Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development.
Steroid hormones are responsible for the coordination and regulation of many biological events during development of multicellular organisms. In all species, steroid hormones are synthesized from cholesterol and/or other phytosterols by multiple steroidogenic enzymes, epitomized by the members of the steroidogenic cytochrome P450 monooxygenases. High-level steroid hormone biosynthesis generally occurs in specialized steroidogenic tissues. Thus, an important condition for achieving tissue-specificity of steroid biosynthesis is providing a regulatory mechanism that ensures tissue-specific expression of the steroidogenic enzyme genes. In vertebrates, major sites of steroid hormone biosynthesis are the adrenal cortex, gonads and placenta, that express steroidogenic enzyme genes such as Cyp11a1, P450c17a, 3β-HSD and 17β-HSD [1]. Key transcriptional regulators for these genes are the orphan nuclear receptors NR5A1 and NR5A2, also known as Ad4BP/Steroidogenic Factor 1 (SF-1) and Liver receptor homolog-1 (LRH-1), respectively [2–5]. Ad4BP/SF-1 and LRH-1 are predominantly expressed in the steroidogenic cells. A collective body of previous studies has established that Ad4BP/SF-1 controls steroid hormone biosynthesis through the transcriptional regulation of all steroidogenic genes [3,5]. Moreover, forced expression of this gene is sufficient to differentiate embryonic stem cells and human induced pluripotent stem cells into the steroidogenic cells [6,7] and to induce ectopic adrenal formation [8], indicating that Ad4BP/SF-1 acts as a master regulator for steroid hormone biosynthesis in vertebrates. In insects, the principal steroid hormones are ecdysteroids, including ecdysone and its active derivative 20-hydroxyecdysone (20E), which plays pivotal roles in controlling a number of developmental and physiological events, especially in guiding transition from one developmental stage to the next via molting and metamorphosis [9–13]. During larval and pupal development, ecdysone is synthesized from dietary cholesterol in a specialized endocrine organ called the prothoracic gland (PG). After release from the PG, ecdysone is converted to 20E in the peripheral tissues through the action of Shade, the terminal P450 monoxygenase in the biosynthetic pathway [14]. In the last 15 years, a number of genes encoding essential ecdysteroidogenic enzymes acting in the PG have been identified and characterized, including noppera-bo [15–17], neverland (nvd) [18,19], Cyp307a1/spook (spo) [20,21], Cyp307a2/spookier (spok) [21], non-molting glossy/shroud (sro) [22], Cyp306a1/phantom (phm) [23,24], Cyp302a1/disembodied (dib) [25,26] and Cyp315a1/shadow (sad) [26]. All of these enzymes (except nvd and spok) are collectively referred to as the Halloween genes [13,27]. Previous studies have identified multiple transcription factors essential for ecdysteroidogenic functions in the PG. For example, the Ecdysone receptor-Ultraspiracle complex and several other ecdysteroid-regulated transcription factors such as βFTZ-F1, Broad, E75A and DHR4 are involved in both forward and feedback regulation of cyclic ecdysteroid production [28–35]. Ecdysteroid biosynthesis is also transcriptionally regulated by other factors including Without children [36], Molting defective (Mld) [37], the CncC-dKeap1 complex [38], Ventral veins lacking (Vvl) [39,40], Knirps [39] and FOXO [35]. Importantly, it has been reported that Broad, CncC, dKeap1, Vvl and Knirps directly bind to enhancer regions of some ecdysteroidogenic enzyme genes [31,33,38,39]. However, it should be noted that, unlike vertebrate Ad4BP/SF-1 and LHR-1, all of the identified steroidogenic transcription factors in insects are highly expressed not only in the PG, but also in many other non-ecdysteroidogenic tissues. Furthermore, some of these transcription factors have important functions other than ecdysteroid biosynthesis. For example, FOXO is well characterized as the primary transcriptional mediator of the insulin/insulin-like peptide signaling pathway in almost all cells [41]. Similarly, the CncC-dKeap1 complex is known to regulate xenobiotic responses [42] while Vvl and Knirps play key roles in cellular differentiation and morphogenesis of several tissues during embryogenesis including the PG (i. e. [43,44]). More notably, βFTZ-F1, the insect homolog of vertebrate Ad4BP/SF-1, plays a crucial role in ecdysteroid-dependent transcriptional cascades in not only the PG but also many other tissues [4,9]. In contrast to the broad roles that all these steroidogenic factors play in other tissues during development, we describe here a much more specific role for the transcription factor coded by the gene ouija board (ouib). Ouib is a C2H2-type zinc finger transcription factor, that is specifically expressed in the Drosophila PG and our genetic analysis clearly demonstrates that ouib is only essential for the expression of Spookier (Spok), a potential rate-limiting enzyme in the ecdysone biosynthetic pathway. Most remarkable, however, is that spok appears to be the essential target of ouib since resupply of a Spok paralog in PG tissue rescues ouib mutants to viability. Since orthologs of ouib and spok are found only in Drosophiladae genomes, this study also suggests a presence of insect clade-specific transcriptional regulatory mechanisms of ecdysone biosynthesis.
We identified CG11762, designated ouija board (ouib), as a gene predominantly expressed in the PG primordia in the embryonic in situ gene expression pattern database of the Berkeley Drosophila Genome Project Experiment ID RT01107 [45]. We confirmed the PG restricted expression of ouib in embryos using RNA in situ hybridization (Figs 1A, 1B and S1). Additional RNA in situ hybridization and quantitative reverse-transcription (qRT) -PCR experiments revealed that ouib is also predominantly expressed in the ring gland including the PG cells during larval development (Fig 1C, 1D and 1E). These results suggest that ouib may be involved in ecdysteroid biosynthesis. The predicted open reading frame of ouib encodes a protein that belongs to the family of the zinc-finger associated domain (ZAD) containing C2H2 zinc-finger proteins (ZFPs) [46,47]. The ZAD-ZFP family constitutes the largest subgroup of C2H2 ZFPs especially in insect species, and are characterized by an N-terminal ZAD consisting of ∼75 amino acid residues that are thought to serve as a protein-protein interaction domain [48]. In D. melanogaster, there are 98 independent loci encoding ZAD-ZFPs [47]. At least some of ZAD-ZFPs are thought to act as transcription factors, since several of them have been reported to bind DNA [49,50]. Notably, 5 paralogs of ouib are duplicated at the 85A9 cytological position of the third chromosome in D. melanogaster genome (S2 Fig), and one of the paralogs designated M1BP codes for a general transcription factor [51], raising the possibility that Ouib acts as a transcription factor in the PG. Orthologs of ouib are found in genomes of 11 other Drosphilidae species (S1 Table) [52]. FlyBase (http: //flybase. org/reports/FBgn0037618. html) also indicates the presence of potential orthologs of ouib in the mosquito species Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. However, a reciprocal BLAST search does not support the idea that the mosquito genomes have true ouib orthologs. In addition, a standard BLAST search did not detect any orthologous counterparts of ouib in any organisms other than Drosophilidae species. This is consistent with the previous report [47], that no orthologs of ouib are found in genomes of the silkworm Bombyx mori and the beetle Tribolium castatenum. Taken together, these results suggest that ouib is a Drosophilidae-specific ecdysteroidogenic component. To assess the in vivo functional importance of ouib, we generated ouib loss-of-function alleles by a CRISPR/Cas9-dependent genome editing technology [53]. We succeeded in isolating two independent mutant alleles, ouib29 and ouib74, each of which had a small deletion induced by different CRISPR single guide RNAs (sgRNAs; Fig 2A and 2B). Both ouib29 and ouib74 alleles led to premature stop codons in the putative coding sequence of ouib, eliminating all 5 zinc-finger domains in the C-terminal region of Ouib (Fig 2C). Embryos transheterozygous for ouib29/ouib74 completed embryogenesis, hatched normally, and showed no apparent morphological defects after hatching. However, ouib29/ouib74 transheterozygotes arrested development in the first instar larval stage and, even 108 hours after egg laying (AEL) or later, never molted into second instars and (Fig 3A and 3B). Eventually all ouib29/ouib74 transheterozygous animals died by 144 hours AEL retaining the first instar larva-type morphology. In contrast, the majority of control ouib29/+ or ouib74/+ heterozygous animals became pupae (Fig 3A and 3B) by this time. To rule out the possibility that the observed phenotype was due to off-target mutations by CRISPR/Cas9 system, we combined ouib29 or ouib74 allele with a deficiency (Df) line that deletes a genomic region containing ouib locus. Similar to ouib29/ouib74 transheterozygotes, ouib29/Df or ouib74/Df animals died in the first instar stage, while +/Df animals were fully viable. This result provides evidence that ouib locus is responsible for the lethal phenotype. These results demonstrate that Ouib is essential for larval development. We next examined whether the larval arrest and lethality phenotype of ouib mutant animals was due to the loss of ecdysteroids. An ELISA assay revealed that the ecdysteroid titer in ouib29/ouib74 transheterozygotes was significantly reduced compared to control animals (Fig 3C). Consistent with this observation, the expression of E75A, which is an early ecdysteroid-inducible gene, was greatly reduced in ouib29/ouib74 transheterozygotes (Fig 3D). Moreover, when ouib29/ouib74 animals or ouib74/Df animals were fed yeast paste containing 20E after hatching, they molted to the second instar larval stage or later, as judged by the anterior spiracular morphologies (Table 1). These results suggest that loss of ouib mutant phenotype is due to ecdysteroid deficiency and that ouib regulates ecdysteroid production in the PG during normal development. As described above, we expect that Ouib acts as a transcription factor. Considering the spatial expression pattern and the loss-of-function phenotype of ouib, we wondered whether loss of ouib resulted in changes in the expression levels of any ecdysteroidogenic genes in the PG. To address this issue, we conducted qRT-PCR experiment to examine expression levels of 6 ecdysteroidogenic genes in the first instar larvae of control and ouib29/ouib74 transheterozygotes. Among the 6 genes, the expression of one gene Cyp307a2/spok was drastically reduced in ouib29/ouib74 transheterozygotes as compared to control animals (Fig 4A). An immunohistological analysis using anti-Spok antibody also revealed a strong decrease of Spok protein level in ouib29/ouib74 larvae compared to control animals, but not that of the Sro protein, another ecdysone biosynthetic enzyme expressed in the PG. (Fig 4B). We also found that expression of Cyp302a1/dib and Cyp315a1/sad, two other ecdysone biosynthetic P450 genes, were also lower than in ouib29/ouib74 animals compared to control animals, but their reduction was just on the threshold of significance (Fig 4A). On the basis of the observation that the mutants cannot induce the expression of “spookier, ” we named CG11762 “ouija board” since this is an instrument for calling ghosts in western countries. We also examined whether there was a correlation between expression of ouib and spok during larval development. Overall the expression of both genes was relatively low during early stages and gradually became higher in late stages. We also found that the temporal expression profile of ouib closely correlates to that of spok in the late third instar stage (Fig 4C). Curiously, the temporal expression profile did not always correlates to the dynamics of ecdysteroid titer during the third instar stage (S3 Fig). For example, ouib expression did not increase prior to white prepupal stage, when the level of ecdysteroid titer was high. This result suggests that the ouib-spok coordinated transcriptional relationship does not fully account for the temporal dynamics of ecdysteroid biosynthesis during development. A previous study reported that Spok plays a crucial role in the “Black Box”, which consists of the conversion steps from 7-dehydrocholesterol (7dC) to 5β-ketodiol (5βkd) in the ecdysteroid biosynthetic pathway [21]. The participation of Spok in the “Black box” reactions was inferred by the observation that the larval arrest phenotype of spok RNAi animals was rescued by oral administration of 5βkd, but not 7dC or the most upstream precursor cholesterol [21]. Indeed, the same tendency as observed in spok RNAi animals was found in ouib loss-of-function animals. When ouib29/ouib74 transheterozygotes were fed yeast paste supplemented with cholesterol or 7dC, the larvae still arrested at the first instar larval stage (Fig 4D and Table 1). In contrast, we found that the first instar larval arrest phenotype of ouib29/ouib74 transheterozygotes was rescued when the animals were fed yeast paste supplemented with 5βkd (Fig 4D and Table 1). These results suggest that loss of ouib function specifically impairs the catalytic conversion that takes place during the “Black Box” reactions. These results also imply that the moderate reduction seen in dib and sad expression does not contribute in a major way to the ouib mutant phenotype. In addition to the feeding rescue experiment, we examined whether the ouib mutant phenotype was rescued by forced expression of spok using GAL4-UAS binary gene expression system. We first established UAS-spok transgenic strains to drive spok expression in the PG cells under control of phm-GAL4#22 driver. However, for an unknown reason, none of our UAS-spok transgenes was expressed in the PG of first and second instar larvae with the phm-GAL4#22 driver and thus these constructs were not suitable for our experimental purpose. Therefore, we decided to examine whether the ouib mutant phenotype could be rescued by forced expression of Cyp307a1/spo, a paralog of spok that appears to provide the same enzymatic activity but only in embryos and in the follicular cells of the ovary [20,21]. We confirmed that spo was functionally equivalent to spok in vivo, as spo overexpression rescued the first instar larval arrest phenotype of spok RNAi animals (S2 Table). Indeed, spo overexpression in the PG rescued the larval arrest phenotype of ouib29/ouib74 transheterozygotes, and some of the animals grew up to the adult stage (Table 2). These results strongly suggest that the developmental arrest phenotype of ouib mutant is due solely to loss of spok expression in the PG. Our data therefore support the idea that Ouib is a special transcription factor primarily required for inducing expression of one biosynthetic gene spok, and no other essential gene during development. To address whether Ouib protein acts directly on the spok enhancer region to induce spok expression, we initially searched for an Ouib-response element in the enhancer/promoter region of spok. We first identified a ~1. 4 kb genomic region upstream of the spok coding sequence that was sufficient to mimic the expression of spok in the PG when fused to a GFP reporter (Figs 5A and S4). The GFP expression driven by the ~1. 4 kb spok enhancer region was almost completely abolished in ouib29/ouib74 transheterozygotes (Fig 5A), suggesting that the ~1. 4 kb element contains a Ouib-response element. In order to identify the cis-regulatory element (s) responsible for the Ouib-mediated control of spok expression, we conducted a promoter/enhancer characterization analysis in a heterologous cell culture system. We generated DNA constructs carrying the upstream region of spok fused with a luciferase (luc) gene cassette and then transfected Drosophila Schneider 2 (S2) cells using these DNA constructs with or without a plasmid for overexpressing FLAG-ouib. We identified a 300 bp genomic region corresponding to the region from -331 bp to -32 bp upstream of the ATG start codon of spok that drives expression of the luc reporter in S2 cells in an Ouib-dependent manner (S5 Fig). The 300 bp region was also sufficient to drive expression of a GFP reporter in the PG cells (S4 Fig). To narrow down the element (s) responsible for the Ouib-dependent expression of spok, we tested several constructs carrying the upstream region of spok with a range of deletions within the 300 bp region (S5 Fig). We first generated the deletion constructs in 50 bp increments from 5´ terminus of the 300 bp region and found that the region from -181 to -131 bp was crucial for the Ouib-dependent luc reporter activity (S5 Fig). We then generated the deletion constructs in 10 bp increments from 5´ terminus of the -181 to -32 region. The construct carrying the -151 to -32 region did not show any induction in luc reporter activity even in the presence of Ouib (Fig 5B). The construct carrying the longer 10 bp 5´ extension (-161 to -32) still retained statistical significant Ouib-dependent luc reporter activity. However, the fold induction of luc reporter activity with the -161 to -32 region was slightly reduced as compared to the -171 to -32 region or longer (Fig 5B). From these results, we hypothesized that the Ouib-response element lay between -166 to -152 bps (Fig 5C). To clarify the importance of this 15 bp region for Ouib-dependent control of gene expression, we introduced transversion mutations of the entire 15 bp sequence. This mutated construct exhibited no luc reporter induction in the presence of Ouib upon transfection into S2 cells (Fig 5D). We also conducted subsequent reporter assays using constructs carrying various mutations in the 15 bp sequence. None of the constructs carrying any of several 3 bp substitutions within the 15bp sequence eliminated the responsive to Ouib (S6 Fig). Therefore, we conclude that Ouib binding tolerates degeneracy throughout the 15 bp sequence (5´-AGCTTTATTATTTAG-3´). We also examined the evolutionary conservation of the Ouib-response elements in putative spok enhancer regions in 12 Drosophilide species whose genome sequences have been determined [52]. EMBOSS Matcher, an algorithm to identify local similarities between two sequences [54], found sequence motifs similar to the D. melanogster Ouib-response element in almost all of the Drosophilidae species (S7 Fig). In particular, the D. yakuba putative spok enhancer contains exactly the same 15 bp sequence motif. In addition, in the species belonging to the subgenus Sophophora, which includes D. melanogaster, the Ouib-response element-like motifs are found in proximity (within 500 bp) to the spok coding region (S7 Fig). These data suggest that Ouib-like response elements are also evolutionarily conserved to some degree. We sought to further establish if Ouib binds directly to the Ouib-response element by performing a DNA/protein binding assay. We first examined the physical interaction between the Ouib-response element sequence and Ouib protein by an ABCD assay, which uses biotin conjugated, double-stranded oligonucleotides containing the Ouib-response element sequences. Nuclear extracts obtained from S2 cells expressing FLAG-ouib were mixed with the biotin-labeled oligonucleotide, and then the protein-oligonucleotide complexes were pulled down using streptavidin beads. We found that FLAG-Ouib protein bound strongly to the wild type Ouib-response element probe, but not to the mutated probe (Fig 6A). In the control experiments, a biotinylated probe corresponding to M1BP (another ZAD-ZFP homolog of Ouib) binding sequence in the enhancer of smoothened locus [51] did not efficiently precipitate FLAG-Ouib. Conversely, FLAG-M1BP protein did not bind to the Ouib-response element, while it bound to M1BP binding element (Fig 6A). To exclude the possibility that FLAG-Ouib protein isolated from cultured cells, indirectly associated with the probe through a complex containing some other endogenous transcription factor unrelated to Ouib, we prepared an E. coli produced recombinant protein containing the C-terminal 5 zinc finger domains (Ouib-Zf), and performed electrophoretic mobility shift assays (EMSAs) between the recombinant protein and the 15 bp Ouib-response element. We utilized 45 bp radiolabeled DNA probes, whose sequences corresponded to the spok enhancer region containing the 15 bp Ouib-response element. We found that the wild type oligonucleotide probes formed DNA/protein complexes with GST-Ouib-Zf, but not with GST alone (Fig 6B and 6C). In contrast, such DNA/protein complexes were not detected when radiolabeled mutated Ouib-response element sequences or sequences corresponding to the M1BP site [51] (Fig 6D) were used as probes, thereby confirming the specificity of the binding. Moreover, the complexes with the wild type 45 bp probes were outcompeted by unlabeled 45 bp DNA probes with the wild type Ouib-response element sequences, but not by the unlabeled mutated DNA probes or by the unlabeled M1BP probes (Fig 6B and 6C). Taken together, these findings strongly support the idea that Ouib specifically regulates spok transcription by direct binding to the Ouib-response element in the spok enhancer. Previous studies found that the increase of spok expression in the late third instar larvae is positively controlled by prothoracicotropic hormone (PTTH) [55,56]. We therefore examined whether ouib expression changed in response to down regulation of PTTH signaling. However, when the levels of the PTTH receptor gene torso were knocked down in the PG by RNAi, we observed no change in ouib expression, suggesting that PTTH regulation of spok is not mediated through ouib (S8 Fig).
In this study, we have demonstrated that the ZAD-ZFP Ouib is required for ecdysteroid biosynthesis in the PG during D. melanogaster development. The following points summarize our finding. First, ouib is predominantly expressed in the PG during embryonic and larval stages. Second, ouib null mutants exhibit early (first instar) larval developmental arrest due to a low ecdysteroid titer. Third, the larval arrest phenotype is caused by a failure of spok expression in the PG, and is rescued by sole overexpression of a spok paralog. Finally, a specific Ouib-response element that binds Ouib was identified in the enhancer region of spok. Our study reports on the discovery of the first invertebrate tissue-specific, steroidogenic transcription factor. ouib mutants exhibit a drastic reduction of spok expression. However, we point out that ouib mutants also show a mild statistically-significant reduction of dib and sad (Fig 4A). In fact, while no DNA sequences exactly matching the spok Ouib-response element (5´-AGCTTTATTATTTAG-3´) are found elsewhere in D. melanogaster genome, a number of degenerate sequences do exist in the genome, including the regions upstream of ecdysteroidogenic gene coding regions (S9 Fig). Considering the fact that the luciferase constructs carrying any of several 3 bp substitutions within the 15bp sequence are still responsive to Ouib (S6 Fig), we cannot completely rule out the possibility that Ouib is also involved in direct transcriptional regulation of genes other than spok, particularly dib and sad. Nevertheless, our results indicate that the impairment of expression of dib and sad seems not to contribute to the ouib phenotype in a major way. First, the arrest during the first instar larval stage of ouib mutants is rescued by oral administration of 5βkd. Since Dib and Sad play roles in the terminal hydroxylation steps downstream of the conversion of 5βkd to ecdysone [23,24,26], this finding suggests that the enzymatic levels of Dib and Sad are still sufficient to make functional levels of ecdysone. Second and more importantly, the first instar larval arrest phenotype of ouib mutants is rescued by a sole overexpression of spo, which is functionally equivalent to spok. Therefore, in addition to the PG specificity, we would argue that the key additional feature of Ouib is its specific role in spok expression. To further clarify the extent to which Ouib regulates other genes, and the functional importance of these genes, will require additional studies including transcriptome analysis/ChIP-seq analysis together with eventual mutational analysis of any identified targets. Curiously, the presence of ouib only in the Drosophilidae genomes is concordant with the Drosophilidae-specific duplication of Cyp307a P450 subfamily. While members of the Cyp307 P450 subfamily, which includes spok, are found in all arthropod species examined so far [20,21,57,58], Drosophilidae Cyp307 genes have been duplicated within the Drosophila radiation [21,59]. In the case of D. melanogaster, the duplicated Cyp307 genes are Cyp307a1/spo and Cyp307a2/spok, which are sub-functionally divergent in terms of gene expression pattern; spo is expressed in early embryogenesis and oogenesis, while spok is expressed in the PG cells in late embryogenesis as well as the larval and pupal stages [20,21]. Our data demonstrate that the spatiotemporal expression pattern of ouib closely matches that of spok but not spo. Notably, neither ouib nor spok transcripts are detected in embryonic stages 5–9 when the embryonic ecdysteroid titer is maximal [21,45], indicating that these genes do not contribute to producing embryonic ecdysteroids. Therefore, an acquisition of ouib might be a critical event for the sub-functionalization of two Cyp307 genes by changing the regulation of their expression during the Drosophilidae evolution. In terms of evolution, it is worth mentioning that there is a case where evolution changed the activity of a single ecdysteroidogenic enzyme (Nvd) dramatically limiting the food source of Drosophila pachea to a single species of cactus [60]. Further assessment of the biological and evolutionary roles of ouib and spok will require determining which transcription factors are involved in the transcriptional regulation of D. melanogaster spo and Cyp307a genes in other insects. Since there are many divergent ZAD-ZFP genes in each insect genome and they are expanded in insect lineage-specific manner [47], it is possible that a different ZAD-ZFP gene whose primary structure is not orthologous to ouib could be a transcription factor for other Cyp307a genes. Regarding the evolutionarily aspect of ouib, it is important to recognize that spok expression is regulated by another ZAD-ZFP called Molting defective (Mld) [21,37,39]. Interestingly, just like ouib, mld genes are also found only in genomes of Drosophilidae but not other insects [21,37]. In contrast to ouib, Mld does not appear to be specific for the regulation of spok expression. First, Mld, unlike ouib, is expressed in several other tissues during development besides the PG [37]. Second, Mld is essential for regulating expression Nvd as well as spok and perhaps other genes [39]. Third and most important, the mld loss–of-function phenotype is not rescued by overexpressing either spo or spok [21]. Therefore, Ouib and Mld overlap in their regulation of spok expression, but also have distinct functions during development. While it is still unclear whether Mld is a transcription factor, it would be intriguing to examine a functional relationship between Ouib and Mld for induction of spok expression in the PG. According to our qRT-PCR data, it is less likely that Mld controls ouib expression in the PG (S10 Fig). Another question to be answered is how ouib expression is regulated during larval development. As shown above, it does not seem to be by PTTH. However, recent work as shown that spok and other ecdysteroidogenic enzyme genes are also influenced by humoral factors such as TGFβ/Activin [61] and monoaminergic tropic factors [62,63]. It will be interesting to determine whether these factors affect spok expression in the PG through modulation of ouib levels. An additional significant aspect of this work is to provide the first evidence for the existence of a catalytic step-specific transcriptional regulation of steroid hormone biosynthesis in organisms. Whereas the substrate of Spok and its product have not yet been identified, Spok appears to play a crucial role in the “Black Box” step of ecdysteroid biosynthetic pathway, and it is a strong candidate for acting as a rate-limiting enzyme in the pathway [10,21,64]. Interestingly, a recent study has reported that pre-mRNA splicing of spok, but not any other ecdysteroidogenic genes expressed in the PG, seems to specifically depend on a protein encoded by ecdysoneless (ecd), whose mutant phenotype includes ecdysteroid deficiency [65]. Thus, a rate-limiting step of ecdysteroid biosynthesis catalyzed by Spok could be under tight control by both specific transcriptional and post-transcriptional mechanisms. Currently, it is unknown whether such catalytic-specific transcriptional and/or posttranscriptional mechanisms also exist in other organisms including vertebrates. Similar to ecdysteroids, vertebrate steroid hormones are synthesized via several intermediates by multiple steroidogenic enzymes. Among them, the rate-limiting step in vertebrate steroid hormone productions is the delivery of substrate cholesterol from the outer mitochondrial membrane to the inner one and the subsequent conversion of cholesterol to pregnenolone by CYP11A1. It is attractive to hypothesize that the rate-limiting step in vertebrate steroid hormone biosynthesis is also specifically regulated by unidentified transcriptional and/or splicing regulator (s). Whereas no apparent orthologs of ouib are found in vertebrates, their genomes possess a ZAD-ZFP gene called ZFP276, which is a tumor suppressor gene [66]. Interestingly a ecd ortholog is also found in humans and may also contribute to the malignancy of certain tumor types [65]. It would be worth examining roles of these genes in steroid hormone biosynthesis in vertebrates.
Drosophila melanogaster flies were reared on standard agar-cornmeal medium at 25°C under a 12: 12 h light/dark cycle. w1118, yw and Oregon R were used as the wild type strain. phm–GAL4#22 [55] and w; UAS-dicer2; phm-GAL4#22/TM6 Ubi-GFP was used as the strain to drive forced gene expression in the PG. UAS-spo [20] and UAS-spok-IR [21] transgenic flies were obtained from Hiroshi Kataoka (The University of Tokyo) and Hajime Ono (Kyoto University), respectively. y1 v1 nos-phiC31; attP40, v1 and y2 cho2 v1; attP40{nos-Cas9}/CyO [53] were obtained from National Institute of Genetics, Japan. The w; snaSco/CyO; P{w+mC = tubP-GAL80ts}7 (stock number #130453) and w1118; Df (3R) ED5330/TM6C Sb1, a deficiency strain that deletes a genomic region including the ouib locus (stock umber #150241) [67], were obtained from Drosophila Genetic Resource Center. UAS-torso-IR (stock number #101154) and UAS-mld-IR (stock number #17329) were obtained from the Vienna Drosophila RNAi center. Digoxygenin (DIG) -labeled antisense RNA probes were synthesized using DIG RNA labeling mix (Roche) and T3 and T7 RNA polymerase (Fermentas). To generate the ouib probe, the ouib ORF was amplified by PCR with cDNA derived from whole bodies of Oregon R larvae and the primers described in S3 Table. PCR product was inserted into SmaI-digested pBluescript II SK (-), and then used as the templates for synthesizing RNA probes. Fixation, hybridization and detection were performed as [23,68]. RNA was isolated using the RNAiso Plus reagent (TaKaRa). Genomic DNA digestion and cDNA synthesis were performed using the ReverTra Ace qPCR RT Kit (TOYOBO). qRT-PCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) or Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 or TP870 system (TaKaRa). Serial dilutions of a plasmid containing the ORF of each gene were used as a standard. The expression levels of the target genes were normalized to an endogenous control ribosomal protein 49 (rp49) in the same sample. The primers for quantifying D. melanogaster ouib and E75A are described in S3 Table. Primers amplifying nvd, sro, spok, phm, dib, sad and rp49 were previously described [22,55]. Tissue dissections were performed in PBS followed by fixation in 4% PFA for 20 minutes at room temperature. For this study, the following primary antibodies were: mouse anti-FLAG M5 (1: 1,000) (Sigma); rabbit anti-Phm (1: 200) [30], guinea pig anti-Spok (1: 200) [61]; guinea pig anti-Sro (1: 1,000) [62]. Tissues were incubated over night with primary antibodies at 4°C. Fluorescent conjugated secondary antibodies used in this study, goat anti-mouse Alexa Fluor 488, goat anti-guinea pig Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555 and goat anti-guinea pig Alexa Fluor 555, were purchased from Life Technologies. Secondary antibodies were diluted 1: 500 and incubated for 1 hour at room temperature. Confocal images were captured using Carl Zeiss LSM 700 laser scanning microscope. The GAL4-UAS system [69] was used to overexpress genes in D. melanogaster. To generate pUAST vector to overexpress ouib, specific primers including a sequence coding FLAG tag at N terminal were used for PCR to add EcoRI and XbaI sites to the 5´ and 3´ ends, respectively (S3 Table). Template cDNAs were reverse transcribed using total RNA of the ring gland from D. melanogaster using ReverTra Ace qPCR RT Kit (TOYOBO). PCR was performed using KOD Plus Neo (TOYOBO). The amplified CDS region of ouib was digested with EcoRI and XbaI, and then ligated into a pWALIUM10-moe vector [70]. Transformants were established by BestGene, Inc. Generation of the ouib allele was carried out by CRISPR/Cas9 system using the pBFv-U6. 2 vector [53] provided by the National Institute of Genetics, Japan. We selected 2 independent target sites (target#1 and target#2 as shown in Fig 2). To minimize off-target effects of CRISPR/Cas9 system, we confirmed by BLAST search that no 15 nucleotide stretches within the selected target sequence (23 nucleotides including PAM motif) matched any other sequence on the 3rd chromosome. Sense and antisense oligonucleotides corresponding to sgRNA target sequences (S3 Table) were annealed and inserted into BbsI-digested pBFv-U6. 2 vector. The ouib sgRNA vectors were injected into the embryos of the y1 v1 nos-phiC31; attP40 strain. The nos-Cas9-based gene targeting was carried out as previously described [53]. Males carrying nos-Cas9 and a sgRNA transgene were crossed to wild-type flies by mass mating. From their progeny, 10 and 50 single males for the target#1 and target#2 sites, respectively, were isolated. Each male was crossed with w; TM3 Sb/TM6 Tb females and then the independent isogenized strains were established. Among them, we surveyed the strains showing homozygous lethality and eventually 1 target#1 and 29 target#2 lethal strains were selected. To confirm indel mutations at ouib locus in each strain, we performed the T7EI assay as previously described [53]. In this assay, genome DNA from the heterozygous adults of each strain was extracted as previously described [53]. To amplify the DNA fragment including Cas9 target sites, PCR was conducted with KOD FX Neo (TOYOBO), the extracted genome DNA, and the primers listed in S3 Table [53]. The PCR products were treated with T7 endonuclease (NEB). The reacted samples were analyzed by agarose gel electrophoresis. Out of 30 total candidate strains, 1 target#1 and 8 target#2 strains were selected as candidate flies possessing indel mutations in ouib region. The PCR products from the 9 strains were subcloned into a SmaI-digested pBluescript II (Promega) and then sequenced with T3 and T7 primers. We detected small deletions in 8 out of the 9 strains. The minimal and maximal deletion sizes were 1 bp and 13 bp, respectively. We chose 1 strain for each target sites for further analyses and renamed them ouib29 and ouib74, both of which caused frameshift mutations for ouib locus (Fig 2). ouib29/TM3 Act-GFP flies, ouib74/TM3 Act-GFP flies and w1118 flies were crossed each other. Eggs were laid on grape plates with yeast pastes at 25°C for 8 hours. 36 hours AEL, 100 hatched GFP negative (ouib29/+, ouib74/+ and ouib29/ouib74) first instar larvae were transferred into vials with standard cornmeal food (25 animals per vial). Every 24 hours, developmental stages were scored by tracheal morphology as previously described [22]. For the rescue experiments, 20 mg of dry yeast was mixed with 38 μl H2O and 2 μl ethanol or supplemented with 2 μl of the following sterols dissolved in ethanol: cholesterol (Wako; 150 mg/ml), 7-dehydrocholesterol (Sigma; 150 mg/ml), 5β-ketodiol (kindly gifted from Yoshinori Fujimoto, Tokyo Institute of Technology; 150 mg/ml) and 20-hydroxyecdysone (Sigma; 50 mg/ml). We crossed ouib29/TM3 Ser1 GMR2 Act-GFP flies with ouib74/TM3 Ser1 GMR2 Act-GFP flies. Eggs were laid on grape plates with yeast pastes at 25°C for 12 hours. At 36 hours AEL, 50 hatched GFP negative (ouib29/ouib74) first instar larvae were transferred to the yeast paste on grape plates and kept at 25°C. Every 24 hours, developmental stages were scored by tracheal morphology as previously described [22]. For the rescue experiments of ouib mutant by ouib overexpression, ouib29 phm-GAL4#22/TM3 Act-GFP was established by chromosomal recombination. The flies of UAS-FLAG-ouib-1M; ouib74/TM6 Ubi-GFP were crossed with the flies of ouib29 phm-GAL4#22/TM3 Act-GFP, the flies of ouib74/TM3 Act-GFP were crossed with the flies of ouib29 phm-GAL4#22/TM3 Act-GFP, and the flies of UAS-FLAG-ouib-1M; ouib74/TM6 Ubi-GFP were crossed with the flies of ouib29/TM3 Act-GFP. Eggs were laid on grape plates with yeast pastes at 25°C for 12 hours. At 36 hours AEL, 50 hatched GFP negative (UAS-FLAG-ouib-1M/+; ouib29 phm-GAL4#22/ouib74, ouib29 phm-GAL4#22/ouib74 and UAS-FLAG-ouib-1M/+; ouib29/ouib74) first instar larvae were transferred to the standard agar-cornmeal medium. Developmental stages were scored 108 hours AEL by tracheal morphology as previously described [22]. For the rescue experiments of spok RNAi by spo overexpression, UAS-spok-IR UAS-spo was established by chromosomal recombination on third chromosome. The flies of UAS-spok-IR UAS-spo strain was crossed with w; UAS-dicer2; phm-GAL4#22/TM6 Ubi-GFP flies. Eggs were laid on standard agar-cornmeal medium at 25°C for 24 hours. After 7 days, developmental stages of the animals on the wall were scored by presence of TM6 balancer. For the rescue experiments of ouib mutant by spo overexpression, Roi/CyO; ouib29 phm-GAL4#22/TM6, Roi/CyO; ouib29 UAS-spo/TM6 and Roi/CyO; ouib74 UAS-spo/TM6 were established by chromosomal recombination on third chromosome. The flies of Roi/CyO; ouib29 phm-GAL4#22/TM6 were crossed with Roi/CyO; ouib74 UAS-spo/TM6, the flies of Roi/CyO; ouib29 phm-GAL4#22/TM6 were crossed with Roi/CyO; ouib74/TM6 and Roi/CyO; ouib74/TM6 were crossed with Roi/CyO; ouib29 UAS-spo/TM6. Eggs were laid on standard agar-cornmeal medium at 25°C for 24 hours. After 7 days, developmental stages of the animals on the wall were scored by presence of TM6 balancer. ouib29/TM3 Ser1 GMR2 Act-GFP flies and w1118 flies were crossed with ouib74/TM3 Ser1 GMR2 Act-GFP flies. Eggs were laid on grape plates with yeast pastes at 25°C and the hatched larvae were cleared. After 8 hours, GFP negative (ouib74/+ and ouib29/ouib74) first instar larvae were transferred into vials with standard cornmeal food. At 12 hours AH, whole larvae were rinsed in water and homogenized in 50 μl methanol and supernatant was collected following centrifugation at 14,000 rpm at 4°C. The remaining tissue was re-extracted in 50 μl methanol over night at 4°C. The supernatants were evaporated using a EYELA CVE-2000 (Tokyo Rikakikai) and redissolved in 50 μl EIA buffer [0. 1 M PBS/0. 1% BSA, 0. 4 M NaCl, 1 mM EDTA and 0. 01% NaN3]. ELISA was performed according to manufacturer’s instructions using 20-Hydroxyecdysone EIA Antiserum, 20-Hydroxyecdysone AChE Tracer and Ellman’s Reagent (Cayman Chemical) that detects 20-hydroxyecdysone with the same affinity. Standard curves were generated using 20E (Sigma). Absorbance was measured at 415 nm on a plate reader, Multiskan GO (Thermo Scientific) using the SkanIt Software 3. 2 (Thermo Scientific). To generate the spok>GFP reporter construct, a ~1. 4 kb fragment immediately upstream of the spok transcription unit was amplified from yw genomic DNA using the primers 1. 45spok-p_F and 1. 45spok-p_R (S3 Table). This fragment was first subcloned into the pCR2. 1-TOPO vector (Life Technologies) and then removed as an EcoRI fragment and cloned into the Drosophila transformation vector pH-Stinger [71]. To refine the location of the PG enhancer, seven 250–300 bp overlapping fragments that covered the entire 1. 4 kb fragment were derived through PCR and each cloned into hH- Stinger. The only fragment that gave expression in the PG of transgenic animals was the ~300 bp fragment immediately upstream of the transcriptional start site. This fragment was generated using the primers 300spok-p_F and 300spok-p_R (S3 Table). Transgenic lines were generated through standard means using a w1118 host background. The GFP reporter strains of spok>GFP; ouib29/TM6 Ubi-GFP and spok>GFP; ouib74/TM6 Ubi-GFP were established and crossed each other. Eggs were laid on grape plates with yeast pastes at 25°C for 4 hours. The first instar larvae were dissected 36 hours AEL and immunostained. The upstream regions of spok were amplified from Oregon R genomic DNA by specific primers to add SacI and BglII sites to the 5´ and 3´ ends, respectively. PCR was performed using KOD Plus Neo (TOYOBO). The amplified upstream regions of spok were digested with SacI and BglII, and then ligated into a pGL3-Basic vector luciferase reporter plasmid (Promega). Reporter plasmids carrying mutated regions were constructed from the pGL3-Basic plasmid containing WT upstream 300 bp region by inverse PCR. The primers for PCR are listed in S3 Table. S2 cells were seeded in 1 ml Schneider’s Drosophila Medium (GIBCO) in a 24-well plate (greiner bio-one) 1 day before transfection. Transfection of S2 cells was performed using the Effectene Transfection Reagent (Qiagen). GFP-pUAST [23] and FLAG-ouib-pWALIUM10-moe plasmids were transfected, respectively, along with the Actin5C-GAL4 construct (a gift from Yasushi Hiromi, National Institute of Genetics) and the luciferase reporter plasmids. The Copia Renilla Control plasmid (addgene; #38093) [72] was used as the reference. The cells were incubated for 2 days after transfection. Then they were processed by using the Dual-Luciferase Reporter Assay System (Promega) in accordance with the manufacturer’s instructions and were analyzed with Flash’n glow LB 955 (Berthold Technologies). S2 cells overexpressing FLAG-ouib or FLAG-M1BP were collected and washed with TBS. Cells were then centrifuged at 4000 g at 4°C for 5 min. The pellet was suspended and vortexed with 400 μl Buffer A [10 mM Hepes pH 7. 9,10 mM KCl, 1 mM DTT and 1 unit Complete Mini (Roche) ] and 25 μl 10% NP-40. Then sample was centrifuged at 1500 g at 4°C for 5 min. The pellet was suspended and vortexed with 50 μl Buffer C [20 mM Hepes pH7. 9,400 mM NaCl, 2 mM MgSO4,1mM DTT and Complete Mini (Roche) ], then shaked at 4°C for 30 min. After shaking, sample was centrifuged at 14,000 rpm at 4°C for 5 min and supernatant was collected. Samples were boiled with SDS sample buffer [150 mM Tris-HCl pH 6. 8,0. 6% SDS, 15% glycerol, 0. 009 mg/μl Bromophenol blue, 5% 2-mercaptoethanol and 1 unit Complete Mini (Roche) ] for 5 min, and loaded on 12% polyacrylamide gel followed by transfer onto PVDF membrane (GE Healthcare). Anti-FLAG M5 monoclonal antibody (1: 1,000; Sigma) was used for primary antibody and ECL Peroxidase labeled anti-mouse antibody (1: 10,000; GE Healthcare) was used for secondary antibody. The band was detected by ECL Ultra Lumigen TMA-6 (GE Healthcare) and Ez-Capture MG (ATTO). Preparation of S2 cell nuclear extracts is described in the Supplemental Materials. ABCD assay was conducted essentially as previously described [73]. Biotin-labeled DNA probes were purchased from Life Technologies. The probes were incubated with Dynabeads M-280 Streptavidin (Life Technologies) at room temperature for 15 min. DNA-beads complexes were mixed with nuclear extracts and ABCD Binding Buffer [50 mM Hepes pH 7. 9,150 mM NaCl, 0. 5% Triton X-100,20 ng/μl poly (dI/dC) ], and incubated at 4°C for 1 hour. After incubation, the beads were washed with ABCD Binding Buffer. The biotin-labeled oligonucleotides are listed in S3 Table. GST proteins fused with or without 150–313 amino acid residues of Ouib (GST-Ouib-Zf) containing 5 zinc finger domains were expressed using pGEX-4T-3 vector system (GE Healthcare) in Escherichia coli BL-21 strain. E. coli cells were harvested and crashed with sonication. GST alone and GST-Ouib-Zf were purified from the supernatant with AKTA start equipped with GSTrap affinity column (GE Healthcare). Electrophoretic mobility shift assay was conducted as previously described [74,75]. 45 bp double-stranded oligonucleotide probes containing wild type M1BP binding site, wild type and mutated (transversion) Ouib response element were prepared by annealing single-strand oligonucleotides listed in S3 Table. The wild type M1BP binding site was derived from the smoothened promoter [51]. Double-stranded DNA fragment was end-labeled by using T4 polynucleotide kinase (TOYOBO) and [γ-32P]ATP. GST or GST-Ouib fusion proteins (400 ng) were incubated for 30 min at 4°C in the reaction mixture [12 mM Hepes, pH 7. 9,1 mM dithiothreitol, 1 mM EDTA, 60 mM KCl, 4 mM MgCl2,2 mM ZnSO4,50 ng/ul poly (dI-dC), 1 mg/ml BSA and 12% Glycerol] in the presence or absence of 100–200-fold molar excess of specific double-stranded competitor DNA. A radiolabeled DNA probe (0. 3 ng, 40,000 cpm) was added, and the incubation was continued for 20 min at 4°C. The incubation mixture was directly loaded on a 5% non-denaturing polyacrylamide gel in 1 × TBE buffer [89 mM Tris-HCl, pH 8. 0,89 mM boric acid, and 2 mM EDTA], and electrophoresed at 4°C with buffer circulation. The gels were dried and analyzed with a bio-imaging analyzer Typhoon 8600 (Amersham Pharmacia Biotech Inc). The competitor oligonucleotides used are listed in the S3 Table.
Steroid hormones are crucial for development and reproduction in multicellular organisms. The spatially-restricted expression of almost all steroid biosynthesis genes is key to the specialization of steroid producing cells. In the last decade, insects have become the focus for research on the biosynthesis of the principal steroid hormones, ecdysteroids. However, the transcriptional regulatory mechanisms controlling the ecdysteroid biosynthesis genes are largely unknown. Here we show that a novel zinc finger transcription factor Ouija board (Ouib) is essential for activating the expression of one ecdysteroid biosynthesis gene, spookier, in the ecdysteroid producing cells. Ouib is the first invertebrate transcription factor that is predominantly expressed in the steroidogenic organs and essential for development via inducing expression of the steroidogenic gene. In addition, this is the first report showing the catalytic step-specific control of steroid hormone biosynthesis through transcriptional regulation.
The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier