InteractiveFly: GeneBrief

ftz transcription factor 1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

FTZ-F1 was first identified as a protein that binds to the zebra element, a 740 base pair DNA sequence upstream of the fushi tarazu (ftz) transcriptional start site. The zebra element is responsible for regulating fushi tarazu expression in seven stripes of alternating segment (pair rule) periodicity in the early embryo. Mutational alteration in the FTZ-F1 binding site results in a lack of expression of ftz in stripe 1, weaker, but detectable expression in stripes 2, 3 and 6, and normal staining in stripes 4, 5 and 7.

FTZ-F1 is found in both early and late forms, corresponding to an early protein found during the first few hours of embryonic development and a late protein, migrating on gels at a faster rate, appearing between 16 and 19 hours of development. DNA binding specificity cannot be distinguished between early and late forms. Besides binding to several sites in the zebra element, FTZ-F1 binds to additional sites within the ftz gene (Ueda, 1989)

FTZ-F1 also influences molting. Chromosomal puffing in the late prepupal salivary gland polytene chromosomes allows for an assessment of the order of gene activation. Puffing is evidence of a 'loosening' of the chromatin holding a gene together, and is thought to accompany gene activation. FTZ-F1 is directly involved in the regulation of the gene activation hierarchy in salivary gland chromosomes. Antibodies directed against FTZ-F1 protein detect staining of 166 loci in the late prepupal salivary gland polytene chromosomes, suggesting that FTZ-F1 regulates transcription of many genes active in polytene chromosomes. It is presumed that FTZ-F1 functions to regulate expression of the gene puffs to which it binds. 51 of these loci represent ecdysone-regulated puffs. Of 33 puffs that show increased activity after the peak of the 75CD puff (responsible for FTZ-F1 synthesis), 17 show reproducable staining for FTZ-F1. These include two prominent late prepupal puffs (74EF and 75B) encoding respectively ets-related and steroid receptor superfamily DNA binding proteins. These late prepupal puffs occur in the latter part of the early phase of the puffing hierarchy. Both 74EF and 75B are induced directly by the late larval and prepupal pulses of ecdysone. These results suggest that FTZ-F1 contributes to a significant fraction of the genes in the late prepupal phase of the molting regulatory hierarchy. Of interest is the observation that FTZ-F1 binds to the 75CD puff itself, raising the possibility of an autoregulatory interaction. Among the 25 puffs that show decreased activity in late prepupae, 7 are bound by FTZ-F1. It is therefore possible that FTZ-F1 may also participate in the repression of these puff loci (Lavorgna, 1993).

Where does FTZ-F1 fit into the hierarchy of regulatory genes expressed during metamorphosis? It has been suggested that its action may serve as a bridge between early and late gene expression during the process of metamorphosis. Early genes include Ecdysone receptor, the master regulator whose dimerization partner is Ultraspiracle. A third early protein, coded for by E75A, is another nuclear receptor superfamily member. These proteins are induced during the third instar larval period beginning during the fourth day of fly development. These early genes both repress their own expression and induce a large set of late genes. The induction of two late genes, E78A and DHR3, is delayed, relative to that of the early genes, apparently owing to an additional requirement for early ecdysone-induced protein synthesis. FTZ-F1, expressed during the fifth day of fly development, is repressed by both itself and ecdysone, thus restricting its expression to the brief interval of low ecdysone titer in midprepupae. FTZ-F1 appears to provide the competence for the "early genes" E54A, E75A, Broad Complex and E93 (an early gene expressed later in prepupal development at 5.5 to 6 days). These latter genes regulate the expression of late genes, expressed in late prepupal development and the pupal phase, beginning 5.5 days after fertilization (Woodard, 1994 and Thummel, 1995). Thus FTZ-F1 acts as a bridge between expression of the earliest genes involved in metamorphosis (Ecdysone receptor and E75) and the late genes.

Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development

Many of the 21 members of the nuclear receptor superfamily in Drosophila are transcriptionally regulated by the steroid hormone ecdysone and play a role during the onset of metamorphosis, including the EcR/USP ecdysone receptor heterodimer. The temporal patterns of expression for all detectable nuclear receptor transcripts were examined throughout major ecdysone-regulated developmental transitions in the life cycle: embryogenesis, a larval molt, puparium formation, and the prepupal-pupal transition. An unexpected close temporal relationship was found between DHR3, E75B, and betaFTZ-F1 expression after each major ecdysone pulse examined, reflecting the known cross-regulatory interactions of these genes in prepupae and suggesting that they act together at other stages in the life cycle. In addition, E75A, E78B, and DHR4 are expressed in a reproducible manner with DHR3, E75B, and betaFTZ-F1, suggesting that they intersect with this regulatory cascade. Finally, known ecdysone-inducible primary-response transcripts are coordinately induced at times when the ecdysteroid titer is low, implying the existence of novel, as yet uncharacterized, temporal signals in Drosophila (Sullivan, 2003).

Total RNA was isolated from two independent collections of embryos staged at 2-h intervals throughout the 24 h of Drosophila embryonic development. Five Northern blots were prepared using equal amounts of RNA from each time point. These blots were sequentially hybridized, stripped, and rehybridized with radioactive probes derived from each of the 21 nuclear receptor genes encoded by the Drosophila genome. This approach allowed the generation of time courses of nuclear receptor gene expression that could be directly compared between family members. The transcripts detected are consistent with reported sizes. Transcripts from eight nuclear receptor genes were not detectable during embryonic development: E75C, E78, CG16801, DHR38, DHR83, dsf, eg, and svp (Sullivan, 2003).

Transcripts from nine nuclear receptor genes can be detected at the earliest time point (0-2 h): usp, EcR-A, FTZ-F1, DHR39, DHR78, DHR96, dERR, dHNF-4, and tll. This expression is consistent with the known maternal contribution of usp, EcR, and FTZ-F1. The observation that transcripts from DHR39, DHR78, DHR96, dERR, and dHNF-4 are undetectable by the next time point examined (2-4 h) suggests that these mRNAs are maternally loaded and rapidly degraded. EcR-B and usp transcripts are induced in early embryos, up-regulated at 6-8 h after egg laying (AEL), and maintain expression through the end of embryogenesis, with down-regulation of EcR-B in late embryos. EcR-A, in contrast, is expressed for a relatively brief temporal window, at 8-14 h AEL (Sullivan, 2003).

Six nuclear receptor genes are expressed in brief intervals during midembryonic stages. DHR39 and E75A are initially induced at 4-6 and 6-8 h AEL, respectively, and peak at 8-12 h AEL. This is followed by induction of DHR3, DHR4, and E75B at 8-12 h AEL, followed by ßFTZ-F1 expression at 12-18 h AEL. DHR39 appears to exhibit an expression pattern reciprocal to that of ßFTZ-F1, with lowest levels of mRNA at 14-16 h AEL and reinduction at 16-18 h as ßFTZ-F1 is repressed. This is followed by a second peak of E75A transcription at 18-22 h AEL (Sullivan, 2003).

A second group of nuclear receptors, DHR78, DHR96, dHNF-4, and dERR, is more broadly expressed at low levels throughout embryogenesis. DHR78 accumulates above its constant low level of expression between 8 and 14 h AEL. dERR exhibits an apparent mRNA isoform switch between 14 and 18 h AEL. dHNF-4 regulation also appears complex, with two size classes of mRNA induced at approximately 8-10 h AEL. While the 4.6-kb dHNF-4 mRNA is expressed throughout embryogenesis, the 3.3-kb mRNA is down-regulated at 14-16 h AEL. This timing is consistent with observations that dHNF-4 is expressed primarily in the embryonic midgut, fat body, and Malpighian tubules. Finally, nuclear receptors known to exert essential functions in patterning the early embryo, tll, kni, and knrl, are expressed predominantly during early stages (Sullivan, 2003).

Two genes that are not members of the nuclear receptor superfamily, BR-C and E74, were also examined in this study, as transcriptional markers for ecdysone pulses during development. Unexpectedly, both of these genes are induced late in embryogenesis, several hours after the rise in ecdysone titer at 6 h AEL. An approximately 7-kb BR-C transcript is induced at 10-12 h AEL and is present through the end of embryogenesis while E74B is induced at 14-16 h AEL and repressed as E74A is expressed from 16-20 h AEL. This BR-C expression pattern is consistent with the identification of the BR-C Z3 isoform in specific neurons of the embryonic CNS (Sullivan, 2003).

First-instar larvae were synchronized as they molted to the second instar, aged and harvested at 4-h intervals throughout second-instar larval development. Two Northern blots were prepared using equal amounts of total RNA isolated from a single collection of animals. Each blot was sequentially hybridized, stripped, and rehybridized to detect nuclear receptor transcription. The following transcripts were not detectable during the second instar: E75C, dERR, CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).

EcR-B expression is induced in mid-second-instar larvae, but does not reach maximum levels until 68-72 h AEL, just before the molt. In contrast, usp is expressed throughout the instar. A sequential pattern of nuclear receptor expression is observed that resembles the pattern seen in midembryogenesis. DHR39 and E75A are expressed in the early second instar. This is followed by induction of E75B, E78B, DHR3, and DHR4, followed by expression of ßFTZ-F1 at the end of the instar. DHR39 again shows a pattern that is approximately reciprocal with ßFTZ-F1, with highest levels during the first half of the instar. Similarly, DHR78, DHR96, and dHNF-4 exhibit broad expression patterns throughout second-instar larval development. E74A, E75A, and DHR38 are coordinately up-regulated with EcR-B at the end of the instar, between 64-72 h AEL. Finally, an approximately 9-kb BR-C transcript is detected throughout the second-larval instar (Sullivan, 2003).

Nuclear receptor gene expression was also examined throughout the third larval instar and into the early stages of metamorphosis, encompassing the ecdysone-triggered larval-to-prepupal and prepupal-to-pupal transitions. Third-instar larvae were staged relative to the molt from the second instar and harvested at 4-h intervals throughout the 48 h of the instar. Prepupae were synchronized relative to puparium formation (±15 min) and harvested at 2-h intervals up to 16 h after puparium formation (APF). Total RNA was isolated from whole animals and analyzed by Northern blot hybridization. Five blots were prepared from two independent collections of animals. These blots were sequentially hybridized, stripped, and rehybridized to detect nuclear receptor gene expression. The following transcripts were not detectable during third-instar larval or prepupal stages: CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).

Most nuclear receptor genes show little or no detectable expression in early and mid-third-instar larvae, a time when the ecdysone titer is low. Similar to the pattern seen in second-instar larvae, usp is expressed at relatively low levels throughout the instar and up-regulated at puparium formation, while EcR-B is induced at approximately 100 h AEL and rapidly down-regulated at puparium formation. This is followed by a sequential pattern of nuclear receptor expression similar to that seen at earlier stages. DHR39, E75A, and E78B are induced at 116-120 h AEL, in concert with the late larval ecdysone pulse, followed by maximum accumulation of E75B, DHR3, and DHR4 at 0-4 h APF. ßFTZ-F1 is expressed from 6-10 h APF, with a pattern that is approximately reciprocal to that of DHR39. EcR-A is expressed in parallel with E75B, DHR3, and DHR4 in midprepupae, similar to their coordinate expression during embryogenesis (Sullivan, 2003).

DHR78, DHR96, and dHNF-4 continue to exhibit broad expression profiles throughout third-instar larval and prepupal development. An E75 isoform not detected in embryos or second-instar larvae, E75C, is also detectable at low levels throughout most of the third instar and up-regulated in correlation with the late-larval and prepupal pulses of ecdysone. DHR38 is detectable at very low levels in early third-instar larvae, in synchrony with the early induction of E74B and BR-C. E74B is repressed, E74A is induced, and BR-C transcripts are up-regulated in late third-instar larvae, in synchrony with the late-larval ecdysone pulse. The prepupal pulse of ecdysone occurs at 10-12 h APF, marking the prepupal-to-pupal transition. EcR-A, E75A, E78B, DHR4, dERR, E75C, dHNF-4, and E74A are all induced at 10-12 h APF, in apparent response to this hormone pulse. These results are consistent with a microarray analysis of gene expression at the onset of metamorphosis where the temporal profiles of about half of these genes have been reported (Sullivan, 2003).

Most nuclear receptors can be divided into one of four classes based on this study: (1) those that are expressed exclusively during early embryogenesis (kni, knrl, tll); (2) those that are expressed throughout development (usp, DHR78, DHR96, dHNF-4); (3) those that are expressed in a reproducible temporal cascade at each stage tested (E75A, E75B, DHR3, DHR4, FTZ-F1, DHR39), and (4) those that are undetectable in these assays (CG16801, DHR83, dsf, eg, svp) (Sullivan, 2003).

Three nuclear receptor genes appear to be expressed exclusively during early embryogenesis: kni, knrl, and tll. This restricted pattern of expression fits well with the functional characterization of these genes, which have been shown to act as key determinants of embryonic body pattern. Eight genes (usp, EcR, FTZ-F1, DHR39, DHR78, DHR96, dERR, and dHNF-4) were identified that appear to have maternally deposited transcripts and thus possible embryonic functions. Indeed, maternal functions have been defined for usp, EcR, and alphaFTZ-F1 (Sullivan, 2003).

Four nuclear receptor genes are broadly expressed through all stages examined: usp, DHR78, DHR96, and dHNF-4. dHNF-4 mRNA is first detectable at 6-10 h AEL, as the ecdysone titer begins to rise. In addition, peaks of dHNF-4 expression are seen at 0, 12, and 16 h APF, in synchrony with the E74 and E75C early ecdysone-inducible genes. These observations raise the interesting possibility that this orphan nuclear receptor is regulated by ecdysone (Sullivan, 2003).

DHR38 transcripts are difficult to detect in these assays. This is consistent with studies which used RT-PCR or riboprobes for this purpose. Nonetheless, DHR38 mRNA can be detected during third-instar larval development, consistent with the widespread expression reported in earlier studies. DHR38 expression peaks at late pupal stages, consistent with its essential role in adult cuticle formation (Sullivan, 2003).

dERR and E75C display related temporal profiles of expression that do not fit with other nuclear receptor genes described in this study. Both of these genes are specifically transcribed during prepupal development, with increases in expression at 0 and 10-12 h APF. dERR, but not E75C, is also expressed during embryogenesis, with an initial induction at approximately 6 h AEL. These increases occur in synchrony with ecdysone pulses, suggesting that these orphan nuclear receptor genes are hormone inducible, although in a stage-specific manner. Further studies of dERR regulation, as well as a genetic analysis of this locus, are currently in progress (Sullivan, 2003).

Interactions between the DHR3 and E75B orphan nuclear receptors contribute to appropriate ßFTZ-F1 regulation during the onset of metamorphosis. DHR3 is both necessary and sufficient to induce ßFTZ-F1 and appears to exert this effect directly, through two response elements in the ßFTZ-F1 promoter. E75B can heterodimerize with DHR3 and is sufficient to block the ability of DHR3 to induce ßFTZ-F1. These three factors thus define a cross-regulatory network that contributes to the timing of ßFTZ-F1 expression in midprepupae. ßFTZ-F1, in turn, acts as a competence factor that directs the appropriate genetic and biological responses to the prepupal pulse of ecdysone. The patterns of DHR3, E75B, and ßFTZ-F1 expression observed at the onset of metamorphosis are consistent with these regulatory interactions as well as the expression patterns reported in earlier studies (Sullivan, 2003).

Unexpectedly, the tight linkage of DHR3, E75B, and ßFTZ-F1 expression seen at the onset of metamorphosis is recapitulated at earlier stages, after each of the major ecdysone pulses examined, in midembryogenesis and second-instar larval development. This observation suggests that the regulatory interactions between these receptors is not restricted to metamorphosis, but rather may recur in response to each ecdysone pulse during development. It is possible that this regulatory cascade contributes to cuticle deposition, which is dependent on ecdysone signaling in embryos, larvae, and prepupae. In support of this proposal, DHR3 and ßFTZ-F1 mutants exhibit defects in larval molting, suggesting that they act together to regulate this early ecdysone response (Sullivan, 2003).

Three other orphan nuclear receptor genes, E75A, DHR4, and DHR39, are expressed in concert with DHR3, E75B, and ßFTZ-F1, after the embryonic, second-instar, and third-instar ecdysone pulses. A peak of E75A expression marks the start of each genetic cascade, correlating with the rising ecdysone titer in 6- to 8-h embryos, the first half of the second instar, and in late third-instar larvae. This is followed by DHR3, E75B, and DHR4 expression which, in turn, is followed by a burst of ßFTZ-F1 expression. E78B is expressed in synchrony with DHR4 in late second and third-instar larvae, but not in embryos. These patterns of expression raise the interesting possibility that E75A, DHR4, and E78B may intersect with the cross-regulatory network defined for DHR3, E75B, and ßFTZ-F1. E75B and E78B are related to the Rev-erb vertebrate orphan nuclear receptor and are both missing their DNA binding domain. E75B and E78B null mutants are viable and fertile, suggesting that they exert redundant regulatory functions. E75A mutants die during larval stages, with no known direct regulatory targets. DHR4 mutants have not yet been described, although recent work indicates that this gene exerts essential roles in genetic and biological responses to the late larval ecdysone pulse. Further functional studies of these nuclear receptor genes should provide insight into their possible contribution to the regulatory circuit defined by DHR3, E75B, and ßFTZ-F1 (Sullivan, 2003).

Interestingly, DHR39 displays a reproducible pattern of expression that is inversely related to that of ßFTZ-F1, defining possible repressive interactions. DHR39 and ßFTZ-F1 have a similar DNA binding domain (63% identity) and bind to identical response elements, suggesting that they may exert cross-regulatory interactions. Moreover, DHR39 can repress transcription through the same response element that is activated by ßFTZ-F1. It would be interesting to determine whether the reciprocal patterns of DHR39 and ßFTZ-F1 expression during development is of functional significance (Sullivan, 2003).

The transcription of BR-C, EcR, E74, and E75 has been extensively characterized during the onset of metamorphosis, due to their rapid and direct regulation by the steroid hormone ecdysone at this stage in development. Surprisingly, however, their expression appears to be disconnected from the high-titer ecdysteroid pulses during embryonic and second-instar larval stages. As expected, EcR is induced early in embryonic development, in coincidence with the rising ecdysone titer at 4-10 h AEL, with EcR-B transcripts appearing first followed by EcR-A. BR-C mRNA, however, is not seen until 10-12 h AEL and E74B mRNA is induced even later, at 14-16 h AEL, when the ecdysteroid titer has returned to a basal level. Both EcR-B and E74B are repressed from 16-20 h AEL as E74A and E75A are induced, a switch that has been linked to the high-titer ecdysone pulse in late third-instar larvae; however, this response occurs during late embryogenesis when the ecdysteroid titer is low. A similar observation has been made for E75A expression in the Manduca dorsal abdominal epidermis, where a brief burst of E75A mRNA is detected immediately before pupal ecdysis, after the ecdysteroid titer has returned to basal levels (Sullivan, 2003).

It thus seems likely that the second instar ecdysone pulse occurs during the first half of the instar. This profile is consistent with the early induction of E75A. EcR-B and E74A, however, are not induced until the second half of the second instar, with a peak at the end of the instar. BR-C mRNA levels remain steady throughout the second instar. Finally, EcR-B, E74B, and BR-C are induced in early to mid-third-instar larvae, a time when one or more low-titer ecdysone pulses may occur. It is curious that E74B is poorly expressed relative to E74A during embryonic and second-instar larval stages, disconnecting its expression from that of EcR. This pattern is not seen in studies that focused on the onset of metamorphosis. Taken together, the temporal profiles of early gene expression (EcR, BR-C, E74, E75A) during late embryonic and late second-instar larval stages appear to be unlinked to the known ecdysteroid pulses at these stages. This could indicate that these promoters are activated in a hormone-independent manner at these stages in the life cycle. Alternatively, these ecdysone primary-response genes may be induced by a novel temporal signal that remains to be identified (Sullivan, 2003).

Several lines of evidence indicate that 20-hydroxyecdysone is not the only temporal signal in Drosophila. A major metabolite of this hormone, 3-dehydro-20-hydroxyecdysone, was shown to be as effective as 20-hydroxyecdysone in inducing target gene transcription in the hornworm, Manduca sexta. Similarly, 3-dehydro-20-hydroxyecdysone is more efficacious than 20-hydroxyecdysone in inducing Fbp-1 transcription in the Drosophila larval fat body. A high-titer pulse of alpha-ecdysone, the precursor to 20-hydroxyecdysone, can drive the extensive proliferation of neuroblasts during early pupal development in Manduca. This is the first evidence that alpha-ecdysone is responsible for a specific response in insects. It is unlikely, however, that this signal is transduced through the EcR/USP heterodimer, which shows only very low transcriptional activity in response to this ligand. Rather, recent evidence indicates that alpha-ecdysone may activate DHR38 through a novel mechanism that does not involve direct hormone binding (Sullivan, 2003).

Studies of ecdysteroid-regulated gene expression in Drosophila have also provided evidence for hormone signaling pathways that may act independently of 20-hydroxyecdysone. Several studies have identified a large-scale switch in gene expression midway through the third larval instar, an event that has been referred to as the mid-third-instar transition. It is not clear whether this response is triggered by a low-titer ecdysteroid pulse, another hormonal signal, or in a hormone-independent manner. Similarly, the let-7 and miR-125 micro-RNAs are induced at the onset of metamorphosis in Drosophila in tight temporal correlation with the E74A early mRNA, but not in apparent response to 20-hydroxyecdysone. These studies indicate that 20-hydroxyecdysone cannot act as the sole temporal regulator during the Drosophila life cycle (Sullivan, 2003).

Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation

Tissue remodeling is a crucial process in animal development and disease progression. Coordinately controlled by the two main insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), tissues are remodeled context-specifically during insect metamorphosis. Previous work has discovered that two matrix metalloproteinases (Mmps) cooperatively induce fat body cell dissociation in Drosophila. However, the molecular events involved in this Mmps-mediated dissociation are unclear. This study reports that JH and 20E coordinately and precisely control the developmental timing of Mmps-induced fat body cell dissociation. During the larval-prepupal transition, the anti-metamorphic factor Kr-h1 was found to transduce JH signaling, which directly inhibited Mmps expression and activated expression of tissue inhibitor of metalloproteinases (timp), and thereby suppressed Mmps-induced fat body cell dissociation. It is also noted that upon a decline in the JH titer, a prepupal peak of 20E suppresses Mmps-induced fat body cell dissociation through the 20E primary-response genes, E75 and Blimp-1, which inhibited expression of the nuclear receptor and competence factor βftz-F1. Moreover, upon a decline in the 20E titer, βftz-F1 expression was induced by the 20E early-late response gene DHR3, and then βftz-F1 directly activated Mmps expression and inhibited timp expression, causing Mmps-induced fat body cell dissociation during 6-12 hrs after puparium formation. In conclusion, coordinated signaling via JH and 20E finely tunes the developmental timing of Mmps-induced fat body cell dissociation. These findings shed critical light on hormonal regulation of insect metamorphosis (Jia, 2017).

MMPs and tissue inhibitor of metalloproteinases (TIMPs) play crucial roles in regulating tissue remodeling in both vertebrates and Drosophila. Previous work has demonstrated the collaborative functions of Mmp1 and Mmp2 in inducing fat body cell dissociation in Drosophila. timp mutant adults show autolyzed tissue in the abdominal cavity and inflated wings, a phenotype consistent with the role of timp in BM integrity and remodeling. The current study clarified the role of timp in inhibiting the enzymatic activity of Mmps and thus, Mmp-induced fat body cell dissociation. In mammals, Mmps activity in vivo is controlled at different levels, including the regulation by gene expression, the zymogens activation, and the inhibition of active enzymes by TIMPs. These studies unify the important inhibitory roles of timp/TIMP in regulating tissue remodeling in both Drosophila and mammals. In addition to regulating Mmp expression, JH and 20E signals differentially regulate timp expression, with the stimulatory role of Kr-h1 and the inhibitory role of βftz-F1. Because timp inhibits the enzymatic activity of Mmps in the Drosophila fat body, it is concluded that JH and 20E coordinately control Mmps activity at both the mRNA and enzymatic levels (Jia, 2017).

Previously work has show the requirement of both JH and its receptors to inhibit fat body cell dissociation in Drosophila. This study demonstrated the ability of Kr-h1 to transduce JH signaling to decrease Mmp expression and to induce timp expression during larval-prepupal transition. Moreover, a Kr-h1-binding sites (KBS) was identified in the Mmp1 promoter, indicating that Kr-h1 directly represses Mmp1 expression. Interestingly, Kr-h1 expression gradually increases from initiation of wandering (IW) to 3 h APF when induced by JH and 20E in an overlapping manner, thus inhibiting the enzymatic activity of Mmps and Mmp-induced fat body cell dissociation during the larval-prepupal transition. Moreover, Kr-h1 acts as an anti-metamorphic factor by inhibiting 20E signaling. It is proposed, in addition to directly affecting the expression of Mmps and timp, that Kr-h1 might also indirectly regulate their expression by inhibiting 20E signaling (Jia, 2017).

Two consecutive 20E pulses control timely metamorphosis in Drosophila. Together with previous findings, the current results show that the conserved 20E transcriptional cascade precisely controls the timing of Mmp-induced fat body cell dissociation. In general, the first 20E signal pulse plays an inhibitory role during the larval-prepupal transition; however, it is a prerequisite for the expression of βftz-F1, which induces the second 20E signal pulse during the prepupal-pupal transition and the expression of Mmps. Because of the requirement for the first 20E signal pulse, blockade of the 20E receptor prevents fat body cell dissociation. When JH titer declines, the prepupal peak of 20E activates expression of two 20E primary-response genes, E75 and Blimp-1, to inhibit fat body cell dissociation: E75 represses DHR3 transactivation of βftz-F1 expression, and Blimp-1 directly represses βftz-F1 expression. During the prepupal-pupal transition, DHR3 directly induces βftz-F1 expression from 6 h APF to 12 APF. Before pupation, βftz-F1 induces Mmp expression and represses timp expression. Moreover, an FBS was identified in the Mmp2 promoter, demonstrating that βftz-F1 directly induces Mmp2 expression. Finally, within 6 h before pupation, Mmp1 and Mmp2 cooperatively induce fat body cell dissociation, with each assuming a distinct role (Jia, 2017).

Insect metamorphosis is coordinately controlled by JH and 20E, whereas the hormonal control of tissue remodeling is strictly context-specific. Different larval tissues and adult organs might have distinct, yet precise, developmental fates and timing. Knowledge regarding this question is poor. Based on previous preliminary information, this study clarified the detailed molecular mechanisms by which JH and 20E precisely control the developmental timing of Mmp-induced fat body cell dissociation at both mRNA and enzymatic levels in Drosophila, and a working model is provided of hormonal control of tissue remodeling in animals (see Model showing developmental timing of Mmp-induced fat body cell dissociation is coordinately and precisely controlled by JH and 20E in Drosophila). In summary, at first, Kr-h1 transduces JH signaling to inhibit Mmp-induced fat body cell dissociation during larval-prepupal transition. Then when JH titer declines, the prepupal peak of 20E suppresses Mmp-induced fat body cell dissociation through E75 and Blimp-1, which inhibit βftz-F1 expression. Finally, until 20E titer declines, DHR3 induces βftz-F1 expression, and βftz-F1 covers the 20E-triggered transcriptional cascade to activate Mmp-induced fat body cell dissociation within 6 h before pupation. This study provides an excellent sample for better understanding the hormonal regulation of insect metamorphosis (Jia, 2017).

REGULATION

Maternal Nanos inhibits Importin-alpha2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. This study shows that Nos represses somatic gene expression by inhibiting translation of maternal importin-alpha2 (impalpha2) mRNA. Mis-expression of Impalpha2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, it is proposed that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a 'double-lock' mechanism to inhibit somatic gene expression in germline progenitors (Asaoka, 2019).

How germ cell fate is established and maintained is a century-old question in developmental, cellular, and reproductive biology. Metazoan species have two distinct modes of germline specification. In some species, germline progenitors are characterized by inheritance of a specialized ooplasm, or the germ plasm, which contains maternal factors necessary and sufficient for germline development. In other species, germline progenitors are specified by inductive signals from surrounding tissues. Irrespective of the mode of germline specification, transcriptional repression of somatic genes is common in germline progenitors, implying that this phenomenon is critical for separation of the germline from the soma (Asaoka, 2019).

In Drosophila, the germ plasm is localized in the posterior pole of cleavage embryos (stage 1-2), and is partitioned into germline progenitors called pole cells (stage 3-4). In pole cells of blastoderm embryos (stage 4-5), the genes required for somatic differentiation are transcriptionally repressed by two maternal proteins in the germ plasm, Polar granule component (Pgc) and Nanos (Nos). Pgc is a Drosophila-specific peptide that suppresses RNA polymerase II-dependent transcription in pole cells by inhibiting the function of positive transcriptional elongation factor b (P-TEFb, a dimer of Cyclin dependent kinase 9 and Cyclin T). By contrast, Nos is an evolutionarily conserved protein that plays an essential role in germline development in various animals. For example, in Drosophila, pole cells lacking Nos (nos pole cells) can adopt a somatic, rather than a germline, fate. Furthermore, depletion of Nos is reported to show ectopic expression of somatic genes, such as fushi tarazu (ftz), even-skipped (eve), and the sex-determination gene Sex lethal (Sxl), in pole cells. Thus, maternal Nos is required in pole cells for repression of somatic genes and establishment of the germ/soma dichotomy. However, the mechanism by which Nos represses somatic gene expression remains unknown (Asaoka, 2019).

Nos acts as a translational repressor of mRNAs that harbor a discrete sequence motif called Nanos Response Element (NRE) in the 3' UTR. NRE contains an evolutionarily conserved Pumilio (Pum)-binding sequence, UGU trinucleotide. In abdominal patterning, Pum represses translation of maternal hunchback (hb) mRNA by binding to NREs in its 3' UTR and recruiting Nos to the RNA/protein complex. Deletion of the NREs from hb mRNA causes its ectopic translation in the posterior half of embryos, which in turn suppresses abdomen formation. Furthermore, deletion of NREs causes hb translation in pole cells, suggesting that NRE-dependent translational repression occurs in pole cells. Indeed, Nos represses translation of head involution defective (hid) mRNA in pole cells in an NRE-like-sequence-dependent manner. In addition, Nos and Pum repress Cyclin B translation in pole cells by binding to a discrete sequence containing two UGU trinucleotides (Cyclin B NRE) These findings led to a speculation that Nos, along with Pum, represses somatic gene expression in pole cells by suppressing translation of mRNAs containing NRE or UGU in their 3' UTRs (Asaoka, 2019).

This study reports that, in pole cells, Nos, along with Pum, represses translation of importin-α2 (impα2)/Pendulin/oho31/CG4799 mRNA, which contains an NRE-like sequence in its 3' UTR. The impα2 mRNA encodes a Drosophila Importin-α homologue that plays a critical role in nuclear import of karyophilic proteins. Nos inhibits expression of a somatic gene, ftz, in pole cells by repressing Impα2-dependent nuclear import of the transcriptional activator, Ftz-F1. Based on these observations, it is proposed that Nos-dependent inhibition of nuclear import of transcriptional activators and Pgc-dependent global transcriptional silencing act as a 'double-lock' mechanism to repress somatic gene expression in pole cells (Asaoka, 2019).

Maternally supplied impα2 mRNA is distributed throughout cleavage embryos. When embryos develop to the blastoderm stage, impα2 mRNA is degraded in the somatic region, but not in pole cells, resulting in enrichment of impα2 mRNA in pole cells. However, this study found that expression of Impα2 protein was at background levels in pole cells. Because impα2 mRNA contains a sequence very similar to the NRE (hereafter, NRE-like sequence) in its 3' UTR, it was assumed that impα2 mRNA is a target of Nos/Pum-dependent translational repression in pole cells. To investigate this possibility, the expression of the Impα2 protein was first monitored in pole cells of embryos lacking maternal Nos or Pum (nos or pum embryos, respectively). In these pole cells, expression of Impα2 protein was higher than in those of control (nos/+) embryos. Because neither nos nor pum mutation affected the impα2 mRNA level in pole cells, these observations show that Nos and Pum repress protein expression from the impα2 mRNA in pole cells (Asaoka, 2019).

Whether this repression is mediated by the NRE-like sequence in the impα2 3' UTR was investigated. To this end, impα2 mRNA, with or without the NRE-like sequence (impα2 WT and impα2 ΔNRE, respectively), was maternally supplied to embryos, and their protein expression was examined in pole cells at the blastoderm stage. Because a triple Myc tag sequence was inserted at the C-terminal end of the coding sequence, protein expression from these mRNAs could be monitored using an anti-Myc antibody. When impα2 WT mRNA was supplied to normal (y w) embryos, the tagged protein was expressed at low levels in the soma, but was barely detectable in pole cells. By contrast, the tagged protein from impα2 ΔNRE mRNA was detected in normal pole cells. Similar protein expression was observed in pole cells lacking Nos (nos pole cells), when impα2 WT mRNA was supplied, as well as when impα2 ΔNRE mRNA was supplied. Because the frequency of tagged protein expression from impα2 ΔNRE mRNA did not increase in cells lacking Nos, these results indicate that the NRE-like sequence mediates Nos-dependent repression of Impα2 protein expression in pole cells (Asaoka, 2019).

The NRE-like sequence of impα2 mRNA contains two UGU trinucleotides. The UGU trinucleotide is a core sequence of an RNA motif (Nos-Pum SEQRS motif: 5'-HWWDUGUR) that was highly enriched in a SEQRS (in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes) analysis of the Nos-Pum-RNA ternary complex. Hence, it was asked whether Pum and Nos form a ternary complex with impα2 mRNA in an NRE-like sequence-dependent manner. To address this question, electrophoretic mobility shift assay (EMSA) was performed using the Pum RNA-binding domain and the Nos protein containing Zn finger motifs and C-terminal region, which are reported to form a Nos-Pum-target RNA ternary complex in vitro. Nos and Pum together, but neither alone, formed a complex with impα2 RNA containing an NRE-like sequence (WT), whereas alteration of the NRE-like sequence (mut) abolished this interaction. These results demonstrate that Nos and Pum are able to interact with the impα2 3' UTR in an NRE-like sequence-dependent manner. The observations described above led to a conclusion that Nos, along with Pum, directly represses impα2 translation in pole cells (Asaoka, 2019).

Impα2 is a Drosophila homologue of Importin-α that mediates nuclear import of karyophilic proteins with classical nuclear localization signal (NLS). It was predicted that ectopic production of Impα2 in nos pole cells would cause aberrant nuclear import of NLS-containing karyophilic proteins. To explore this possibility, this study focused on a transcriptional activator, Ftz-F1, which contains a classical NLS and is expressed throughout early embryos, including pole cells. In normal embryos, Ftz-F1 was enriched in the cytoplasm of pole cells, although it was in the nuclei of somatic cells. In the absence of maternal Nos, the percentage of embryos with Ftz-F1 signal accumulating in pole-cell nuclei was higher than in normal embryos. Furthermore, the nuclear/cytoplasmic ratio of Ftz-F1 signal intensities in nos pole cells was higher than in normal pole cells. To determine whether this aberrant concentration of Ftz-F1 was caused by mis-expression of Impα2, this study expressed Impα2 ectopically in pole cells of normal embryos. To this end, impα2 mRNA in which the 3' UTR was replaced with the nos 3' UTR, was maternally supplied under the control of the nos promoter; the mRNA was localized to the germ plasm and pole cells under the control of the nos 3' UTR. The percentage of these embryos (impα2-nos3'UTR embryos) with Ftz-F1 in pole-cell nuclei and the nuclear/cytoplasmic ratio of Ftz-F1 intensities in their pole cells were higher than those of normal pole cells. These observations suggest that mis-expression of Impα2 in pole cells caused by depletion of maternal Nos results in aberrant nuclear import of Ftz-F1 (Asaoka, 2019).

Depletion of maternal Nos results in ectopic expression of the somatic genes ftz, eve and Sxl in pole cells. Because Ftz-F1 is required for proper expression of ftz in the soma, it was asked whether mis-expression of Impα2 causes ectopic expression of ftz in pole cells. In normal embryos, ftz mRNA was expressed in seven stripes of somatic cells, but never expressed in pole cells. By contrast, in impα2-nos3'UTR embryos, ftz mRNA was rarely detectable in pole cells. It is assumed that this low frequency of ftz expression was due to Pgc-mediated silencing of global mRNA transcription. To test this idea, Impα2 was expressed in pole cells of embryos lacking maternal Pgc (pgc impα2-nos3'UTR embryos); the frequency of ftz expression was drastically increased, compared to those of impα2-nos3'UTR embryos and the embryos lacking Pgc (pgc embryos). A similar situation was observed in embryos lacking both Pgc and Nos activities (pgc nos embryos). The percentage of embryos expressing ftz in pole cells was 82.8%, an increase relative to 35.8% in pgc embryos. Furthermore, ectopic ftz expression in pgc nos pole cells was suppressed by injecting double-stranded RNA (dsRNA) against impα2. Therefore, it is concluded that ectopic expression of ftz in pole cells is cooperatively repressed by Nos-dependent suppression of Impα2 production and Pgc (Asaoka, 2019).

In addition to ftz expression, eve was expressed ectopically in pole cells of pgc impα2-nos3'UTR embryos. Ectopic eve mRNA and its protein expression were significantly higher in pgc impα2-nos3'UTR pole cells than pgc or impα2-nos3'UTR pole cells (S3 Fig). Expression of the sex-determination gene Sxl was examined in early pole cells, because Sxl is also repressed by nos in both male and female pole cells. In males, Sxl mRNA expression was rarely detectable in pole cells of nos, impα2-nos3'UTR, pgc, and pgc impα2-nos3'UTR embryos. By contrast, in females, the percentage of embryos expressing Sxl mRNA in pole cells was significantly higher in pgc impα2-nos3'UTR embryos than in impα2-nos3'UTR, and pgc embryos. These results indicate that eve and Sxl, like ftz, are cooperatively repressed in pole cells by Impα2 depletion and Pgc-dependent transcriptional silencing. Because there is no evidence for the involvement of Ftz-F1 in eve and Sxl expression, it is likely that Impα2 mediates nuclear import of other transcriptional activator(s) for eve and/or Sxl in pole cells (Asaoka, 2019).

Nos is required in pole cells for mitotic quiescence, repression of apoptosis, and proper migration to embryonic gonads. Hence, it was asked whether mis-expression of Impα2 causes defects in these processes. First, using an antibody against a phosphorylated form of histone H3 (PH3), a marker of mitosis, whether pole cells enter mitosis in stage 7-9 embryos was investigated. Premature mitosis was detected in pole cells of nos embryos, as described previously, but never in pole cells of impα2-nos3'UTR or pgc impα2-nos3'UTR embryos. Second, using an antibody against cleaved Caspase-3, a marker of apoptosis, whether pole cells enter apoptosis in stage 10-16 embryos was investigated. Pole cells never expressed the apoptotic marker in impα2-nos3'UTR embryos, whereas in pgc impα2-nos3'UTR embryos, 20.4% of pole cells expressed the apoptotic marker. The latter was statistically indistinguishable from pgc pole cells, which have been reported to enter apoptosis. These data indicate that mis-expression of Impα2 does not affect apoptosis of pole cells even in the absence of pgc function. Last, whether mis-expression of Impα2 affects pole cell migration was investigated. The ability of pole cells to migrate properly into the embryonic gonads was never impaired in impα2-nos3'UTR embryos, and the percentage of pole cells entering the gonads in pgc impα2-nos3'UTR embryos was statistically indistinguishable from that of pgc pole cells, which has been reported to exhibit migration defect. These observations indicate that mis-expression of Impα2 does not induce premature mitosis, apoptosis, or mis-migration of pole cells. This can be partly explained by the facts that Cyclin B and hid mRNAs are the targets for Nos-dependent translational repression regulating mitosis and apoptosis in pole cells, respectively (Asaoka, 2019).

During the course of the experiments described above, it was observed that impα2-nos3'UTR interacts genetically with the pgc mutation to cause dysgenic gametogenesis. Because almost all of the ovaries in females derived from pgc mothers mated with y w males were agametic, the effect of impα2-nos3'UTR in pgc/+ background was examined. The percentage of dysgenic ovaries in pgc/+ impα2-nos3'UTR females derived from pgc/+ impα2-nos3'UTR mothers mated with y w males was significantly higher than those in pgc/+ and impα2-nos3'UTR females. In the dysgenic ovaries, almost all of the egg chambers fail to complete the vitellogenic stage, and consequently only a few mature oocytes were present. Furthermore, the percentages of dysgenic and agametic testes in pgc impα2-nos3'UTR males derived from pgc impα2-nos3'UTR mothers mated with y w males were higher than those in pgc and impα2-nos3'UTR males. In these testes, the abundance of Vasa-positive germline cells was reduced (dysgenic) or absent (agametic). Because dysgenic and agametic gonads were barely detectable in females and males derived from reciprocal crosses, the data suggest that mis-expression of Impα2 from maternal transcript, concomitant with maternal pgc depletion in pole cells, causes defects in gametogenesis. However, it cannot be tested whether concomitant depletion of maternal Nos and Pgc causes a similar phenotype because nos pole cells degenerate before adulthood, even when apoptosis in these cells is genetically repressed (Asaoka, 2019).

Expression of Importin-α subtypes is spatio-temporally regulated in the soma during development in multiple animal species, including Drosophila, and they control nuclear transport of unique karyophilic proteins to activate different sets of somatic genes. Drosophila genome contains three Importin-α family genes: impα1, 2, and 3. impα1/Kap-α1/CG8548 mRNA is not detectable in pole cells during early embryogenesis, and its protein product is ubiquitously expressed at a very low level throughout embryogenesis. By contrast, maternal impα3/Kap-α3/CG9423 mRNA is detectable in germ plasm during pole cell formation, and production of Impα3 protein is upregulated during the blastoderm stage. Because Impα3 production was independent of maternal nos activity, it is likely that Nos-dependent repression of Impα2 production is solely responsible for suppression of somatic gene expression in pole cells. By contrast, pole cells become transcriptionally active during gastrulation, when Impα2 is undetectable in these pole cells. Thus, the onset of zygotic transcription in pole cells may require Impα3-dependent nuclear import of transcription factors, in addition to the disappearance of Pgc and the alteration in chromatin-based regulation. After gastrulation, maternal impα2 mRNA is rapidly degraded in pole cells, and neither impα2 mRNA nor protein is detectable in the germline before adulthood. This suggests that maternal impα2 is dispensable for germline development, and that maternal impα2 mRNA partitioned into early pole cells must be silenced by Nos and Pum in order to suppress mis-expression of somatic genes (Asaoka, 2019).

Depletion of maternal Nos activities caused mis-expression of ftz in pole cells. Although ftz expression was barely observed in pole cells lacking only maternal Nos, it was partially derepressed in pole cells in the absence of Pgc alone, probably because a trace amount of Ftz-F1 enters pole cell nuclei even in the absence of the impα2 translation. Therefore, it is proposed that a subset of somatic genes, including ftz and eve, are repressed in pole cells by two distinct mechanisms: Nos-dependent repression of nuclear import of transcriptional activators and Pgc-dependent silencing of mRNA transcription. Pgc inhibits P-TEFb-dependent phosphorylation of Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of RNA polymerase II, a modification that is critical for transcriptional elongation; thus, mRNA transcription in pole cells is globally suppressed by Pgc. By contrast, Nos inhibits transcription of particular genes by repressing Impα2-dependent nuclear import of the corresponding transcriptional activators (Asaoka, 2019).

Nos is evolutionarily conserved and expressed in the germline progenitors of various animal species. In C. elegans, nos-1 and -2 are essential for rapid turnover of maternal lin-15B mRNA, which encodes a transcription factor that would otherwise cause inappropriate transcriptional activation in primordial germ cells. In the germline progenitors of Xenopus embryos, Nos-1, along with Pum, destabilizes maternal VegT mRNA and represses its translation to inhibit somatic (endodermal) gene expression, which is activated by VegT protein. Furthermore, in the germline progenitors (small micromeres) of sea urchin embryos, Nos silences maternal mRNA encoding a deadenylase, CNOT6, to stabilize other maternal mRNAs inherited into small micromeres. This study demonstrated that Nos inhibits translation of maternal impα2 mRNA in pole cells in order to suppress nuclear import of a transcriptional activator for somatic gene expression. Based on these observations, it is proposed that Nos silences maternal transcripts that are inherited into germline progenitors but deter the proper germline development. In addition to Nos-dependent silencing of maternal transcripts, transient suppression of RNA polymerase II elongation is observed during germline development of a wide range of animals, including Drosophila, C. elegans, Xenopus, and an ascidian, Halocynthia roretzi. Therefore, it is proposed that the 'double-lock' mechanism achieved by Nos and global suppression of RNA polymerase II activity plays an evolutionarily widespread role in germline development (Asaoka, 2019).

Promoter

SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a 'nucleosomal barrier' (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both 'repressive' and 'transient' Pol II pausing (Vorobyeva, 2012).

The mechanism of ftz-f1 transcription activation has been analyzed in S2 cells. Sequential addition and removal of ecdysone allows the DHR3 and ftz-f1 genes in these cells to be activated in accordance with their expression pattern in vivo. This system is of considerable interest, since only a few Drosophila models of activated transcription are available. It also provides the possibility of studying the mechanism of pausing in the active and repressed transcription states of the same gene, whereas previous such studies have been performed with different genes (Vorobyeva, 2012).

Pol II pausing on ftz-f1 occurs at about 1.5 kb downstream of the promoter, i.e. at a much greater distance than that described for other genes (from +30 to +100 nt). Future studies will show how widespread is this mode of pausing. It is of interest in this context that a case of Pol II pausing at 800 bp downstream of the promoter was described for the β-actin gene (Vorobyeva, 2012).

The ftz-f1 activation at the molecular level is a several-stage process. At the first stage, when the ecdysone titer and DHR3 expression are high, DHR3, SAYP, TFIID, Brahma and Pol II accumulate at the promoter. Transcription is initiated, but Pol II is paused 1.5 kb downstream of the promoter; DHR3, SAYP and Brahma are also present at this site, where a nucleosomal barrier is formed. At the next stage, ~1 h after ecdysone removal, promoter-bound factors remain at the same levels, except for SAYP (its level on the promoter decreases). Pol II and associated factors disappear from the site of pausing, and the nucleosomal barrier is eliminated, but the transcription level does not increase. The following stage is characterized by rapid intensification of transcription, which reaches a maximum within several hours; the level of Pol II increases in the body of the gene, and its pausing is observed again, with SAYP and Brahma being present at the corresponding position. In addition, the level of SAYP on the promoter is recovered, indicating that it is highly regulated at different transcription stages. The DHR3 activator is present at the site of pausing, and its level does not change upon SAYP knockdown. This is evidence that DHR3 may participate in SAYP recruitment for subsequent nucleosomal barrier formation and Pol II pausing (Vorobyeva, 2012).

The region of high nucleosome density (nucleosomal barrier) is specific for the repression stage, at which the DHR3 activator induces the assembly of the Pol II preinitiation complex on the promoter and makes paused Pol II competent for transcription initiation. Nucleosomal barrier disruption by SAYP knockdown leads to the full-length transcript synthesis, indicating that the nucleosomal barrier contributes to preventing the entry of Pol II to the transcribed region. The data show that SAYP and Brahma play the crucial role in organization of the nucleosomal barrier: this barrier coincides in location with the peak of these coactivators and disappears after SAYP knockdown, which leads to elimination of Brahma from the gene. Thus, SAYP and Brahma at the stage of repressed transcription have an important role in blocking the synthesis of full-length transcripts. Although the transcription increases upon SAYP depletion and elimination of the nucleosomal barrier, its level remains low, compared with that in the permissive state. This is evidence for the existence of different mechanisms of Pol II pausing regulation, which also correlates with the fact that the depletion of NELF, an important factor of Pol II pausing, causes a 2.5-fold increase in the transcription of hsp70 or hsp26 gene in the repressed state, which, however, does not reaches the level characteristic of a fully activated gene (Vorobyeva, 2012).

The question arises as to the structure of the nucleosomal barrier. As shown previously, the human SWI/SNF complex can not only erase nucleosomes from the template but also produce a stable remodeled dimer of mononucleosome core, with this complex being also needed for converting this product back to the cores. One may suggest that the Drosophila Brahma complex operates in the same way. In the current experiments, the level of histone H3 increased ~2-fold in the region of the nucleosomal barrier, compared with its general level on the gene, which agrees with the assumption concerning the presence of a nucleosome dimer. The fact that the region of nucleosomal barrier is significantly enriched in sequences with a high nucleosome-positioning probability indicates that DNA sequences probably contribute to organization of this barrier (Vorobyeva, 2012).

Previous experiments have revealed a relationship between Pol II pausing and the nucleosomal structure of the template. It has been shown that Pol II stops at the site where the nucleosome density is restored to the average level characteristic of the gene. However, no specific nucleosome-dense regions preventing Pol II transcription have been described as yet (Vorobyeva, 2012).

The transition to the transcription-permissive state correlates with significant rearrangements in the promoter-distal region (disappearance of Brahma, SAYP, Pol II and nucleosomal barrier at the site of Pol II pausing). However, no increase in the ftz-f1 transcription level has been observed within the first 30 min after this transition. As shown in the study on estradiol (ER)-mediated gene expression, productive transcription is preceded by an unproductive cycle (~40 min) that is necessary for promoter preparation to this process. This may be the case for ftz-f1, with a certain period of time being required for rearrangements preceding its active transcription (Vorobyeva, 2012).

At the (+;-) stage, the level of SAYP on the promoter is recovered within 2-3 h after the onset of transcription, with SAYP RNAi influencing the Brahma and TFIID levels on the promoter. Pol II pausing correlating with its Ser2 modification is again observed as the transcription level increases. Although SAYP and Brahma occur again together with paused Pol II, their function appears to be different from that at the repression stage. The nucleosomal barrier is not restored, and SAYP depletion has only a slight effect on chromatin structure (Vorobyeva, 2012).

However, SAYP depletion severely disturbs transient pausing, interfering with Ser2 phosphorylation. This impairs proper transition to productive elongation and leads to a decrease in Pol II level on the body of the gene. Thus, SAYP knockdown not only affects the level of ftz-f1 activation but also shifts the timing of its expression. The slower kinetics of transcription induction together with the slight decrease in the Pol II level on the promoter upon SAYP knockdown are evidence for the retarded Pol II passage in the coding region of the gene and, hence, for disturbances in the elongation mechanisms. Similar consequences are observed for other genes regulating on pausing mechanisms (Vorobyeva, 2012).

The results of this study show that SAYP is important for proper timing of ftz-f1 transcription during Drosophila metamorphosis. The ftz-f1 gene is a major regulator of metamorphosis, that is why its precise activation in time is crucial during development. On the whole, the data provide evidence for the important role of pausing in sequential activation of genes in cascades and indicate that this mechanism may have a general role in development (Vorobyeva, 2012).

In addition, these results also support the idea that Pol II pausing may require not only NELF and DSIF but also other factors, such as nucleosome-remodeling complexes. Interestingly, the depletion of NELF proved to result in an increased nucleosome occupancy at the promoters of some genes (Vorobyeva, 2012).

In summary, this study has found that Pol II pausing is dependent on the interplay of several molecular mechanisms, including the formation of a specific chromatin structure via the action of coactivators. These results indicate that, although Pol II pausing is a genome-wide phenomenon, the specific molecular mechanism controlling paused Pol II activity on individual genes may vary significantly (Vorobyeva, 2012).

Transcriptional Regulation

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

In the early stages of Drosophila metamorphosis Hr46/DHR3represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. DHR is shown to interact directly with the Ecdysone receptor. The mechanism of DHR3 repression may involve an interaction between the DHR3 and Ecdysone receptor ligand binding domains. DHR also induces ßFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The DNA binding domain of DHR3, and perhaps sequences NH2-terminal to it, are necessary for the activating function of DHR3. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming a complex with DHR3 on the ßFTZF1 promoter, thereby providing a timing mechanism for ßFTZF1 induction that is dependent on the disappearance of E75B. DHR3 has two high-affinity binding sites approximately 300 base pairs apart, that lie downstream of the transcription start site of ßFTZF1. DHR3 appears to bind as a monomer to these sites, since sequencing and footprinting analysis have uncovered single consensus DHR3 sites at each of these DNA sites. E75B fails to bind DNA in the absence of DHR3. Thus E75B acts like a co-repressor with DHR3, rather than as a competitor with DHR3 for DNA binding; the restricted temporal expression of E75B apparently acts as a precise timer for the onset of ßFTZF1 expression (White, 1997).

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

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

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

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

DHR4 plays an essential role in ßFTZ-F1 regulation

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

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

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

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

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

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

Steroid hormone inactivation is required during the juvenile-adult transition in Drosophila

Steroid hormones are systemic signaling molecules that regulate juvenile-adult transitions in both insects and mammals. In insects, pulses of the steroid hormone 20-hydroxyecdysone (20E) are generated by increased biosynthesis followed by inactivation/clearance. Although mechanisms that control 20E synthesis have received considerable recent attention, the physiological significance of 20E inactivation remains largely unknown. This study shows that the cytochrome P450 Cyp18a1 lowers 20E titer during the Drosophila prepupal to pupal transition. Furthermore, this reduction of 20E levels is a prerequisite to induce βFTZ-F1, a key factor in the genetic hierarchy that controls early metamorphosis. Resupplying βFTZ-F1 rescues Cyp18a1-deficient prepupae. Since Cyp18a1 is 20E-inducible, it appears that the increased production of steroid is responsible for its eventual decline, thereby generating the regulatory pulse required for proper temporal progression of metamorphosis. The coupling of hormone clearance to βFTZ-F1 expression suggests a general mechanism by which transient signaling drives unidirectional progression through a multistep process (Rewitz, 2010; full text of article).

One proposed route for 20E inactivation is through 26-hydroxylation catalyzed by the cytochrome P450 Cyp18a1. Interestingly, Cyp18a1 was first identified based on its inducibility by 20E, consistent with the 20E-inducible 26-hydroxylase activity. If this is the case, inactivation is dependent on the concentration of the hormone itself, representing an elegant feedback regulation of steroid levels (Rewitz, 2010).

The aim of the present study was to examine the functional importance of steroid pulses during development by studying the role of Cyp18a1 in the decline of 20E levels. Evidence is presented that Cyp18a1 is required for the decline of the 20E titer and that failure to reduce 20E levels after the late larval 20E peak disrupts metamorphic development and leads to animal death. Furthermore, it was shown that these animals die because elevated 20E levels repress the expression of the mid-prepupal gene βFTZ-F1, a factor necessary for providing competence to respond to 20E in a stage-specific manner during metamorphosis (Rewitz, 2010)

Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster

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

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

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

Targets of Activity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Stripy Ftz target genes are coordinately regulated by Ftz-F1

During development, cascades of regulatory genes act in a hierarchical fashion to subdivide the embryo into increasingly specified body regions. This has been best characterized in Drosophila, where genes encoding regulatory transcription factors form a network to direct the development of the basic segmented body plan. The pair-rule genes are pivotal in this process as they are responsible for the first subdivision of the embryo into repeated metameric units. The Drosophila pair-rule gene fushi tarazu (ftz) is a derived Hox gene expressed in and required for the development of alternate parasegments. Previous studies suggested that Ftz achieves its distinct regulatory specificity as a segmentation protein by interacting with a ubiquitously expressed cofactor, the nuclear receptor Ftz-F1. However, the downstream target genes regulated by Ftz and other pair-rule genes to direct segment formation are not known. In this study, candidate Ftz targets were selected by virtue of their early expression in Ftz-like stripes. This identified two new Ftz target genes, drumstick (drm) and no ocelli (noc), and confirmed that Ftz regulates a serotonin receptor (5-HT2). These are the earliest Ftz targets identified to date and all are coordinately regulated by Ftz-F1. Engrailed (En), the best-characterized Ftz/Ftz-F1 downstream target, is not an intermediate in regulation. The drm genomic region harbors two separate seven-stripe enhancers, identified by virtue of predicted Ftz-F1 binding sites, and these sites are necessary for stripe expression in vivo. It is proposed that pair-rule genes, exemplified by Ftz/Ftz-F1, promote segmentation by acting at different hierarchical levels, regulating first, other segmentation genes; second, other regulatory genes that in turn control specific cellular processes such as tissue differentiation; and, third, 'segmentation realizator genes' that are directly involved in morphogenesis (Hou, 2009).

This study identified Ftz targets based on a search for genes expressed in striped patterns in the early Drosophila embryo. Each of these Ftz-dependent genes is also regulated by Ftz-F1, an orphan nuclear receptor previously shown to interact with Ftz in vitro and in vivo. Unlike Ftz, which is expressed in a striped pattern in the Drosophila blastoderm, Ftz-F1 is expressed ubiquitously, in all somatic cells at the blastoderm stage. The finding in this study that all three additional Ftz-dependent genes, identified by virtue of their striped expression patterns, require Ftz-F1 for expression in stripes lends support to the model that interaction with Ftz-F1 is the key to Ftz functional specificity as a segmentation protein. The three genes characterized in this study, 5-HT2, noc and drm, are the earliest identified downstream targets of Ftz. Expression in stripes was observed at the cellular blastoderm stage when Ftz-F1 is highly expressed throughout the embryo and the seven Ftz stripes are at their peak levels. These early target gene stripes were lost in ftz and also in ftz-f1 mutants. In addition, ectopic expression was observed at early stages when Ftz was ectopically expressed throughout the embryo. En, long thought to be a major mediator of Ftz function in segmentation, is expressed later than these target genes, and it was verified that En is not required for the Ftz-dependent stripe expression of noc or drm. These findings suggest that Ftz and Ftz-F1 directly regulate expression of these three target genes. This new study brings to seven the targets of Ftz that appear to be directly co-regulated by Ftz and Ftz-F1: ftz itself, en, apt, Dsulf1, 5HT-2, noc and drm. For each gene, multiple potential Ftz-F1 binding sites were found within a 15-20 kb genomic region. In all cases, multiple potential Ftz binding sites surround the Ftz-F1 binding sites that could mediate cooperative interactions between Ftz and Ftz-F1. Many of these sites have been maintained during evolution and are present in distant Drosophila species. Other Ftz targets, such as Ubx, prd, odd and tsh are also likely to be co-regulated by Ftz-F1 (Hou, 2009).

The seven Ftz/Ftz-F1 target genes identified to date play diverse roles in segmentation and act at different levels of the embryonic hierarchy. First, Ftz acts in a cross-regulatory fashion to modulate expression of other pair-rule genes: it interacts with Ftz-F1 in autoregulation and also has been shown to regulate the pair-rule genes prd, odd and slp. Second, Ftz/Ftz-F1 directly regulate components of the segment polarity system: first, they activate en expression in alternate stripes, and, second, they regulate Dsulf1, thought to modulate Wg activity. Ftz has also been shown to repress wg expression. Ftz/Ftz-F1 thus indirectly control compartment border formation, via regulation of En and Wg. Third, Ftz/Ftz-F1 regulate transcription factors that in turn control the differentiation of specific cell types: apt, noc, drm. drm encodes an odd-skipped family zinc finger transcription factor that it is required for patterning the dorsal epidermis, thus regulating the differentiation of specific cell types. noc plays a role in trachael morphogenesis with mutants displaying defects in branch migration and expanded expression of trachael-specific genes. Similarly, apt is involved in this process as a regulator of the migration of trachael precursor cells. Finally, Ftz/Ftz-F1 regulate a target gene more directly involved in morphogenesis, 5HT-2. 5-HT2 encodes a serotonin receptor that demonstrates specific ligand binding in transfected cells and in Drosophila embryo extracts. Phenotypic analysis suggested a role for 5-HT2 and other genes involved in serotonin biosynthesis in morphogenetic movements during gastrulation: deficiency embryos lacking 5HT-2 displayed delayed and incomplete movements during germband extension accompanied by mislocalization of Armadillo protein, suggestive of abnormalities in adherens junctions. It will be of interest in the future to determine whether other pair-rule genes direct expression of additional cell surface proteins that coordinate these processes (Hou, 2009).

This study has identified enhancers of drm by combining bioinformatics with enhancer-reporter gene expression analysis in vivo. Fragments chosen for the in vivo analysis contained one or more matche(s) to a Ftz-F1 binding site. Three of the four fragments directed expression in drm-like patterns in vivo. The upstream fragment, drm1, harbors a late stage enhancer that directs segmental expression of drm. drm2 directed expression in seven strong stripes. drm34 harbors enhancers for the region-specific expression of drm in the proventriculus and hindgut, expression that is important for the development of the fore- and hindgut, as well as an early 7-stripe enhancer. Whether any of these enhancers also direct expression in the leg imaginal discs was not investigated. Two of the fragments, drm2 and drm34, directed expression in 7-stripe patterns. Surprisingly, for each of them, the set of seven stripes is in register with Ftz, suggesting that both regulate expression of the drm-primary stripes. Although unexpected, this phenomenon has been observed in other cases where it was suggested that enhancers directing the same or similar expression patterns function as shadow enhancers to enhance the precision of expression patterns and facilitate the rapid evolution of cis-regulatory sequences. Point mutations of either or both of the predicted Ftz-F1 binding sites in the drm34 Early 7-Stripe Enhancer abolished expression of lacZ fusion genes. Stripe expression was decreased but not completely abolished by mutation of the single predicted Ftz-F1 binding site in the drm2 7-Stripe Enhancer, suggesting additional inputs into regulation of the drm primary stripes by this enhancer. Together, these results suggest that Ftz-F1 activates expression in the primary drm stripes via the drm34 Early 7-Stripe Enhancer. It is speculated that following this initial activation, autoregulation by Drm may augment Ftz-F1 activation of stripes via the drm2 7-Stripe Enhancer to raise levels of transcription in drm primary stripes (Hou, 2009).

Drosophila ftz is a typical pair-rule gene: ftz mutant embryos die lacking even-numbered body segments. How this wild type function of ftz, and other pair-rule genes, is executed is not yet known. As for other segmentation mutants, the pair-rule mutant phenotype results from cell death. However, this cell death appears to be an indirect effect. Similarly, pair-rule genes regulate segment border formation indirectly, via activation of the segment polarity genes such as en and wg. In addition to this, segment-polarity-independent roles for the pair-rule genes in morphogenesis have been revealed by careful studies from the Wieschaus lab. For example, it was found that cell intercalation and germ band extension are regulated by the pair-rule genes, independent of segment polarity genes. Similarly, cellular studies defined two subtle morphogenetic processes that occur before gastrulation - one, controlled by the pair-rule gene paired. More recently, studies have shown that the planar polarity and organization of intercalating cells during germ band extension are controlled by the striped expression patterns of eve and runt and that the longitudinal division of cells during germ band extension is controlled by eve. These studies are suggestive of direct roles for the pair-rule system in cell shape changes and rearrangements during germ band extension. Together, these studies support the notion that combinatorial expression of early patterning genes assigns unique identities in the blastoderm at a single cell level. This study has shown that the pair-rule gene ftz regulates target genes prior to and independently of En. These findings support the model that the stripes of pair-rule genes play active roles in patterning the embryo rather than serving solely as intermediary patterns whose function is to produce the segmental stripes of segment polarity genes. One role for these pair-rule stripes may be to establish differential adhesiveness to groups of cells in the blastoderm embryo. Future work identifying additional pair-rule targets will be required to explain the fundamental biological roles of pair-rule patterning and to understand how the assignment of positional identities by pair-rule genes, prior to morphogenesis, translates into the development and differentiation of body segments (Hou, 2009).

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-beta/BMP signaling and orphan nuclear receptors

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. This study describes the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, this study shows that phagocytes have a key role in this process. Importantly, the presence of peripheral glial cells is shown close to the neuro-muscular junction that retracts before the motor neuron. In muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. This study interrupted TGF-β signaling in motor neurons using expression of dominate negative Wishful thinking. It is proposed that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation (Boulanger, 2012).

It is a general feature of maturing brains, both in vertebrates and in invertebrates, that neural circuits are remodeled as the brain acquires new functions. In holometabolous insects, the difference in lifestyle is particularly apparent between the larval and the adult stages. These insects possess two distinct nervous systems at the larval and adult stages. A class of neurons is likely to function in both the larval and the adult nervous systems. The neuronal remodeling occurring during this developmental period is expected to be necessary for the normal functioning of the new circuits (Boulanger, 2012).

The pruning of an axon can involve a retraction of the axonal process, its degeneration or both a retraction and degeneration. The MB γ axon is pruned through a local degeneration mechanism. In contrast, axons may retract their cellular processes from distal to proximal in the absence of fragmentation and this mechanism is called retraction. Interestingly, the two mechanisms can occur sequentially in the same neuron, as in the case of the dendrites of the da neurons, where branches degenerate and the remnant distal tips retract (Boulanger, 2012).

This study provides evidence that the motor neuron innervating larval muscle 4 (NMJ 4) is pruned predominantly through a retraction mechanism. The first morphological indication of motor neuron retraction is the absence of fragmentation observed with anti-HRP staining at the level of the presynapse in all the developmental stages analyzed, together with a decrease in perimeter size observed after 2 h APF. The continuity of this HRP staining is in contrast to the pronounced interruptions between blebs observed with an antibody against mCD8 in γ axons. A molecular indication of motor neuron retraction in these studies is the fact that β-Spectrin disappears at the synapse 5 h APF, before motor neuron pruning takes place. Indeed, it has been shown using an RNA interference approach that loss of presynaptic β-Spectrin leads to presynaptic retraction and synapse elimination at the NMJ during larval stages. The modifications of the microtubule morphology that were observed, such as an increase in microtubule thickness and withdrawal, provide additional evidence of axonal retraction during NMJ remodeling. Finally, a strong argument in favor of a motor neuron retraction mechanism is the fact that Tubulin is present at the NMJ throughout all stages of axonal pruning at the start of metamorphosis (0-7 h APF). This stands in clear contrast to the abolition of Tubulin expression observed before the first signs of γ axon degeneration. It is also interesting to note that the motor neuron retraction observed in this study at metamorphosis and at larval stages are morphologically different. During metamorphosis, retraction bulbs or postsynaptic footprints, which have been reported at larval stages, were never visualized. The fact that the postsynapse dismantles at metamorphosis before motor neuron retraction might explain these discrepancies. Worth noting is the mechanistic correlation between accelerated debris shedding observed here for NMJ pruning at the start of metamorphosis and axosome shedding occurring during vertebrate motor neuron retraction (Boulanger, 2012).

In vertebrates, glia play an essential role in the developmental elimination of motor neurons. In Drosophila, the role of glia in sculpting the developing nervous system is becoming more apparent. Clear examples of a role for engulfing glial cells in axon pruning are well documented during the MB γ axon degeneration at metamorphosis. Also, glia are required for clearance of severed axons of the adult brain. A distinct protective role of glia has been recently discovered during the patterning of dorsal longitudinal muscles by motor neurobranches. This study describes the presence of glia processes close to the end of the pupal NMJ. The observations suggest that the glial extensions retract at 5 h APF, just before motor neuron retraction is observed. When the glial dynamic is blocked, the NMJ dismantling might be also blocked. It is hypothesized that during development in larvae and early pupae, glial processes have a protective role and aid in the maintenance of the NMJ. Then, between 2 and 5 h APF, glial retraction would be a necessary initial step that allows NMJ dismantling. In accordance with this hypothesis, glia play a protective role in the maintenance of NMJ during pruning of second order motor neuron branches 31 h APF (Boulanger, 2012).

Disruption of shi function specifically in glial cells results in an unpruned mushroom body γ neuron phenotype and prevents glial cell infiltration into the mushroom body (Awasaki, 2004). One can note that at the NMJ the role of the glia is proposed to be essentially opposite from its role in MB γ axons pruning but in both cases blocking the glia dynamics results in a similar blocking of the pruning process (Boulanger, 2012).

In vertebrates, phagocytes are recruited to the injured nerve where they clear, by engulfment, degenerating axons. In Drosophila, phagocytic blood cells engulf neuronal debris during elimination of da sensory neurons. This study shows that blocking phagocyte dynamics with shi produces a strong blockade of the NMJ dismantling process. One possibility is that phagocytes attack and phagocytose the postsynaptic material, a process blocked by compromising shi function resulting in postsynaptic protection. In accordance, it has been shown that phagocytes attack not only the da dendrites to be pruned, but also the epidermal cells that are the substrate of these dendrites (Boulanger, 2012).

During NMJ dismantling, the muscle has an instructive role for motor neuron retraction. In all the situations where postsynapse dismantling is blocked, the corresponding presynaptic motor neuron retraction is also blocked. Therefore, it is sufficient to propose that both glial cells and phagocytes affect only the postsynaptic compartment. Nevertheless, one cannot rule out that these two cell types both act directly at the pre and at the postsynapse (Boulanger, 2012).

ECR-B1 is highly expressed and/or required for pruning in remodeling neurons of the CNS. MB γ neurons and antennal lobe projection neurons remodeling require both the same TGF-β signaling to upregulate EcR-B1. In the MBs only neurons destined to remodel show an upregulation of EcR-B1. At least two independent pathways insure EcR-B1 differential expression. The TGF-β pathway and the nuclear receptor pathway are thought to provide the necessary cell specificity of EcR-B1 transcriptional activation. This study shows that in the motor neuron pruning these two pathways are also necessary to activate EcR-B1. Noteworthy, showing an analogous requirement of ftz-f1/Hr39 pathway in two different remodeling neuronal systems unravels the fundamental importance of this newly described pathway (Boulanger, 2012).

The following model is proposed for the sequential events that are occurring during NMJ dismantling at early metamorphosis. First, EcR-B1 is expressed in the muscle under the control of FTZ-F1. FTZ-F1 activates EcR-B1 and represses Hr39. This repression is compulsory for EcR-B1 activation. Importantly, TGF-β/BMP signaling does not appear to be required for EcR-B1 activation in this tissue, however, a result of EcR-B1 activation in the muscle would be the production of a secreted TGF-β family ligand. Then, this secreted TGF-β family ligand reaches the appropriate receptors and activates the TGF-β signaling in the motor neuron. Finally, TGF-β signaling in association with the nuclear receptor pathway activates EcR-B1 expression resulting in motor neuron retraction. Since glial cells and phagocytes are required for the dismantling process, it is possible that a TGF-β/BMP family ligand(s) be produced by one or both of these cell types and not by the postsynaptic compartment. Noteworthy, a recent study shows that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila MBs (Awasaki, 2011). Nevertheless, one can note that the requirement of the two pathways in the motor neuron provides a simple molecular explanation of the instructive role of postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation. It was proposed that in the MBs, the association of these two pathways provides the cell (spatial) specificity of pruning. In this paper, this association is proposed to provide the temporal specificity of the events. Future studies will be necessary to understand how EcR-B1 controls the production of a TGF-β/BMP ligand(s) in the muscle, the reception of this signal by the motor neuron and the ultimate response by the motor neuron to initiate retraction. These steps will be necessary to unravel the molecular mechanisms underlying the NMJ dismantling process and related phenomenon in vertebrate NMJ development and disease. Interestingly, it appears that TGF-β ligands on the one hand are positive regulators of synaptic growth during larval development and on the other hand, they are positive regulators of synaptic retraction, at the onset of metamorphosis. In both situations signaling provides a permissive role, sending a signal from the target tissue to the neuron. The consequence of this signal would be dependent on developmental timing thus, on a change in context (Boulanger, 2012).

Protein interactions

The orphan nuclear receptor alpha Ftz-F1, which is deposited in the egg during oogenesis, is an obligatory cofactor for Fushi tarazu. Mutation of Ftz-F1 causes a pair-rule phenotype even though the maternal alphaFTZ-F1 gene product is uniformly distributed through the embryo. Surprisingly, patterns of FTZ mRNA and protein expression in the alphaFTZ-F1 mutants are indistinguishable from wild type. In Ftz-F1 mutant embryos, as in ftz mutant embryos, Ftz-dependent engrailed stripes fail to be expressed, and wingless stripes expand. Thus alphaFtz-F1 is required for all Ftz activities tested except that for which it was first identified: regulation of the ftz promoter. Given this result, a test was made for direct interaction between Ftz protein and alphaFtz-F1. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. The conserved motif is independent of the Ftz homeodomain and is located in the central portion of the protein, flanked by prolines. Deletion of this motif disrupts all but one of the the Ftz activities described above, that is, it is still capable of broadening endogeneous ftz expression stripes. Thus removal of the alphaFtz-F1 interaction domain from the Ftz polypeptide results in the same loss of Ftz activities as removal of alphaFtz-F1. The Ftz-mediated repression of wingless requires both Paired and alphaFtz-F1. This interaction could involve either simultaneous or competitive interactions among the three proteins, as Prd also contacts residues 101-150 of Ftz. Paired may be a cofactor of Ftz or Ftz-F1 that is required for target genes that are repressed by Ftz, because Prd is required for Ftz-dependent wingless repression, but not for Ftz-dependent activation of engrailed or ftz auto-regulation (Guichet, 1997).

A native binding site for the homeodomain protein Fushi tarazu, the upstream regulatory region of the ftz gene itself, was used to isolate Ftz-F1, a protein of the nuclear hormone-receptor superfamily and a new Ftz cofactor. Ftz and Ftz-F1 are present in a complex in Drosophila embryos. The upstream regulatory region contains five Ftz binding sites and three Ftz-F1 binding sites. One Ftz-F1 site is adjacent to a medium-affinity Ftz binding site. Ftz-F1 facilitates the binding of Ftz to DNA, allowing interactions with weak-affinity sites at concentrations of Ftz that alone would bind only high-affinity sites. By virtue of mutual binding to the same regulatory region, Ftz-f1 and Ftz are both implicated in Ftz autoregulation. Embryos lacking Ftz-F1 display ftz-like pair-rule cuticular defects, and engrailed transcription sites are not present in Ftz-f1 mutant embryos. The pair-rule phenotype is a result of abnormal ftz function, because ftz is expressed but fails to activate downstream genes (Yu, 1997).

One Drosophila Tbp interaction with transcription factors involves a coactivator of the transcription factor FTZ-F1. The coactivator, Multiprotein bridging factor 1 (MBF1), makes possible a connection, or bridges, the TATA box-binding protein (Tbp) and the nuclear hormone receptor FTZ-F1. MBF1 is functional in interactions with Tbp and a positive cofactor MBF2. MBF1 makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the binding of FTZ-F1 to its recognition site. The central region of MBF1 (residues 35-113) is essential for the binding of FTZ-F1, MBF2, and Tbp. MBF1, in the presence of MBF2, and FTZ622 bearing the FTZ-F1 DNA-binding domain, support selective transcriptional activation of the fushi tarazu gene. Mutations that disrupt the binding of FTZ622 to DNA or MBF1, or an MBF2 mutation that disrupts the binding to MBF1, all abolish the selective activation of transcription. These results suggest that tethering the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription (Takemaru, 1997).

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (Ftz-f1) protein is a conserved member of the nuclear receptor superfamily, but has been thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. It is shown here that Ftz-f1 does in fact have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (Ftz). Ftz contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins (Schwartz, 2001).

Ftz-f1 deletion constructs were sequenced to ensure that no errors had been introduced during the course of PCR and cloning procedures. These analyses revealed a discrepancy between the sequences in this study and those of the published Ftz-f1 sequence. An 8 bp repeat beginning at position 3006 of the alphaFtz-f1 sequence was contained in the sequences obtained in this study. The predicted coding region of the revised sequence diverges at amino acid 1003 and encodes an additional 24 amino acids. This revised sequence contains an AF2 consensus that is highly similar to the sequences of vertebrate Ftz-f1 homologs. Confirmation of this revised sequence has recently been provided by the Drosophila genome sequencing project. The sequence of the Ftz-f1 protein of B. mori has also been reported to lack an AF2 motif. A brief examination of the DNA coding sequence reveals that the addition of a single nucleotide in an appropriate location would change the reading frame and yield an AF2 nearly identical to that of the Drosophila protein. Hence, a Bombyx Ftz-f1 cDNA was obtained and sequenced. An additional adenosine nucleotide was found that follows the thymidine at position 1659. The shifted reading frame generates a new C-terminus with 27 of the 34 predicted amino acids identical to the Drosophila sequence. An additional four residues are conservative substitutions. The C-terminal AF2s of the two insect proteins are identical at 15 of 16 positions. It is concluded that both insect proteins contain highly conserved AF2 motifs (Schwartz, 2001).

It is not yet known how Ftz and Ftz-f1 regulate their target genes. Interestingly, Ftz and Ftz-f1 are known to function as both transcriptional activators and repressors. Thus, Ftz may be able to act as both a coactivator and corepressor for Ftz-f1 function. Alternatively, these differential activities may depend on the actions of other cofactors, some of which may be target gene specific. The fact that Ftz-f1 utilizes Ftz as a coregulator of target genes, and that Ftz contains a nuclear receptor box, suggests the possibility that, like other nuclear receptor cofactors, Ftz may help recruit histone acetyltransferase or deacetylase complexes. Alternatively, Ftz may act via regulatory factors or complexes that are quite different from these. For example, Ftz target genes such as engrailed and homeotic genes of the Antennapedia and bithorax gene clusters are regulated by chromatin-organizing Polycomb and Trithorax group protein complexes. The Ftz-Ftz-f1 complex may play a role in recruiting these larger protein complexes (Schwartz, 2001).

Does Ftz-f1 have a ligand? There are several observations that argue both for and against the existence of a Ftz-f1 ligand. Motifs and residues required for ligand binding by other receptors are conserved in Ftz-f1. These include residues in the AF2 helix that, based on other receptor structures, are expected to contact the ligand-binding pocket. Ftz-f1 also contains a conserved residue in the third helix of the LBD (N840) that has been shown in other receptors to be a key ligand-contacting residue. Although a study with the Ftz-f1 homolog SF-1 suggests that its transcriptional activity could indeed be enhanced in the presence of certain oxysterols, subsequent studies have been unable to validate this finding. A more general observation in favor of a ligand is that while there are a large number of orphan nuclear receptors whose ligands have not been identified, there are currently no clear examples of receptors that are fully functional as activators in the absence of ligand (Schwartz, 2001 and references therein).

Observations that argue for the absence of a Ftz-f1 ligand include the avid binding of Ftz to Ftz-f1 observed in vitro in the absence of ligand. For most nuclear receptors with known ligands, the ligand is required to position the overlying AF2 in the proper orientation for coactivator binding. The ability of Ftz to bind in the absence of ligand suggests that coactivator binding to Ftz-f1 may be constitutive, and that temporal and spatial regulation of Ftz-f1 activity is controlled by the presence or absence of cofactors such as Ftz rather than by ligands. Alternatively, conditions in the cell may be more stringent, making the Ftz-Ftz-f1 interaction ligand dependent (Schwartz, 2001).

The Drosophila homeodomain protein Fushi Tarazu (Ftz) and its partner, the orphan receptor Ftz-F1, are members of two distinct families of DNA binding transcriptional regulators. Ftz and Ftz-F1 form a novel partnership in vivo as a Hox/orphan receptor heterodimer. The murine Ftz-F1 ortholog SF-1 functionally substitutes for Ftz-F1 in vivo, rescuing the defects of ftz-f1 mutants. This finding identified evolutionarily conserved domains of Ftz-F1 as critical for activity of this receptor in vivo. These domains function, at least in part, by mediating direct protein interactions with Ftz. The Ftz-F1 DNA binding domain interacts strongly with Ftz and dramatically facilitates the binding of Ftz to target DNA. This interaction is augmented by a second interaction between the AF-2 domain of Ftz-F1 and the N-terminus of Ftz via an LRALL sequence in Ftz that is reminiscent of LXXLL motifs in nuclear receptor coactivators. It is proposed that Ftz-F1 serves as a cofactor for Ftz by facilitating the selection of target sites in the genome that contain Ftz/Ftz-F1 composite binding sites. Ftz, in contrast, influences Ftz-F1 activity by interacting with the Ftz-F1 AF-2 domain in a manner that mimics a nuclear receptor coactivator (Yussa, 2001).

The orphan receptor Ftz-F1 and homeodomain (HD) protein Ftz cooperate to promote the development of alternate body segments, presumably by activating expression of downstream target genes such as engrailed. Ftz and Ftz-F1 are each sequence specific DNA binding proteins with strong transcriptional activation domains. However, neither is apparently able to function in Drosophila embryos to regulate target gene expression in the absence of its partner, since mutation of either protein results in lethality accompanied by identical pair-rule defects. Each protein is necessary but neither Ftz nor Ftz-F1 alone is sufficient to select and activate target gene transcription in the embryo. Why do Ftz and Ftz-F1 require partners in vivo to regulate gene expression? By analogy to the function of beta ftz-f1 as a competence factor for the ecdysone response, alpha ftz-f1 can be seen as a competence factor for a 'pulse' of Ftz expression in seven stripes in the blastoderm embryo (Yussa, 2001).

Why does Ftz-F1 fail to activate Ftz-F1/Ftz (F1F) targets in the absence of Ftz protein? Ftz-F1 proteins are strong transcriptional activators in a variety of cell systems. In Drosophila, ftz-f1 is maternally deposited and Ftz-F1 is found in all somatic nuclei before Ftz is expressed in seven stripes. Yet, Ftz-F1 does not detectably activate transcription in the absence of Ftz either temporally (before Ftz is expressed zygotically) or spatially (in regions of the embryo outside the seven Ftz stripes). Thus the presence of Ftz functions as an apparent 'on' versus 'off' switch to enable Ftz-F1 to activate transcription. Two mechanisms are proposed that may contribute Ftz-F1's requirement for Ftz to activate transcription. (1) Ftz-F1 might require Ftz for stable DNA binding. In vitro, Ftz-F1 activates transcription of the ftz proximal enhancer (323-fPE) and F1F reporter constructs more strongly than does Ftz, consistent with the in vitro DNA binding properties of the two proteins. In fact, the order of magnitude of synergy of transcriptional activation of Ftz-F1 compared to Ftz-F1 + Ftz (5-10 fold) is similar to the enhancement of DNA binding conferred by Ftz on Ftz-F1. While this mechanism likely contributes to Ftz-F1 activation, the enhancement seen in vitro may not translate into the apparent 'on' vs. 'off' state of Ftz-F1 in Ftz-expressing vs. Ftz-non-expressing cells in vivo (Yussa, 2001). Therefore, (2) a model in which Ftz-F1 is actively repressed in the absence of Ftz, even if it is bound to cognate DNA target sites, is favored. Ftz-F1 is present in all somatic nuclei before Ftz is expressed and may bind cognate DNA sequences, but remains quiescent until it is activated by interaction with Ftz protein. It is proposed that interaction of Ftz with Ftz-F1 through its LXXLL motif displaces corepressor molecules, allowing productive transcription complexes to form. The candidates for repressor molecules that keep Ftz-F1 in an 'off' state are the corepresssors that inhibit activity of other nuclear receptor family members. The corepressors identified in Drosophila that are expressed throughout the blastoderm embryo include Alien, which interacts with Ftz-F1 in GST-pulldown assays and SMRTER. A corepressor might bind directly to Ftz-F1. It is also possible that another partner of Ftz-F1 recruits a corepressor to the complex, as has been shown for the murine ortholog of Ftz-F1 (SF-1) which is regulated by interaction with the nuclear receptor Dax-1 that recruits corepressors and inhibits SF-1 activity (Yussa, 2001).

Why does Ftz require Ftz-F1 for target gene regulation in vivo? Ftz and Ftz-F1 bind cooperatively to DNA to select specific target sites that are composite binding sites for the two proteins. The binding of Ftz and Ftz-F1 to composite sites is stabilized by protein-protein interactions mediated by at least two regions of each partner protein. The composite nature of the binding site raises the selectivity of Ftz binding by requiring a heterodimeric site for productive interaction. The affinity of Ftz for composite sites is dramatically increased by Ftz-F1. Ftz protein can bind to monomeric 'ATTA' core sites in vitro and it appears to associate with a wide array of sites in the genome, as determined by UV cross-linking experiments. However, it is proposed that in vivo, binding of Ftz to monomeric 'ATTA' sites is transient, during the time that Ftz scans the genome for cognate DNA binding sites. This relatively unstable binding does not allow Ftz to effectively activate transcription on its own, consistent with the finding that concatamerized Ftz binding sites do not mediate a Ftz-dependent pattern of gene expression in vivo. Ftz binds productively to composite heterodimeric sites, where it is stabilized by protein-protein interactions with Ftz-F1. This notion is also consistent with reports that fusion of Ftz to a strong VP16 activator does not alter target gene regulation in vivo. Thus, specificity of gene regulation by Ftz protein is achieved at the level of DNA binding/target site selection, as a result of interaction with DNA binding cofactors such as Ftz-F1 (Yussa, 2001).

Future studies are required to elucidate the detailed mechanism whereby transcription is activated by the Ftz/ Ftz-F1 complex. One candidate mechanism is direct contact of TFIIB by Ftz and/or Ftz-F1. Ftz was shown to directly contact TFIIB, activating transcription via a C-terminal region that does not appear to be involved in contacting Ftz-F1. Similarly, SF-1 interacts with TFIIB via the Ftz-F1 box and adjacent proline-rich region, neither of which appear to be necessary for interaction with Ftz. Thus, these regions of the proteins could be available in the DNA bound ternary complex to directly contact the basal transcription machinery (Yussa, 2001).

Does Ftz interaction obviate a requirement for a Ftz-F1 coactivator? The novel possibility is suggested that Ftz substitutes for coactivator function for Drosophila Ftz-F1. For mammalian SF-1, standard coactivator interactions have been demonstrated in vitro and in cell culture, suggesting that SRC and CBP/p300 family proteins are partners of SF-1. Drosophila CBP has been well characterized and one p160/SRC-type coactivator, Taiman, was recently identified. Like Ftz, Drosophila CBP has one LXXLL motif while Tai has four such motifs, as is typical for mammalian coactivators. However, it is unlikely that interaction with either of these coactivators is sufficient to activate Ftz-F1 since both dCBP and Tai are expressed ubiquitously in the embryo, including the cells where Ftz-F1 is apparently inactive. One interesting possibility is that dCBP acts as a corepressor in the context of Ftz-F1, as it has been shown to do for TCF. Thus dCBP might silence Ftz-F1 by interacting with its AF-2 domain via an LXXLL motif. This interaction could be displaced by Ftz because of an intrinsically higher affinity of its LXXLL motif as compared to that of dCBP. Note that peptides with variations of the LXXLL motif have different affinities for AF-2 domains of nuclear receptors. Alternatively, Ftz may interact preferentially with Ftz-F1 because of the additional protein-protein and protein-DNA interactions that bring high levels of Ftz protein in close proximity to Ftz-F1, driving the interaction of its LXXLL motif with Ftz-F1 (Yussa, 2001).

In addition to its role in DNA binding, the Ftz HD is involved in direct protein-protein interactions with its cofactor Ftz-F1. These findings underscore the importance of the HD, which is absolutely required for the wild type function of Ftz, as it is for other Hox proteins. Some years ago, a 'HD-independent' activity of Ftz was described. These studies made use of a protein carrying a deletion within the HD (DHD) that removes helix 2 and portions of helices 1 and 3. It was shown that this protein is able to cause 'anti-ftz' phenotypes that result from mis-expression of Ftz with a heat inducible promoter and it was suggested that this protein can rescue ftz-dependent cuticle when similarly overexpressed. In contrast to these results, Ftz DHD was unable to rescue ftz mutants when the protein was expressed under control of endogenous ftz regulatory elements. In addition, even subtle mutations within this region of the HD abolish rescue potential. Finally, FtzDHD was unable to rescue any cuticular defects associated with ftz mutations when expressed using native ftz regulatory elements. Thus it is likely that under conditions of overexpression, interactions with Ftz-F1 through the AF-2/LXXLL domains of the proteins can to some extent overcome the endogenous requirement for the HD by positioning Ftz on the DNA of some target elements, allowing for gene activation (Yussa, 2001).

Given the highly conserved nature of the HD, the finding that it is involved in the Ftz/Ftz-F1 interaction led the authors to ask if other Hox proteins could interact with Ftz-F1. Preliminary results indicate that a number of Hox proteins coordinately activate transcription in conjunction with Ftz-F1 in cells. Experiments are underway to determine whether these interactions -- which are less potent than Ftz -- are strong enough to support interactions between Ftz-F1 and Hox proteins during Drosophila development. Such interactions would have been masked in previous genetic studies by the fact that the earliest function of Ftz-F1 in segmentation results in a phenotype which precludes analysis of Ftz-F1 function at slightly later stages when Hox genes such as Ubx, Antp and Scr are active. An additional question is whether conserved Hox proteins are partners of mammalian Ftz-F1 proteins. Co-expression of and interaction between Hox proteins and Ftz-F1 have not been investigated in mammals. Several partners of SF-1 have been characterized, including the nuclear receptor Dax-1, the zinc finger transcription factor WT-1, and the Bcd-family HD protein Ptx-1. For Ptx-1, the interaction domain with SF-1 maps outside of the HD. However, interactions between other nuclear receptors and the HDs of other proteins have been reported. Since ftz-f1 but not ftz genes are found in the vertebrate lineage, an intriguing possibility consistent with the results discussed above is that vertebrate Ftz-F1 proteins interact with HDs of Hox proteins that are conserved throughout evolution, to execute unique functions as nuclear receptor/Hox protein heterodimers (Yussa, 2001).

Orphan receptors for whom cognate ligands have not yet been identified form a large subclass within the nuclear receptor superfamily. To address one aspect of how they might regulate transcription, the mode of interaction between the Drosophila orphan receptor FTZ-F1 (NR5A3) and a segmentation gene product, Fushi tarazu (FTZ), was investigated. Strong interaction between these two factors was detected by use of the mammalian one- and two-hybrid interaction assays without addition of ligand. This interaction requires the AF-2 core and putative ligand-binding domain of FTZ-F1 and the LXXLL motif of FTZ. The requirement of these elements has been further confirmed by examination of their target gene expression in Drosophila embryos and observation of a cuticle phenotype in transgenic fly lines that express mutated factors (Suzuki, 2001).

In Drosophila cultured cells, FTZ is required for FTZ-F1 activation of a FTZ-F1 reporter gene. These results reveal a resemblance in the mode of interaction between FTZ-F1 and FTZ and that of nuclear receptor-coactivator and indicate that direct interaction is required for regulation of gene expression by FTZ-F1. Thus, it is proposed that FTZ may represent a category of LXXLL motif-dependent coactivators for nuclear receptors (Suzuki, 2001).

The general structure of the LBD of nuclear receptor superfamily members is composed mainly of 12 helices. Interaction with ligand induces allosteric changes in conformation, especially in the configuration of helix 12 at the C terminus of the LBD, leading to transcriptional activation or repression. Helix 12 is often referred to as the AF-2 core (or AF-2 activation domain, tauc or tau4) that serves in some receptors as a conserved domain essential for ligand-dependent transcriptional activation. Transcriptional coactivators such as CBP/p300, TRAP220, and p160 family factors, SRC-1/NcoA-1, TIF2/GRIP1/NcoA-2, and p/CIP/ACTR/AIB1, have been shown to mediate activating signals through binding to nuclear receptors in a ligand-dependent manner. For this receptor-coactivator interaction, conserved sequences containing a short signature motif of LXXLL (where L is leucine and X can be any amino acid) have been implicated. The conserved leucines in these so-called LXXLL motifs, or NR boxes, appear indispensable for interaction with nuclear receptors. In nuclear receptors, the importance of helices 3, 5, and 12 (AF-2 core) in the LBD has been demonstrated, and computational modeling studies have predicted that helices 3, 5, and an appropriately realigned helix 12 form an interacting surface for the LXXLL. The majority of nuclear receptors, however, are 'orphans', for which cognate ligands have not yet been identified and the molecular mechanisms of their transcriptional regulation remain unclear. From an evolutionary aspect, the extension of the structural conservation to domains including the LBD strongly suggests a functional significance, raising a naive question. Despite large-scale ligand screenings that have been undertaken by many groups, why are there still so many 'orphans' remaining? One answer may be that the structural conservation in the LBD implicates an importance for interactions with various intracellular factors other than small lipophilic molecules (Suzuki, 2001).

Ubiquitous expression of FTZ in early embryos under control of the heat shock promoter broadens even-numbered engrailed (en) stripes, represses alternate wg stripes, and results in a so-called anti-ftz cuticle phenotype, in which roughly reciprocal segments are missing compared with the ftz larval cuticle phenotype. However, ectopic en induction or wg repression was not observed by expressing a construct (mutFTZ) containing substitutions in the tandem leucines, indicating that the LXXLL motif in FTZ is necessary for the ectopic expression of en and the repression of wg. FTZ-dependent en induction and wg repression were also observed by expression of both FTZ-F1 and FTZ under control of the heat shock promoter in the ftz-f1 mutant embryo but not when FTZ-F1DeltaAF2C was used instead of FTZ-F1. An anti-ftz cuticle phenotype was produced by forced expression of wild-type FTZ but not by expression of mutFTZ. In ftz-f1 mutant embryos, anti-ftz phenotypes were obtained when FTZ-F1 and FTZ were coexpressed under heat shock control but not upon replacement of FTZ-F1 with FTZ-F1DeltaAF2C. These observations indicate that interaction through the LXXLL motif in FTZ and AF-2 core in FTZ-F1 is necessary for producing an anti-ftz phenotype and further support the results of the one- and two-hybrid assay (Suzuki, 2001).

Thus, results using embryos strongly suggest that FTZ-F1 activates engrailed in vivo through the AF-2-LXXLL-dependent direct interaction. In early fly embryos, FTZ-F1 seems to function as an activator for engrailed only in regions where FTZ is also present despite the uniform expression of FTZ-F1. Such situations mimic that of the requirement for a ligand by a nuclear receptor in controlling its function and specificity in gene expression. The characteristic cooperation of FTZ-F1 and FTZ provides a novel example of transcriptional regulation by a nuclear receptor, which may be an alternative pathway to the conventional one using lipophilic ligands. From an evolutionary aspect, it has been proposed that the ancestral nuclear receptor had no ligand and the ability to bind a ligand was acquired by a subset of descendent receptors later in evolution. It has also been presumed that FTZ-F1 is one of the most ancient receptors based on its distribution among species. It is believed that transcriptional activation by FTZ-F1 through binding to FTZ might represent a primitive style of regulation by nuclear receptors before the acquisition of ligand-binding ability. The existence is presumbed of yet unidentified corresponding factors for other orphan receptors as well as for ligand-responsive receptors, which may form a new group of nuclear receptor coactivators and play critical roles for development and metabolism (Suzuki, 2001).

Bonus interacts with FTZ-F1 and inhibits FTZ-F1-dependent transcription

The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the activator function AF-2 domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo (Beckstead, 2001).

bon was isolated in a screen for mutations affecting embryonic peripheral nervous system (PNS) development. Three independent P-element alleles, bonS024108 (bon²⁴¹), bonS024912 (bon²⁴⁹), and bonS048706 (bon⁴⁸⁷), which mapped to 92E8-14, fail to complement each other. An additional bon allele, bon^21B, was generated by imprecise excision of bon²⁴¹. This allele fails to complement all bon alleles and a deficiency, Df(3R)H^B79, which removes chromosomal region 92E (Beckstead, 2001).

To establish the strength of each allele, complementation tests were performed and the lethal phase associated with each allelic combination was defined. Df(3R)H^B79/bon^21B animals exhibit the earliest stage of lethality, while homozygous bon²⁴¹/bon²⁴¹ animals display the least severe phenotype, with 34% of the expected animals surviving to pharate adults. In all genetic combinations, some first instar larvae survive up to a week and fail to molt into second instar larvae. No male third instar larvae, pharate adults or adults survived in any bon genetic background, indicating that loss of bon has a more deleterious effect on males than females. Based on the complementation data, the bon alleles were ordered as follows: Df(3R)H^B79 > bon^21B > bon⁴⁸⁷ > bon²⁴¹ = bon²⁴⁹ (Beckstead, 2001).

To pinpoint the phenotypes associated with bon mutations, morphological defects associated with different allelic combinations were analyzed. Df(3R)H^B79/bon^21B mutant embryos fully develop. However, many are unable to hatch from their egg case, and both Df(3R)H^B79/bon^21B embryos and first instar larvae exhibit disrupted fluid-filled trachea. The embryonic/first instar lethality probably corresponds to the zygotic null or a severe loss of function phenotype as there is very little maternal protein remaining in mature Df(3R)H^B79/bon^21B embryos (Beckstead, 2001).

Less severe loss of Bon function results in pupal defects. The majority of bon⁴⁸⁷/bon^21B mutant pupae display an almost complete lack of pigmentation. They initiate but fail to complete development of legs, wings, head, eyes, and cuticle. Salivary glands, which normally undergo apoptosis at 12 hr postpupariation, are present in 4 days post-pupariation bon⁴⁸⁷/bon^21B pupae and are similar in size to third instar larval glands. In bon²⁴¹/bon⁴⁸⁷ animals, defects in cuticle and bristle development are observed. The abdominal cuticle of these flies appears immature and the tergite and sternal bristles are severely reduced or absent. Bristles of the anterior wing margin of bon²⁴¹/bon⁴⁸⁷ pharate adults are almost entirely lacking. Finally, there is a dramatic reduction in pigmentation in the mutant wing cuticle. This data indicates that bon is required for numerous developmental processes, including control of larval molting, cuticle deposition and pigmentation, bristle development, and elimination of salivary glands by cell death (Beckstead, 2001).

To determine the effect of complete loss of bon on the development of adult tissues, the FLP/FRT system was used to create mutant clones in the developing eye imaginal disc with the eyeless enhancer driving FLP. Flies heterozygous for FRT bon^21B and a cell lethal gene marked with w⁺ (FRT w⁺ cl3R) expressing FLP in the eye disc generate clones of bon^21B/bon^21B. Loss of bon in the eye causes a loss of all mutant photoreceptors. A small patch of red photoreceptors remains because of a limited number of bon^21B/cl3R cells. This indicates that bon is required for cell viability or proliferation of photoreceptors. In addition, much of the head cuticle is missing, indicating that most or all cells of the eye disc that produce cuticle are also lacking. In addition, no adult mutant bon clones were observed in FLP/FRT experiments using heat shock-FLP; FRT82 bon^21B animals, even though numerous wild-type twin spots were observed. Hence, early and complete loss of bon is lethal to cells or disrupts proliferation (Beckstead, 2001).

To clone bon, genomic DNA flanking bon²⁴¹ was isolated and used to identify cDNAs. The bon cDNA (AF210315) permitted isolation of genomic phages and determination of the structure of the locus. Flanking sequences from bon²⁴¹, bon²⁴⁹, and bon⁴⁸⁷ were used to map the P-elements. Sequencing of bon^21B revealed a deletion of most of exon 1 and the 5' end of intron 1 (Beckstead, 2001).

Database searches have revealed that bon encodes the only Drosophila homolog of mammalian TIF1s. Bon exhibits 29% identity with mouse TIF1alpha and mouse TIF1beta, and 26% identity with human TIF1gamma. The overall identity between Bon and TIF1s is similar to the identity observed between the TIF1 members. A higher degree of identity is seen in the N- and C-terminal regions spanning the conserved domains. At the N terminus, a C3HC4 zinc-finger motif or RING finger is followed by two cysteine-rich zinc binding regions (B-boxes) and a coiled coil domain forming a tripartite motif designated RBCC. At the C terminus, a bromodomain is preceded by a C4HC3 zinc-finger motif or PHD finger (Beckstead, 2001 and references therein).

Northern analysis demonstrates that bon produces one predominant 6 kb transcript and two 4 kb transcripts, which each encode a protein of ~140 kDa. The two 4 kb transcripts are only present in 0-3 hr embryos and adult females. It is therefore possible that the 4kb mRNAs are maternal components. bon is expressed throughout embryogenesis and in first instars. Its levels increase in 9-12 hr embryos and are low during the second instar stage. bon is upregulated in late third instar larvae. The upregulation of bon during midembryogenesis and prior to pupariation correlates well with known high titer pulses of ecdysone (Beckstead, 2001).

Immunohistochemical staining of numerous tissues show that Bon is a nuclear protein expressed in most and possibly all cells during embryogenesis, in fat body, imaginal discs, salivary glands, brain, gut, Malpighian tubules, and trachea. Bon is a chromatin-associated protein that localizes to ~10%-15% of the polytene chromosome bands. This pattern is highly reproducible (Beckstead, 2001).

To determine whether the defects seen in bon mutants are due to disruptions in the ecdysone-regulated pathway, the expression of several ecdysone-regulated genes were examined in y w and bon²⁴¹/bon²⁴¹ larvae, prepupae, and pupae. In bon²⁴¹/bon²⁴¹ animals, levels of betaFTZ-F1, EcR-A, EcR-B, E74A, E74B, and BR-C are reduced. It appears that each gene is upregulated in response to the ecdysone pulse, but is unable to maintain expression in the bon mutants. However, DHR3 transcripts are prematurely expressed and the overall level of expression is elevated in bon²⁴¹/bon²⁴¹ animals when compared to y w control animals. In addition, the EcR-A transcript levels appear slightly reduced in bon²⁴¹/bon²⁴¹ animals, while the EcR-B transcript levels are severely reduced when compared to controls. Similar observations were made for all of the above genes in bon^21B/bon⁴⁸⁷ animals, except that DHR3 transcript levels are also reduced. Based on these effects on gene expression, defects in larval molting and metamorphosis, and the temporal expression pattern of Bon, it is proposed that Bon plays an important role in the regulation of genes in the ecdysone response pathway (Beckstead, 2001).

To better characterize the function of Bon, interacting proteins were sought. A Drosophila embryonic cDNA library was screened using Bon as bait. Isolated cDNAs were classified as positive when retested in another version of the two-hybrid system using the DNA binding domain of the estrogen receptor fused to Bon (DBD-Bon) and an ERE-URA3 reporter gene. One positive clone encoded the 488 C-terminal residues of betaFTZ-F1 (amino acids 315-802). Coexpression of DBD-Bon with AAD-betaFTZ-F1(315-802) transactivates the URA3 reporter. Hence, Bon is able to interact with betaFTZ-F1(315-802) in yeast cells (Beckstead, 2001).

To test whether Bon interacts with betaFTZ-F1 as well as other Drosophila nuclear receptors in vitro, binding assays were performed using purified recombinant proteins. Glutathione-S transferase (GST)-fused betaFTZ-F1, alphaFTZ-F1 (amino acids 154-1029), Seven-up (SVP), DHR3, USP, and EcR were immobilized on glutathione-Sepharose and incubated with purified N-terminally His-tagged Bon (His-Bon). His-Bon binds to GST-betaFTZ-F1, GST-alphaFTZ-F1, GST-DHR3, GST-SVP, GST-USP, and GST-EcR, but not to GST alone. Thus, Bon can bind directly to many members of the nuclear receptor family in vitro (Beckstead, 2001).

To define the domain(s) of betaFTZ-F1 responsible for Bon interaction, a deletion analysis of betaFTZ-F1 was performed using the yeast two-hybrid system. Various segments of betaFTZ-F1 were fused to the VP16 AAD and assayed for DBD-Bon interaction. No increase in reporter activity was observed with the N-terminal A/B region or with a fusion protein containing residues 270-631, which include the DNA binding domain and the hinge region of the receptor. In contrast, a 7-fold activation was detected in the presence of AAD-betaFTZ-F1(555-802), indicating that the E region encompassing the putative ligand binding domain (LBD) is sufficient for interaction with Bon (Beckstead, 2001).

Sequence analysis of the E region of betaFTZ-F1 has revealed a conserved transcriptional activation domain 2 core motif (AF-2 AD core) between residues 791 and 797. To investigate its activity, an expression vector encoding the E region of betaFTZ-F1 fused to the yeast GAL4 DNA binding domain was cotransfected into Drosophila Schneider (S2) cells together with a GAL4 reporter plasmid. An increase in reporter gene activity was observed, whereas no transactivation was detected with a GAL4-betaFTZ-F1 construct lacking the AF-2 AD core. Deletion of the AF-2 AD core also abolished Bon interaction with the betaFTZ-F1 E region in yeast. Thus, the LBD of betaFTZ-F1 contains an AF-2 activation domain, whose integrity is required for Bon interaction (Beckstead, 2001).

To determine which domain of Bon interacts with betaFTZ-F1, a series of DBD-Bon deletion constructs were generated and assayed for interaction with the E region of betaFTZ-F1. No significant increase in reporter activity was observed when fusion proteins of the RBCC motif (1-450) and the PHD/bromodomain (891-1133) of Bon were coexpressed with AAD-betaFTZ-F1 E region. In contrast, a 15-fold enhancement was observed in the presence of DBD-Bon (527-700) domain. Analysis of this region has revealed a predicted alpha-helical segment extending from residues 561 to 570. This domain contains an LxxLL consensus sequence, originally identified in the nuclear receptor-interacting domain of TIF1alpha and subsequently found in many other AF-2 mediators. In the presence of AAD-betaFTZ-F1 (E) wild-type, but not AAD-betaFTZ-F1(E)DeltaAF-2 AD-core, residues 561 to 570 of Bon fused to the ERalpha DBD activate the reporter gene ~8-fold above the level of unfused AAD. Thus, Bon contains an LxxLL motif that is sufficient to interact with the LBD of betaFTZ-F1 in an AF-2-integrity-dependent manner. To investigate whether Bon actually binds betaFTZ-F1 through this LxxLL motif, mutations in Bon were generated that eliminate the conserved leucine residues at positions 566 and 567. The replacement of these leucines by alanine residues abolishes the interaction with the LBD of betaFTZ-F1 in yeast. Hence, Bon interacts with the AF-2 of betaFTZ-F1 through an LxxLL motif (Beckstead, 2001).

betaFTZ-F1 plays an important role in the stage-specific response to the prepupal ecdysone pulse by positively regulating the expression of E74A, E75B, BR-C, EDG84A, and E93, and negatively regulating its own expression. Mutant betaFTZ-F1 animals display variable defects in early pupal events such as adult head eversion, leg elongation, and salivary gland cell death. Similar phenotypes are observed in bon mutant pupae (Beckstead, 2001).

The phenotypes associated with betaFTZ-F1^ex17/Df(3L)Cat^Dh104 mutants have been categorized into three lethal pupal classes: 38% die as pharate adults with short malformed legs; 45% undergo head eversion, but arrest early in pupal development; and 17% fail to undergo head eversion, but continue developing into cryptocephalic pharate adults. All betaFTZ-F1 mutants have deformed legs.

Because Bon is able to interact with betaFTZ-F1 in vitro, attempts were made to establish whether Bon interacts with betaFTZ-F1 in vivo. Flies were generated with either bon²⁴¹ or bon⁴⁸⁷ in the Df(3L)Cat^Dh104/betaFTZ-F1^ex17 or betaFTZ-F1^ex17/betaFTZ-F1^ex17 mutant backgrounds and assessed for their effect on betaFTZ-F1 phenotypes. Loss of one copy of bon is able to suppress the phenotypes associated with loss of betaFTZ-F1. In the Df(3L)Cat^Dh104/betaFTZ-F1^ex17 background, partial loss of Bon rescues the majority of mutant animals to pharate adult stages: 87% for bon⁴⁸⁷ and 70% for bon²⁴¹. In the betaFTZ-F1^ex17 homozygotes, partial loss of Bon dramatically increases the number of adult escapers: 72% for bon⁴⁷⁸ and 60% for bon²⁴¹, compared to 31% in a wild-type background. In addition, one mutant copy of bon also strongly suppresses the leg phenotypes associated with loss of betaFTZ-F1. In summary, these data indicate that partial loss of Bon suppresses the phenotypes associated with a partial loss of betaFTZ-F1 (Beckstead, 2001).

betaFTZ-F1^ex17 has been shown to be a hypomorphic allele that is the result of a deletion of a positive regulatory element. Northern analysis has demonstrated that betaFTZ-F1^ex17/betaFTZ-F1^ex17 animals exhibit low levels of betaFTZ-F1 transcripts. It was therefore hypothesized that bon suppression of the betaFTZ-F1^ex17 phenotypes may be the result of betaFTZ-F1 upregulation. To test this hypothesis, Northern analysis was performed on betaFTZ-F1^ex17/+, betaFTZ-F1^ex17/betaFTZ-F1^ex17, and betaFTZ-F1^ex17 bon⁴⁸⁷/betaFTZ-F1¹⁷ staged prepupae and the levels of betaFTZ-F1 expression was estimated. One mutant copy of bon⁴⁸⁷ results in a 1.8- and a 1.9-fold upregulation of betaFTZ-F1 in betaFTZ-F1^ex17 mutants. These results suggest that suppression by bon is at least partially due to the upregulation of betaFTZ-F1 and that Bon seems to play a direct role in repressing betaFTZ-F1 expression. These data appear in contrast to, but are not inconsistent with the general loss of Bon function that affects the transcription of most nuclear receptors negatively. Because betaFTZ-F1 is a downstream effector in the ecdysone pathway, the specificity of the interaction between betaFTZ-F1 and Bon is probably masked in a severe loss of function bon animal (Beckstead, 2001).

To determine whether Bon is able to repress transcription, the coding sequence of Bon was fused to the yeast GAL4 DNA binding domain. The resulting fusion protein was tested for its ability to repress transcription activated by ER(C)-VP16, a chimeric activator containing the DBD of ERalpha fused to VP16. GAL4-Bon and ER(C)-VP16 were transiently transfected into S2 cells with a reporter containing a GAL4 binding site (17M) and an estrogen response element (ERE) in front of a thymidine kinase (tk) promoter-CAT fusion (17M-ERE-tk-CAT). GAL4-Bon efficiently represses transcription in a dose-dependent manner. In contrast, coexpression of Bon without the GAL4 DNA binding domain causes a reproducible increase in CAT activity, indicating that repression by Bon is entirely dependent on DNA binding (Beckstead, 2001).

To map the domain of Bon responsible for transcriptional repression, a set of N- and C-terminally truncated derivatives were assayed for their ability to repress VP16-activated transcription in S2 cells. In the absence of the RBCC motif, the GAL4-Bon fusion protein, GAL4-Bon [471-1133]) fails to repress transcription, indicating that the N-terminal region of Bon is required for repression. However, this region is not sufficient for full repression. Consistent with this, a C-terminal truncation, GAL4-Bon (1-890), is a less potent repressor, indicating that the C-terminal residues of the protein including the PHD finger and the bromodomain also contribute to the repression potential of Bon. However, this domain on its own exhibits little repression. A 3- to 4-fold increase in CAT activity is observed with the central region between the coiled-coil and the PHD finger, suggesting that Bon may also contain a 'masked' activation domain. Note, however, that no significant activation was observed with GAL4-Bon (471-890) tested in the absence of ER(C)-VP16. Taken together, these results indicate that most of the repression activity of Bon resides within the N-terminal RBCC domain (Beckstead, 2001).

To investigate functional consequences of the Bon-betaFTZ-F1 interaction, the transcriptional activity of betaFTZ-F1 AF-2 was assayed alone or in combination with overexpressed Bon in transiently transfected cells. Bon and the GAL4-betaFTZ-F1(E) derivative were cotransfected into S2 cells together with the GAL4-responsive reporter, 17M-ERE-tk-CAT. GAL4-betaFTZ-F1(E) exerts a trans-stimulation activity that is repressed by the addition of Bon. Taken together, these results provide support for the hypothesis that Bon plays a role in downregulating betaFTZ-F1-dependent transcription (Beckstead, 2001).

Bon and TIF1s contain an N-terminal RBCC (RING finger/B boxes/coiled coil) motif. In the absence of the RBCC motif, the GAL4-Bon protein, unlike the full-length protein, fails to repress transcription. The TIF1beta RBCC domain has been shown to be necessary for the oligomerization of TIF1beta and KRAB binding. Because Bon is able to homodimerize, this domain may be involved in formation of protein complexes (Beckstead, 2001).

The PHD finger and bromodomain are characteristic features of nuclear proteins known to be associated with chromatin and/or to function at the chromatin level. For instance, the chromosomal proteins Trithorax and Polycomb-like contain multiple PHD fingers, while the histone acetyltransferases CBP and GCN5 as well as the chromatin-remodeling factor SWI2/SNF2 are also bromodomain containing proteins. Bromodomains have been shown to bind to acetyl-lysine and specifically interact with the amino-terminal tails of histones H3 and H4, suggesting a chromatin-targeting function for this highly evolutionarily conserved domain. Because Bon is localized to hundreds of chromatin bands on Drosophila polytene chromosomes, it is probably involved in chromatin-mediated regulation of transcription of numerous genes (Beckstead, 2001).

Bon can repress both basal and activated transcription when recruited to the promoter region of a target gene, similar to TIF1alpha, -beta, and -gamma. For TIF1alpha and TIF1beta, a link between silencing and histone modification has been established, and TIF1beta is part of a large multiprotein complex that possesses histone deacetylase activity. Moreover, TIF1beta was also reported to colocalize and interact directly with members of the heterochromatin protein 1 (HP1) family. Similar to TIF1beta, TIF1alpha can bind the HP1 proteins in vitro. However, TIF1alpha-mediated repression in transfected cells does not require the integrity of the HP1 interaction domain, nor is there any significant subnuclear colocalization of HP1alpha and TIF1alpha. No interactions were observed between Bon and HP1 in a yeast two-hybrid assay, nor was any evidence found for genetic interactions. However, in a yeast two-hybrid screen, Bon interacted with members of the Polycomb group, suggesting that Bon may also be part of heterochromatin-like complexes and/or may require some of the members of the Polycomb group genes to repress transcription. This would imply that Bon has a dual role, similar to some members of the Polycomb group family: transcriptional repression and heterochromatin formation. Both of these roles may be required in transcriptional repression (Beckstead, 2001).

Upon ecdysone binding, the EcR/USP complex upregulates the expression of a group of transcription factors, many of which are nuclear receptors. During this ecdysone regulatory cascade, both induction and repression of transcription are required to regulate the timing and the response to the ecdysone signal. Bon is able to interact with many members of the nuclear receptor family, suggesting it may have a role in multiple steps during metamorphosis and affect expression of many ecdysone regulated genes. For example, DHR3, a key component of the ecdysone response, is required for patterning and integrity of the adult cuticle, and DHR3 mutant clones exhibit a loss of pigmentation, cuticle defects, and missing bristles, similar to a partial loss of Bon. In addition, mutations in betaFTZ-F1, E74B, and BR-C exhibit malformed legs, which are a result of failure in the ecdysone response pathway. Again, very similar defects are observed in bon mutants. Salivary glands in betaFTZ-F1, BR-C, and bon mutant pupae also fail to undergo apoptosis. The ability of bon mutations to cause phenotypes that resemble defects associated with mutations with multiple members of the pathway suggests that Bon is interacting with several members of the pathway at several stages, in agreement with the biochemical observations (Beckstead, 2001).

The interaction of Bon with nuclear receptors is similar to TIF1alpha but unlike TIF1beta and TIF1gamma. This interaction requires the integrity of the nuclear receptor AF-2 activation domain and is mediated by the Bon/TIF1alpha LxxLL motif. These observations suggest that Drosophila nuclear receptors and Bon have co-evolved to maintain their interaction. It is therefore likely that the biological role of this interaction has been conserved in mammals (Beckstead, 2001).

The data provide genetic evidence for the biological relevance of the interaction between Bon and the nuclear receptor betaFTZ-F1. Reduction in the level of Bon, but not the complete loss of Bon, which affects the entire pathway, suppresses the phenotypes associated with a regulatory loss of function mutation of betaFTZ-F1. This suppression is likely to be the result of an increase in the transcription of betaFTZ-F1, suggesting that Bon plays a role in the repression of betaFTZ-F1. Because betaFTZ-F1 represses its own transcription, it is likely that a protein complex containing betaFTZ-F1 and Bon is required for this repression. Removal of a copy of Bon may therefore lead to an up-regulation of betaFTZ-F1 transcription. Although these data appear to contrast the loss of betaFTZ-F1 transcription in bon²⁴¹/bon²⁴¹ mutants, they are not inconsistent. In the bon²⁴¹/bon²⁴¹ mutant background, loss of two copies of bon severely affects the entire ecdysone pathway. This is clearly not the case when one copy of bon is mutated. Therefore, removal of one copy of bon in the betaFTZ-F1 mutant background allows for the detection of protein:protein interactions between betaFTZ-F1 and Bon. Thus the phenotypic suppression and the S2 cell transcription data are in agreement with Bon functioning as a negative regulator of betaFTZ-F1-dependent transcription. It is therefore tempting to speculate by analogy that TIF1alpha may also interact with and inhibit transactivation by nuclear receptors in mammals. A model is favored in which Bon (or TIF1alpha), once recruited to particular regions of chromatin containing acetylated histones via Bon's bromodomain, interacts via Bon's bromodomain LxxLL motif with the AF-2 domain of DNA-bound nuclear receptors. This complex then represses transcription from cognate target genes, possibly via an effect on chromatin structure (Beckstead, 2001).

rigor mortis encodes a nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. A novel nuclear receptor interacting protein is described, encoded by rigor mortis (rig), that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation -- phenotypes that resemble those seen in EcR, usp, E75A and ßFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and ßFTZ-F1. The ligand binding domain of ßFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development (Gates, 2003).

Mutations in rig result in prolonged second and third instar larval stages, defects in molting, larval lethality and duplicated mouth parts. These phenotypes are characteristic of defects in ecdysone signaling, suggesting a critical role for rig in ecdysone responses during larval development. Two classes of genes produce mutant phenotypes that resemble those seen in rig mutant animals: those required for ecdysone biosynthesis or release -- including ecdysoneless (ecd), dare and itpr -- and those encoding nuclear receptors that mediate the ecdysone signal -- EcR, usp, E75A, and ßFTZ-F1. Unlike ecdysone-deficient mutants, the lethal phenotypes of rig mutants cannot be rescued by feeding 20E, indicating that ecdysone is not limiting in these animals and that rig acts downstream from hormone biosynthesis or release. Rather, it is proposed that Rig is functioning as a nuclear receptor cofactor, based on five lines of evidence. (1) The lethal phenotypes of rig mutants are very similar to those defined for EcR, usp, E75A and ßFTZ-F1, although all of these nuclear receptor genes are expressed in an essentially normal manner in rig mutant larvae. (2) rig mutants display a defect in the ecdysone-triggered switch in E74 isoform expression that is characteristic of reduced ecdysone signaling, indicating that rig is required for the appropriate expression of specific ecdysone-inducible genes. (3) These effects on gene expression are likely to be indirect as the predicted Rig protein sequence contains multiple protein-protein interaction domains and no known DNA-binding motifs. (4) Rig protein can interact physically with several Drosophila nuclear receptors, including EcR, USP and ßFTZ-F1, all of which have mutant phenotypes in common with rig mutants. (5) Rig protein shuttles between the cytoplasm and nucleus of larval cells in a manner similar to the active subcellular redistribution that has been reported for known Drosophila and vertebrate nuclear receptor cofactors (Gates, 2003).

Five Drosophila nuclear receptor cofactors have been identified to date: Alien, SMRTER, MBF1, Taiman and Bonus. Of these, only bonus appears to have activities in common with rig, although relatively limited genetic studies have been undertaken for most of these cofactors. No mutants have been characterized for SMRTER or Alien, which act as co-repressors in tissue culture transfection assays. MBF1 null mutants are viable and display a strong genetic interaction with tdf/apontic mutants: this indicates a role in tracheal and nervous system development. Somatic clones of taiman mutants reveal a role in border cell migration during oogenesis. In contrast, bonus mutants display first instar larval lethality as well as defects in salivary gland cell death and cuticle and bristle development, implicating a role for bonus in ecdysone responses during development. Also like rig, bonus mutations result in gene-specific defects in ecdysone-regulated transcription, and Bonus protein can interact with a range of Drosophila nuclear receptors, including EcR, USP, SVP, DHR3 and FTZ-F1. Bonus, however, interacts with these receptors in an AF-2-dependent manner, unlike Rig. Moreover, the larval lethal phenotypes of rig mutants do not resemble those reported for bonus mutants and, unlike Rig, Bonus protein appears to be exclusively nuclear in both larval and imaginal tissues. Further work is required to determine whether bonus and rig might act together to regulate ecdysone response pathways (Gates, 2003).

Rig is distinct from all known Drosophila nuclear receptor cofactors in that it is not part of an evolutionarily conserved protein family. Alien, SMRTER, MBF1, Taiman and Bonus all have vertebrate homologs, and Taiman and Bonus are the fly orthologs of the well characterized vertebrate nuclear receptor cofactors AIB1 and TIF1, respectively. In contrast, Rig does not contain identifiable enzymatic activities nor the conserved functional domains that define most nuclear receptor cofactors. BLAST searches with the Rig protein sequence did not reveal any closely related sequences in other organisms, although the top hits, which show limited homology in the WD-40 repeats, are in factors known to modify chromatin, including human histone acetyltransferase type B subunit 2 (RBBP-7) and chromatin assembly factor 1 (CAF-1) (Gates, 2003).

The WD-40 repeats that comprise about half of the Rig protein sequence are likely to play an important role in its activity. Consistent with this proposal, an N-terminal fragment of Rig, containing two WD-40 repeats but missing the LXXLL motif (amino acids 1-300), is capable of interacting with GST-DHR3 and GST-USP, suggesting that these repeats are sufficient for Rig-nuclear receptor interactions. WD-40 repeats provide multiple surfaces for protein-protein interactions and have been identified in over 150 proteins that function in a wide range of processes, including cytoskeleton assembly, transcriptional regulation, and pre-mRNA processing. In Drosophila, WD-40 repeats are associated with several transcriptional regulators, including the p85 subunit of TFIID, the Polycomb group protein encoded by extra sex combs, and the Groucho corepressor. In addition, a WD-40 repeat protein, TBL1, has been identified as part of a multiprotein complex with thyroid hormone receptor that contains the SMRT nuclear receptor corepressor and HDAC-3. The presence of these sequences in Rig may thus provide a scaffold for protein-protein interactions that could mediate the formation of multiprotein transcriptional complexes on ecdysone-regulated promoters. Further biochemical studies of Rig should provide insights into the significance of its WD-40 repeats as well as a foundation for understanding how Rig exerts its effects on transcription (Gates, 2003).

It is not clear how Rig expression in the brain, imaginal discs and salivary glands of second and third instar larvae is related to the lethal phenotypes of rig mutants, although neuroendocrine signaling is clearly required for molting, a process that is defective in rig mutant larvae. The subcellular localization of Rig protein at later stages, however, correlates with the distinct fates of larval and imaginal cells during metamorphosis. Rig protein appears to be restricted to the cytoplasm of cells that are fated to form parts of the adult fly, including neuroblasts, imaginal discs, and the imaginal islands of the larval midgut. In contrast, Rig shows dynamic changes in its subcellular distribution in larval salivary gland and midgut cells, both of which undergo steroid-triggered programmed cell death during metamorphosis. It is possible that these differences in subcellular localization could contribute to the distinct fates of these tissues in response to ecdysone signaling (Gates, 2003).

In addition to this spatial correlation, there is also a temporal correlation between the times at which Rig protein shuttles between the cytoplasm and nucleus in larval tissues and the coordinated changes in ecdysone-regulated gene expression that occur during the third instar. The switch from cytoplasmic to nuclear localization in larval salivary glands and midguts occurs at approximately the same time, 24-30 hours after the second-to-third instar larval molt, suggesting that Rig may be responding to a common temporal signal. Cell type-specific factors, however, must also contribute to this regulation as Rig is localized to the nucleus of only a subset of cells in the larval midgut. Interestingly, this protein redistribution correlates with a poorly understood event that is represented by widespread changes in ecdysone-regulated gene expression, called the 'mid-third instar transition.' It is possible that the cytoplasmic-to-nuclear transport of Rig in larval tissues contributes to the regulation of this response, which prepares the animal for metamorphosis one day later. Similarly, Rig returns to the cytoplasm of salivary gland cells at puparium formation, in synchrony with the widespread changes in ecdysone-regulated gene expression associated with the onset of metamorphosis. This translocation, however, is not seen in the larval midgut, where Rig protein remains in the nucleus of some cells. Rig shuttling thus appears to be differentially controlled in both a temporally and spatially restricted manner, correlating with major switches in ecdysone-regulated transcription. The observation that the first of these shifts in subcellular distribution occurs during the major lethal phase of rig mutants -- the mid-third instar -- suggests that these intracellular movements contribute to the critical functions of Rig during development (Gates, 2003).

Interestingly, several recent reports have described the subcellular redistribution of nuclear receptor cofactors in both vertebrate and Drosophila cells. The p/CIP vertebrate nuclear receptor coactivator is differentially distributed within the cells of the mouse female reproductive organs. For example, p/CIP is detected primarily in the nuclei of highly proliferative follicular cells while it is most abundant in the cytoplasm of terminally differentiated cells of the corpus luteum. p/CIP displays active nucleocytoplasmic shuttling in response to growth factors in cell culture, and interacts directly with the microtubule network in the cytoplasm. Similarly, MEK-1 kinase-mediated phosphorylation of the SMRT mammalian corepressor leads to the translocation of this factor from the nucleus to the cytoplasm in cell culture transfection assays. The functional homolog of this protein in flies, SMRTER, also shows active redistribution from the nucleus to the cytoplasm in response to a MAP kinase pathway, in this case mediated by EGFR/Sno/Ebi in the Drosophila eye. In both of these systems, regulated phosphorylation of SMRT/SMRTER results in dissociation of a repressor complex and derepression of target gene transcription (Gates, 2003).

These observations raise the possibility that the subcellular location of Rig could determine its regulatory function in different cell types. For example, by analogy with SMRT/SMRTER, loss of Rig from the nucleus of larval cells might disrupt a corepressor complex on specific promoters, leading to coordinate target gene derepression. This is consistent with the proposal that the ecdysone receptor exerts critical repressive functions during larval development. Alternatively, Rig protein in the cytoplasm may tether one or more nuclear receptors, preventing them from acting on their cognate target genes in the nucleus. This model is not favored, however, because antibody stains reveal an exclusively nuclear localization for EcR, USP and ßFTZ-F1 at the onset of metamorphosis. It is also interesting to note that Rig protein appears to localize to discrete regions within the nuclei of larval midgut cells that do not contain chromosomes while Rig co-localizes with the giant polytene chromosomes in larval salivary gland cells. Rig may thus exert some functions in the nucleus that are independent of chromatin binding. Further biochemical studies of Rig, including the identification of additional proteins that interact with this factor, should provide insights into the significance of the subcellular localization of Rig protein as well as a mechanistic understanding of how Rig contributes to ecdysone responses during Drosophila larval development (Gates, 2003).

DEVELOPMENTAL BIOLOGY

Embryonic

FTZ-F1 mRNA of 5.2 kb is likely to be of maternal origin, present from 0 to 4 hour embryos, consistent with the period of FTZ-F1 activity and the expression of fushi tarazu in early embryos. FTZ-F1 mRNA is not detectable from 4 to 14 hours of development, but reappears in 14-22 hour embryos. The late mRNA species are slightly different in size (5.6 and 4.8 kb), suggesting that they are modified at the transcriptional or posttranscriptional level. The reappearance of FTZ-F1 DNA binding activity at a time when ftz is silent suggests that FTZ-F1 has a function distinct from the activation of ftz (Lavorgna, 1993).

Larval and Pupal

FTZ-F1, a member of the nuclear receptor superfamily, has been implicated in the activation of the segmentation gene fushi tarazu during early embryogenesis of Drosophila. An isoform of FTZ-F1, ßFTZ-F1, is expressed in the nuclei of almost all tissues slightly before the first and second larval ecdysis and before pupation. The tissue distribution of ßFTZ-F1 protein was examined by immunostaining. An antibody against ßFTZ-F1 stains the nuclei of most larval tissues at 44-46 hours AEL -- for example, the salivary gland, fat body, trachea, ring gland, epidermis, guts and Malpighian tubules. Staining of gonads was not detectable. Similar nuclear staining patterns are observed in stage-16 embryos, larvae at approximately 72 hours AEL and prepupae at 9 hours APF. No staining was observed in either prepupal tissues at 4 hours APF or larval tissues at 60-63 hours AEL, consistent with the temporal expression profile described above. These results clearly show that ßFTZ-F1 is expressed in most tissues during particular stages and that the protein is localized to the nucleus (Yamada, 2000).

Severely affected ftz-f1 mutants display an embryonic lethal phenotype, but can be rescued by ectopic expression of ßFTZ-F1 during the period of endogenous ßFTZ-F1 expression in the wild type. The resulting larvae are not able to molt, but this activity is rescued again by forced expression of ßFTZ-F1, allowing progression to the next larval instar stage. However, premature expression of ßFTZ-F1 in wild-type larvae at mid-first instar or mid-second instar stages causes defects in the molting process. Sensitive periods were found to be around the time of peak ecdysteroid levels and slightly before the start of endogenous ßFTZ-F1 expression. A hypomorphic ftz-f1 mutant that arrests in the prepupal stage can also be rescued by ectopic, time-specific expression of ßFTZ-F1. Failure of salivary gland histolysis, one of the phenotypes of the ftz-f1 mutant, is rescued by forced expression of the ftz-f1 downstream gene Br-C during the late prepupal period. These results suggest that ßFTZ-F1 regulates genes associated with ecdysis and metamorphosis, and that the exact timing of its action in the ecdysone-induced gene cascade is important for proper development (Yamada, 2000).

FTZ-F1 functions in cuticle formation. The insect cuticle is composed of layers of film. Sequential formation of different layers (cuticlin, epicuticle and endocuticle), is observed beginning approximately 12 hours before the next ecdysis. ßFTZ-F1 is expressed after a new epicuticle layer for the next instar appears. Premature expression of ßFTZ-F1 induces disruption of the epicuticle. These observations highlight the importance of ßFTZ-F1 in the formation of normal cuticle structure and suggest that some of the target genes of ßFTZ-F1 are involved in the process of cuticle formation. In particular, the importance of the timing of expression of these genes is demonstrated. It has been shown that some pupal cuticle proteins are expressed in a stage-specific manner during prepupal periods. ßFTZ-F1 regulates the EDG78E and EDG84A genes, which encode putative pupal cuticle proteins. These observations suggest that ßFTZ-F1 is responsible for the stage-specific expression of cuticle proteins during the prepupal stage (Yamada, 2000).

Analysis of FTZ-F1 transcription during larval and prepupal development shows the appearance of the 5.6- and 4.8-kb FTZ-F1 RNAs (corresponding to the late mRNA species) at 6-8 hours of prepupal development, identical to the timing and level of puffing at puff 75CD (Lavorgna, 1993).

Temporally tuned neuronal differentiation supports the functional remodeling of a neuronal network in Drosophila

During insect metamorphosis, neuronal networks undergo extensive remodeling by restructuring their connectivity and recruiting newborn neurons from postembryonic lineages. The neuronal network that directs the essential behavior, ecdysis, generates a distinct behavioral sequence at each developmental transition. Larval ecdysis replaces the cuticle between larval stages, and pupal ecdysis externalizes and expands the head and appendages to their adult position. However, the network changes that support these differences are unknown. Crustacean cardioactive peptide (CCAP) neurons and the peptide hormones they secrete are critical for ecdysis; their targeted ablation alters larval ecdysis progression and results in a failure of pupal ecdysis. This study demonstrates that the CCAP neuron network is remodeled immediately before pupal ecdysis by the emergence of 12 late CCAP neurons. All 12 are CCAP efferents that exit the central nervous system. Importantly, these late CCAP neurons were found to be entirely sufficient for wild-type pupal ecdysis, even after targeted ablation of all other 42 CCAP neurons. Evidence indicates that late CCAP neurons are derived from early, likely embryonic, lineages. However, they do not differentiate to express their peptide hormone battery, nor do they project an axon via lateral nerve trunks until pupariation, both of which are believed to be critical for the function of CCAP efferent neurons in ecdysis. Further analysis implicated ecdysone signaling via ecdysone receptors A/B1 and the nuclear receptor ftz-f1 as the differentiation trigger. These results demonstrate the utility of temporally tuned neuronal differentiation as a hard-wired developmental mechanism to remodel a neuronal network to generate a scheduled change in behavior (Veverytsa, 2012; full text of article).

Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles

One of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of the understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. This study analyzed the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24h of metamorphosis. Ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis (Zirin, 2013)

There are three different isoforms of the EcR gene, EcR A, EcR B1, and EcR B2, each sharing the same DNA binding and ligand binding domains, but with a unique amino terminus. The different temporal and spatial expression patterns of EcR A and EcR B isoforms are thought to reflect their distinct functions during development. EcR B1 is expressed primarily in larval cells that are destined for histolysis, while EcR A is expressed primarily in imaginal tissues destined for differentiation into adult structures. Thus the response of salivary glands and midgut to ecdysone during metamorphosis is dependent on EcR B1. Nonetheless, EcR A mutants also have a defect in salivary gland histolysis, suggesting that the isoform might also contribute to this process. Furthermore, some neurons in the ventral nerve cord and brain that strongly express EcR A undergo apoptosis in response to ecdysone soon after eclosion, suggesting that ecdysone induced PCD is not strictly a function of EcR B1 signaling (Zirin, 2013)

This study examined expression of both EcR A and EcR B1 isoforms and found that only EcR B1 was detectable in the dorsal internal oblique muscles (DIOMs) and DEOMs during pupariation. Consistent with its expression pattern, knockdown of EcR B1 specifically in the muscle inhibited DEOM histolysis. The inhibition achieved with the EcR B1 isoform RNAi was not as strong as with RNAi targeting all isoforms, or with overexpression of the dominant negative EcR B1. This could be due to either differences in the efficiency of knockdown or to a role for EcR B2 in DEOM degradation. Taken together these data strongly supports the view that, like in salivary glands and midgut, ecdysone signals through EcR B1 to induce cell death in abdominal muscles. However, given that both DIOMs and DEOMs express EcR B1 at the same time, the presence of the receptor is not sufficient to explain why only the latter muscles are degraded. Another important player in the timing of salivary glands cell death is the orphan nuclear hormone receptor gene ftz fl, which is transcribed midway through prepupal development, when the ecdysone titer is relatively low. FTZ F1 has been hypothesized as a competence factor, directing the subsequent genetic responses to the ecdysone pulse at the prepupal/pupal transition. Thus FTZ F1 is required for the induction of salivary gland histolysis. In contrast, the midgut does not express ftz fl prior to its earlier ecdysone induced cell death, indicating that FTZ F1 is not required for all histolysis during Drosophila metamorphosis. Despite the fact that the timing of muscle histolysis is similar to that of the midgut, the function of FTZ F1 was more like in the salivary gland, as FTZ F1 was observed in the DEOMs starting at ~ 5 h APF, prior to caspase activation at 8 h APF. Furthermore, ftz fl was required for proper muscle histolysis, as knockdown cell autonomously delayed caspase activation and death in the DEOMs (Zirin, 2013)

These results raised the possibility that the presence of FTZ F1 in the DEOMs, but not in the DIOMs, determined the different response of these muscles to ecdysone. However, overexpression of FTZ F1 in the OJOMs, while causing severe muscle degeneration, was unable to induce caspase activity or cell death. Nor could the presence of HR39 in the DIOMs account for the different response of the muscles to ecdysone. Although a reciprocity of HR39 and FTZ F1 expression was observed in the DIOMs and DEOMs, Hr39 mutant DJOMs still persist through metamorphosis. It is concluded that FTZ F1 and HR39 expression determine the timing of the abdominal muscle response to ecdysone but that these factors do not change the nature of the response (Zirin, 2013)

It was recently shown that EcR B1 expression is regulated by FTZ F1 and HR39 in mushroom body neurons and abdominal motor neurons during metamorphosis. The current observation that EcR B1 promotes muscle degeneration is consistent with the finding that EcR B1 promotes post synaptic dismantling in the motor neuron, and supports the notion that muscle degeneration is instructive on motor neuron retraction. However, it was show that even though both ftz f1 and EcR B1 are essential for the proper histolysis of DEOMs, there was no change in EcR B1 staining upon ftz fl knockdown as was observed in the mushroom body system. This suggests that changes observed in the muscle synapse due to ftz fl knockdown are not the result of a downstream effect on EcR B1 expression in the muscle cell. Thus, the regulatory relationship between FTZ F1, HR39 and EcR B1 in early pupal abdominal muscles is distinct from the relationship reported in neurons (Zirin, 2013)

This suggests that there is an additional unknown factor whose expression dictates the fate of the OJOMs or DEOMs. This factor is unlikely to be either of the nuclear proteins EAST or Chromator (Chro ), which were previously identified as having opposing effects on the destruction of the abdominal DEOMs during metamorphosis. Breakdown of DEOMs was incomplete in Chro mutants, and promoted in east mutants, leading to the proposal that Chro activates and EAST inhibits tissue destruction and remodeling. However, neither east nor chro alleles cause histolysis of the DIOMs, nor do they alter caspase activation in either DEOMs or DIOMs. Rather these genes may affect the timing of muscle histolysis through a function downstream of PCD induction. It is proposed that there must be an additional factor present in the DIOMs which inhibits EcRB1 signaling from inducing PCD, or alternatively, a factor present in the DEOMs, which permits EcRB1 to activate a death program. The identification of this factor will be a focus of future studies (Zirin, 2013)

The previously reported cleaved caspase 3 staining in the DEOMs is the only data addressing the nature of abdominal muscle PCD prior to this study. This study addressed whether muscle histolysis is apoptotic, autophagic or some combination of both. During salivary gland histolysis, several autophagy related genes (ATGs) are upregulated, and mutations or knockdown of these ATGs specifically in the salivary gland inhibit the destruction of the tissue. Caspase activation also occurs in the histolyzing salivary glands, but overexpression of the caspase inhibitor p35 only partially blocks salivary gland degradation. Simultaneous inhibition of both autophagy and apoptosis in the salivary gland produces the strongest inhibition of death, suggesting that both pathways contribute to histolysis of this tissue). In contrast to salivary gland histolysis, midgut histolysis requires autophagy but not caspase activity. Mutations or knockdown of ATGs inhibit midgut death, but p35 expression has no effect. Based on these two model systems, it appears that there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis (Zirin, 2013)

These data serves to further highlight how distinctive PCD for each of the tissues undergoing histolysis. DEOMs stain positive for cleaved caspase 3, consistent with previous reports. TUNEL positive staining and chromatin condensation was also observed in the DEOMs at 8 h APF, both markers of apoptosis. Importantly, it was possible to suppress DEOM degradation by overexpression of the pan caspase inhibitor p35, indicating that unlike the midgut, muscle histolysis is apoptotic. To determine whether the muscle PCD was autophagic in nature, the DEOMs were examined by EM. Although some autophagic vesicles were observed in the dying muscles, they were not abundant, nor were GFP Atg8 localization to autophagosomes examined by confocal microscopy. Several essential components of the autophagic machinery were knocked down, and no effect was observed on the timing or extent of DEOM histolysis. Although knockdown efficiency is always a concern with RNAi experiments. each of the transgenes was able to strongly inhibit autophagosome formation in larval muscles. It can be said therefore with confidence that autophagy is not required for DEOM PCD, putting the abdominal muscle in the unique category of non autophagic histolysis. In future studies it will be interesting to compare muscles, salivary gland, and midgut to determine why each tissue has a distinctive type of PCD (Zirin, 2013)

Function of the nuclear receptor FTZ-F1 during the pupal stage in Drosophila melanogaster

The nuclear receptor betaFTZ-F1 is expressed in most cells in a temporally specific manner, and its expression is induced immediately after decline in ecdysteroid levels. This factor plays important roles during embryogenesis, larval ecdysis, and early metamorphic stages. However, little is known about the expression pattern, regulation and function of this receptor during the pupal stage. This study analyzed the expression pattern and regulation of ftz-f1 during the pupal period, as well as the phenotypes of RNAi knockdown or mutant animals, to elucidate its function during this stage. Western blotting revealed that betaFTZ-F1 is expressed at a high level during the late pupal stage, and this expression is dependent on decreasing ecdysteroid levels. By immunohistological analysis of the late pupal stage, FTZ-F1 was detected in the nuclei of most cells, but cytoplasmic localization was observed only in the oogonia and follicle cells of the ovary. Both the ftz-f1 genetic mutant and temporally specific ftz-f1 knockdown using RNAi during the pupal stage showed defects in eclosion and in the eye, the antennal segment, the wing and the leg, including bristle color and sclerosis. These results suggest that betaFTZ-F1 is expressed in most cells at the late pupal stage, under the control of ecdysteroids and plays important roles during pupal development (Sultan, 2014).

EFFECTS OF MUTATION

Ectopic expression of ftz-f1 at first instar, late second instar or early prepupal periods causes developmental defects. The sensitive stages slightly precede the endogenous ftz-f1 expression times. Premature expression at late second instar causes a failure in the second ecdysis, though third instar mouthooks and anterior spiracles form. Premature expression of ftz-f1 induces the Edg78E and Edg84A genes, which contain strong FTZ-F! binding sites upstream of their transcription start sites (Ueda, 1995).

In Drosophila, fluctuations in 20-hydroxyecdysone (ecdysone) titer coordinate gene expression, cell death, and morphogenesis during metamorphosis. It has been hypothesized that ßFTZ-F1 (an orphan nuclear receptor) provides specific genes with the competence to be induced by ecdysone at the appropriate time, thus directing key developmental events at the prepupal-pupal transition. This study examines the role of ßFTZ-F1 in morphogenesis. A detailed study has been made of morphogenetic events during metamorphosis in control and ßFTZ-F1 mutant animals. Leg development in ßFTZ-F1 mutants proceeds normally until the prepupal-pupal transition, when final leg elongation is delayed by several hours and significantly reduced in the mutants. ßFTZ-F1 mutants fail to fully extend their wings and to shorten their bodies at the prepupal-pupal transition. ßFTZ-F1 mutants are unable to properly perform the muscle contractions that drive these processes. The muscular contractions believed to drive leg extension, as well as head eversion and wing extension, are thought to do so by causing an increase in the internal pressure of the animal. The inflation and extension of the legs and wings is thought to require the generation of greater pressure inside the developing legs than outside. Several defects can be rescued by subjecting the mutants to a drop in pressure during the normal time of the prepupal-pupal transition. These findings indicate that ßFTZ-F1 directs the muscle contraction events that drive the major morphogenetic processes during the prepupal-pupal transition in Drosophila (Fortier, 2003).

Therefore, wildtype ßFTZ-F1 function is not required for morphogenetic processes that occur during the late larval and early prepupal stages. At 0 h APF, ßFTZ-F1 mutant leg discs appear normal, suggesting that ßFTZ-F1 has no role in leg disc development prior to the beginning of metamorphosis. Leg discs examined at 6 h APF also show no defects in length or in the cell shape changes required for the first phase of elongation, indicating that ßFTZ-F1 is not involved in these early metamorphic events. The ßFTZ-F1 allele used in this study, FTZ-F1¹⁷, is hypomorphic and is expressed at very low levels. It is possible that these cell shape changes require only a minute amount of ßFTZ-F1 and would not occur normally in the complete absence of this protein. Although this is a formal possibility, the evidence indicates that it is unlikely given the developmental expression pattern of ßFTZ-F1. ßFTZ-F1 is expressed during the last larval molt (approximately 48 h before puparium formation), but is not expressed again until the mid-prepupal stage, beginning at about 5 h APF (Fortier, 2003).

ßFTZ-F1 mutant legs develop normally until the prepupal-pupal transition. In the mutant, the anterior translocation and subsequent extension of the legs are delayed by several hours and are incomplete. All subsequent leg development in the mutant appears to occur normally, indicating that the abnormalities seen in these mutants are in fact due to stage-specific defects, rather than to general weakness or ill-health (Fortier, 2003).

In wild-type animals, during the prepupal-pupal transition, contractions of larval muscles shorten the prepupal body, translocate the mid-abdominal gas bubble to the posterior end of the pupal case, and then move the gas to the anterior, providing a space into which the head can evert. These contractions have long been thought to generate hydrostatic pressure, inflating and elongating the legs and wings in the animal. Detailed observation of the ßFTZ-F1 mutants in this study reveal defects in each of these developmental processes. Furthermore, in ßFTZ-F1 mutants, the muscle contractions that drive these events are much less deliberate, vigorous, and consistent than in controls (Fortier, 2003).

Thus bubble translocation, leg and wing elongation, and head eversion can be rescued by exposing mutant prepupae to decreased external pressure. This indicates that these defects result from failure to generate sufficient internal pressure at the appropriate time. This also provides direct evidence that hydrostatic pressure does in fact drive the major extensions of legs and wings at the prepupal-pupal transition. The observation that a drop in pressure can completely rescue leg elongation in some ßFTZ-F1 mutants suggests that there are no defects in the leg imaginal discs of these animals and indicates that ßFTZ-F1 is required for the muscle contractions that drive major morphogenetic events at the prepupal-pupal transition. To test the possibility that abnormalities in muscle morphology account for these contractile defects, both light microscopy and transmission electron microscopy (TEM) studies were performed, comparing musculature of control and ßFTZ-F1 mutant animals up to 12 h APF. No muscle differences have been detected between control and mutant animals (Fortier, 2003).

The increase in hydrostatic pressure that inflates and elongates the wings and legs during pupation normally occurs when the surrounding pupal cuticle is still incomplete. During wild-type metamorphosis, the development of the pupal cuticle is completed shortly after the prepupal-pupal transition. Therefore, in ßFTZ-F1 mutants, it is possible that the delay in muscle contraction results in this transition taking place within a rigid pupal cuticle, which does not allow complete head eversion, or extension of wings and legs. Another possibility is that abnormal cuticle formation or deposition may affect some events at pupation. The expression of EDG78E and EDG84A, genes that encode pupal cuticle proteins, is reduced and delayed in ßFTZ-F1 mutants. If these proteins are not expressed properly, the cuticle may be abnormally rigid at the end of the prepupal stage. Thus, a greater force of contraction would be required to elongate the legs and wings to their normal length during the prepupal-pupal transition. Defective cuticle does not explain failed gas bubble translocation, however, so it appears that, in the ßFTZ-F1 mutant, muscle contractions are insufficient. Nonetheless, the notion of increased cuticular rigidity is an interesting concept that merits future exploration (Fortier, 2003).

These findings indicate that the major morphogenetic defects seen in ßFTZ-F1 mutants result from ineffective muscular contractions at the normal time of the prepupal-pupal transition. ßFTZ-F1 regulates the expression of several genes during the late-prepupal stage, including BR-C, E74A, E75A, and E93. The defects seen during pupation in the ßFTZ-F1 mutant are possibly due to the reduced expression of one or more of these, or other, target genes. Attempts will be made to determine which ßFTZ-F1 target genes direct these morphogenetic events. ßFTZ-F1 is ubiquitously expressed in mid-prepupae. Further characterization of this expression pattern will be helpful in understanding the function of ßFTZ-F1 in morphogenesis. As a competence factor that enables genes to respond to ecdysone at the right time in the proper cells, ßFTZ-F1 has a pivotal position in directing the transformation from larva to adult. Elucidating the role of ßFTZ-F1 in Drosophila metamorphosis will be an important step toward understanding how steroid hormones coordinate the complex events of animal development (Fortier, 2003).

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes ßFTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death (Martin, 2004).

Proteolysis and changes in the assembly of the cytoskeleton both appear to be involved in the regulation of changes that occur during autophagic cell death of salivary glands. Although caspases play an important role in the cell death of salivary glands, several lines of evidence suggest that some changes in the structure of the cytoskeleton may occur in a caspase-independent manner. First, whereas changes in filamentous Actin localization occur in synchrony with changes in proteins such as nuclear Lamins that are cleaved by caspases, changes in Actin protein levels are delayed by 4 hours. Second, mutations in steroid-signaling genes, such as ßFTZ-F1, that prevent expression of active caspase-3 and cleavage of nuclear Lamins do not prevent changes in filamentous Actin localization. Third, although inhibition of caspases by expression of either p35 or a dominant-negative form of Dronc is sufficient to prevent changes in nuclear Lamins and alpha-Tubulin, these inhibitors are not sufficient to block changes in filamentous Actin. These data are further supported by the observation that numerous small GTPases increase their expression immediately prior to salivary gland cell death. Although previous studies have suggested that changes in the Actin cytoskeleton are required for autophagic cell death, the failure to distinguish between cytoskeleton proteolysis and rearrangement has made it difficult to interpret the potential significance of maintenance of the cytoskeleton during cell death (Martin, 2004).

Studies of salivary glands indicate that caspases play an important role in their autophagic cell death. The caspase-encoding genes dronc and drice show an increase in their transcription following the rise in steroid that triggers salivary gland autophagic cell death. This increase in caspase transcription corresponds to the increase in active caspase protein levels and in the cleavage of substrates such as nuclear Lamins in dying salivary glands. Mutations in the steroid-regulated ßFTZ-F1, E93 and BR-C genes, which prevent salivary gland cell death, exhibit little or no active Caspase-3/Drice expression, and have altered alpha-Tubulin, alpha-Spectrin and nuclear Lamin expression in salivary glands. Although E74A mutants prevent salivary gland cell death, they have elevated Caspase-3/Drice levels and degraded nuclear Lamins. Although these data are consistent with the partially degraded morphology of E74A mutant salivary glands, it remains unclear what factor(s) E74A may regulate that are required for normal cell death. However, the data indicate that ßFTZ-F1, E93 and BR-C play a crucial role in determining caspase levels in dying salivary gland cells, and this is supported by the impact of these genes on the transcription of dronc. Significantly, inhibition of caspases by expression of either p35 or dominant-negative Dronc is sufficient to prevent DNA fragmentation, changes in nuclear Lamins and alpha-Tubulin, and death of salivary glands (Martin, 2004).

The Drosophila nuclear receptors DHR3 and βFTZ-F1 control overlapping developmental responses in late embryos

Studies of the onset of metamorphosis have identified an ecdysone-triggered transcriptional cascade that consists of the sequential expression of the transcription-factor-encoding genes DHR3, βFTZ-F1, E74A and E75A. Although the regulatory interactions between these genes have been well characterized by genetic and molecular studies over the past 20 years, their developmental functions have remained more poorly understood. In addition, a transcriptional sequence similar to that observed in prepupae is repeated before each developmental transition in the life cycle, including mid-embryogenesis and the larval molts. Whether the regulatory interactions between DHR3, βFTZ-F1, E74A and E75A at these earlier stages are similar to those defined at the onset of metamorphosis, however, is unknown. This study turned to embryonic development to address these two issues. It was shown that mid-embryonic expression of DHR3 and βFTZ-F1 is part of a 20-hydroxyecdysone (20E)-triggered transcriptional cascade similar to that seen in mid-prepupae, directing maximal expression of E74A and E75A during late embryogenesis. In addition, DHR3 andβFTZ-F1 exert overlapping developmental functions at the end of embryogenesis. Both genes are required for tracheal air filling, whereas DHR3 is required for ventral nerve cord condensation and βFTZ-F1 is required for proper maturation of the cuticular denticles. Rescue experiments support these observations, indicating that DHR3 has essential functions independent from those of βFTZ-F1. DHR3 and βFTZ-F1 also contribute to overlapping transcriptional responses during embryogenesis. Taken together, these studies define the lethal phenotypes of DHR3 and βFTZ-F1 mutants, and provide evidence for functional bifurcation in the 20E-responsive transcriptional cascade (Ruaud, 2010).

The regulatory interactions between DHR3, αFTZ-F1 and E74A/E75A that are described in this study in embryos are indistinguishable from those seen in prepupae. First, DHR3 expression in embryos is dependent on 20E signaling. Second, DHR3 mutants display reduced levels of αFTZ-F1, E74A and E75A expression at both stages in the life cycle, and αFTZ-F1 mutants have reduced levels of E74A mRNA and no detectable E75A expression. Taken together with studies that show that ectopic αFTZ-F1 is sufficient to drive maximal expression of E74A and E75A, these results indicate that DHR3 exerts its effect on these genes through its induction of αFTZ-F1 in embryos. Third, a loss of DHR3 function during embryogenesis does not eliminate αFTZ-F1 expression. This is probably due to other upstream factors that contribute to this response. One candidate for this function is the DHR4 nuclear receptor, which is coexpressed with DHR3 in both embryos and prepupae. DHR4 mutants have no effect on DHR3 expression, but display significantly reduced levels of αFTZ-F1 mRNA in prepupae. These mutants, however, have no effect on embryonic development, suggesting that DHR4 does not play a major role in αFTZ-F1 induction at this early stage in the life cycle (Ruaud, 2010).

The late larval pulse of 20E both directly and indirectly induces DHR3 and represses αFTZ-F1. Taken together with the inductive effect of DHR3 on αFTZ-F1 expression, this regulation ensures that the peak of αFTZ-F1 expression will be delayed until the proper time during development. The observation that the embryonic 20E pulse, at ~8 hours AEL, immediately precedes DHR3 expression suggests that similar regulatory interactions are acting in embryos. However, unlike prepupae, there is no known hormone peak in late embryos that could account for the coordinated induction of E74A and E75A mRNA at this time, as is known to occur in late prepupae. It is possible that these transcripts are fully dependent on trans-acting factors such as αFTZ-F1 for their expression in embryos. Alternatively, these 20E primary-response genes might be induced by a novel temporal signal that remains to be identified (Ruaud, 2010).

It is interesting to note that a similar temporal profile of DHR3, αFTZ-F1 and E74A/E75A expression is also seen in larvae. A burst of DHR3 expression in mid-second instar larvae immediately follows the peak in the 20E titer and precedes the transient expression of αFTZ-F1, which is followed by co-expression of E74A and E75A at the end of the instar. Curiously, E75A, but not E74A, is expressed at an earlier time as well, in apparent synchrony with the 20E pulse, recapitulating the timing seen in embryos. It is thus likely that a common set of regulatory interactions function in both embryos and larvae to dictate the precise timing of these expression patterns at each stage in the life cycle, prior to the third instar. Moreover, the observation that EcR, E75A and αFTZ-F1 mutants display defects in larval molting indicates that their expression is essential for proper progression through these stages in development (Ruaud, 2010).

DHR3 and αFTZ-F1 null mutations lead to fully penetrant embryonic lethality, with relatively minor and partially penetrant phenotypes reported in DHR3 mutant embryos and no phenotypic description of αFTZ-F1 mutant embryos. The studies described in this paper define both common and unique functions for these two nuclear receptors during embryogenesis. DHR3 and αFTZ-F1 null mutants both display a highly penetrant defect in air filling of the tracheal tree. In addition to this common function, αFTZ-F1 is required for the proper differentiation of the denticles in the ventral cuticle and DHR3 is required for VNC condensation. Both DHR3 and αFTZ-F1 mutants display apparently normal muscle movements at the end of embryogenesis, indicating that only some developmental responses are blocked at this stage. These processes of cuticle differentiation, tracheal air filling, muscular movements and VNC condensation represent the major developmental events that can be described in late embryos. Defects in three of these four pathways thus define a central role for DHR3 and αFTZ-F1 in late embryonic development. In addition, unlike prepupae, in which DHR3 and αFTZ-F1 mutants have essentially identical phenotypes, these studies establish independent functions for these two nuclear receptors during development. Together with the previously identified early embryonic roles of the 20E receptor EcR in dorsal closure, head involution and midgut morphogenesis, these data indicate that each step of the 20E-induced transcriptional cascade controls sequential developmental programs during embryogenesis. Moreover, the observation that this transcriptional cascade is also required for larval molting suggests that it represents a stereotypic 20E response that is required for progression through each major transition in the life cycle (Ruaud, 2010).

Ectopic expression of wild-type αFTZ-F1 is sufficient to rescue the lethality of αFTZ-F1 mutants, but has no effect on the viability of DHR3 mutants, indicating that DHR3 exerts essential functions independently of its downstream partner. The causes of lethality in DHR3 and αFTZ-F1 mutant embryos, however, remain unclear. Strong loss-of-function mutations in the signal peptide peptidase (Spp) gene result in tracheal air-filling defects; however, Spp mutant embryos hatch normally and die as first or second instar larvae. Similarly, embryos with severe defects in VNC condensation can hatch into first instar larvae and survive to later stages of development. These results indicate that the lethality of DHR3 and αFTZ-F1 mutant embryos cannot be directly attributed to defects in these pathways. Rather, DHR3 and αFTZ-F1 may participate in a developmental checkpoint necessary to trigger the last steps of embryogenesis required for hatching and survival (Ruaud, 2010).

The microarray study revealed that a number of 20E-responsive genes are misregulated in DHR3 mutants, consistent with studies in prepupae that indicate a crucial role for DHR3 in 20E signaling. The microarray analysis also identified several genes that are involved in chitin metabolism and protein secretion, which could account for the defects in tracheal gas filling seen in DHR3 mutants. These included the chitinase genes Idgf5 (-8.6-fold) and kkv (+2.4-fold), the CBP Cht12 (+2.6-fold) and the COPII coat subunit sec13 (+2.5-fold). This study also identified a number of genes that play a role in axon guidance. Interestingly, most of these genes have dose-dependent effects, whereby either reduced or increased expression can disrupt nervous system development. Failure of DHR3 mutant embryos to express these genes at normal levels could thus contribute to the PNS defects (Ruaud, 2010).

Northern blot hybridization studies to examine the effects of DHR3 and αFTZ-F1 mutants on selected DHR3-regulated genes confirm and extend phenotypic studies of these mutants. Some genes, such as retn, E93 and kkv, display similar transcriptional responses in DHR3 and αFTZ-F1 mutants, whereas E74A and E75A are more significantly affected in αFTZ-F1 mutants and Idgf5 is selectively reduced in DHR3 mutants. These transcriptional effects support phenotypic studies and provide further evidence that DHR3 and αFTZ-F1 exert common and independent regulatory roles during embryogenesis. This conclusion is consistent with experimental and theoretical studies of gene regulatory networks, which indicate that transcriptional cascades provide an effective means of amplifying signals and integrating multiple cues to provide specificity in biological responses. Transcriptional cascades can also direct temporal programs of successive gene expression, as observed in the formation of flagella in Escherichia coli and the specification of anteroposterior patterning in the Drosophila embryo. In addition, the DHR3-αFTZ-F1 transcriptional cascade involves nuclear receptors that could potentially act as ligand-regulated transcription factors, introducing an additional level of control by small lipophilic compounds. These observations support the proposal that the sequential expression of DHR3 and αFTZ-F1 at multiple stages of development can specify successive biological programs that promote appropriate progression through the life cycle. By combining insect endocrinology with the predictive power of genetics, the 20E-triggered transcriptional cascades in Drosophila provide an ideal context to define how a repeated systemic signal can be refined into precise stage-specific temporal responses during development (Ruaud, 2010).

DHR3 is required for VNC condensation, a terminal step in embryonic nervous system morphogenesis that is dependent on nervous system activity, glial cell function and apoptosis. In addition, previous studies have identified roles for DHR3 in PNS development. Interestingly, these functions, which are specific for DHR3 and are not shared with its direct target, αFTZ-F1, parallel the role of the mammalian DHR3 homolog RORα in brain development. RORα was initially identified as the gene associated with the spontaneous staggerer mutation in mice, which display ataxia associated with cerebellum developmental defects and degeneration. The cerebellum in staggerer mutants is dramatically smaller than in controls, containing fewer of the two major cell types: granule cells and Purkinje cells. Further investigation showed that this phenotype arises primarily from reduced expression in Purkinje cells of Sonic hedgehog (Shh), a mitogenic signal for granule cells. These data support the hypothesis that there is an evolutionarily conserved role for the ROR/DHR3 family of nuclear receptors in nervous system development and suggest that further functional studies of DHR3 may provide new insights into its ancestral functions in this pathway (Ruaud, 2010).

Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila

The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. This study analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. Expression patterns of two EcR isoforms, EcR-A and EcR-B1, were examined in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, EcR function were genetically analyzed in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. betaFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression (Hara, 2013).

This study analyzed the requirement of ecdysone in the optic lobe cell death. The role of ecdysone in cell death during metamorphosis has been examined in the salivary gland, larval midgut, and two types of neurons in the VNC, vCrz neurons and RP2 neurons. In the salivary gland, ecdysone triggers cell death in vitro, and the cell death required some components of ecdysone cascade, BR-C, E93, E74 and βFTZ-F1. In the midgut, cell death was induced by injection of ecdysone, and required BR-C and E93. For the cell death in vCrz neurons and RP2 neurons, ecdysone requirement was shown using EcR mutants. Among these tissues, a requirement for EcR isoforms was addressed only in the vCrz neurons and RP2 neurons. In the vCrz neurons, the cell death occurred in EcR-A or EcR-B1 mutants, but not in EcR-B1 and EcR-B2 mutant, indicating that EcR-B2 is required for this cell death. In RP2 neurons, cell death did not require EcR-A, but EcR-B1, EcR-B2 or both. Here, it was shown that the optic lobe cell death included ecdysone-independent and dependent tissues. The ecdysone-dependent cell death required EcR-B1 (Hara, 2013).

The number of dying cells in the optic lobe of EcR-B1 mutant animals at 24 and 36 h APF was much smaller than that in wild-type animals, but the number in EcR-A mutant animals was not. This finding showed that cell death in the optic lobe at these stages required EcR-B1, but not EcR-A (Hara, 2013).

Dying cells were examined in the following structures; LAD, lamina anterior dying cells; LPD, lamina posterior dying cells; LUD, lamina underlying dying cells; MALD, medulla anterolateral dying cells; MAMD, medulla anteromedial dying cells; MCD, medulla cortex dying cells; MCLD, medulla cortex lateral dying cells; MCMD, medulla cortex medial dying cells; MLBD, medulla-lamina boundary dying cells; MPLD, medulla posterolateral dying cells; PMCD, posterior medulla cortex dying cells; PMD, posteromedial dying cells; T/C, dying cells in the T/C region; LopD, dying cells in the lobula plate cortex; MMC, abnormal dying cells in the medial side of the medulla cortex; MLopD, abnormal dying cells in the medial side of the lobula plate cortex. The dependence on EcR-B1 was common among all dying cells in all clusters, except the MCMD. At 24 h APF, dying cells in the LAD, LPD, LUD, MCLD and T/C region were absent in optic lobes of most EcR-B1 mutants. In these mutants at 36 h APF, dying cells in the LUD, MLBD, MPLD and the lobula plate cortex were not evident. Similarly, at 48 h APF, MPLDs were absent from the EcR-B1 mutants. These results indicated that cell death in most clusters required EcR-B1 at stages after 24 h APF. It could not be determined whether death of MCMD was dependent on EcR-B1 because it was not clear whether the abnormal dying cells included MCMD in the medial side of the medulla cortex in EcR-B1 mutants (Hara, 2013).

In EcR-B1 mutant, a significant number of dying cells was constantly observed after 24 h APF. However, this fact does not mean that the loss of EcR-B1 delayed the timing of cell death. From 24 to 72 h APF, dying cells were mostly located in the medial side of the medulla cortex in EcR-B1 mutants and they did not include those in the clusters which would have been normally observed from 24 to 48 h APF, i.e., the LAD, LPD, LUD, MCLD, MPLD, MLBD, and dying cells in the T/C region and the lobula plate cortex. This fact strongly suggests that the optic lobe cell death was not delayed but suppressed by EcR-B1 mutation (Hara, 2013).

In some EcR-B1 mutants, enormous dying cells were observed at 72 h APF at positions where cell death would have normally occurred: the LAD, MCLD, MPLD, MLBD and T/C region. This suggests that delayed cell death can be induced at normal position without EcR-B1 at 72 h APF in these samples. It is known that experimental suppression of cell death can lead a delayed cell death by another complementally cell death mechanism. Therefore, it is possible that a complementary mechanism was induced in these samples (Hara, 2013).

It has been shown that a cell death initiator caspase, Dronc, had a EcRE in its promoter and EcR-B1 could induce Dronc expression. Indeed, the optic lobe cell death was suppressed in Dronc mutant in a preliminary experiment. Altogether, it is most likely that EcR-B1 directly controls cell death and consistently induces the death at right time in the optic lobe (Hara, 2013).

In this study, requirement of EcR-B2 function was not suggested. When the functions of all EcR isoforms were inhibited after puparium formation, the number of dying cells was larger than that in EcR-B1 mutant at 24 h APF (638.6 versus 363.8). If EcR-B2 was required for the optic lobe cell death, the number would have been less than that in the mutant. However, a function of EcR-B2 in the ecdysone-dependent cell death can still not be entirely excluded since there was a possibility that RNAi was insufficient to entirely inhibit EcR function in this experimental condition (Hara, 2013).

The number of dying cells in the optic lobes of EcR-A and EcR-B1 mutants was the same as that in wild-type animals at 0 h APF. When distribution of dying cells was examined, all clusters that were present in wild-type animals (specifically the LAD, MALD, MCD, MAMD, PMCD and dying cells in the T/C region) were also observed in the mutants. These results strongly indicated the cell death at 0 h APF was independent of both EcR-A and EcR-B1. EcR-B2 was also not required for the cell death because concurrent knockdown of all EcR isoforms via expression of hs-EcRi-11 resulted in no reduction in the number of dying cells at 0 h APF (Hara, 2013).

The above argument is relevant only for the zygotic not with maternal EcR. The contribution of maternal EcR should be tested. The result of the heat shock-inducible EcR RNAi denied the contribution of maternal EcR mRNA of all EcR isoforms. As with maternal proteins, there was no detectable EcR-B1 in any cluster or region at 0 h APF. In contrast, EcR-A was weakly expressed in all cluster regions, and these is no information about EcR-B2. Therefore, possible roles of maternal EcR-A and EcR-B2 protein cannot be excluded. However, there are no published reports of a requirement for maternal EcR proteins during metamorphosis. Taken together, these findings indicated that cell death in the optic lobe at 0 h APF is independent of any EcR. Furthermore, it seems likely this cell death is also independent of ecdysone because the number of dying cells gradually increased from 0 to 6 h APF rather than decreased, but the ecdysone titer rapidly drops and is very low during this period (Hara, 2013).

The period around 12 h APF may be a transient period when the ecdysone-dependence of cell death changes. In many EcR-B1 mutant optic lobes, the number of dying cells was the same as that in the wild type. In contrast, the number was reduced in a few mutant optic lobes and dying cells were absent in many of the clusters. These findings indicated that most of the cell deaths in many optic lobes was independent of EcR-B1, but some had become EcR-B1-dependent (Hara, 2013).

There is no previous report on ecdysone-independent cell death during metamorphosis. The ecdysone-independent cell death was limited to the early phase of metamorphosis in the optic lobe. However, this timing does not necessarily indicate that all cell death is independent of ecdysone because the larval midgut and vCrz neurons die ecdysone-dependently during this period. Therefore, ecdysone independence is a unique feature of the cell death that occurs in the optic lobe. There has been no report that the cell death that occurs during embryogenesis and larval development depends on ecdysone. Hence, it is proposed that the same mechanisms that mediate cell death during embryogenesis or larval development work for cell death during the early phase of optic lobe development (Hara, 2013).

Based on findings from many previous studies, every cell death that occurred during metamorphosis was part of the degeneration of a larval tissue and was dependent on ecdysone. These finding are understandable because ecdysone orchestrates the entire developmental process of metamorphosis. However, cell death within the optic lobe was independent of ecdysone during an early phase and then this cell death became ecdysone dependent later. This unique feature of the optic lobe cell death may be due to the fact that cell death in the optic lobe takes place during metamorphosis and is simultaneously involved in the organogenesis. So two cell death mechanisms, i.e., an organogenesis-accompanied (ecdysone-independent) mechanism and a metamorphosis-accompanied (ecdysone-dependent) mechanism may have evolved to cooperate during the optic lobe development (Hara, 2013).

The expression pattern of EcR-A and EcR-B1 was examined in this study. Expression of each isoform altered during development in isoform specific manner. However, there was no direct relationship between EcR-B1 expression and the emergence of the cell death. At 0 h APF, when cells die independent of ecdysone, EcR-B1 was not expressed in any region with clusters of dying cells. In contrast, EcR-B1 was expressed in all regions with clusters of dying cells at 12 h APF, although cell death, at this stage, was, for the most part, ecdysone independent in all clusters, except PMD. Thus, there was a temporal gap between EcR-B1 expression and ecdysone-dependent cell death. This indicates that the expression of EcR-B1 was not a direct cause that shifted cell death from an ecdysone-independent to an ecdysone-dependent one. EcR-B1 expression would be one of the requisites to make cells competent to undergo ecdysone-dependent cell death at a later time point and another mechanism following EcR-B1 expression would be required for the shift (Hara, 2013).

Although cell death in the optic lobe after 24 h APF required EcR-B1, the level of EcR-B1 expression varied among cluster regions during this period. For example, at 24 h APF, EcR-B1 was expressed weakly in the anterior region of the lamina cortex where LAD was located. On the lateral side of the medulla cortex where MCLD were present, EcR-B1 was expressed moderately. EcR-B1 was strongly expressed in the T/C region where many dying cells were present. The expression levels also varied among cluster regions at 36 and 48 h APF. All these findings indicate that the death decision, even for the ecdysone-dependent cell death, was not simply related to high EcR-B1 levels. This decision would be made within specific context of each cluster (Hara, 2013).

EcR-B1 expression was correlated with glial ensheathment in the lamina cortex, medulla cortex and T/C region. In these regions, newly-born neurons derived from the OOA or IOA compose pre-ensheathed domains. As development proceeds, they become to be surrounded by glial membrane and compose ensheathed domains of mature neurons. In particular, the ensheathed domain in lamina cortex corresponds to a region with columnar structures. In the lamina cortex, EcR-B1 was weakly expressed in the pre-ensheathed domain, while strongly expressed in the ensheathed domain. In the medulla cortex and T/C region, it was not expressed in the pre-ensheathed domains, but expressed in the ensheathed domains. These facts suggest a possibility that the glial ensheathment promotes or initiates EcR-B1 expression in the process of neuronal differentiation in these regions. This possibility is supported by the fact that EcR-B1 expression became stronger after the ensheathment as development proceeded (Hara, 2013).

With regard to cell death, the LAD, MCLD and MALD were always located near the border of the pre-ensheathed and ensheathed domains. Therefore, cell death may be linked to the entry of glial membrane in these clusters. Since this positional relationship was observed from 0 to 24 h APF, the glial ensheathement and ecdysone signaling via EcR-B1 may cooperate to induce cell death in the clusters after 12 h APF, when cell death become dependent on EcR-B1 (Hara, 2013).

Secretory competence in a gateway endocrine cell conferred by the nuclear receptor βFTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a 'gateway cell', whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline (Cho, 2014).

Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy

This study defines TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (Ras^V12) and loss of the tumor suppressor Scribble (scrib¹). Malignant transformation of the ras^V12scrib¹ tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to ras^V12scrib¹ tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in ras^V12scrib¹ tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like ras^V12scrib¹ tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While ras^V12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, this study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes. (Kulshammer, 2015).

Genome-wide transcriptome profiling in the Drosophila epithelial tumor model has generated a comprehensive view of gene expression changes induced by defined oncogenic lesions that cause tumors of an increasing degree of malignancy. These data allowed discovery of how a network of collaborating transcription factors confers malignancy to Ras^V12scrib¹ tumors (Kulshammer, 2015).

This study revealed that the response of transformed Ras^V12scrib¹ epithelial cells is more complex in comparison to those with activated Ras^V12 alone with respect to both the scope and the magnitude of expression of deregulated genes. Aberrant expression of more than half of the genes in Ras^V12scrib¹ tumors requires JNK activity, highlighting the significance of JNK signaling in malignancy. Importantly, the tumor-associated, JNK-dependent transcripts cluster with biological functions and processes that tightly match the phenotypes of previously described tumor stages. Furthermore, the Ras^V12scrib¹ transcriptome showed significant overlap (27% upregulated and 15% downregulated genes) with microarray data derived from mosaic EAD in which tumors were induced by overexpressing the BTB-zinc finger TF Abrupt (Ab) in scrib¹ mutant clones as well as with a transcriptome of scrib¹ mutant wing discs. It is proposed that 429 misregulated transcripts (e.g. cher, dilp8, ets21c, ftz-f1, mmp1, upd), shared among all the three data sets irrespective of epithelial type (EAD versus wing disc) or cooperating lesion (Ras^V12 or Ab), represent a 'polarity response transcriptional signature' that characterizes the response of epithelia to tumorigenic polarity loss. Genome-wide profiling and comparative transcriptome analyses thus provide a foundation to identify novel candidates that drive and/or contribute to tumor development and malignancy while unraveling their connection to loss of polarity and JNK signaling (Kulshammer, 2015).

In agreement with a notion of combinatorial control of gene expression by an interplay among multiple TFs, this study identified overrepresentation of cis-acting DNA elements for STAT, GATA, bHLH, ETS, BTB, bZIP factors and NRs in genes deregulated in Ras^V12scrib¹ mosaic EAD, implying that transcriptome anomalies result from a cross-talk among TFs of different families. Many of the aberrantly expressed genes contained binding motifs for AP-1, Ets21c and Ftz-F1, indicating that these three TFs may regulate a common set of targets and thus cooperatively promote tumorigenesis. This is consistent with the occurrence of composite AP-1-NRRE (nuclear receptor response elements), ETS-NRRE and ETS-AP-1 DNA elements in the regulatory regions of numerous human cancer-related genes, such as genes for cytokines, MMPs (e.g., stromelysin, collagenase) and MMP inhibitors (e.g., TIMP) (Kulshammer, 2015).

Interestingly, Drosophila ets21c and ftz-f1 gene loci themselves contain AP-1 motifs and qualify as polarity response transcriptional signature transcripts. Indeed, this study has detected JNK- and Fos-dependent upregulation of ets21c and ftz-f1 mRNAs in Ras^V12scrib¹ tumors. While JNK-mediated control of ftz-f1 transcription has not been reported previously, upregulation of ets21c in the current tumor model is consistent with JNK requirement for infection-induced expression of ets21c mRNA in Drosophila S2 cells and in vivo. Based on these data, it is proposed that Ftz-F1 and Ets21c are JNK-Fos-inducible TFs that together with AP-1 underlie combinatorial transcriptional regulation and orchestrate responses to cooperating oncogenes. Such an interplay between AP-1 and Ets21c is further supported by a recent discovery of physical interactions between Drosophila Ets21c and the AP-1 components Jun and Fos (Rhee, 2014). Whether regulatory interactions among AP-1, Ets21c and Ftz-F1 require their direct physical contact and/or the presence of composite DNA binding motifs of a particular arrangement to control the tumor-specific transcriptional program remains to be determined (Kulshammer, 2015).

Importantly, some of the corresponding DNA elements, namely AP-1 and STAT binding sites, have recently been found to be enriched in regions of chromatin that become increasingly accessible in Ras^V12scrib¹ mosaic EAD relative to control. This demonstrates that comparative transcriptomics and open chromatin profiling using ATAC-seq and FAIRE-seq are suitable complementary approaches for mining the key regulatory TFs responsible for controlling complex in vivo processes, such as tumorigenesis (Kulshammer, 2015).

The prototypical form of AP-1 is a dimer comprising Jun and Fos proteins. In mammals, the Jun proteins occur as homo- or heterodimers, whereas the Fos proteins must interact with Jun in order to bind the AP-1 sites. In contrast to its mammalian orthologs, the Drosophila Fos protein has been shown to form a homodimer capable of binding to and activating transcription from an AP-1 element, at least in vitro (Kulshammer, 2015).

The role of individual AP-1 proteins in neoplastic transformation and their involvement in pathogenesis of human tumors remain somewhat elusive. While c-Jun, c-Fos and FosB efficiently transform mammalian cells in vitro, only c-Fos overexpression causes osteosarcoma formation, whereas c-Jun is required for development of chemically induced skin and liver tumors in mice. In contrast, JunB acts as a context-dependent tumor suppressor. Thus, cellular and genetic context as well as AP-1 dimer composition play essential roles in dictating the final outcome of AP-1 activity in tumors (Kulshammer, 2015).

This study shows that, similar to blocking JNK with its dominant-negative form, Bsk, removal of Fos inhibits ets21c and ftz-f1 upregulation, suppresses invasiveness, improves epithelial organization and differentiation within Ras^V12scrib¹ tumors and allows larvae to pupate. Strikingly, depletion of Jun had no such tumor-suppressing effects. It is therefore concluded that in the malignant Ras^V12scrib¹ tumors, Fos acts independently of Jun, either as a homodimer or in complex with another, yet unknown partner. A Jun-independent role for Fos is further supported by additional genetic evidence. Fos, but not Jun, is involved in patterning of the Drosophila endoderm Ÿand is required for expression of specific targets, e.g., misshapen (msn) and dopa decarboxylase (ddc), during wound healing. Future studies should establish whether the JNK-responsive genes containing AP-1 motifs, identified in this study, are indeed regulated by Fos without its 'canonical' partner (Kulshammer, 2015).

The current data identify Fos as a key mediator of JNK-induced MMP1 expression and differentiation defects in Ras^V12scrib¹ tumors. Only Fos inhibition caused clear suppression of MMP1 levels and restoration of neurogenesis within clonal EAD tissue, thus mimicking effects of JNK inhibition. Improved differentiation and reduced invasiveness are, however, not sufficient for survival of animals to adulthood, because interfering with Fos function in Ras^V12scrib¹ clones always resulted in pupal lethality (Kulshammer, 2015).

The systems approach of this paper, followed by genetic experiments, identified Ets21c and Ftz-F1 as being essential for Ras^V12scrib¹-driven tumorigenesis. It was further shown that mutual cooperation of both of these TFs with Fos is required to unleash the full malignancy of Ras^V12scrib¹ tumors (Kulshammer, 2015).

TFs of the ETS-domain family are key regulators of development and homeostasis in all metazoans, whereas their aberrant activity has been linked with cancer. ets21c encodes the single ortholog of human Friend leukemia insertion1 (FLI1) and ETS-related gene (ERG) that are commonly overexpressed or translocated in various tumor types. While FLI1 is considered pivotal to development of Ewing's sarcoma, ERG has been linked to leukemia and prostate cancer. As for Ftz-F1 orthologs, the human liver receptor homolog-1 (LRH-1) has been associated with colonic, gastric, breast and pancreatic cancer, whereas steroidogenic factor 1 (SF-1) has been implicated in prostate and testicular cancers and in adrenocortical carcinoma. However, the molecular mechanisms underlying oncogenic activities of either the ERG/FLI1 or the SF-1/LRH-1 proteins are not well understood (Kulshammer, 2015).

This study shows that removal of Ftz-F1 markedly suppressed invasiveness of Ras^V12scrib¹ tumors, restoring the ability of tumor-bearing larvae to pupate. Additionally, and in contrast to Fos, Ftz-F1 inhibition also partly reduced tumor growth in the third-instar EAD and allowed emergence of adults with enlarged, rough eyes composed predominantly of non-clonal tissue. The reduced clonal growth coincided with downregulation of the well-established Yki target, expanded, implicating Ftz-F1 as a potential novel growth regulator acting on the Hpo/Yki pathway. It is further speculated that reduced viability of Ras^V12scrib¹ftz-f1RNAi clones and induction of non-autonomous compensatory proliferation by apoptotic cells during the pupal stage could explain the enlargement of the adult eyes. The precise mechanism underlying compromised growth and invasiveness of Ras^V12scrib¹ftz-f1RNAi tumors and improved survival of the host remains to be identified (Kulshammer, 2015).

In contrast, effects of Ets21cLONG knockdown in Ras^V12scrib¹ tumors appeared moderate relative to the clear improvement conferred by either Fos or Ftz-F1 elimination. ets21c^LONG RNAi neither reduced tumor mass nor suppressed invasiveness, and pupation was rescued only partly. However, unlike ftz-f1RNAi, ets21c^LONG RNAi significantly reduced expression of dilp8 mRNA. Based on abundance of Ets21c binding motifs in the regulatory regions of tumor-associated genes and the normalized expression of >20% of those genes upon removal of Ets21c, it is further suggested that Ets21c acts in Ras^V12scrib¹ tumors to fine-tune the tumor gene-expression signature (Kulshammer, 2015).

Dilp8 is known to be secreted by damaged, wounded or tumor-like tissues to delay the larval-to-pupal transition. This study has corroborated the role of JNK in stimulating dilp8 expression in Ras^V12scrib¹ tumor tissue, and has further implicated Ets21c and Fos as novel regulators of dilp8 downstream of JNK. However, the data also show that elevated dilp8 transcription per se is not sufficient to delay metamorphosis. Unlike the permanent larvae bearing Ras^V12scrib¹ tumors, those with Ras^V12scrib¹ftz-f1RNAi tumors pupated despite the excessive dilp8 mRNA. Likewise, pupation was not blocked by high dilp8 levels in larvae bearing EAD clones overexpressing Abrupt. As Dilp8 secretion appears critical for its function, it is proposed that loss of Ftz-F1 might interfere with Dilp8 translation, post-translational processing or secretion (Kulshammer, 2015).

Consistent with the individual TFs having unique as well as overlapping functions in specifying properties of Ras^V12scrib¹ tumors, knocking down pairwise combinations of the TFs had synergistic effects on tumor suppression compared with removal of single TF. This evidence supports the view that malignancy is driven by a network of cooperating TFs, and elimination of several tumor hallmarks dictated by this network is key to animal survival. An interplay between AP-1, ETS-domain TFs and NRs is vital for development. For example, the ETS-factor Pointed has been shown to cooperate with Jun to promote R7 photoreceptor formation in the Drosophila adult eye. In mosquitoes, synergistic activity of another ETS-factor, E74B, with the ecdysone receptor (EcR/USP) promotes vitellogenesis. It is thus proposed that tumors become malignant by hijacking the developmental mechanism of combinatorial control of gene activity by distinct TFs (Kulshammer, 2015).

Despite the minor impact of ets21c^LONG knockdown on suppressing Ras^V12scrib¹ tumors, Ets21c^LONG is the only one of the tested TFs that was capable of substituting for loss of scrib in inducing malignant clonal overgrowth when overexpressed with oncogenic Ras^V12 in EAD. While invasiveness of such Ras^V12ets21c^LONG tumors required JNK activity, JNK signaling appeared dispensable for tumor growth. Importantly, the overgrowth of Ras^V12ets21c^LONG tumors was primarily independent of a prolonged larval stage, because dramatic tumor mass expansion was detected already on day 6 AEL. How cooperativity between Ets21c^LONG and Ras^V12 ensures sufficient JNK activity and the nature of the downstream effectors driving tumor overgrowth remain to be determined. In contrast, co-expression of either Ftz-F1 or Fos with Ras^V12 resulted in a non-invasive, Ras^V12-like hyperplastic phenotype (Kulshammer, 2015).

Why does Ets21c^LONG exert its oncogenic potential while Fos and Ftz-F1 do not? Simple overexpression of a TF may not be sufficient, because many TFs require activation by a post-translational modification (e.g., phosphorylation), interaction with a partner protein and/or binding of a specific ligand. Full activation of Fos in response to a range of stimuli is achieved through hyperphosphorylation by mitogen-activated protein kinases (MAPKs), including ERK and JNK. Indeed, overexpression of a FosN-Ala mutated form that cannot be phosphorylated by JNK was sufficient to phenocopy fos deficiency, indicating that Fos must be phosphorylated by JNK in order to exert its oncogenic function. Consistent with the current data, overexpression of FosN-Ala partly restored polarity of lgl mutant EAD cells. It is therefore conclude that the tumorigenic effect of Fos requires a certain level of JNK activation, which is lacking in EAD co-expressing Fos with Ras^V12. Nevertheless, the absence of an unknown Fos-interacting partner cannot be excluded (Kulshammer, 2015).

Interestingly, MAPK-mediated phosphorylation also greatly enhances the ability of SF-1 and ETS proteins to activate transcription. Two potential MAPK sites can be identified in the hinge region of Ftz-F1, although their functional significance is unknown. Whether Ets21c or Ftz-F1 requires phosphorylation and how this would impact their activity in the tumor context remains to be determined. Genetic experiments demonstrate that at least the overgrowth of Ras^V12ets21c^LONG tumors does not require Ets21c phosphorylation by JNK (Kulshammer, 2015).

In addition, previous crystallography studies revealed the presence of phosphoinositides in the ligand binding pocket of LHR-1 and SF-1 and showed their requirement for the NR transcriptional activity. Although developmental functions of Drosophila Ftz-F1 seem to be ligand independent, it is still possible that Ftz-F1 activity in the tumor context is regulated by a specific ligand. An effect of Ftz-F1 SUMOylation cannot be ruled out (Kulshammer, 2015).

In summary, this work demonstrates that malignant transformation mediated by Ras^V12 and scrib loss depends on MAPK signaling and at least three TFs of different families, Fos, Ftz-F1 and Ets21c. While their coordinated action ensures precise transcriptional control during development, their aberrant transcriptional (Ets21c, Ftz-F1) and/or post-translational (Fos, Ftz-F1, Ets21c) regulation downstream of the cooperating oncogenes contributes to a full transformation state. The data implicate Fos as a primary nuclear effector of ectopic JNK activity downstream of disturbed polarity that controls ets21c and ftz-f1 expression. Through combinatorial interactions on overlapping sets of target genes and acting on unique promoters, Fos, Ftz-F1 and Ets21c dictate aberrant behavior of Ras^V12scrib¹ tumors. Although originally described in Drosophila, detrimental effects of cooperation between loss of Scrib and oncogenic Ras has recently been demonstrated in mammalian tumor models of prostate and lung cancer. This study and further functional characterization of complex TF interactions in the accessible Drosophila model are therefore apt to provide important insight into processes that govern cancer development and progression in mammals (Kulshammer, 2015).

EVOLUTIONARY HOMOLOGS

Evolution of the hormone receptor superfamily

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3" and human RORalpha. Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

From a database containing sequences of published nuclear hormone receptors (NRs), an alignment of the C, D and E domains of NR transcription factors was constructed. Using this alignment, tree reconstruction was performed using both distance matrix and parsimony analysis. The robustness of each branch was estimated using bootstrap resampling methods. The trees constructed by these two methods gave congruent topologies. From these analyses six NR subfamilies were derived: (I) a large clustering of thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptors (VDRs) and ecdysone receptors (EcRs) as well as numerous orphan receptors such as RORs or Rev-erbs; (II) retinoid X receptors (RXRs) together with COUP, HNF4, tailless, TR2 and TR4 orphan receptors; (III) steroid receptors; (IV) NGFIB orphan receptors; (V) FTZ-F1 orphan receptors; and finally (vi) only one gene (to date), the GCNF1 orphan receptor. The relationships between the six subfamilies are not known except for subfamilies I and IV, which appear to be related. Interestingly, most of the liganded receptors appear to be derived when compared with orphan receptors. This suggests that the ligand-binding ability of NRs has been gained by orphan receptors during the course of evolution to give rise to the presently known receptors. The distribution into six subfamilies correlates with the known abilities of the various NRs to bind to DNA as homo- or hetero-dimers. For example, receptors heterodimerizing efficiently with RXR belong to the first or the fourth subfamilies. It is suggested that the ability to heterodimerize evolved once, just before the separation of subfamilies I and IV and that the first NR was able to bind to DNA as a homodimer. From the study of NR sequences existing in vertebrates, arthropods and nematodes, two major steps of NR diversification have been defined: one that took place very early, probably during the multicellularization event leading to all the metazoan phyla, and a second occurring later on, corresponding to the advent of vertebrates. In vertebrate species, the various groups of NRs accumulated mutations at very different rates (Laudet, 1997).

An Ftz-f1 homolog in C. elegans

Nuclear receptors are essential players in the development of all metazoans. The nematode C. elegans possesses more than 200 putative nuclear receptor genes, several times more than the number known in any other organism. Very few of these transcription factors are conserved with components of the steroid response pathways in vertebrates and arthropods. Ftz-F1, one of the evolutionarily oldest nuclear receptor types, is required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila. Two complementary approaches, direct mutagenesis and RNA interference, were used to explore the developmental role of nhr-25, a C. elegans ortholog of Ftz-F1. Deletion mutants show that nhr-25 is essential for embryogenesis. RNA interference reveals additional requirements throughout the postembryonic life, namely in molting and differentiation of the gonad and vulva. All these defects are consistent with the nhr-25 expression pattern, determined by in situ hybridization and GFP reporter activity. These data link the C. elegans Ftz-F1 ortholog with a number of developmental processes. Significantly, its role in the periodical replacement of cuticle (molting) appears to be evolutionarily shared with insects and thus supports the monophyletic origin of molting (Asahina, 2000).

Inactivation of nhr-25 function, either by a mutation deleting the essential regions of nhr-25 or by RNAi, causes embryonic lethality. The introduction of an nhr-25 plasmid into the (delta2389) mutants partially restores hatching, indicating that the embryonic lethal phenotype is due to a loss-of-function of nhr-25. Incomplete rescue may be due to the detrimental effects of excessive copies of a functional gene or to the mosaic partition of the extrachromosomal array during development. nhr-25 is expressed in embryonic epithelial cells. Although early descendants of the E blastomere are the first cells to express nhr-25, this expression seems to be transient and is soon followed by lasting expression, mainly in the progeny of ABp and C cells, forming the hyp7 syncytium, seam and other hypodermal cells. Consistent with the nhr-25 expression in hypodermal cells, both the nhr-25 mutant and RNAi affected embryos fail to elongate, arresting at the 1.5-fold stage. These embryos are not properly enclosed, showing extruding tissues, particularly at their posterior. This suggests that nhr-25 is required for the secretion of cuticle by the hypodermis (Asahina, 2000).

In situ hybridization shows strong expression in the germ-line and maternal deposition of the mRNA. Although dsRNA is known to interfere efficiently with maternal genes, defects earlier than those seen in nhr-25 mutant embryos were not seen using RNAi. It is assumed that either sufficient nhr-25 protein is also deposited into the eggs or that the maternal RNA is not essential (Asahina, 2000).

Post-embryonic expression of nhr-25 suggests that this gene plays a role in larvae and adults. RNAi causes lethality in L1-L2 but not in older larvae, corresponding with nhr-25 expression and its decline in the hypodermis past L2 stage. Defects in cuticle replacement (molting) and morphogenesis, consistent with the spatial expression, again point to an nhr-25 requirement in the hypodermis. Lesions and irregularities of the integument show that not only the shedding of the old cuticle, but also synthesis of a new one are compromised (Asahina, 2000).

Those larvae that reached L3-L4 instars usually formed sterile adults, invariably with tumorous gonads and often with a missing or abnormal vulva and deformed tail. Since genetic data are lacking with respect to the involvement of nhr-25 in pathways controlling gonadal and vulval differentiation, it is difficult at this point to discuss how nhr-25 participates in these events. The hyperplasia of the RNAi-affected gonad shows that excessive mitoses occur in the germ-line. Similar gonad appearance results from loss of cul-1, a cullin gene necessary for cell cycle exit in C. elegans. Unlike the case with cul-1, however, no extra neurons were found in RNAi treated worms carrying the mec-7::GFP marker, suggesting that nhr-25 is not a general regulator of the cell cycle. Similar defects of gonadal differentiation also result from mutations in the gon-1 metalloprotease gene, implicated in the shaping of extracellular matrix. Gonadal hyperplasia is also caused by a constitutive activation of the Notch relative GLP-1, required for signaling between the distal tip cell and the germ-line. It is speculated that nhr-25, which is expressed strongly in the germ-line, might be a downstream component of such a pathway (Asahina, 2000).

Contrasting with nhr-25 is Drosophila Ftz-F1, expressed in perhaps all organs except for the gonads. Consistently, ftz-f1 mutant phenotypes do not suggest defects in reproduction. In the case of vulval development, nhr-25 expression in hypodermal vulva precursor cells may be required for their competence to form vulva. In this sense, ßFtz-F1 has been shown to render Drosophila tissues competent to undergo changes in response to the ecdysteroid signal. The posterior end defects possibly result from faulty expression of cuticle constituents, such as the sqt-1 and rol-6 collagen genes (Asahina, 2000).

Both arthropods and nematodes need steroids for molting. In insects, precursors such as cholesterol are converted into ecdysteroids and used as extracellular signals to synchronize the molting of distant body parts. In nematodes, the absence of dietary sterols compromises molting. More directly, both sterol starvation and mutations in a megalin-related protein LRP-1, which presumably mediates sterol endocytosis by hyp7 hypodermis, have been shown to prevent the shedding and degradation of the old cuticle at all C. elegans molts. Whether ecdysteroids are required for nematode molting is not clear, since their synthesis from cholesterol has not been demonstrated (Asahina, 2000).

In the epidermis of insects, molting is regulated by ecdysteroids acting through an ecdysone receptor complex EcR/USP and a cascade of transcription factors, including ßFtz-F1, on the expression of stage-specific genes. ßFtz-F1 is induced by an increase and a consequent decline of ecdysteroid titer and is likely to control genes acting in cuticle formation. In Drosophila, ßFtz-F1 is necessary for metamorphosis and for activation of at least one pupal cuticle gene (EDG84A). The importance of ßFtz-F1 for the larval molt is suggested by a rescue of ftz-f1 mutants with heat shock induction of ßFtz-F1 around the molting period. Expression of Drosophila ßFtz-F1 is activated by another nuclear receptor, DHR3 (Asahina, 2000 and references therein).

How can nhr-25 act in the molting of C. elegans? Since nhr-25 and NHR-23 are conserved with Ftz-F1 and DHR3, it is possible that they are part of a pathway analogous to that which exists in insects. Significantly, RNAi targeting of nhr-23 causes molting defects that were similar to those shown for nhr-25. An antibody is being prepared to test whether the genetic epistasis between nhr-23 and nhr-25 is the same as in Drosophila. However, the upstream part of this pathway is unclear, because C. elegans lacks orthologs of EcR and USP, which are thus far the only known receptors capable of conveying the ecdysteroid signal to gene expression. There is an alternative possibility: instead of being downstream of a steroid receptor, nhr-25 could mediate the conversion of dietary sterols to compounds active in molting. Encouraging this idea is the fact that Ftz-F1 type proteins direct steroid conversion in mammals (Asahina, 2000).

This analysis of nhr-25 function suggests that the roles of ftz-f1 orthologs in Drosophila and C. elegans molting are parallel and supports the monophyletic origin of molting. It is proposed that Ftz-F1/nhr-25 and DHR3/NHR-23 are the central, evolutionarily oldest regulators of genes executing cuticle replacement in all Ecdysozoa. While these orphan receptors are sufficient to control the molting program in small, simple forms, such as C. elegans, the synchronized molting of distant and diversified body parts in arthropods demands coordination by an extracellular signal (a molting hormone) and its receptor (Asahina, 2000).

The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25

The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. The paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family (Hayes, 2006).

The C. elegans genes nhr-23 and nhr-25 encode orphan nuclear hormone receptors orthologous, respectively, to DHR3 and ßFTZ-F1, which are related to mammalian ROR/RZR/RevErb and SF-1, respectively. Both receptors are essential for completion of the larval molts, suggesting that particular functions of nhr-23/DHR3 and nhr-25/ ßFTZ-F1 might be conserved and, further, that regulation by steroid hormones might be a common feature of molting in C. elegans and Drosophila. However, a steroid hormone regulating molting of C. elegans has not yet been identified and the genome lacks orthologs of ECR or USP (Hayes, 2006).

A genetic model is presented for the function of mir-84 and let-7 in epithelial differentiation, as related to the molting cycle. The let-7 miRNA targets lin-41 mRNA and also hbl-1 mRNA, in combination with paralogous miRNAs. During early larval development, LIN-41 and HBL-1 together repress production of the zinc-finger transcription factor LIN-29. Expression of let-7 and related miRNAs late in larval development represses lin-41 and hbl-1, thereby activating LIN-29. LIN-29 promotes expression of col-19 and possibly other collagen genes characteristic of an adult cuticle and also represses expression of col-17 and possibly other collagen genes characteristic of larval cuticle. LIN-29 is likely to regulate additional genes that control the molting cycle that have not yet been identified (Hayes, 2006).

Inactivation of either one of the nuclear hormone receptor genes nhr-23 or nhr-25 is sufficient to prevent the aberrant supernumerary molt caused by reduced levels of mir-84 and let-7. NHR-23 and NHR-25 thus serve as key downstream effectors of the miRNAs in regulation of the molting cycle. One model is that LIN-29, or a transcription factor regulated by LIN-29, represses nhr-23 and nhr-25 following the fourth molt. Accordingly, GFP expression from an nhr-23 reporter gene increases fourfold in the hypodermis of let-7 mir-84 adults. The relationship between nhr-23 and nhr-25 in C. elegans remains to be determined; however, DHR3 stimulates transcription of ßFTZ-F1 in flies (Hayes, 2006).

The identification of sites in the 3' UTR of nhr-25 that are complementary to let-7 family members and are also conserved in other nematodes suggests that the let-7 family targets the nhr-25 message to negatively regulate production of NHR-25 in adults. Consistent with this model, increasing the abundance of mir-84 partly suppresses the supernumerary molt caused by a probable null mutation in the lin-29 gene. Also, in preliminary experiments RNA species attributable to cleavage of the nhr-25 message upon binding of let-7-like miRNAs were detected in extracts from wildtype adults. Steroid hormones and co-factors probably also regulate activity of NHR-23 and NHR-25 during the life cycle (Hayes, 2006).

Regulation by miRNAs thus converges on transcription factors upstream in the genetic networks regulating molting. NHR-23 coordinates several aspects of larval molting by promoting expression of genes required for patterning the new cuticle and ecdysis, including, respectively, the collagen gene dpy-7 and the collagenase gene nas-37. Inactivation of either nhr-23 or nhr-25 abrogates the reiterated expression of gfp reporters for mlt-10 and nas-37 caused by mutation of let-7 and mir-84. NHR-25 might promote expression of the corresponding genes during larval development, even though RNAi of nhr-25 is not sufficient to abrogate expression of the gfp reporters in wild-type larvae. Interestingly, inactivation of nhr-23 or nhr-25 causes an earlier blockade in the molting program in let-7 mir-84 adults than in wild-type larvae, such that the mutant adults do not enter lethargus or attempt to ecdyse. Parallel pathways might drive early steps of molting during larval development (Hayes, 2006).

Intriguingly, adults with reduced levels of mir-84 and let-7 are unable to shed their cuticle to complete the supernumerary molt. One possibility is that particular genes required for ecdysis are not induced. Whereas the hypodermis and seam cells retain some larval character in let-7 mir-84 adults, other cells, perhaps particular neurons or specialized epithelia, might be fully differentiated and therefore unable to coordinate with the molting program. Consistent with this idea, let-7 mir-84 adults spend an atypically long time in lethargus, suggesting a failure to exit the behavioral program. Alternatively, particular structural features of the fifth cuticle might be physically incompatible with shedding the exoskeleton (Hayes, 2006).

Considering an aberrant ecdysis as the terminal phenotype of let-7 mir-84 mutants, it is intriguing to speculate that the let-7 family and possibly other miRNAs regulate aspects of the larval molting cycle. Indeed, increased expression of either mir-84 or let-7 causes some larvae to arrest development, trapped inside partly shed cuticle, indicating that levels of let-7-like miRNAs can impact molting of larvae (Hayes, 2006).

Mechanisms that set the pace of the molting cycle are not well understood, although physiologic cues such as nutritional status and environmental cues such as temperature impact the duration of larval stages. Interestingly, let-7 and let-7 mir-84 mutants initiate the supernumerary molt in synchrony, rather than in a stochastic fashion, relative to the time of hatching. Thus, a timing mechanism for molting persists in these particular miRNA mutants (Hayes, 2006).

The let-7 gene is perfectly conserved throughout bilaterian phylogeny, and vertebrate genomes specify many miRNAs homologous to let-7. Vertebrate let-7 and protein-coding genes orthologous to targets of let-7 identified in C. elegans play crucial roles in development. Moreover, reduced expression of human let-7 correlates with shortened survival in lung cancer patients, and let-7 might regulate the RAS oncogene. The possibility of functional conservation among homologs of let-7 in humans and worms intimates the importance of understanding how let-7 and its paralogs function in C. elegans. This work shows how analysis of double mutants can reveal how the many miRNAs that form paralogous families work together to regulate their targets (Hayes, 2006).

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, the apoptosis of the prothoracic gland was analyzed of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, prothoracic gland degeneration was shown to be effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species (Mané-Padrós, 2010).

In Drosophila, a similar ecdysteroid-dependent cascade directs the stage-specific destruction of the salivary glands. In this insect, however, the cascade converges in the induction of rpr and hid, and the consequent massive caspase activation and cell death. Interestingly, RPR and HID homologous proteins have not been reported in any insect outside Drosophila and closely related species, suggesting that although the role of 20E as inducer of cell death is conserved, the mechanisms by which the hormone controls such process would be different in hemimetabolous insects (Mané-Padrós, 2010).

This study has demonstrated that BgFTZ-F1 is a critical factor to specifically induce the degeneration of the prothoracic gland of B. germanica after the imaginal molt. Several observations support this: (1) the prothoracic gland of BgFTZ-F1 knockdown adults failed to degenerate; (2) ectopic expression of BgFTZ-F1 in mid-last nymphal instar, mediated by the reduction of BgE75, prematurely induced the degeneration of the prothoracic gland; (3) the prothoracic gland of last instar nymphs that had been simultaneously knockdown for BgE75 and BgFTZ-F1, thus preventing the ectopic expression of BgFTZ-F1 during mid-nymphal instar, also failed to degenerate; (4) the expression of BgFTZ-F1 in the prothoracic gland during the imaginal molt is significantly higher (12-fold) than during nymphal transitions, and its expression is maintained during the first days of the adult stage until the onset of prothoracic gland degeneration (Mané-Padrós, 2010).

The nuclear receptor FTZ-F1 has also been implicated in the apoptotic response in Drosophila. In this insect, βFTZ-F1 acts as a transcriptional competence factor that provides the stage-specificity for salivary gland degeneration at the onset of the pupal development. Moreover, its activity is required for the destruction of the cytoplasm, nuclear DNA fragmentation and controlling caspase levels in salivary gland cells (Mané-Padrós, 2010).

In addition, it was also demonstrated that the pro-apoptotic effect of BgFTZ-F1 is restricted to the last nymphal instar due to the anti-apoptotic effect exerted by JH before the last nymphal instar. Although JH regulates a number of developmental events in insects, its molecular mechanism of action still remains a matter of debate. The death inhibitor effect of JH has been also described in relation to the midgut remodelling that occurs during metamorphosis of the mosquito A. aegypti and the moth H. virescens. Recently, it has been also shown that the larval fat body of JH-deficient Drosophila undergoes premature degeneration and that this response can be prevented by the application of methoprene. Interestingly, however, the anti-apoptotic effect of methoprene in Drosophila was based on the inhibition of two bHLH-PAS transcription factors involved in JH action, Methoprene-tolerant and Germ-cell expressed, but was not related, in contrast to B. germanica, to the suppression of the ecdysteroid-dependent transcriptional cascade (Mané-Padrós, 2010).

In summary, these results led a model for developmentally regulated PCD in the hemimetabolous B. germanica. In this model, JH is responsible to hold back the degeneration of the prothoracic gland during nymphal-nymphal transitions. The low levels of BgFTZ-F1 observed at the end of the penultimate nymphal instar would be below a critical threshold necessary to overcome the BgIAP1-mediated inhibition of prothoracic gland degeneration. During the last nymphal instar, however, the specific and dramatic up-regulation of BgFTZ-F1 in the prothoracic gland at the end of the instar (12 fold higher than in the penultimate instar) would overcome, in the absence of JH, the inhibitory effect of BgIAP1 making the prothoracic gland competent to execute the cell death program (Mané-Padrós, 2010).

This study shows that prothoracic gland degeneration in the nymphal-adult transition of B. germanica show conserved but also divergent features with respect to the highly derived and thoroughly studied species Drosophila melanogaster. This underlines the importance of investigating basal, less modified species, in order to understand the evolutionary trends and mechanisms that led to the highly sophisticated holometabolan mode of metamorphosis (Mané-Padrós, 2010).

Ftz-f1 homologs in insects and other invertebrate species

Coactivators MBF1 (Drosophila homolog: see Multiprotein bridging factor 1) and MBF2 mediate Bombyx mori FTZ-F1-dependent transcriptional activation in vitro by interconnecting BmFTZ-F1, TATA binding protein TBP, and TFIIA. Temporal and spatial expression patterns of MBF2 have been examined during embryonic and larval development of the silkworm Bombyx mori. MBF2 is detected in unfertilized eggs and embryos until stage 26. In stage 22 embryos, MBF1, MBF2, and BmFTZ-F1 colocalize in neural cells. During the larval stage, MBF2 is not expressed in the fat body and trachea. In the silk gland, MBF2 mRNA is constitutively expressed, but MBF2 protein appears in the period between the second day and the molting D3 stage in both the third and the fourth instars and then disappears. MBF2 is also detected on the second and third days of the fifth instar. Immunostaining during the fourth molt shows that MBF1, MBF2, and BmFTZ-F1 localize in the nucleus only at the D3 stage, while the two cofactors are present in the cytoplasm at other stages. Immunoprecipitation experiments suggest that MBF1, MBF2, and BmFTZ-F1 form a complex at the D3 stage. Transient expression of these factors in Schneider cell line 2 reveals that MBF1 and MBF2 localize to the nucleus and enhance BmFTZ-F1-dependent transcription only when all three factors are present. These data illustrate the functional regulation of MBF1 and MBF2 at the step of nuclear transport and implicate MBF2 in tissue- and stage-specific transcription (Liu, 2000).

The most interesting finding in the present study is the dramatic change in the subcellular localization of MBF1 and MBF2 during development. The two cofactors are found in the nucleus only in the molting D3 stage and in the cytoplasm in other stages. Coimmunoprecipitation experiments suggest that MBF1 and MBF2 are present as a complex in the cytoplasm at the molting D1 stage and then enter the nucleus and form a ternary complex with BmFTZ-F1 at the molting stage D3. Transient expression of these factors in S2 cells shows that simultaneous expression of BmFTZ-F1 is essential for the nuclear localization of MBF1 and MBF2. It is possible that the ternary complex is formed in the cytoplasm and transported into the nucleus together with BmFTZ-F1. Alternatively, the MBF1-MBF2 complex may be constitutively excluded from the nucleus through a nuclear export system but it may stay in the nucleus once the ternary complex with BmFTZ-F1 is formed. MBF1 contains a nuclear export signal (NES)-like sequence in its C-terminal region, which is conserved among eukaryotes. For example, amino acid residues 119-130 of B. mori MBF1, LGKIERAIGIKL, and the corresponding region of human MBF1, LGIERAIGLKL, are similar to the leucine-rich NES in HIV Rev protein, LPPLERLTL, and protein kinase inhibitor, LALKLAGLDI. Whatever the mechanism may be, a regulation of the action of these coactivators has been revealed at the step of nuclear transport (Liu, 2000).

Tissue staining of embryos shows the colocalization of BmFTZ-F1, MBF1, and MBF2 in neural cells, suggesting that these transcription factors play a role in the embryonic neural cells. During larval development, BmFTZ-F1, MBF1, and MBF2 form a nuclear complex at the molting stage D3. Transient expression of a reporter gene in S2 cells demonstrated that MBF1 and MBF2 enhance BmFTZ-F1-dependent transcription. These observations support the model that MBF1 and MBF2 serve as coactivators that mediate BmFTZ-F1-dependent transcriptional activation. However, BmFTZ-F1 expression only begins from the D3 stage and culminates later at the E1 and E2 stages, when MBF2 has already disappeared and MBF1 resides in the cytoplasm. It is possible that MBF1 and MBF2 are required only to initiate expression of putative BmFTZ-F1 target genes in D3 stage. The small amount of BmFTZ-F1 in D3 stage may be enough to play the role because MBF1 can significantly stabilize BmFTZ-F1 binding to DNA. When present at high levels, BmFTZ-F1 no longer requires the aid of MBF1 and MBF2 to maintain the expression of its target gene(s) and these cofactors may be excluded from the system to turn off the expression rapidly upon decline in the level of BmFTZ-F1. This may allow immediate turn on and off of the BmFTZ-F1-dependent gene(s) (Liu, 2000).

Why is FTZ-F1 function under precise temporal control? FTZ-F1 appears to regulate its target genes, which should be expressed only within a limited time. For example, Drosophila betaFTZ-F1 governs stage-specific expression of the EDG84A gene, which encodes a cuticle protein. Manduca sexta FTZ-F1 is likely to regulate genes acting in cuticle formation, including the dopadecarboxylase gene, which is responsible for melanization of cuticle. Insect cuticle consists of layers of film. Different layers contain different kinds of proteins. These proteins are expressed in a stage-specific manner and are deposited systematically starting from epicuticle to endocuticle. Any disturbance in the order of protein deposition and melanization would be detrimental to the cuticle formation. Indeed, disruption of the cuticle structure takes place upon ectopic expression of FTZ-F1 when endogenous ßFTZ-F1 is absent. These observations support the notion that temporally restricted action of FTZ-F1 is critical for development) (Liu, 2000).

During the last larval molt in Manduca sexta, a number of transcription factors are sequentially expressed. MHR4 (Drosophila homolog: Hr4) is a transcription factor that belongs to the nuclear receptor superfamily and is a homolog of germ cell nuclear factor (GCNF)-related factors (GRFs) of Bombyx mori and Tenebrio molitor and is similar to a sequence found in the Drosophila genome. Unlike E75A and MHR3, whose mRNAs are induced when the ecdysteroid titer increases, the expression of MHR4 mRNA occurs transiently at the onset of the decline of ecdysteroid titer followed by ßFTZ-F1 mRNA expression when the ecdysteroid titer becomes low. When day 2 fourth epidermis is exposed to 20-hydroxyecdysone (20E) in vitro, MHR4 mRNA appears between 12 and 21 h, peaks at 24 h, and then declines. Using the protein synthesis inhibitors cycloheximide and anisomycin both in vivo and in vitro, it has been found that the MHR4 transcript is directly induced by 20E and requires the presence of 20E for its expression. The accumulation of MHR4 mRNA, however, does not occur until a 20E-induced inhibitory protein(s) disappears. This control of MHR4 expression is unique among the ecdysone-induced transcription factors. When the epidermis is cultured with 20E, ßFTZ-F1 mRNA is not induced until after the removal of 20E as previously found for Drosophila and the silkworm Bombyx mori. The presence of juvenile hormone had no effect on accumulation of either transcript (Hiruma, 2001).

In Drosophila, DHR3 activates ßFTZ-F1 mRNA expression and represses ecdysteroid-induced early gene expression such as that of E74A, E75A, and BRC. Yet in Manduca, relatively little MHR3 is present between 10 and 19 h after HCS when ßFTZ-F1 mRNA is increasing. Possibly MHR3 is initially activating the ßFTZ-F1 gene, but the presence of MHR4 suppresses this expression. Then when the 20E level declines below the threshold for MHR4 expression, ßFTZ-F1 mRNA can appear. This scenario would be similar to the complex control of MHR4 mRNA expression that has been shown in this study (Hiruma, 2001).

BmFTZ-F1 is a sequence-specific DNA-binding factor in the silkworm Bombyx mori sharing similar biochemical characteristics with Drosophila FTZ-F1. Amino acid sequences in the zinc finger DNA-binding region and the putative ligand-binding domain of BmFTZ-F1 showed strong similarity to not only FTZ-F1 but also its mammalian homologs, LRH-1, ELP, and Ad4BP, suggesting the importance of each region for the function of these proteins. Northern blot analyses of RNA isolated from the middle and posterior silk glands and fat bodies show the presence of a 6.1-kb BmFTZ-F1 mRNA. Expression of BmFTZ-F1 mRNA is intermittent, being high during larval molting and both the larval-pupal and the pupal-adult transformations. Injection of 20-hydroxyecdysone at the third day of the 5th instar larvae induces BmFTZ-F1 mRNA in the posterior silk gland after 24 hr. When 5th instar silk glands are cultured in vitro, BmFTZ-F1 mRNA is induced by a 6-hr exposure to 20-hydroxyecdysone followed by 6 hr in hormone-free medium. This suggests that BmFTZ-F1 is inducible by decline in the ecdysteroid titer and may play an important role in the development of the silkworm as a transcription factor (Sun, 1994).

Transcriptional activation by many eukaryotic sequence-specific regulators appears to be mediated through transcription factors that do not directly bind to DNA. MBF1 and MBF2 are two polypeptides that form a heterodimer and mediate activation of in vitro transcription from the fushi tarazu promoter by BmFTZ-F1. Neither MBF1, MBF2, nor a combination of them binds to DNA. MBF1 interacts with BmFTZ-F1 and stabilizes the BmFTZ-F1-DNA complex. MBF1 also makes direct contact with TATA-binding protein (TBP). Both MBF1 and MBF2 are necessary to form a complex between BmFTZ-F1 and TBP. A model has been proposed in which MBF1 and MBF2 form a bridge between BmFTZ-F1 and TBP and mediate transactivation by stabilizing the protein-DNA interactions (Li, 1994).

To study the role of ecdysone and the ecdysone inducible gene in the regulation of molting and development in crustaceans, a cDNA encoding an orphan nuclear receptor family member was cloned from the eyestalk of the shrimp Metapenaeus ensis. The size of the cDNA is 4.3 kb with the longest open reading frame encoding a protein of 545 amino acid residues. The deduced amino acid sequence of the shrimp cDNA consists of regions that are characteristic of those of the nuclear hormone receptors. It shows a high degree of amino acid sequence identity in the DNA binding domain, ligand binding domain and the FTZ box, as compared to those of other invertebrates and vertebrates. Unlike the insects Drosophila melanogaster and Bombyx mori, an AF2 transactivation domain is present in the shrimp FTZ-F1. Northern blot analysis using total RNA indicates that the FTZ-F1 mRNA can also be detected in the mature ovary. Northern blot analysis and RT-PCR analysis shows that the shrimp FTZ-F1 transcripts can be detected in the ovary, newly hatched nauplius, testis, eyestalk and epidermis of the adult shrimp. Although the cDNA clone was isolated from the eyestalk library, the shrimp FTZ-F1 appears to express most abundantly in the mature oocytes. The presence of abundant FTZ-F1 specific maternal message in the late vitellogenic ovary and early nauplius indicates that it may be important for the early embryonic and larval development of the shrimp. Interestingly, shrimp FTZ-F1 can also be found in testis of the male shrimp. The presence of FTZ-F1 in other tissues such as epidermis suggests that it may also be involved in other physiological processes such as molting (Chan, 1999).

The expression and function of the Caenorhabditis elegans gene nhr-25, a member of the widely conserved FTZ-F1 family of nuclear receptors, has been analyzed. The gene encodes two protein isoforms, only one of which has a DNA binding domain. nhr-25 is transcribed during embryonic and larval development. A nhr-25::GFP fusion gene is expressed in the epidermis, the developing somatic gonad, and a subset of other epithelial cells. RNA-mediated interference indicates a requirement for nhr-25 function during development: disruption of nhr-25 function leads to embryonic arrest due to failure of the epidermally mediated process of embryo elongation. Animals that survive to hatching arrest as misshapen larvae that occasionally exhibit defects in shedding molted cuticle. In addition, somatic gonad development is defective in these larvae. These results further establish the importance of FTZ-F1 nuclear receptors in molting and developmental control across evolutionarily distant phyla (Gissendanner, 2000).

The NHR-25 LBD has an apparent AF-2 core motif, a conserved sequence element that contributes to both ligand binding and NR interactions with coactivators. This motif is absent from the known insect FTZ-F1 family members, suggesting that the function of the NHR-25 LBD may be more similar to those of the vertebrate FTZ-F1 family members than to that of insect FTZ-F1. The cuticle shedding defect exhibited by some nhr-25(RNAi) larvae is similar to that resulting from disruption of nhr-23, another NR gene that also functions in hypodermal development. The requirement of both nhr-23 and nhr-25 for proper execution of the molt is particularly intriguing since the Drosophila orthologs of both genes (DHR3 and FTZ-F1, respectively) have been shown to function in the metamorphic response to the molting hormone 20-hydroxyecdysone. It is speculated that nhr-23 and nhr-25 likewise function in a regulatory cascade for direct regulation of nematode molting. Conservation of such a regulatory cascade would be consistent with the recent proposal of molting as a defining evolutionary trait for a phylogenetic grouping of nematodes, insects, and other molting animals. The molting defects observed in RNAi animals could result from a direct disruption of this proposed regulatory cascade or indirectly from defects in epidermal differentiation or function. Dissection of the exact roles of nhr-23 and nhr-25 in the regulation of nematode molting must await a more detailed genetic analysis. A full comparison of the functions of the C. elegans genes with those of their apparent Drosophila orthologs will also require a more comprehensive analysis of the early zygotic and larval phenotypes of the corresponding Drosophila mutants. Nevertheless, the results described here establish an essential function for nhr-25 in nematodes, adding another phylum to those in which members of the ancient FTZ-F1 NR family are known to perform key developmental roles (Gissendanner, 2000).

Ftz-f1 homologs in fish and frogs

Steroidogenic factor 1, a member of the Fushi tarazu factor 1 (FTZ-F1) subfamily of nuclear receptors, is a key regulator in mammalian reproduction. From an embryonic complementary DNA library, the zebrafish homolog of FTZ-F1 (zFF1A) and an alternatively spliced variant (zFF1B) were isolated. zFF1B represented a C-terminally truncated version of zFF1A. Both zFF1A and B transcripts are present in the developing pituitaries, adult fish brain, gonads, and liver, albeit zFF1B messenger RNA is absent in testis. Comparison of the primary sequences of zFF1 with those of other FTZ-F1 subfamily members shows a close structural relationship between the mouse liver receptor homolog, which activates the alpha1-fetoprotein gene in rodent liver. Similar to mouse steroidogenic factor 1, zFF1A regulates chinook salmon gonadotropin IIbeta subunit gene expression. zFF1B, which can bind a consensus gonadotrope-specific element with an affinity similar to that of zFF1A, lacks both the trans-activation function and synergistic interaction with the estrogen receptor. Furthermore, cotransfection studies in HeLa cells show that zFF1B is a strong competitor for the action of zFF1A on the chinook salmon gonadotropin IIbeta subunit gene promoter. This investigation suggests that (1) zFF1 represents an ancestor protein of the vertebrate FTZ-F1 homologs; (2) the antagonistic relationship between zFF1A and -B may dictate the expression of the FTZ-F1 target genes in a variety of tissues, including the pituitary, and (3) the naturally occurring zFF1B provides evidence that the C-terminal portion of zFF1A (80 amino acid residues) contains a major trans-activation function and a protein-protein interface (Liu, 1997).

SF-1/Ad4BP is a transcriptional factor that was originally found to be a mammalian homolog of Drosophila Ftz-F1. Ftz-F1 gene-deficient mice lack adrenal glands and gonads. Besides mammals, however, the SF-1/Ad4BP cDNA has only been isolated to date in fish and birds. To understand its role(s) for adrenal and gonadal development in vertebrates, cloning of this gene in animals other than mammals is required. Frog (Rana rugosa) SF-1/Ad4BP cDNA has been isolated from a testis lambdagt10 cDNA library. It encodes a protein of 468 amino acids, and its open reading frame shares 70% similarity with that of chicken OR2.1 (a SF-1/Ad4BP homolog) and 62% with bovine SF-1/Ad4BP. SF-1/Ad4BP mRNA is expressed in the testes, brains, adrenals/kidneys and spleens, but not ovaries, of adult frogs. In addition, the 5'-untranslated region (4.6kb) of the SF-1/Ad4BP gene was cloned with exons I and II. Genomic structure analysis has shown that frog SF-1/Ad4BP is also transcribed from the same gene as that of mammals. However, many Ftz-F1-related proteins have been reported so far. The Ftz-F1 gene does not encode all of those Ftz-F1-related proteins. Thus, the name of Ftz-F1 is not adequate for the gene coding SF-1/Ad4BP. The use of SF-1/Ad4BP instead of Ftz-F1 is proposed for the gene that encodes SF-1/Ad4BP in vertebrates (Kawano, 1998).

A homolog (rrFTZ-F1alpha) of the FTZ-F1 gene of Drosophila has been cloned from the frog Rana rugosa. The frog gene is expressed at high levels in the testis. The FTZ-F1alpha mRNA level in adult frogs does not change throughout the year, even during hibernation. However, when immunohistological studies using the anti-rrFTZ-F1alpha antibody were employed to examine which testicular cells expressed this gene, Sertoli cells were found to produce rrFTZ-F1alpha in two seasons: the breeding season (from March through May) and the pre-hibernating season (from October through November). Interstitial cells, however, expressed the gene only in the breeding season (from April through May). Taken together, the results suggest that the rrFTZ-F1alpha expression is regulated at the post-transcriptional step, and that the rrFTZ-F1alpha may play an important role(s) in the seasonal activities of Sertoli and interstitial cells in the frog testis (Takase, 2001).

Vertebrate Ftz-f1 homologs: Gene structure

The human homolog of the Drosophila melanogaster orphan nuclear receptor Fushi tarazu factor 1 (Ftz-F1), NR5A2 (hB1F), was initially identified as a regulatory factor that binds and activates enhancer II of hepatitis B virus. NR5A2 (hB1F) is expressed specifically in pancreas and liver, playing important roles in the regulation of several liver-specific genes. A detailed analysis on the genomic structure and promoter activity will greatly promote future studies on the function of the NR5A2 (hB1F) gene. In this report, a bacterial artificial chromosome clone and several phage clones covering the NR5A2 (hB1F) gene were isolated and the complete genomic sequence was obtained. Alignment of different cDNAs of the NR5A2 (hB1F) gene with the genomic sequence facilitated the delineation of its structural organization, which spans over 150 kb and consists of eight exons interrupted by seven introns. RT-PCR and 3'-RACE reveals that utilization of two polyadenylation signals results in 3.8 and 5.2 kb transcripts. The transcription start site of the NR5A2 (hB1F) gene maps downstream of a canonical TATA box. An upstream fragment containing binding sites for several liver-specific and ubiquitous transcription factors exhibits hepatocyte-specific promoter activity. Transient transfections indicated that hepatocyte nuclear factors HNF1 and HNF3beta can activate NR5A2 (hB1F) promoter (Zhang, 2001).

Vertebrate Ftz-f1 homologs: Domain structure and protein interactions

Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, its domain function was investigated by mutagenesis and functional analyses. The putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation (Li, 1999).

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), plays an important role in the development of the adrenal gland and in sexual differentiation. SF-1 regulates the transcription of variety of genes, including several steroidogenic enzymes, Mullerian inhibiting substance, and gonadotropin genes. Attempts have been made to identify domains in SF-1 that are required for transactivation and to determine whether SF-1 interacts with a subset of known coactivators. Natural variants of the FTZ-F1 locus include embryonal long terminal repeat-binding protein (ELP)-1, ELP-2, and SF-1, all of which share the DNA-binding domain. Analyses of the transcriptional activity of these variants reveal that the activity of ELP-2 and SF-1 is much greater than ELP-1, which contains a distinct carboxy terminus. Further studies were performed using GAL4-SF-1 fusion proteins that were constructed by replacement of the zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding domain of GAL4. Elimination of the putative AF-2 domain at the carboxy terminus of GAL4-SF-1 proteins results in a complete loss of transactivation. Several lines of evidence demonstrate that SF-1 interacts with steroid receptor coactivator-1 (SRC-1). Full-length SRC-1 enhances GAL4-SF-1-mediated transactivation, whereas a dominant negative form of SRC-1, consisting of its interaction domain alone, inhibits the activity of GAL4-SF-1. In mammalian two-hybrid assays, fusion of the VP16 activation domain to the interaction domain of SRC-1 confirms the interaction between SRC-1 and GAL4-SF-1 and demonstrates that the AF-2 domain is required for interaction with SRC-1. Furthermore, SRC-1, together with the cAMP responsive element binding protein (CBP) or a closely related factor, p300, synergistically enhance transcriptional activity of GAL4-SF-1. It is concluded that the carboxy-terminal AF-2 region of SF-1 functions as an activation domain and that SRC-1 and CBP/p300 are components of the coactivator complex with SF-1 (Ito, 1998)

Multiprotein bridging factor 1 (MBF1) is a coactivator that mediates transcriptional activation by interconnecting the general transcription factor TATA element-binding protein and gene-specific activators such as the Drosophila nuclear receptor FTZ-F1 or the yeast basic leucine zipper protein GCN4. The human homolog of MBF1 (hMBF1) has been identified but its function, especially in transcription, remains unclear. Reported here is the cDNA cloning and the functional analysis of hMBF1. Two isoforms, termed hMBF1alpha and hMBF1beta, have been identified. hMBF1alpha mRNA is detected in a number of tissues, whereas hMBF1beta exhibits tissue-specific expression. Both isoforms bind to TBP and Ad4BP/SF-1, a mammalian counterpart of FTZ-F1, and mediate Ad4BP/SF-1-dependent transcriptional activation. While hMBF1 is detected in the cytoplasm by immunostaining, coexpression of the nuclear protein Ad4BP/SF-1 with hMBF1 induces accumulation of hMBF1 in the nucleus, suggesting that hMBF1 is localized in the nucleus through its binding to Ad4BP/SF-1. hMBF1 also binds to ATF1, a member of the basic leucine zipper protein family, and mediates its activity as a transcriptional activator. These data establish that the coactivator MBF1 is functionally conserved in eukaryotes (Kabe, 1990).

PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization has been demonstrated. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested in a ligand-dependent manner, including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR). Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. A coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC functions as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. A 23-amino acid sequence in the carboxy-terminal region, amino acids 278-300, is critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. The two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins (Zhou, 2000).

PNRC2 (proline-rich nuclear receptor co-regulatory protein 2) was identified using mouse steroidogenic factor 1 (SF1) as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC2 is an unusual coactivator in that it is the smallest coactivator identified so far, with a molecular weight of 16 kDa, and interacts with nuclear receptors using a proline-rich sequence. In yeast two-hybrid assays PNRC2 interacted with orphan receptors SF1 and estrogen receptor-related receptor alpha1 in a ligand-independent manner. PNRC2 was also found to interact in a ligand-dependent manner with the ligand-binding domains of estrogen receptor, glucocorticoid receptor, progesterone receptor, thyroid receptor, retinoic acid receptor and retinoid X receptor. A functional activation function 2 domain is required for nuclear receptors to interact with PNRC2. Using the yeast two-hybrid assay, the region amino acids 85-139 were found to be responsible for the interaction with nuclear receptors. This region contains an SH3 domain-binding motif (SEPPSPS) and an NR box-like sequence (LKTLL). A mutagenesis study has shown that the SH3 domain-binding motif is important for PNRC2 to interact with all the nuclear receptors tested. These results reveal that PNRC2 has a structure and function similar to PNRC, a previously characterized coactivator. These two proteins represent a new type of nuclear receptor co-regulatory proteins (Zhou, 2001).

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is a critical developmental regulator in the urogenital ridge, because mice targeted for disruption of the SF-1 gene lack adrenal glands and gonads. SF-1 was recently shown to interact with DAX-1, another orphan receptor whose tissue distribution overlaps that of SF-1. Naturally occurring loss-of-function mutations of the DAX-1 gene cause the human disorder X-linked adrenal hypoplasia congenita (AHC), which resembles the phenotype of SF-1-deficient mice. Paradoxically, however, DAX-1 represses the transcriptional activity of SF-1, and AHC mutants of DAX-1 have lost repression function. To further investigate these findings, the interaction between SF-1 and DAX-1 was characterized and it was found that their interaction indeed occurs through a repressive domain within the carboxy terminus of SF-1. Furthermore, DAX-1 recruits the nuclear receptor corepressor N-CoR to SF-1, whereas naturally occurring AHC mutations of DAX-1 permit the SF-1-DAX-1 interaction, but markedly diminish corepressor recruitment. Finally, the interaction between DAX-1 and N-CoR shares similarities with the interaction between the nuclear receptor RevErb and N-CoR, because the related corepressor SMRT is not efficiently recruited by DAX-1. Therefore, DAX-1 can serve as an adapter molecule that recruits nuclear receptor corepressors to DNA-bound nuclear receptors like SF-1, thereby extending the range of corepressor action (Crawford, 1998).

Products of steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. In males, SF-1 participates in sexual development by regulating expression of the polypeptide hormone Mullerian inhibiting substance (MIS). WT1-KTS isoforms [the KTS isoform is an alternative splice variant that has an insertion of three amino acids (KTS) between the third and fourth zinc fingers] associate and synergize with SF-1 to promote MIS expression. In contrast, WT1 missense mutations, associated with male pseudohermaphroditism in Denys-Drash syndrome, fail to synergize with SF-1. Additionally, the X-linked, candidate dosage-sensitive sex-reversal gene, Dax-1, antagonizes synergy between SF-1 and WT1, most likely through a direct interaction with SF-1. It is proposed that WT1 and Dax-1 functionally oppose each other in testis development by modulating SF-1-mediated transactivation (Nachtigal, 1998).

Vertebrate Ftz-f1 homologs: Transcriptional regulation

Ad4BP, also known as SF-1, is a steroidogenic tissue-specific transcription factor that is also essential for adrenal and gonadal development. Two mechanisms for the transcriptional regulation of the mammalian FTZ-F1 gene encoding Ad4BP in adrenocortical cells have been proposed: (1) the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F1 genes, and (2) a possible autoregulatory mechanism of the rFTZ-F1 gene by Ad4BP itself through binding to the Ad4 (or SF-1) site in the first intron. The transcriptional regulation of the human FTZ-F1 gene in adrenocortical cells has been investigated from several angles, including the above two mechanisms. Using a series of deletion analyses of the 5'-flanking region of the hFTZ-F1 gene and site-directed mutagenesis for transient transfection studies, an E box element, CACGTG at -87/-82 from the transcriptional start site, was found to be essential for the transcription of the hFTZ-F1 gene in mouse or human adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite the presence of a corresponding Ad4 site, CCAAGGCC at +163/+156 in the first intron of the hFTZ-F1 gene, an autoregulatory mechanism through the Ad4 site was found to be unlikely in the hFTZ-F1 gene mainly due to site-directed mutagenesis. In addition, the forced expression of Ad4BP has little effect on hFTZ-F1 gene transcription in non-steroidogenic CV-1 cells. Such Ad4BP-independent regulation of the hFTZ-F1 gene is in striking contrast to the regulation of steroidogenic CYP genes, such as the human CYP11A gene, in which the proximal promoter activity is Ad4BP-dependent and the transactivation by Ad4BP is silenced by DAX-1. Even though the Ad4BP-dependent transcriptional regulation of the DAX-1 gene has been reported, DAX-1 did not affect the transcriptional activity of the hFTZ-F1 gene in this study. Taken together, these observations suggest that the E box is indeed required for the expression of the FTZ-F1 gene, at least in mammalian species, but may not determine the tissue-specific expression of the hFTZ-F1 gene, and that, unlike the steroidogenic CYP gene, the regulation of the hFTZ-F1 gene appears to be independent of both Ad4BP and DAX-1 (Oba, 2000).

Vertebrate Ftz-f1 homologs: Translational regulation

The nuclear receptor steroidogenic factor-1 (SF-1) is essential for development of the gonads, adrenal gland, and the ventromedial hypothalamic nucleus. It also regulates the expression of pivotal steroidogenic enzymes and other important proteins in the reproductive system. A domain located C-terminal to the DNA binding domain of SF-1, exhibits transcriptional repression function. Point mutations in this domain markedly potentiate the transcriptional activity of native SF-1. Using an SF-1 region that spans this proximal repression domain as bait in a yeast two-hybrid system, an SF-1 interacting protein has been cloned that is homologous to human DP103, a member of the DEAD box family of putative RNA helicases. DP103 directly interacts with the proximal repression domain of SF-1, and mutations in this domain abrogate its interaction with DP103. DP103 is expressed predominantly in the testis and is also expressed at a lower level in other steroidogenic and nonsteroidogenic tissues. Functionally, DP103 exhibits a native transcriptional repression function that localizes to the C-terminal region of the protein and represses the activity of wild-type, but not mutant, SF-1. Together, the physical and functional interaction of DP103 with a previously unrecognized repression domain within SF-1 represents a novel mechanism for regulation of SF-1 activity (Ou, 2001).

Vertebrate Ftz-f1 homologs regulate steroidogenesis

The sequence of the putative DNA-binding domain for steroidogenic factor 1 (SF-1) exactly the corresponding sequence of the mouse homolog of the Drosophila transcription factor Fushi tarazu-factor I. SF1 coordinately regulates the expression of three enzymes that are required for the biosynthesis of corticosteroids: cholesterol side chain cleavage enzyme, steroid 21-hydroxylase, and the aldosterone synthase isozyme of steroid 11 beta-hydroxylase. SF-1 interacts with the related promoter elements from these steroidogenic enzymes. SF-1 binds six steroidogenic regulatory elements. This strongly supports the model that a steroidogenic cell-selective protein interacts with related promoter elements from three steroidogenic enzymes to regulate their coordinate expression. The recognition sequence of SF-1 closely resembles the sequences of nuclear hormone receptor family members, suggesting that SF-1 may belong to this supergene family (Lala, 1992).

The cytochrome P450 steroid hydroxylases are coordinately regulated by SF-1. SF-1 coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contains both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defines shared exons encoding common regions and alternative promoters and 3'-exons leading to difference ELP transcripts. Transcripts are not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development (Ikeda, 1993).

SF-1, a nuclear receptor that regulates gene expression of the cytochrome P450 steroid hydroxylases, and ELP, an embryonal protein that suppresses expression of the Moloney murine leukemia virus LTR, are isoforms transcribed from the same gene by alternative promoter usage and splicing. SF-1 is the mammalian homolog of the Drosophila fushi-tarazu factor 1 (FTZ-F1) gene. The mouse and human genes are located in the homologous regions of mouse Chr 2 and human Chr 9, respectively (Taketo, 1995).

An E box is found in the transcriptional element in the 5'-upstream region of the rat SF-1 gene. There is also a steroidogenic cell-specific transcriptional element in the first intron of the gene. SF-1 itself binds to the intronic element (SF-1 site). Thus, two elements are essential for the full transcriptional activity of the SF-1 gene. The chromatin structure around the SF-1 site and the E box is "open up" in the adrenal glands and Y-1 cells. In the liver, the same structure is "closed down." These observations indicate that the mftz-f1 gene is controlled by an autoregulatory loop in the steroidogenic tissues. The autoregulatory mechanism seems to be necessary to keep the mftz-f1 gene activated and thus maintain the tissue differentiation (Nomura, 1996).

Cholesterol 7alpha-hydroxylase is the first and rate-limiting enzyme in a pathway through which cholesterol is metabolized to bile acids. The gene encoding cholesterol 7alpha-hydroxylase, CYP7A, is expressed exclusively in the liver. Overexpression of CYP7A in hamsters results in a reduction of serum cholesterol levels, suggesting that the enzyme plays a central role in cholesterol homeostasis. A hepatic-specific transcription factor that binds to the promoter of the human CYP7A gene has been identified. This factor has been designated CPF, for CYP7A promoter binding factor. Mutation of the CPF binding site within the CYP7A promoter abolishes hepatic-specific expression of the gene in transient transfection assays. A cDNA encoding CPF was cloned and identified as a human homolog of the Drosophila orphan nuclear receptor Ftz-F1. Cotransfection of a CPF expression plasmid and a CYP7A reporter gene results in specific induction of CYP7A-directed transcription. These observations suggest that CPF is a key regulator of human CYP7A gene expression in the liver (Nitta, 1999).

The high density lipoprotein (HDL) receptor mediates the uptake of cholesterol and cholesteryl esters, substrates for steroidogenesis, from an HDL particle in the adrenal gland and gonads. Treatment of rat luteal cells with 1 mM (Bu)2cAMP for 24 h dramatically induces (118-fold) HDL receptor messenger RNA levels. The rat HDL receptor promoter contains a steroidogenic factor-1 (SF-1)-binding site (SFBd; 5'-TCAAGGCC-3') through which SF-1 protein binds and activates transcription of this gene in both human HTB9 bladder carcinoma and mouse Y1 tumor cells, an effect that is enhanced by cAMP. These observations demonstrate that this motif is required for both basal and cAMP-induced regulation of the HDL receptor gene. Cotransfection studies in Kin 8 cells, a Y1 cell line resistant to cAMP activation as a result of a mutation in the protein kinase A (PKA) regulatory subunit, shows that a functional PKA is required for cAMP induction of HDL receptor gene transcription. Deleting the activation function-2 domain (amino acids 448-461) or mutating Ser430, a potential consensus phosphorylation site for PKA in the SF-1 protein, decreases both basal and cAMP-induced activation of the HDL receptor promoter. These data suggest that these regions within the SF-1 protein are required for both basal and cAMP-induced regulation of the HDL receptor gene. The mediation of cAMP responsiveness of the HDL receptor gene by SF-1 suggests how important this trans-acting factor is in steroid hormone synthesis by assuring that all required elements (substrate and enzymes) are present when they are needed for maximal steroid production (Lopez, 1999).

Using a mouse Leydig tumor cell line, the mechanisms involved in thyroid hormone-induced steroidogenic acute regulatory (StAR) protein gene expression, and steroidogenesis has been investigated. Triiodothyronine (T3) induces an approximately 3.6-fold increase in the steady-state level of StAR mRNA, which parallels the level of the acute steroid response ( approximately 4.0-fold), as monitored by quantitative reverse transcriptase-polymerase chain reaction assay and progesterone production, respectively. The T3-stimulated progesterone production is effectively inhibited by actinomycin-D or cycloheximide, indicating the requirement of on-going mRNA and protein synthesis. T3 displays the highest affinity of [125I]iodo-T3 binding and is most potent in stimulating StAR mRNA expression. In accordance, T3 significantly increases testosterone production in primary cultures of adult mouse Leydig cells. The T3 and human chorionic gonadotropin (hCG) effects on StAR expression are similar in magnitude and additive. Cells expressing steroidogenic factor 1 (SF-1) show marginal elevation of StAR expression, but coordinately increase T3-induced StAR mRNA expression and progesterone levels. In contrast, overexpression of DAX-1, a repressor of SF-1 activity, markedly diminishes the SF-1 mRNA expression, and concomitantly abolishes T3-mediated responses. Noteworthy is the fact that T3 augments the SF-1 mRNA expression, while inhibition of the latter by DAX-1 strongly impairs T3 action. Northern hybridization analysis reveals four StAR transcripts that increase 3-6-fold following T3 stimulation. These observations clearly identify a regulatory cascade of thyroid hormone-stimulated StAR expression and steroidogenesis that provides novel insight into the importance of a thyroid-gonadal connection in the hormonal control of Leydig cell steroidogenesis (Manna, 1999).

The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. In steroidogenic tissues, StAR expression is acutely regulated by trophic hormones through a cAMP second messenger pathway, leading to increased StAR mRNA levels within 30 min, reaching maximal levels after 4-6 h of stimulation. The molecular mechanisms underlying such regulation remain unknown. The StAR promoter was examined for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormone-induced context. Two putative CCAAT/enhancer binding protein (C/EBP) DNA elements have been identified at -113 (C1) and -87 (C2) in the mouse StAR promoter. C/EBP beta binds with high affinity to C1, but C2 proves to be a low-affinity C/EBP site. Functional analysis of these sites in the murine StAR promoter show that mutation of one or both of these binding sites decreases both basal and (Bu)2cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation [(Bu)2cAMP-stimulated/basal] of the promoter. These two C/EBP binding sites are required for steroidogenic factor-1 (SF-1)-dependent transactivation of the StAR promoter in a nonsteroidogenic cell line. These data indicate that in addition to SF-1, C/EBP beta is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis (Reinhart, 1999).

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. It is proposed this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction (Hammer, 1999).

Other Ftz-F1 homolog targets

The 5'-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRepsilon3) gene has been cloned and the cis- and trans-activating regulatory elements responsible for its tissue specific activity have been characterized. By using a native epsilon3-promoter/lacZ-construct and various 5'-deletion constructs, beta-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal epsilon3-gene-expressing HT-4 cells were compared. Large parts of the epsilon3 promoter are shown to be responsible for the repression of the epsilon3 gene in non-neuronal cells. Deletion of exon 1 sequences leads to an enhancement of epsilon3 transcription, suggesting a role for the 5'-untranslated region in epsilon3 gene regulation. Sequence analysis of the promoter region reveals potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologs, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirm specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal epsilon3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of epsilon3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site (Pieri, 1999).

The Dax-1 gene encodes a protein that is structurally related to members of the orphan nuclear receptor superfamily. Dax-1 is coexpressed with another orphan nuclear receptor, steroidogenic factor-1 (SF-1), in the adrenal, gonads, hypothalamus, and pituitary gland. Mutations in Dax-1 cause adrenal hypoplasia congenita, a disorder that is characterized by adrenal insufficiency and hypogonadotropic hypogonadism. These developmental and endocrine abnormalities are similar to those caused by disruption of the murine Ftz-F1 gene (which encodes SF-1), suggesting that these nuclear receptors act along the same developmental cascade. Cloning of the murine Dax-1 gene revealed a candidate SF-1-binding site in the Dax-1 promoter. In transient expression assays in SF-1-deficient JEG-3 cells, SF-1 stimulates expression of the Dax-1 promoter. However, deletion or mutation of the consensus SF-1-binding site does not eliminate SF-1 stimulation. Further analyses have revealed the presence of a cryptic SF-1 site that creates an imperfect direct repeat of the SF-1 element. When linked to the minimal thymidine kinase promoter, each of the isolated SF-1 sites is sufficient to mediate transcriptional regulation by SF-1. Mutation of both SF-1 sites eliminates SF-1 binding and stimulation of the Dax-1 promoter. Unexpectedly, mutation of either half of the composite SF-1 sites increases basal activity in JEG-3 cells, suggesting interaction of a repressor protein. Gel shift analyses of the composite response element reveals an additional complex that is not supershifted by SF-1 antibodies. This complex was eliminated by mutation of either half-site, and it was supershifted by antibodies against chicken ovalbumin upstream promoter-transcription factor (COUP-TF). It is proposed that Dax-1 is stimulated by SF-1, and that SF-1 and COUP-TF provide antagonistic pathways that converge upon a common regulatory site (Yu, 1998).

Enhancer II (ENII) of hepatitis B virus (HBV) is one of the essential cis-elements for the transcriptional regulation of HBV gene expression. Its function is highly liver-specific, suggesting that liver-enriched transcriptional factors play critical roles in regulating the activity of ENII. In this report, a novel hepatocyte transcription factor, which binds specifically to the B1 region (AACGACCGACCTTGAG) within the major functional unit (B unit) of ENII, has been cloned from a human liver cDNA library by yeast one-hybrid screening, and has been demonstrated to trans-activate ENII via the B1 region. This factor has been named hB1F, for human B1-binding factor. Amino acid analysis revealed this factor structurally belongs to the nuclear receptor superfamily. Based on the sequence similarities, hB1F is characterized to be a novel human homolog of the orphan receptor FTZ-F1. A splicing isoform of hB1F (hB1F-2) was identified, which has an extra 46 amino acid residues in the A/B region. Examination of the tissue distribution has revealed an abundant 5.2-kilobase transcript of hB1F is present specifically in human pancreas and liver. Interestingly, an additional transcript of 3.8 kilobases was found to be present in hepatoma cells HepG2. Fluorescent in situ hybridization has mapped the gene locus of hB1F to the region q31-32.1 of human chromosome 1. Altogether, this study provides the first report that a novel human homolog of FTZ-F1 binds and regulates ENII of HBV. The potential roles of this FTZ-F1 homolog in tissue-specific gene regulation, in embryonic development, as well as in liver carcinogenesis are discussed (Li, 1998).

Mutation of vertebrate Ftz-f1 homologs

Studies in adrenocortical cells have implicated the orphan nuclear receptor SF-1 in the gene regulation of the steroid hydroxylases. Targeted disruption of the Ftz-F1 gene, which encodes SF-1, was used to examine its role in intact mice. Despite normal survival in utero, all Ftz-F1 null animals die by postnatal day 8; these animals lack adrenal glands and gonads and are severely deficient in corticosterone, supporting adrenocortical insufficiency as the probable cause of death. Male and female Ftz-F1 null mice have female internal genitalia, despite complete gonadal agenesis. These studies establish that the Ftz-F1 gene is essential for sexual differentiation and formation of the primary steroidogenic tissues (Luo, 1994).

Associated with these dramatic developmental abnormalities, all Ftz-F1-disrupted mice die in the immediate postnatal period and have very low glucocorticoid levels. Treatment with corticosteroids markedly prolongs survival of the Ftz-F1-disrupted mice, proving that steroid hormone deficiency causes their death. SF-1-specific knockout mice were generated with a targeting construct that specifically disrupts the SF-1 coding sequence without impairing the ELP protein. The phenotype of the SF-1-specific knockout mice is indistinguishable from that observed in Ftz-F1-disrupted mice that lack both SF-1 and ELP. Taken together, these results indicate that SF-1 is the Ftz-F1-encoded protein that is required for multiple aspects of endocrine development and for postnatal survival (Luo, 1995).

While serum levels of corticosterone in SF-1-deficient mice are diminished, levels of adrenocorticotropic hormone (ACTH) are elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appears normal, and they have normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. SF-1 was examined for whether it is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc. In contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 has no effect on P450scc mRNA levels in vivo. Although the region targeted by disruption is shared by SF-1 and by ELP ( a hypothesized alternatively spliced product), it is thought that the observed phenotype reflects absent SF-1 alone, as PCR analysis fails to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established (Sadovsky, 1995).

The spleen has two main functions. The first is to provide a proper microenvironment to lymphoid and myeloid cells, whereas the second involves clearance of abnormal erythrocytes. Ad4BP/SF-1, a product of the mammalian FTZ-F1 gene (mFTZ-F1), was originally identified as a steroidogenic, tissue-specific transcription factor. Immunohistochemical examination of the mammalian spleens confirms the expression of Ad4BP/SF-1 in endothelial cells of the splenic venous sinuses and pulp vein. In mFtz-F1 gene-disrupted (KO) mice, several structural abnormalities are detected in the spleen, including underdevelopment and nonuniform distribution of erythrocytes. Examination of the spleen of KO fetuses shows failure of development of certain tubular structures during embryogenesis. These structures are normally assembled by Ad4BP/SF-1 immunoreactive cells, and most likely form the vascular system during later stages of development. Other structural abnormalities in the spleen of the KO mice include defects in the tissue distribution of type-IV collagen, laminin, c-kit, and vimentin. These morphologic defects in the vascular system are associated with a decrease in the proportion of hematopoietic cells, although differentiation of these cells is not affected significantly. A high number of abnormal red blood cells containing Howell-Jolly bodies are noted in the KO mice, indicating impaired clearance by the splenic vascular system. The presence of an mRNA-encoding cholesterol side-chain cleavage P450 was detected in the spleen, resembling the findings in steroidogenic tissues such as the gonads and adrenal cortex. The mRNA transcript is not involved in splenic structural defects, since it is detected in the spleens of both normal and KO mice, indicating that the regulatory mechanism of the P450 gene in the spleen is different from that in steroidogenic tissues. These results indicate that a lack of the mFtz-F1 gene in mice is associated with structural and functional abnormalities of the splenic vascular system (Morohashi, 1999).

Ftz-f1 homologs and pituitary development and function

In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones show a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. Pituitaries from Ftz-F1-disrupted mice lack transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibit decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first become detectable at embryonic day 13.5-14.5, precede the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalize with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacts with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice (Ingraham, 1994).

mRNA coding for SF-1 is detected in the hypothalamus and pituitary. The transcription factor is expressed in nuclei of the dorsomedial part of the ventromedial hypothalamus (dmVMH) and in some subpopulation of the adenohypophysial cells. Staining for SF-1 and trophic peptide hormones (FSH, TSH, and ACTH), indicates a restricted localization of SF-1 to the gonadotroph. Disruption of the mouse Ftz-F1 gene induces severe defects in the organization of the dmVMH and the function of the pituitary gonadotroph. However, some of the dm VMH neurons and pituitary gonadotrophs persist, providing a sharp contrast to complete agenesis of the peripheral steroidogenic tissues (adrenal and gonads) in the mutant mouse. Additional abnormalities are seen in the ventrolateral part of the VMH and the dorsomedial hypothalamic nucleus, neither of which expresses SF-1, but both have strong reciprocal fiber-connections with the dmVMH. Aromatase P450-containing cells in the medial preoptico-amygdaloid region, which are devoid of SF-1, persist even in the brain of the gene disrupted mice. The hypothalamic and pituitary SF-1 are clearly seen to be essential for normal development of the functional VMH and gonadotroph through some mechanism distinct from that in the peripheral steroidogenic tissues (Shinoda, 1995).

The nuclear receptor steroidogenic factor 1 (SF-1) regulates the biosynthesis of the two essential mediators of male sexual differentiation: androgens and Mullerian-inhibiting substance. SF-1 is required for adrenal and gonadal development and gonadotropin expression. SF-1 is also expressed in the embryonic ventral diencephalon, subsequently localizing to the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene have normal numbers and location of GnRH neurons but exhibit grossly impaired ventromedial hypothalamic nucleus structure. Despite their apparently normal GnRH neurons, treatment of Ftz-F1-disrupted mice with GnRH restores pituitary gonadotropin expression. These studies define SF-1's essential role within a discrete hypothalamic nucleus previously linked to reproduction (Ikeda, 1995).

The Ptx1 (pituitary homeobox 1; see Drosophila Ptx1) homeobox transcription factor is a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, its expression and function was examined in other pituitary lineages. Ptx1 is expressed in most pituitary-derived cell lines as is the related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the betaLH promoter but not on the alphaGSU (glycoprotein hormone alpha-subunit gene) and betaFSH promoters. Synergism between these two classes of factors is reminiscent of the interaction between the products of the Drosophila genes ftz (fushi tarazu) and ftz-F1. Antisense RNA experiments performed in alphaT3-1 cells that express the alphaGSU gene show that expression of endogenous alphaGSU is highly dependent on Ptx1, whereas many other genes are not affected. Interestingly, the only other gene found to be highly dependent on Ptx1 for expression is the gene for the Lim3/Lhx3 transcription factor. Thus, these experiments place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear to work in a combinatorial code to direct pituitary-, lineage-, and promoter-specific transcription (Tremblay, 1998).

Pituitary gonadotropins are critical regulators of gonadal development and function. Expression and secretion of the mature hormones are regulated by gonadotropin-releasing hormone (GnRH), which is itself secreted from the hypothalamus. GnRH stimulation of gonadotropin expression and secretion occurs through the G-protein-linked phospholipase C/inositol triphosphate intracellular signaling pathway, which ultimately leads to protein kinase C (PKC) activation and increased intracellular calcium levels. Transcription factors mediating the effects of GnRH-induced signals on transcription of gonadotropin genes have not yet been identified. Recent studies have identified three key factors involved in luteinizing hormone beta (LHbeta) gonadotropin gene transcription: the nuclear receptor SF-1, the bicoid-related homeoprotein Ptx1 (Pitx1), and the immediate-early Egr-1 gene. GnRH is a potent stimulator of Egr-1, but not Ptx1 or SF-1, expression. Further, Egr-1 activation of the LHbeta promoter is specifically enhanced by PKC, in agreement with a role for Egr-1 in mediating a GnRH effect on transcription. Egr-1 interacts directly with Ptx1 and with SF-1, leading to an enhancement of Ptx1- and SF-1-induced LHbeta transcription. Thus, Egr-1 is a likely transcriptional mediator of GnRH-induced signals for activation of the LHbeta gene (Tremblay, 1999).

Tissue-specific expression of the mammalian FTZ-F1 gene is essential for adrenal and gonadal development and sexual differentiation. The FTZ-F1 gene encodes an orphan nuclear receptor, termed SF-1 (steroidogenic factor-1) or Ad4BP, which is a primary transcriptional regulator of several hormone and steroidogenic enzyme genes that are critical for normal physiological function of the hypothalamic-pituitary-gonadal axis in reproduction. The objective of the current study was to understand the molecular mechanisms underlying transcriptional regulation of SF-1 gene expression in the pituitary. A series of deletion and point mutations in the SF-1 promoter region was studied for transcriptional activity in alphaT3-1 and L/betaT2 (pituitary gonadotrope), CV-1, JEG-3, and Y1 (adrenocortical) cell lines. Maximal expression of the SF-1 promoter in all cell types requires an E box element at -82/-77. This E box sequence (CACGTG) is identical to the binding element for USF (upstream stimulatory factor), a member of the helix-loop-helix family of transcription factors. Studies of the SF-1 gene E box element using gel mobility shift and antibody supershift assays indicate that USF may be a key transcriptional regulator of SF-1 gene expression (Harris, 1998).

GnRH plays a pivotal role in regulating human reproductive functions. This hypothalamic peptide interacts with its receptor (GnRHR) on the pituitary gonadotropes to trigger the secretion of gonadotropins, which, in turn, regulates the release of sex steroids from the gonads. In light of the importance of GnRHR, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR (hGnRHR) gene become a key issue in understanding human reproduction. In this report, the possible involvement of steriodogenic factor-1 (SF-1) as a key cell-specific regulator for hGnRHR gene expression was examined. By means of transient luciferase reporter gene assays, the wild-type promoter, containing 2.3 kb ofthe hGnRHR gene 5'-flanking region relative to the ATG codon, was able to drive a 3.6 +/- 0.2-fold (P < 0.05) increase in luciferase activity in the mouse alphaT3-1 gonadotropes. Subsequent deletion analysis indicates that the most proximal 173 bp within the first exon of the gene, although not a promoter itself, contains a critical regulatory element(s) essential for the basal expression of the hGnRHR gene. The functional roles of the putative gonadotrope-specific elements [GSE; consensus 5'-CTG(A)/(T)CCTTG-3'] residing at positions -5, -134, and -396 were studied by site-directed mutagenesis, and it was found that only the mutation at position -134 significantly reduces the promoter activity (80% reduction; P < 0.05). The attenuation effect of this GSE mutant is cell specific, since it is restricted to alphaT3-1 cells, but not to COS-7 and human ovarian adenocarcinoma (SKOV-3) cells. Competitive mobility shift assays indicates that SF-1 is able to interact specifically with this GSE element positioned at -134. A SF-1 antibody completely abrogates complex formation in the gel shift assays. The sequences essential for the interaction with SF-1 have been identified [5'-TTG(A)/(T)CCCTG-3']. Overexpression of the SF-1 mRNA was able to enhance promoter activities in all of the cells tested. On the contrary, expression of the antisense SF-1 mRNA reduces the hGnRHR promoter activity only in alphaT3-1 cells, not in COS-7 or SKOV-3 cells. In summary, the data reported here provide conclusive evidence that SF-1 interacts with the GSE motif at position -134 within the first exon of the hGnRHR gene to mediate its cell-specific expression (Ngan, 1999).

The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH beta-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LH beta-subunit promoter in the gonadotrope cell line, alpha T3-1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contains two Egr response elements. Coexpression of Egr1 and SF-1 leads to a synergistic activation of the equine (e)LH beta promoter. Mutation of any of the Egr or SF-1 response elements attenuate this synergism. Endogenous expression of Egr1 in alpha T3-1 cells is not detectable under basal conditions, but is rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicates that these sites are required for GnRH induction. Mutation of both Egr sites within the eLH beta promoter completely attenuates its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLH beta promoter. Finally, GnRH not only induces expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1-induced transcription of the eLH beta promoter (Wolfe, 1999).

Early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) beta deficiency. Egr-1 activates the LHbeta gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for stimulation of LHbeta by gonadotropin-releasing hormone (GnRH), the activity of the LHbeta promoter was examined in the gonadotrope cell line LbetaT2. GnRH markedly stimulates the LHbeta promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHbeta promoter attenuates this stimulation, whereas mutation of both promoter elements abrogates GnRH induction of the LHbeta promoter. Furthermore, GnRH stimulates Egr-1 but not SF-1 expression in LbetaT2 cells. Importantly, overexpression of Egr-1 alone is sufficient to enhance LHbeta expression. Although other Egr proteins are expressed in LbetaT2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 is the most robust. The nuclear receptor DAX-1, a repressor of SF-1 activity, reduces Egr-1-SF-1 synergy and diminishes GnRH stimulation of the LHbeta promoter. It is concluded that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHbeta gene and plays a central role in the dynamic regulation of LHbeta expression (Dorn, 1999).

Ftz-f1 homologs, adrenal gland and gonadal development

FTZ-F1 is a member of the family of orphan nuclear receptors; this is a subfamily of the steroid hormone receptor superfamily. FTZ-F1 plays a role in the blastoderm and nervous system development in Drosophila. Recently, several other FTZ-F1- like genes have been cloned in several species. SF-1/Ad4BPs have been identified as master regulators controlling steroidogenic P-450 genes in mammals and are considered to be the mammalian homologs of FTZ-F1. Moreover, SF-1/Ad4BP plays a critical role in the sexual differentiation of gonads in mammals. In vertebrates other than mammals, the functional homolog of SF-1/Ad4BP had not been previously identified. Two chicken cDNAs (OR2.0 and OR2.1), which encode putative FTZ-F1 family receptors, have been cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). OR2.1 consists of 3255 bp, is expressed in the adrenal glands and gonads, and is considered to be the chicken counterpart of mammalian SF-1/Ad4BP. However, OR2.0 consists of 2945 bp, is expressed in the livers and the adrenal glands, and is considered to be the chicken counterpart of mouse LRH-1, which is a member of the FTZ-F1 family in mammals (Kudo, 1997).

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal gland and gonads and is an important regulator of the expression of cytochrome P-450 steroidogenic enzymes in cultured cells. Targeted disruption of the SF-1 gene in mice shows that it is a critical participant in the genetic program that promotes the development of urogenital mesoderm into the adrenal gland and gonads. To assess the ability of SF-1 to regulate this differentiation pathway, SF-1 was ectopically expressed in murine embryonic stem (ES) cells. Stable expression of SF-1 is sufficient to alter ES cell morphology, permit cyclic AMP (cAMP) and retinoic acid-induced expression of the endogenous side chain cleavage enzyme gene, and consequently, promote steroidogenesis. While steroid production is dependent upon SF-1, cAMP induction of steroidogenesis does not enhance the responsiveness of an SF-1-specific reporter. Furthermore, the activity of a P450SCC promoter/luciferase reporter construct, which is induced by cAMP in steroidogenic cells and ES cells converted by stable expression of SF-1, is not induced by cAMP in wild-type ES cells transiently transfected with SF-1, suggesting that the induction of downstream gene products is required before steroidogenesis can occur. Mutants that disrupt the DNA binding domain or the AF2 transcriptional activation domain of SF-1 do not confer the steroidogenic phenotype to ES cells. Notably, AF2 mutants fused to the VP16 activation domain do confer the steroidogenic phenotype to ES cells, but only in the presence of a portion of the ligand binding domain. These studies extend the role of SF-1 in steroidogenic tissues to that of a dominant regulator of the steroidogenic cell phenotype (Crawford, 1997).

Ad4BP/SF-1 is a transcription factor essential for the development of the adrenal gland and the gonads as well as for the maintenance of their functions through the regulation of tissue-specific gene transcription. In the whole body, hypothalamo-pituitary-gonadal and -adrenal axes are known to play prominent roles in mediating the function of the gonads and adrenal. In this study, the effects of the tropic peptide hormones secreted by the pituitary on the regulation of the rat Ftz-F1 (rFtz-F1) gene encoding Ad4BP/SF-1 were investigated. Immunochemical studies have shown that Ad4BP/SF-1 is expressed even in the adrenal cortex of hypophysectomized rats. Such persistent expression of Ad4BP/SF-1 is also observed in the testes and ovaries of the hypophysectomized animals. In contrast to Ad4BP/SF-1, the expressions of steroidogenic P450s are reduced significantly. The transcriptional activities of the endogenous and transfected rFtz-F1 genes were examined with Y-1 and I-10 cells derived from mouse adrenocortical and testicular Leydig cell tumors, respectively. Neither gene appears to be activated significantly by cAMP, whereas both endogenous and exogenous CYP11A genes encoding P450(SCC) are activated. Taken together, these observations indicate that the expression of the rFtz-F1 gene is mainly regulated by a mechanism independent of the neuroendocrine axes (Nomura, 1998).

The pituitary peptide hormone ACTH regulates transcription of the cholesterol side chain cleavage cytochrome P450 (CYP11A) gene via cAMP and activation of cAMP-dependent protein kinase. A G-rich sequence element conferring cAMP-dependent regulation has been found to reside within region -118 to -100 of the bovine CYP11A promoter. This region has been shown to bind a protein antigenically related to the transcription factor Sp1. The -118/-100 element binds both Sp1 and Sp3, members of the Sp family of transcription factors. Drosophila SL2 cells, which lack endogenous Sp factors, were used to dissect the possible functional roles of Sp1, Sp3, and Sp4. All factors stimulate the activity of cotransfected reporter constructs in which the promoter of the bovine CYP11A gene regulates luciferase expression. Sp3 does not repress Sp1-dependent activation, as has previously been shown for other G-rich promoters. Mutation of the -118/-100 element of CYP11A abolishes Sp1-mediated activation of a CYP11A reporter gene in SL2 cells as well as cAMP responsiveness in human H295R cells. Furthermore, cotransfection of SL2 cells with the catalytic subunit of cAMP-dependent protein kinase, together with Sp1 and a CYP11A reporter construct, enhances Sp1-dependent activation of the reporter 4.2-fold, demonstrating that Sp1 confers cAMP responsiveness in these cells. Thus, introduction of Sp1 alone in an Sp-negative cell such as SL2 is sufficient to achieve the cAMP-dependent regulation observed using the -118/-100 element of CYP11A in adrenocortical cells. The sequence between 70 and 50 also predicts a binding site for steroidogenic factor 1 (SF-1), a nuclear orphan receptor required for normal development of adrenals and gonads and a positive regulator of all steroid hydroxylase genes studied so far. In most cases, the SF-1-binding sites are localized within cAMP-responsive regions, and SF-1 has been proposed to mediate the response in certain cases. In fact, both Sp1 and SF-1 are necessary to achieve cAMP-dependent regulation of the CYP11A gene in Y1 cells as well as in primary bovine luteal cells. Furthermore, a two-hybrid assay has revealed protein-protein interactions between Sp1 and SF-1, although no physical interaction could be demonstrated by co-immunoprecipitation assays. This suggests that the interaction between the two factors could be through interaction with a coactivator. Mutation within the activation function-2 domain of SF-1 transforms the transcription factor into a dominant-negative mutant with respect to cAMP-dependent activation of the bovine CYP17 gene. Also, SF-1 can interact with steroid receptor coactivator 1. It could therefore be envisioned that cAMP might stimulate the interaction of coactivators, e.g. steroid receptor coactivator 1, not only with SF-1, but possibly also with Sp1. In conclusion, these results establish Sp1 as a cAMP-responsive transcription factor in the context of the bovine CYP11A promoter and suggest the interesting possibility that cAMP may influence the interaction of Sp1 with cofactors shared with other transcription factors involved in CYP11A regulation (Ahlgren, 1999 and references).

REFERENCES

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