the Br pattern have two effects on the spatial pattern of Dpp signaling: higher levels of Br lead to higher levels of tkv in the roof cells. Second, the dorsoanterior and midline repression of Br generates a corresponding repression of tkv. (4) As a result, the anteriorly produced Dpp can diffuse over the 'Tkv-free' area to the roof cells. A combination of the arrival of the anteriorly produced ligand and a higher level of receptor expression leads to a higher level of Dpp signaling in the roof cells and subsequent repression of br. Another layer of regulation is provided by Brk, a transcriptional repressor of Dpp signaling, which is induced by Gurken and repressed by Dpp signaling in the dorsal follicle cells. Brk antagonizes the repressive effect of Dpp in the roof cells until the level of Dpp signaling in the roof cells becomes high enough to repress Brk expression (Yakoby, 2008a).

The network characterized in this study can interact with a number of previously discovered feedback loops. For instance, Argos, which provides negative-feedback control of EGFR signaling in the dorsal midline, is a potential target of Dpp signaling. Future work is required to explore the extent to which this feedback loop, which had been proposed to affect dorsal midline patterning, interacts with the mechanism established in this paper (Yakoby, 2008a).

Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila

The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. This study shows that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity (Maurange, 2008).

Initially investigated was whether distinct temporal subsets of neurons are generated throughout the larval CNS. Chinmo and Broad Complex (Br-C), two BTB-zinc finger proteins known to be expressed in the postembryonic CNS, are distributed in complementary patterns in the central brain, thoracic, and abdominal neuromeres at the larval/prepupal transition stage at 96 hr (timings are relative to larval hatching at 0 hr). Chinmo is expressed by early-born neurons located in a deep layer, whereas Br-C marks later-born neurons in a largely nonoverlapping and more superficial layer. The deep Chinmo+ layer comprises most/all neurons born in the embryo plus an early subset of those generated postembryonically. Thoracic postembryonic neuroblasts undergo the Chinmo --> Br-C switch at ~60 hr such that they have each generated an average of 15 Chinmo+ cells expressing little or no Br-C and 39 Chinmo- Br-C+ cells by 96 hr. The Chinmo+ and Br-C+ neuronal identities can be recognized as distinct cell populations on the basis of an ~2-fold difference in cell-body volume. This equates to an average cell-body diameter for Chinmo+ neurons of 4.5 μm, compared to only 3.6 μm for Br-C+ neurons. Plotting cell diameter versus deep-to-superficial position within postembryonic neuroblast clones reveals an abrupt decrease in neuronal size at the Chinmo --> Br-C transition. Together, these results provide evidence that most, if not all, postembryonic neuroblasts sequentially generate at least two different populations of neurons. First they generate large Chinmo+ neurons and then they switch to producing smaller Br-C+ neurons (Maurange, 2008).

To begin dissecting the neuronal switching mechanism, the functions of Chinmo and Br-C were investigated, but neither factor was found to be required for the transition in cell identity and cell size. Next it was asked whether a temporal transcription factor series related to the embryonic Hb --> Kr --> Pdm --> Cas sequence might be involved. Cas is known to be expressed in the larval CNS, and this study shows that many different thoracic neuroblasts progress through a transient Cas+ phase during the 30-50 hr time window. Thoracic neuroblasts transiently express another member of the embryonic temporal series, Svp, during a somewhat later time window, from ~40 to ~60 hr. These results indicate that postembryonic Cas and Svp bursts are observed in many, but probably not all, thoracic progenitors and that their timing varies from neuroblast to neuroblast (Maurange, 2008).

To determine Svp function, thoracic neuroblast clones were generated homozygous for svpe22, an amorphic allele. In ~53% of svpe22 neuroblast clones induced in the early larva (at 12-36 hr), the Br-C+ neuronal identity is completely absent, all neurons express Chinmo, and there is no sharp decrease in neuronal size. The proportion of lineages failing to generate Br-C+ neurons rises to ~70% when clones are induced in the embryo and falls to only ~7% with late-larval (65-75 hr) induction. This is consistent with a previous finding that Svp bursts are asynchronous from neuroblast to neuroblast. The expression and clonal analyses together demonstrate that a progenitor-specific burst of Svp is required in many lineages for the switch from large Chinmo+ to small Br-C+ neurons (Maurange, 2008).

Thoracic neuroblast lineages homozygous for a strong cas allele, cas24, show no obvious defects in the Chinmo --> Br-C transition when induced at 12-36 hr. However, since Cas is expressed in many postembryonic neuroblasts before their first larval division, it can only be removed by inducing clones during embryonic neurogenesis. Such cas24 clones generate supernumerary Chinmo+ neurons and completely lack Br-C+ neurons at 96 hr, although this switching phenotype is restricted to only ~16% of thoracic neuroblasts. Constitutively expressing Cas blocks the Chinmo --> Br-C switch in a similar manner, with a frequency dependent upon whether thoracic UAS-cas clones are induced during embryogenesis (~47%), at early-larval (~10%) or at late-larval (0%) stages. This indicates that the response to Cas misexpression decreases as neuroblasts age. Together, the expression and loss- and gain-of-function analyses demonstrate that Chinmo and Br-C are negative and positive targets, respectively, of Cas and Svp. They also strongly suggest that progression through transient Cas+ and Svp+ states permits many postembryonic neuroblasts to switch from generating large to small neurons (Maurange, 2008).

To investigate whether Cas and Svp regulate neural proliferation as well as neuronal fates, the effector mechanism ending neurogenesis in the central brain and thorax was identified. In these regions, most neuroblasts cease dividing in the pupa at ~120 hr. Correspondingly, neurogenesis in all regions of the wild-type CNS ceases before the adult fly ecloses such that no adult neuroblasts are detected. In contrast to the central abdomen, blocking cell death by removing Reaper, Hid, and/or Grim (RHG) activity in the central brain and thorax does not prevent or delay pupal neuroblast disappearance. However, time-lapse movies of individual thoracic neuroblasts at ~120 hr reveal an atypical mitosis that is much slower than at ~96 hr, producing two daughters of almost equal size. This is largely accounted for by a reduction in the average diameter of neuroblasts from 10.4 μm at 96 hr to 7 μm at 120 hr, as GMC size does not vary significantly during this time window. The end of this atypical progenitor mitosis temporally correlates with reduced numbers of Mira+ cells and disappearance of the M phase marker phosphorylated-Histone H3 (PH3), indicating that it marks the terminal division of the neuroblast (Maurange, 2008).

Next whether late changes in basal complex components might underlie loss of neuroblast self-renewal was addressed. At 120 hr, it was found that Mira becomes delocalized from the cortex to the cytoplasm and nucleus of many interphase neuroblasts. In metaphase neuroblasts, Mira fails to localize to the basal side of the cortex, although it does selectively partition into one daughter during telophase. This late basal restoration resembles the 'telophase rescue' associated with several apical complex mutations. Pros, the Mira-binding transcription factor and GMC-determinant, is not detectable in the neuroblast nucleus at 96 hr, but at 120 hr a burst of Pros was observe in the nucleus of many Mira+ cells of intermediate size indicative of neuroblasts in the interphase preceding the terminal mitosis. Clones lacking Pros activity contain multiple Mira+ neuroblast-like cells. It was found that they do not respect the ~120 hr proliferation endpoint and even retain numerous dividing Mira+ progenitors into adulthood. In addition, GAL80ts induction was used to induce transiently the expression of a YFP-Pros fusion protein well before the normal ~120 hr endpoint. Under these conditions, YFP-Pros can be observed in the nucleus of neuroblasts, most Mira+ progenitors disappear prematurely, and neural proliferation ceases much earlier than normal. Together, these results provide evidence that most neuroblasts terminate activity in the pupa via a nuclear burst of Pros that induces cell-cycle exit. These progenitors are referred to as type I neuroblasts to distinguish them from the much smaller population of type II neuroblasts that terminate via RHG-dependent apoptosis (Maurange, 2008).

Whether the postembryonic pulses of Cas and Svp in type I neuroblasts are implicated in scheduling their subsequent cell-cycle exit was tested. Remarkably, it was observed that many svpe22 clones induced at early-larval stages retain a single Mira+ neuroblast at 7 days into adulthood. The persistent adult neuroblasts in a proportion of these clones also express the M phase marker, PH3, indicating that they remain engaged in the cell cycle and, accordingly, they generate approximately twice the normal number of cells by 3 days into adulthood. Furthermore, superficial last-born neurons in these over proliferating svpe22 adult clones are Chinmo+ Br-C- indicating a blocked Chinmo --> Br-C transition. Similar phenotypes are obtained in some UAS-cas and cas24 clones. This analysis demonstrates that stalling the temporal series not only inhibits the late-larval switch to Br-C+ neuronal identity but also prevents the pupal cell-cycle exit of type I neuroblasts (Maurange, 2008).

To test the regulatory relationship between Pros and the temporal series, svpe22 clones were examined at pupal stages. It was found that mutant interphase neuroblasts fail to switch on nuclear Pros at 120 hr, although svpe22 GMCs express nuclear Pros as normal. This likely accounts for why adult clones lacking Svp retain only a single neuroblast, whereas those lacking Pros contain multiple neuroblast-like progenitors. Importantly, these results demonstrate that nuclear Pros acts downstream of the temporal series in type I neuroblasts. Together, the genetic and expression analyses of Svp and Pros show that the temporal series triggers a burst of nuclear Pros in type I neuroblasts, thus inducing their cell-cycle exit (Maurange, 2008).

To determine how the temporal series is linked to the cessation of progenitor divisions, two transcription factors expressed in neuroblasts in a temporally restricted manner were examined. Dichaete (D), a member of the SoxB family, is dynamically expressed in the early embryo and is required for neuroblast formation. Consistent with the previous studies, it was observed that most or all embryonic neuroblasts progress through a transient D+ phase, but those in the lateral column of the ventral nerve cord initiate expression after their medial and intermediate counterparts. Dichaete subsequently becomes repressed in ~85% of neuroblasts during late-embryonic and postembryonic stages. Grainyhead (Grh) is first activated in neuroblasts in the late embryo and is required for regulating their mitotic activity during larval stages. Blocking early temporal series progression in the embryo, either by persistent Hb or loss of Cas activity, prevents most neuroblasts from downregulating D and also from activating Grh at late-embryonic and postembryonic stages. As forcing premature Cas expression leads to precocious D repression and Grh activation, both factors are likely to be regulated by Cas rather than by a later member of the temporal series. These results demonstrate that transient embryonic Cas activity permanently switches the expression of Grh on and D off. They also identify Grh and D as positive and negative targets, respectively, of the temporal series in many neuroblasts (Maurange, 2008).

Loss of Grh activity in thoracic neuroblasts (here defined as type I neuroblasts) leads to their reduced cell-cycle speed and disappearance during larval stages. At 96 hr, it was observed that 65% of grh370 type I neuroblasts are smaller than normal (~6.4 μm in diameter), delocalize Mira from the cortex to the cytoplasm and nucleus, and strongly express Pros in the nucleus. These events, reminiscent of the 120 hr terminal cell cycle of wild-type progenitors, show that Grh is required to prevent the premature cell-cycle exit of type I neuroblasts. Given this finding, and that Cas is required to activate Grh, the question arises as to how some neuroblasts lacking embryonic Cas activity are able to continue dividing into adulthood. However, cas24 neuroblasts persisting in adults all retain Grh, suggesting that they may derive from clones that lacked only the last of the two Cas pulses observed in some embryonic neuroblasts, perhaps retaining enough Cas activity to support Grh activation but not later progression of the temporal series (Maurange, 2008).

To determine if late temporal series inputs, after embryonic Cas, are also required to maintain the long-lasting postembryonic expression of Grh, svpe22 clones were induced in early larvae. Although type I neuroblasts in these mutant clones have a stalled temporal series, they retain postembryonic Grh expression through to adult stages. Thus, two sequential inputs from the temporal series are required for type I neuroblasts to undergo timely Pros-dependent cell-cycle exit. First, embryonic Cas activity switches on sustained Grh expression, inhibiting premature nuclear Pros and permitting continued mitotic activity. Second, a late postembryonic input, requiring Svp, counteracts this activity of Grh by triggering a pupal burst of nuclear Pros (Maurange, 2008).

Despite undergoing premature cell-cycle exit, it was noticed that grh mutant neuroblasts can still generate both Chinmo+ and Br-C+ neurons. Thus, Grh is not required for the neuronal Chinmo --> Br-C switch. Conversely, neither Chinmo nor Br-C appears to be required postembryonically for neuroblast cell-cycle exit. In summary, the properties of both neuroblasts and neurons are regulated by downstream targets of the temporal series (Maurange, 2008).

Next whether the temporal series and its targets also function in type II neuroblasts, which terminate via RHG-dependent apoptosis rather than Pros-dependent cell-cycle exit, was tested. Focus was placed on one identified type II neuroblast in the central abdomen, called dl, which undergoes apoptosis at 70-75 hr and produces only a small postembryonic lineage of ~10 neurons. It was observed that the dl neuroblast expresses bursts of Cas (~45 to ~60 hr) and Svp (~62 to ~65 hr) and sequentially generates Chinmo+ (~7 deep) and Br-C+ (~3 superficial) neurons. Loss of Cas or Svp activity, or prevention of temporal series progression in several other ways all lead to a blocked Chinmo --> Br-C transition, a failure to die at 70-75 hr, and the subsequent generation of many supernumerary progeny. These results show that the temporal series performs similar functions in type I and type II neuroblast lineages, regulating both the Chinmo --> Br-C neuronal switch and the cessation of progenitor activity. Next, the mechanism linking the temporal series to RHG-dependent death of type II neuroblasts was tested. As for type I neuroblasts, dl progenitors in cas24 clones, induced in the embryo, fail to activate Grh and repress D. Moreover, if grh activity is reduced, or if D is continuously misexpressed, dl progenitors persist long after 75 hr. Therefore, both of these early Cas-dependent events are essential for subsequent type-II neuroblast apoptosis. However, in contrast to members of the temporal series themselves, persistent misexpression of their target D does not block the Chinmo --> Br-C switch in the dl lineage. Thus, the D- Grh+ state of type I and type II neuroblasts, installed via an embryonic Cas pulse, appears to be necessary for progenitor termination but not for Chinmo --> Br-C switching. Nevertheless, dl neuroblasts stalled at a postembryonic stage in svpe22 and UAS-cas clones retain the D- Grh+ code yet still fail to undergo apoptosis. Therefore, as with type I cell-cycle exit, timely type II apoptosis requires both embryonic Cas-dependent and postembryonic Svp-dependent inputs from the temporal series (Maurange, 2008).

Finally, the regulatory relationship between the temporal series and AbdA, a Hox protein transiently expressed in postembryonic type II (but not type I) neuroblasts and required for their apoptosis was dissected. dl neuroblasts lacking postembryonic Svp activity or persistently expressing Cas still retain AbdA expression yet do not die. This suggests that AbdA is unable to kill neuroblasts unless they progress, in a Svp-dependent manner, to a late Cas- temporal state. To test this prediction directly, use was made of the previous finding that ectopic AbdA is sufficient to induce neuroblast apoptosis, albeit only within a late time window. Constitutive AbdA-induced apoptosis is efficiently suppressed by persistent Cas, not only in type II but also in type I neuroblasts. This result demonstrates that, in order to terminate, type II neuroblasts must progress to a late Cas- state, thus acquiring a D- Grh+ Cas- AbdA+ code. It also suggests that AbdA is sufficient to intercept progression of the temporal series in type I neuroblasts, inducing an early type II-like termination (Maurange, 2008).

This study has found that the Drosophila CNS contains two distinct types of self-renewing progenitors: type I neuroblasts terminate divisions by cell-cycle withdrawal and type II neuroblasts via apoptosis. Despite these different exit strategies, both progenitor types use a similar molecular timer, the temporal series, to shut down proliferation and thus prevent CNS overgrowth. These findings demonstrate that the temporal series does considerably more than just modifying neurons; it also has multiple inputs into neural proliferation. The identification and analysis of several pan-lineage targets of the temporal series also begins to shed light on the mechanism by which developmental age modifies the properties of neuroblasts and neurons. Two targets, Chinmo and Br-C, are part of a downstream pathway temporally regulating the size and identity of neurons. Two other temporal series targets, Grh and D, function in neuroblasts to regulate Prospero/RHG activity, thereby setting the time at which proliferation ends. The temporal series regulates both cell proliferation and cell identity; a feedforward mechanism is proposed for generating combinatorial transcription factor codes during progenitor aging (Maurange, 2008).

This study has found that the temporal series regulates a widespread postembryonic switch in neuronal identity. Most, if not all, type I and type II neuroblasts first generate a deep layer of large Chinmo+ neurons and then switch to producing a superficial layer of small Br-C+ neurons. Two lines of evidence argue that this postembryonic neuronal switch is likely to be regulated by a continuation of the same temporal series controlling early/late neuronal identities in the embryo. First, the postembryonic Chinmo --> Br-C neuronal switch is promoted by the transient redeployment of two known components of the embryonic temporal series, Cas and Svp. Second, this switch remains inhibitable by misexpression of the other embryonic temporal factors such as Hb. Since both Cas and Svp are expressed somewhat earlier than the neuronal-size transition, it is likely that they promote bursts of later, as yet unknown, members of the temporal series that more directly regulate Chinmo and Br-C. Although neuronal functions for both BTB zinc-finger targets have yet to be characterized, a progressive early-to-late decrease in postmitotic Chinmo levels is known to regulate the temporal identities of mushroom-body neurons. The current results now suggest that this postmitotic gradient mechanism may be linked to, rather than independent from, the temporal series (Maurange, 2008).

Type I neuroblasts in clones lacking postembryonic Cas/Svp activity or retaining an early temporal factor, fail to express nuclear Pros during pupal stages and thus continue dividing long into adulthood. These overproliferating adult clones each contain only a single neuroblast, sharply contrasting with adult clones lacking Brat or Pros, in which there are multiple neuroblast-like progenitors. Hence, manipulations of the temporal series and its progenitor targets offer the prospect of immortalizing neural precursors in a controlled manner, without disrupting their self-renewing asymmetric divisions (Maurange, 2008).

This study demonstrates that type I and type II neuroblasts must progress through at least two critical phases of the temporal series in order to acquire the D- Grh+ Cas- combinatorial transcription factor code that precedes Pros/RHG activation. The early phase corresponds to embryonic Cas activity switching neuroblasts from D+ Grh- to D- Grh+ status. The equally essential, but less well-defined, late postembryonic phase of the temporal series requires transition to a Cas- state and a late Svp burst. For type I neuroblasts, Grh and a late Cas- temporal identity are both required for timely expression of nuclear Pros and subsequent cell-cycle withdrawal. For type II neuroblasts, these two inputs are also necessary for RHG-dependent apoptosis, with the additional requirement that D must remain repressed. Although the temporal series and its targets are similarly expressed in type I and type II neuroblasts, only the latter progenitors undergo a larval burst of AbdA. This AbdA expression is likely to be the final event required to convert the D- Grh+ Cas- state, installed by the temporal series, into the D- Grh+ Cas- AbdA+ combinatorial code for RHG-dependent apoptosis. This code prevents type II neuroblasts in the abdomen from reaching the end of the temporal series and accounts for why they generate fewer progeny and terminate earlier than their type I counterparts in the central brain and thorax (Maurange, 2008).

The data in this study support an indirect feedforward model for neuroblast aging. Key to this model is the finding that, although members of the temporal series are only expressed very transiently, some of their targets can be activated or repressed in a sustained manner, as observed for Chinmo/Br-C in neurons and also for Grh/D in neuroblasts. In principle, this indirect feedforward allows aging progenitors to acquire step-wise the combinatorial transcription factor codes modulating cell-cycle speed, growth-factor dependence, competence states, and neural potential. Like Drosophila neuroblasts, isolated mammalian cortical progenitors can sequentially generate neuronal fates in the correct in vivo order. These studies suggest that it will be important to investigate whether the transcription factors controlling this process also regulate cortical proliferation and whether their targets include BTB-zinc finger, Grh, SoxB, Prox, or proapoptotic proteins. Some insect/mammalian parallels seem likely, since it is known that Sox2 downregulation and Prox1 upregulation can both promote the cell-cycle exit of certain types of vertebrate neural progenitors (Dyer, 2003, Graham, 2003). Thus, although insect and mammalian neural progenitors do not appear to use the same sequence of temporal transcription factors, at least some of the more downstream components identified in this study might be functionally conserved (Maurange, 2008).

br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. This study used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. A conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter was identified using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development (Xiang, 2010).

Ecdysone hormone produced in the ring gland plays a central role in regulating insect development. This study identified RSE, a ring gland-specific cis-regulatory element, in the promoter of the ecdysone biosynthesis-related gene npc1. In addition, br, an ecdysone early response gene, was found to be a key regulator for the ring gland expression of npc1. Moreover, br seems to regulate the expression of many other ecdysone biosynthesis-related genes in the ring gland (Xiang, 2010).

The functions of br have been studied extensively. As an ecdysone early response gene, it regulates the transcription of many late response genes, including L71, sgs-4, and hsp23, in response to the ecdysone signal. br also regulates the expression of Drosophila caspase Dronc, which is important for programmed cell death during metamorphosis. The null allele of br, brnpr-3 displays an ecdysone-deficient phenotype, arresting at wandering third instar for a long time, which is consistent with its role in mediating the ecdysone response. However, it was found that implanting a wild-type ring gland can partially rescue the brnpr-3 phenotype, implying that br mutants may be partial ecdysone deficient. Previous studies on the role of BR have mainly focused on ecdysone-responding tissues, for instance the salivary gland. It remains unclear whether BR also affects ecdysone biosynthesis. The current findings suggest that besides having a role in triggering ecdysone late response gene expression in larval peripheral tissues, br also has an important role in regulating ecdysone biosynthesis in the ecdysone-producing organ. In addition, the fact that the larval-arrest phenotype in the animals with ring gland-specific knockdown of br was only partially rescued by ecdysone indicates that br may have other roles in the ring gland. In agreement with that, experiments with ring gland-specific overexpression of different br isoforms revealed that br may function in regulating the degeneration of ring gland (Xiang, 2010).

This study implies the existence of a positive feedback loop in which ecdysone regulates the transcriptional expression of the early response gene br and br could subsequently augment the transcription of ecdysone biosynthesis-related genes to further boost ecdysone production in the ring gland. The feedback regulation of ecdysone biosynthesis has been well documented in insects and ecdysone could have both positive and negative roles on ecdysone biosynthesis. What is the significance of such a positive feedback regulation mechanism? During development, ecdysone levels increase and decrease rapidly before molt and after molt, respectively, as well as before pupariation and after pupariation. The mechanism by which such quick changes of ecdysone levels are achieved remains elusive. It is believed that the positive feedback regulation may facilitate the rapid increase in ecdysone biosynthesis. In contrast, this feedback may be involved in the subsequent decline in ecdysone biosynthesis after molt or pupariation. Therefore, this feedback regulation could help to fine-tune ecdysone biosynthesis within a small time window during rapid development (Xiang, 2010).

The initial aim of this study was to identify a ring gland-specific factor that acts on the RSE to regulate npc1 tissue-specific gene expression. However, several known ring gland-specific transcription regulators seem not to be required for npc1 expression. In contrast, BR, which is not a ring gland-specific protein, was proved to be vital for npc1 ring gland expression. These studies suggest that there are unknown factor(s) involved in npc1 transcriptional regulation. Wild-type nuclear extracts led to a high-molecular-weight shift of the RSE core probe, suggesting that these factors may form a large protein complex. Unfortunately, the identity of these unknown factors remains to be determined. More work, for example purifying BR interacting proteins to reveal their identity and to examine whether they are ring gland-specifically expressed, needs to be done (Xiang, 2010).

The RSE identified in this study is the first ring gland-specific element to be discovered. It is conserved through evolution in several Drosophila species. RSEs of other Drosophila species are active in D. melanogaster. Consistently, the regulator BR is also conserved. Moreover, the RSE core BR-Z4 binding site is present in a set of ecdysone biosynthesis genes, suggesting that the RSE is important for ecdysone biosynthesis. In addition, many ring gland-specific Gal4 lines have previously been reported, including 2-286, P0206, May60, and phm. Besides phm-Gal4, the regulatory sequences for these Gal4 lines are unknown. This study found that while the activity of npc1-Gal4 is regulated by br, the activity of P0206-Gal4 is also regulated by br. In contrast, the RSE is likely not the only ring gland-specific element. There are other ring gland-specific transcription regulators, such as ecd, mld, and woc, which do not act on the RSE of npc1. The targeting regulatory elements for these genes have not yet been identified. Finding such elements would undoubtedly advance knowledge of the regulation of ecdysone biosynthesis (Xiang, 2010).

As an evolutionary conserved gene, npc1 is important for cholesterol trafficking in many other systems including mammals. In mice, npc1 is vital for neurosteroid biosynthesis, which is likely a key factor determining the neurodegenerative phenotype of npc1 mutants. These studies on the regulation of Drosophila npc1 tissue-specific expression by br may contribute to studies on the regulation of neurosteroid biosynthesis in higher animals (Xiang, 2010).

Conserved microRNA miR-8 controls body size in response to steroid signaling in Drosophila

Body size determination is a process that is tightly linked with developmental maturation. Ecdysone, an insect maturation hormone, contributes to this process by antagonizing insulin signaling and thereby suppressing juvenile growth. This study reports that the microRNA miR-8 and its target, u-shaped (USH), a conserved microRNA/target axis that regulates insulin signaling, are critical for ecdysone-induced body size determination in Drosophila. The miR-8 level is reduced in response to ecdysone, while the USH level is up-regulated reciprocally, and miR-8 is transcriptionally repressed by ecdysone's early response genes. Furthermore, modulating the miR-8 level correlatively changes the fly body size; either overexpression or deletion of miR-8 abrogates ecdysone-induced growth control. Consistently, perturbation of USH impedes ecdysone's effect on body growth. Thus, miR-8 acts as a molecular rheostat that tunes organismal growth in response to a developmental maturation signal (Jin, 2012).

This study reveals the mechanism by which ecdysone suppresses insulin signaling and thereby decelerates larval growth. During larval development, ecdysone regulates the levels of miR-8 and its target, USH, a PI3 kinase inhibitor, through the EcR downstream pathway, and quantitative regulation of miR-8 by ecdysone leads to determining the final fly size. Since the ecdysone level is low during the larval stage not engaged in the molting process, a mild response of miR-8 increase by EcR inhibition in this stage would be expected. Notably, however, it was found that this increase in miR-8 level by EcRDN is constantly sustained throughout the third instar larval stage, which is the period of exponential growth. Interestingly, among early response genes of EcR signaling, E74 and BR-C are also persistently expressed during this period; these gene products repress miR-8 expression at the transcriptional level. Thus, throughout the third instar larval period, EcR downstream signaling keeps miR-8 (and, concomitantly, insulin signaling) under control. Because the duration of this regulation lasts several days, the effect of miR-8 modulation accumulates and manifests a significant impact on final body size. This cumulative effect accounts for the effect of ecdysone signaling on body size despite only modest changes in the miR-8 level. When EcR signaling was hampered throughout the larval stage, leading to a sustained increase of miR-8, the final body size became noticeably bigger (Jin, 2012).

This function of miR-8 in shaping body size provides a novel example in which an animal uses the inherent ability of miRNAs in fine-tuning target gene expression. Previously, several studies have shown that such a strategy has been used with miRNAs in diverse biological contexts. Specific examples include maintaining the optimal level of miRNA target proteins, which is critical for organismal survival, and setting the thresholds of target gene activity to prevent inappropriate development. The current data show the application of this type of strategy in a continuous process of organismal growth. By tuning the activity of insulin signaling, miRNAs could regulate organismal growth and ensure the attainment of appropriate body size. It is currently unclear whether similar regulatory mechanisms exist in other organisms. However, in humans and rodents, the miR-200 family of miRNAs are predominantly expressed in organs such as the pituitary, thyroid, testes, ovary, and breast, most of which are major target organs of steroid hormones. Moreover, the miR-200 family of miRNAs are significantly down-regulated by the estrogen hormone in breast cancer cells and uterus tissues, suggesting that the miR-200 family may also be controlled by steroids in mammals. It would be interesting to investigate whether a comparable regulatory axis of steroid hormone/miR-200/insulin signaling is conserved through metazoan evolution (Jin, 2012).

Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster

In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. This study provides evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. Early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. It was also shown that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down-regulated and activation and translocation of transcription factor Relish is also affected (Verma, 2015).

The ecdysone and Notch pathways synergistically regulate Cut at the dorsal-ventral boundary in Drosophila wing discs

Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression. In Drosophila, the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation. The determination of the dorsal-ventral (DV) boundary of the wing disc depends on the Notch pathway, which is activated along the DV boundary and induces the expression of the homeobox transcription factor, Cut. This study shows that Broad (Br), a zinc-finger transcription factor, is also involved in regulating Cut expression in the DV boundary region. However, Br expression is not regulated by Notch signaling in wing discs, ecdysone signaling is the upstream signal that induces Br for Cut upregulation. Also, it was found that the ecdysone-Br cascade upregulates cut-lacZ expression, a reporter containing a 2.7 kb cut enhancer region, implying that ecdysone signaling, similar to Notch, regulates cut at the transcriptional level. Collectively, these findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc. Additionally, br was shown to promote Delta, a Notch ligand, near the DV boundary to suppress aberrant high Notch activity, indicating further interaction between the two pathways for DV patterning of the wing disc (Jia, 2016).

Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster
Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. This study manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. Expression was monitored of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, it was found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. It is suggested that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression (Kaieda, 2017).

Developmental regulation of regenerative potential in Drosophila by ecdysone through a bistable loop of ZBTB transcription factors

In many organisms, the regenerative capacity of tissues progressively decreases as development progresses. However, the developmental mechanisms that restrict regenerative potential remain unclear. In Drosophila, wing imaginal discs become unable to regenerate upon damage during the third larval stage (L3). This study shows that production of ecdysone after larvae reach their critical weight (CW) terminates the window of regenerative potential by acting on a bistable loop composed of 2 antagonistic Broad-complex/Tramtrack/Bric-a-brac Zinc-finger (ZBTB) genes: chinmo and broad (br). Around mid L3, ecdysone signaling silences chinmo and activates br to switch wing epithelial progenitors from a default self-renewing to a differentiation-prone state. Before mid L3, Chinmo promotes a strong regenerative response upon tissue damage. After mid L3, Br installs a nonpermissive state that represses regeneration. Transient down-regulation of ecdysone signaling or Br in late L3 larvae enhances chinmo expression in damaged cells that regain the capacity to regenerate. This work unveils a mechanism that ties the self-renewing and regenerative potential of epithelial progenitors to developmental progression (Narbonne-Reveau, 2019).

broad: Biological Overview | Evolutionary Homologs | Transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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