What this is
- This research investigates the roles of nuclear receptor genes HR3 and E75 in the circadian rhythms of the primitive insect firebrat (Thermobia domestica).
- The study identifies HR3 and E75 as essential for the rhythmic expression of the cyc gene, which is crucial for maintaining locomotor rhythms.
- Using (), the researchers demonstrate that silencing these genes disrupts the transcriptional rhythms of key clock genes, affecting locomotor activity.
Essence
- HR3 and E75 are crucial for the in firebrats, affecting the expression of cyc and other clock genes. experiments reveal their role in maintaining locomotor activity rhythms.
Key takeaways
- HR3 and E75 are rhythmically expressed in firebrats, peaking late in the day to early night. Their normal expression is necessary for sustaining locomotor rhythms.
- of HR3 or E75 alters the rhythmic expression of cyc, tim, and Clk genes, suggesting these nuclear receptors are vital for the phase relationship among clock genes.
- Both HR3 and E75 also regulate ecdysis, linking circadian rhythms with developmental processes in firebrats.
Caveats
- The study relies on , which may have off-target effects that could influence the results. Further validation is needed to confirm the specific roles of HR3 and E75.
- The research focuses on a single primitive insect species, limiting the generalizability of findings to other insects or organisms.
Definitions
- circadian rhythm: A biological process that displays an endogenous, entrainable oscillation of about 24 hours, responding to light and dark cycles.
- RNA interference (RNAi): A biological process in which RNA molecules inhibit gene expression or translation, effectively silencing targeted genes.
AI simplified
Introduction
Circadian clocks provide an adaptive advantage by coordinating physiological, behavioral, and biochemical events to occur at an appropriate time of the day [1]. The clock is believed to consist of transcriptional/translational feedback loops. In the fruit fly Drosophila melanogaster, molecular biological studies have shown that the transcriptional activators CLOCK (CLK) and CYCLE (CYC) heterodimerize and bind to the E-box within the promoters of the per and tim genes to activate their transcription [2]–[4]. PER and TIM proteins accumulate and, subsequently, associate with the CLK/CYC complex to repress their own transcription, forming an about 24-h period feedback loop for the rhythmic expression of per and tim. In addition, the transcription of Clk is also under circadian regulation [5], [6]. It is controlled by cyclical and reciprocal activities of the basic leucine zipper transcription factors VRILLE (VRI) and PAR DOMAIN PROTEIN 1ε (PDP1ε). Both VRI and PDP1ε competitively bind to the VRI/PDP1ε binding site (V/P-box) within the Clk promoter to repress or activate transcription, respectively [7]. Mutations in vri and Pdp1ε affect locomotor activity rhythms, as well as the CLK expression levels [6], [8]. The expression of vri and Pdp1ε is under circadian control, which requires CLK/CYC, thereby connecting the PER/TIM and VRI/PDP1ε feedback loops.
The firebrat Thermobia domestica also possesses a circadian clock based on the rhythmic expression of tim under the control of Clk and cyc[9], [10]. However, unlike Drosophila, cyc, but not Clk, was rhythmically expressed in both light–dark cycles (LD) and constant darkness (DD) [9]. Similar patterns of cyc and Clk expression have been reported for Gryllus bimaculatus and Apis mellifera[11]–[13]. While a large body of genetic and biochemical evidence has been accumulated on the regulation of per, tim, and Clk expressions, little is known about the control of cyc. In the mammalian clock, the cyc ortholog Bmal1 is cyclically expressed, and the underlying mechanism has been extensively studied. Two nuclear receptors, RORα and REV-ERBα, are known to be involved in the mechanism. They directly regulate the expression of Bmal1 by binding to a specific ROR/REV-ERB response element in the Bmal1 promoter region [14]. RORα stimulates Bmal1 transcription, whereas REV-ERBα represses it [15], [16]. Therefore, we expected that cyc might be regulated by a mechanism similar to that found in mammals.
In this study, by taking advantage of the efficacy of RNAi, we investigated the role of probable nuclear hormone receptor 3 gene (HR3), a Ror ortholog, and ecdysone induced protein 75 gene (E75), a Rev-erb ortholog, for the first time in the insect circadian clock by using the firebrat, one of the most primitive insect species. Surprisingly, the gene silencing of HR3 by RNAi revealed the rhythmic expression of Clk, suggesting that this ancestral insect possesses a mechanism for the rhythmic expression of both cyc and Clk genes. The RNAi of HR3 and E75 disrupted the expression levels and phase relationship of the clock genes, leading to a loss of the locomotor activity rhythm. HR3 and E75 were also found to be involved in the regulation of ecdysis. Based on the obtained results, we propose a unique clock model for this ancestral insect.
Materials and Methods
Animals
Adult firebrats, Thermobia domestica, were used for all experiments. They were taken from our laboratory culture reared under a light cycle of 12-h light and 12-h dark conditions (LD 12∶12) at a constant temperature of 30°C. They were fed laboratory chow (CA-1, Clea Japan, Tokyo).
cDNA cloning ofand HR3 E75
Total RNA was extracted with the TRIzol Reagent (Invitrogen, Carlsbad, CA) from 5 adult firebrats collected at zeitgeber time (ZT) 10 (ZT0 corresponds to light on and ZT12 to light off). A 5 µg of total RNA was used for reverse transcription to obtain cDNA, using SuperScript III (Invitrogen). Using the obtained cDNA as a template, we performed PCR with degenerate primers deduced from the conserved amino acid sequences among insect HR3 and E75 homologs (Table 1). The amplified fragments were cloned into T-Vector pMD20 (Takara, Ohtsu, Japan) and sequenced with the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The 5′ and 3′ RACEs were performed with the GeneRacer Kit (Invitrogen) and SMARTer RACE cDNA Amplification Kit (Takara), respectively, using Blend Taq Plus (Toyobo, Osaka, Japan) with gene-specific primers as follows: 5′-TTCCTCGGGCACTGATAGTT-3′ for 5′RACE, and 5′-CGAGGTTCGGTTTCACAGAG-3′ for 3′RACE of HR3, and 5′-GACGCCTGCTTTGAGAAGAG-3′ for 5′RACE, and 5′-AAGCTGGACTCGCCTAATGA-3′ for 3′RACE of E75. RACE fragments were purified, cloned, and sequenced as mentioned above. Sequences were analyzed by Genetyx v.6 (Genetic Information Processing Software, Tokyo, Japan) and BioEdit v.7.0.9.0 (Biological Sequence Alignment Editor, Ibis Therapeutic, Carlsbad, CA). Amino acid sequences of HR3 and E75 were analyzed, and a neighbor-joining tree was inferred with ClustalW (http://clustalw.ddbj.nig.ac.jp/↗). Known sequences of insects were obtained from GenBank.
| Genes | Forward | Reverse |
|---|---|---|
| Degenerate PCR | ||
| HR3 | 5′-ccagtcctccgtggtgaaYtaYcaRtg-3′ | 5′-cggggaccatcttggcRaaYtcDat-3′ |
| E75 | 5′-cgcatcctggccgcNatgcaRca-3′ | 5′-acttcttgtggggcttgtaggYYtcRtccat-3′ |
| Quantitative RT-PCR | ||
| HR3 | 5′-CGAGGTTCGGTTTCACAGAG-3′ | 5′-GAGGAGGACGGTTGTTGTTG-3′ |
| E75 | 5′-GAAATGCCCAGCTACACCAC-3′ | 5′-GAACTCAACAACCCCACGAA-3′ |
| Timeless | 5′-TACAAGCCAGGTCCATCACA-3′ | 5′-TCAAGCGTCAATTCAGCATC-3′ |
| Clock | 5′-ATCGCAAGGGTCTGGAAGTG-3′ | 5′-GGAAAACTCGCCAAGACAGG-3′ |
| Cycle | 5′-CGTGTAATCTGTCGTGTTTGGTG-3′ | 5′-GAATCGTCCGCCTTTCCTC-3′ |
| rp49 | 5′-AGTCCGAAGGCGGTTTAAGG-3′ | 5′-TACAGCGTGTGCGATCTCTG-3′ |
| dsRNA synthesis | ||
| HR3#1 | 5′-TAATACGACTCACTATAGGGACTTCTCCTGCCTCGGGTAT-3′ | 5′-TAATACGACTCACTATAGGGCAGTTTTAGCTCCGCCAAAC-3′ |
| HR3#2 | 5′-TAATACGACTCACTATAGGGCGAAAATGGTACCTGGCTTT-3′ | 5′-TAATACGACTCACTATAGGGTCGCTTGAATTTACCCAAGG-3′ |
| E75#1 | 5′-TAATACGACTCACTATAGGGAGTCAAAGTCACCCCGAGAA-3′ | 5′-TAATACGACTCACTATAGGGTCATTAGGCGAGTCCAGCTT-3′ |
| E75#2 | 5′-TAATACGACTCACTATAGGGCAGAACCAATGGTGGTTTCC-3′ | 5′-TAATACGACTCACTATAGGGGCCGCTGTTGTTGTAGAGGT-3′ |
| timeless | 5′-TAATACGACTCACTATAGGGTGCATTTGGTTGTGACTGCT-3′ | 5′-TAATACGACTCACTATAGGGAGAGGCGTGTGCCTTGTACT-3′ |
| Clock | 5′-TAATACGACTCACTATAGGGACCACCAATCGAAAAATGGA-3′ | 5′-TAATACGACTCACTATAGGGCCCAGTTCCCACGAAAACTA-3′ |
| cycle | 5′-TAATACGACTCACTATAGGGAGGGGCTGTTCATTCCTACA-3′ | 5′-TAATACGACTCACTATAGGGCGCCCACGACTTCAAATAAC-3′ |
| DsRed2 | 5′-TAATACGACTCACTATAGGGTCATCACCGAGTTCATGCG-3′ | 5′-TAATACGACTCACTATAGGGCTACAGGAACAGGTGGTGGC-3′ |
Quantitative real-time RT-PCR
Quantitative reverse-transcription PCR (qPCR) was used to measure mRNA levels. Total RNA extraction from adult firebrats was performed using the TRIzol Reagent, and the obtained RNA was treated with DNase I to remove contaminating DNA. Approximately 500 ng of total RNA of each sample was reverse transcribed with random 6mers using the PrimeScript RT Reagent Kit (Takara). Real-time PCR was performed with the Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA) using the Universal SYBR Green Master (Roche, Tokyo, Japan), including SYBR Green with primers for HR3, E75, tim (GenBank/EMBL/DDBJ Accession No. AB644410↗), Clk (AB550828↗), cyc (AB550829↗), and rp49 (AB550830↗) (Table 1). In all cases, a single expected amplicon was confirmed by melting analysis. The results were analyzed using the software associated with the instrument. The quantification of mRNA levels was performed by the standard curve method, and the values were normalized with those for rp49, a housekeeping gene, at each time point. Results of 3–4 independent experiments were pooled to calculate the mean ± SEM. Data were analyzed by t-test or ANOVA.
RNA interference
Double-stranded RNAs (dsRNA) for HR3, E75, tim, Clk, and cyc were synthesized using the MEGAscript High Yield Transcription Kit (Ambion, Austin, TX) as described previously [10]. DsRed2 derived from a coral species (Discosoma sp.) was used for negative control because the firebrat does not possess the gene; its dsRNA was synthesized in the same procedure. Primers fused with the T7 promoter sequence were designed for synthesizing the dsRNAs (Table 1). The dsRNA solution was stored at −80°C until use. A total of 69 nl (10 µM) of the dsRNA solution was injected using the Nanoliter Injector (WPI, Sarasota, FL) into the abdomen of adult firebrats anesthetized with CO2. The injected amount of dsRNA solution corresponded to 10.23–13.95 µg/g (body weight). The total RNA was extracted from the whole body 1 week after the injection when the effect of the RNA was expected to reach its maximal potential [17].
Recording of locomotor activity
To monitor locomotor activity, adult firebrats were individually housed in transparent acrylic rectangular tubes (6×6×70 mm) as previously described [10]. The raw data were displayed as conventional double-plotted actograms to judge the activity patterns and existence of the rhythmicity, and free-running periods were analyzed by the Lomb-Scargle periodogram by using actogramJ [18], [19]. If the peak of the periodogram appeared above the 0.05 confidence level, then the period of the peak was designated as statistically significant.
Hormonal treatment
20 hydroxyecdysone (20E) (Sigma, St Louis, MO, USA) was dissolved in 100% EtOH (0.5 mg/ml) and 69 nl (34.5 ng) was injected into the abdomen of adult firebrats anesthetized with CO2. The control was treated with an equivalent amount of 100% EtOH. The total RNA from whole body was isolated at 1week after the injection to measure the mRNA levels of HR3 and E75.
Results
Cloning and sequencing ofandcDNA HR3 E75
The cDNAs of the nuclear receptors HR3 and E75 were obtained from T. domestica (GenBank Accession Nos. AB829732↗ for HR3 and AB829733↗ for E75). The T. domestica HR3 (Td'HR3) and Td'E75 cDNAs encode proteins of 543 and 848 aa residues, respectively, and have 5′- and 3′-untranslated regions (UTR) of 61 and 228 bp, and 98 and 102 bp, respectively. Fig. 1 shows the alignment of the obtained amino acid sequences of Td'HR3 and Td'E75 with those of several other species. Td'HR3 shares 74% sequence identity with Tribolium castaneum HR3 (Tc'HR3). Its DNA-binding domain (DBD) contains two zinc fingers that share 95–100% identity with the HR3 orthologs from other species (Table 2). The ligand-binding domain (LBD) shared 61–83% identity with those of other insects' HR3 orthologs. Td'E75 shared 45–61% identity with other insects' E75 orthologs and its DBD and LBD were nearly 100% and 69–88% identical, respectively, to those of the E75 orthologs. The DBD was equipped with a zinc finger motif, which shares 100% identity (Fig. 1B; Table 2). These are the first HR3 and E75 orthologs in Thysanura insects.
A phylogenetic tree based on the amino acid sequences of HR3 and E75 from known insects, sea urchins, and some vertebrates revealed three clusters of HR3 and E75. Td'HR3 formed a cluster with the beetle Tribolium castaneum, the cockroach Blattella germanica, the honeybee Apis mellifera, the bumble bee Bombus terrestris, and the ant Harpegnathos saltator, while Td'E75 with the beetle Tribolium castaneum, the cockroach Blattella germanica, and the louse Pediculus humanus corporis (Fig. 1).
Sequence alignments of conserved domains and phylogenetic neighbor-joining tree ofand. HR3 E75 (A, B) Schematic structure of various HR3 (A) or E75 (B) proteins, comparing the organization of the 3 conserved domains, DNA-binding domain (DBD), zinc finger domain (ZIF), and ligand binding domain (LBD). The numbers on the right indicate the number of amino acid residues.;;;. (C, D) A phylogenetic neighbor-joining tree of known animal HR3 or E75 proteins. The GenBank or RefSeq accession numbers are indicated in brackets. A reference bar indicates distance as the number of amino acid substitutions per site. Td, Thermobia domestica Dm, Drosophila melanogaster Dp, Danaus plexippus Mm, Mus musculus
| Species | Identity | Similarity | Identity | |||
|---|---|---|---|---|---|---|
| DBD | Zinc Finger 1 | Zinc Finger 2 | LBD | |||
| HR3 | ||||||
| Daphnia pulex | 65 | 73 | 97 | 100 | 95 | 78 |
| Drosophila melanogaster | 61 | 76 | 97 | 100 | 95 | 61 |
| Pediculus humanus corporis | 66 | 74 | 100 | 100 | 100 | 83 |
| Tribolium castaneum | 74 | 84 | 100 | 100 | 100 | 83 |
| E75 | ||||||
| Daphnia pulex | 55 | 68 | 100 | - | 100 | 77 |
| Drosophila melanogaster | 49 | 61 | 100 | - | 100 | 69 |
| Pediculus humanus corporis | 55 | 65 | 100 | - | - | 87 |
| Tribolium castaneum | 61 | 68 | 100 | - | 100 | 88 |
Temporal expression patterns ofandmRNA Td'HR3 Td'E75
To determine whether the transcripts of Td'HR3 and Td'E75 oscillated in a circadian manner, we measured the levels of their mRNA under LD 12∶12 and on the 2nd day of DD by using qPCR (Fig. 2). In LD, both Td'HR3 and Td'E75 mRNAs were rhythmically expressed, peaking at late day or early night (ANOVA, P<0.03, S1 Table). Their rhythmic expressions persisted in DD (ANOVA, P<0.05, S1 Table), thus, suggesting their circadian control.
Expression profiles ofandmRNA and effects of their dsRNAs on their mRNA levels and the survival rate in firebrats. Td'HR3 Td'E75 Both(A, C) and(B, D) were rhythmically expressed in LD (A, B) and DD (C, D), peaking late (subjective) day to early (subjective) night (black symbols). Ds(pink triangles and squares for #1 and #2, respectively) and ds(green triangles and squares for #1 and #2, respectively) downregulated mRNA levels and disrupted the rhythm of their respective genes (A and B). Treatment with dsas a negative control did not disrupt the rhythmic expression in both genes (A and B, blue symbols), but caused a significant decrease inmRNA levels at ZT 2 and ZT 18 (+<0.05,test vs intact). White, black, and gray bars above each graph indicate day, night/subjective night, and subjective day, respectively. Total RNA was extracted from firebrats collected at 4-h intervals starting 2 h after lights on or 2 h into subjective day (ZT2 or CT2, respectively). The data collected from 3–4 independent experiments were averaged and plotted as the mean ± SEM values relative to the value ofmRNA used as the reference. Values with different letters significantly differ from each other within the same treatment groups (<0.05, ANOVA with Tukey test,). *<0.05, **<0.01,test vs ds(exactvalues are shown in). (E) Survival rate after injection of 10.23–13.95 µg/g ds, ds, or dsinto the abdomen. Dsand dssignificantly reduced the survival rate. For further explanation, see text. Td'HR3 Td'E75 HR3#1,2 E75#1,2 DsRed2 Td'HR3 P t rp49 P P P t DsRed2 P HR3 E75 DsRed2 HR3 E75 S1 Table S2 Table
RNAi ofand Td'HR3 Td'E75
To examine the possible role of Td'HR3 and Td'E75 in the firebrat circadian clock, we examined the effectiveness of RNAi on Td'HR3 and Td'E75 one week after the treatment. We first examined the effects of dsRNA of DsRed2 used as negative control. As shown in Fig. 2A, Td'HR3 was slightly reduced but the reduction was not significant at most time points, except for ZT 2 and ZT 18. No significant effect was observed in Td'E75 transcript levels (Fig. 2B).
For RNAi of Td'HR3 and Td'E75, we used two different dsRNAs synthesized for different regions of the two genes (Table 1). The Td'HR3 RNAi and Td'E75 RNAi firebrats showed a high lethality because of ecdysis failure (Fig. 2E); 50% of the treated firebrats died within two weeks. This may be because these genes are involved in the ecdysone-signaling pathway involved in ecdysis. When examined 1 week after dsRNA treatment, the dsRNAs significantly reduced respective mRNAs (Fig. 2A–B, S2 Table) to near or below basal levels of intact firebrats and the levels were maintained throughout the day with no rhythmic changes (ANOVA, P>0.1, S1 Table). Thus, the expression of Td'HR3 and Td'E75 were successfully suppressed through RNAi. The levels of knockdown were similar to those known for Td'Clk and Td'cyc but less than for Td'tim[9], [10].
dsRNA anddsRNA treatments disrupt circadian locomotor activity and ecdysis Td'HR3 Td'E75
In order to examine the role of Td'HR3 and Td'E75 in the regulation of circadian behavioral rhythms, we compared locomotor rhythms between Td'HR3 RNAi and Td'E75 RNAi firebrats and control firebrats with DsRed2 dsRNA injected in LD and DD (Fig. 3). Because Td'HR3 RNAi and Td'E75 RNAi firebrats showed a high lethality (Fig. 2E), we performed analysis of the locomotor activity rhythm only for firebrats survived for more than two weeks after the injection of dsRNA. The results are summarized in Table 3. Most of the DsRed2 RNAi firebrats exhibited a nocturnal rhythm synchronizing to LD and free running in DD with a period of about 24 h (Fig. 3E and F), similar to intact animals [9], [10]. However, the free-running period considerably varied among individuals, showing rather large standard deviation (Table 3). In the Td'HR3 RNAi and Td'E75 RNAi firebrats, 100% (6/6) and 71% (12/17) of animals, respectively, exhibited, under LD, a rhythmic activity synchronized to the LD (Fig. 3A–D; Table 3), and the remaining animals were arrhythmic (Fig. 3B). In the ensuing DD, 75% (6/8) and 100% (4/4) animals for Td'HR3 dsRNA#1 and #2 treatment, respectively, and 78% (7/9) and 83% (5/6) of animals for Td'E75 dsRNA#1 and #2 treatment, respectively, almost immediately became arrhythmic (Fig. 3A–D; Table 3), suggesting that the rhythm observed in LD was a masking effect of the light. Interestingly, none of Td'HR3 and Td'E75 RNAi firebrats showed ecdysis. Thus, the interruption of the locomotor activity associated with ecdysis was not observed (Fig. 3A–D).
Effects ofanddsRNA on locomotor rhythms in the firebrat. HR3 E75 Double-plotted actograms (left) and Lomb-Scargle periodograms (right) of locomotor rhythms under LD 12∶12 and DD at a constant temperature of 30°C are shown for firebrats injected with(A, B) anddsRNA (C, D) ordsRNA (E, F). White and black bars above the actograms indicate light (white) and dark (black) conditions. Arrowheads indicate the day when the firebrats were transferred from LD to DD. a and b indicated in the periodogram correspond to the analyzed time span, a and b, indicated in the actogram, respectively. An oblique line in the periodogram indicates a significance level of<0.05; a peak value above the line was designated as significant. (A–D) Some of theandRNAi firebrats showed a rhythm in LD, which disappeared upon transfer to DD. (E, F) Control firebrats injected withdsRNA showed a significant rhythm throughout the recording period. No activity was recorded during the period indicated by double headed arrows because of molting. For further explanation, see text. HR3 E75 DsRed2 P HR3 E75 DsRed2
| Treatment | n | No. of insects | Free-running period (mean ± SD) h | |
|---|---|---|---|---|
| Rhythmic (%) | Arrhythmic (%) | |||
| LD | ||||
| dsRNA#1HR3 | 6 | 6 (100) | 0 (0) | 24.0±1.6 |
| dsRNA#1E75 | 17 | 12 (71) | 5 (29) | 23.8±1.0 |
| dsRNADsRed2 | 20 | 18 (90) | 2 (20) | 23.9±0.3 |
| DD | ||||
| dsRNA#1HR3 | 8 | 2 (25) | 6 (75) a | 24.2±0,6 |
| dsRNA #2HR3 | 4 | 0 (0) | 4(100)b | - |
| dsRNA #1E75 | 9 | 2 (22) | 7 (78)b | 24.5±0.4 |
| dsRNA #2E75 | 6 | 1 (17) | 5 (83)b | 24.3 |
| dsRNADsRed2 | 24 | 18 (75) | 6 (25) | 24.1±0.8 |
RNAi andRNAi affect the expression of,, and Td'HR3 Td'E75 Td'cyc Td'tim Td'Clk
We next examined whether Td'HR3 and/or Td'E75 were regulators of Td'cyc transcription by measuring the daily expression profiles of Td'cyc mRNA in Td'HR3 RNAi and Td'E75 RNAi firebrats. For this and the following analyses only Td'HR3 dsRNA#1 and Td'E75 dsRNA#1 were used. Td'E75 RNAi upregulated the Td'cyc mRNA level more than three-fold of the negative control but did not alter its rhythmic expression, while Td'HR3 RNAi reversed the phase of the Td'cyc mRNA rhythm, with a gradual increase throughout the night to peak late at night (Fig. 4A, S3 and S4 Tables). These results demonstrate the involvement of Td'HR3 and Td'E75 in the circadian transcriptional regulation of Td'cyc.
Td'HR3 and Td'E75 RNAi considerably affected the circadian expression of the clock genes Td'tim and Td'Clk. Td'HR3 RNAi upregulated the expression of Td'tim and shifted the rhythms to peak late at night, while Td'E75 RNAi slightly increased the Td'tim mRNA levels to peak during the day (Fig. 4B, S3 and S4 Tables). Surprisingly, Td'HR3 RNAi induced the rhythmic expression of Td'Clk to peak late at night with the upregulation of mRNA levels (Fig. 4C, S3 and S4 Tables), suggesting that the firebrat has a mechanism for the rhythmic expression of Td'Clk, which may be concealed by a mechanism involving Td'HR3. Td'E75 RNAi, however, had no significant effect on Td'Clk mRNA levels (Fig. 4C, S3 Table). Interestingly, in Td'HR3 RNAi firebrats, the rhythms of Td'cyc, Td'tim, and Td'Clk peaked almost in phase late at night, whereas in Td'E75 RNAi firebrats, Td'cyc and Td'tim showed in-phase oscillations and peaked during the day (Fig. 4A–C).
![Effects of(ds, pink symbols) anddsRNA (ds, green symbols) on mRNA levels of,, andin firebrats. HR3 HR3 E75 E75 cyc tim Clk Values for intact and dsinjected control are shown in black and blue symbols, respectively, and replotted in lower panels with an expanded ordinate. (A)normally peaked during the day. dssignificantly upregulated the mRNA levels, while dsshifted the rhythm to peak late at night. (B)showed a rhythm with a peak at late day or early night in intact firebrats. Dsslightly advanced the rhythm while dsupregulated and shifted the rhythm to peak late at night. (C)showed no significant rhythm in intact firebrats. Dshad no significant effect onmRNA levels, while dsinduced a rhythm with a peak at mid–late night. *<0.05, **<0.01,test vs ds(exactvalues are shown in). Values with different letters significantly differ from each other (p<0.05, ANOVA with Tukey test,). Forin firebrats treated with ds(B), significant rhythm was confirmed by the single cosinor methodand significant difference was found between values at ZT6 and ZT22 (-test, P<0.05). For further explanation, see text and. DsRed2 cyc E75 HR3 tim E75 HR3 Clk E75 Clk HR3 P P t DsRed2 P tim HR3 t S3 Table S4 Table [[28]] Fig. 2](https://europepmc.org/articles/PMC4263706/bin/pone.0114899.g004.jpg.jpg "Click to view full size")
Effects of(ds, pink symbols) anddsRNA (ds, green symbols) on mRNA levels of,, andin firebrats. HR3 HR3 E75 E75 cyc tim Clk Values for intact and dsinjected control are shown in black and blue symbols, respectively, and replotted in lower panels with an expanded ordinate. (A)normally peaked during the day. dssignificantly upregulated the mRNA levels, while dsshifted the rhythm to peak late at night. (B)showed a rhythm with a peak at late day or early night in intact firebrats. Dsslightly advanced the rhythm while dsupregulated and shifted the rhythm to peak late at night. (C)showed no significant rhythm in intact firebrats. Dshad no significant effect onmRNA levels, while dsinduced a rhythm with a peak at mid–late night. *<0.05, **<0.01,test vs ds(exactvalues are shown in). Values with different letters significantly differ from each other (p<0.05, ANOVA with Tukey test,). Forin firebrats treated with ds(B), significant rhythm was confirmed by the single cosinor methodand significant difference was found between values at ZT6 and ZT22 (-test, P<0.05). For further explanation, see text and. DsRed2 cyc E75 HR3 tim E75 HR3 Clk E75 Clk HR3 P P t DsRed2 P tim HR3 t S3 Table S4 Table [[28]] Fig. 2
Rhythmic expressions ofandrequire,, and Td'HR3 Td'E75 Td'cyc Td'tim Td'Clk
BMAL1 and CLK have been postulated to be positive regulators of Rorα and Rev-erbα in mammalian clocks [15], [16]. This hypothesis prompted us to examine whether Td'CYC and Td'CLK activate Td'HR3 and Td'E75 transcriptions in the firebrat. Therefore, we measured their daily expression profiles in Td'cyc RNAi and Td'Clk RNAi firebrats. The mRNA levels of Td'HR3 were significantly reduced and its circadian rhythm was abolished in both Td'cyc RNAi and Td'Clk RNAi firebrats (Fig. 5A). However, for Td'E75, its rhythm was disrupted in both dsClk and dscyc treatments but no significant changes were observed in its mRNA levels (Fig. 5B).
We also performed the same measurement in Td'tim RNAi firebrats because we speculated that the PER/TIM heterodimer, similar to the PER/CRY complex in mammals, may suppress Td'HR3 and Td'E75 transcription indirectly by repressing the transcriptional activity of CYC/CLK. The levels of both Td'HR3 and Td'E75 mRNAs were significantly downregulated and their rhythms were disrupted in Td'tim RNAi firebrats (Fig. 5A, B). This result is inconsistent with the mammalian clock hypothesis but is explained by our earlier finding that Td'tim RNAi downregulates Td'cyc transcript levels [10].
Effects of,, anddsRNA (ds, ds, and ds) on mRNA levels ofandin firebrats. cyc Clk tim cyc Clk tim HR3 E75 (A) Ds, ds, and dsabolished the rhythmic expression ofwith significant downregulation of mRNA levels. (B) RNAi of the three genes abolished the rhythm of. Dseffectively downregulatedmRNA but dsand dshad no significant effects, except at ZT18 where a significant increase was observed. *<0.05, **<0.01,test vs ds. Data for dsfirebrats are replotted from. For further explanation, see text and. cyc Clk tim HR3 E75 tim E75 cyc Clk P P t DsRed2 DsRed2 Fig. 2 Fig. 2
20E upregulatesandtranscription Td'HR3 Td'E75
Given that HR3 and E75 genes expression profiles in D. melanogaster strongly correlated with ecdysis [20], we examined the effects of 20E on Td'HR3 and Td'E75 mRNA levels. We injected 34.5 ng of 20E into the abdomen of adult firebrats. Td'HR3 and Td'E75 were evidently upregulated 1 week after 20E injection compared with the control firebrats injected with EtOH (Fig. 6).
Effects of ecdysone on mRNA levels of(A) and(B) in firebrats. Td'HR3 Td'E75 White and blue columns indicate the mRNA levels of EtOH (negative control) and ecdysone injected firebrats, respectively. 100% EtOH (69 nl) or 34.5 ng ecdysone (dissolved in 69 nl of EtOH) was injected and firebrats were collected at ZT 10 (10 h after light on) one week after the injection. The abundance of mRNA was measured by qPCR. The data collected from 4 independent experiments were averaged and plotted as mean ± SEM values relative to the value ofmRNA used as reference.*<0.05,-test. rp49 P t
Discussion
The present study revealed that the mRNAs of the nuclear receptor genes Td'HR3 and Td'E75 were rhythmically expressed in both LD and DD, peaking late day to early night (Fig. 2). The rhythmic expression was comparable to their mammalian orthologs, Rorα and Rev-erbα[15], [16]. Since RNAi of Td'cyc and Td'Clk disrupted the daily expression rhythms of Td'HR3 and Td'E75 genes, and downregulated Td'HR3 mRNA levels (Fig. 5), we assume that the rhythmic expression of Td'HR3 and Td'E75 are regulated by Td'CYC and Td'CLK, similar to their mammalian orthologs.
Our results demonstrated that Td'HR3 and Td'E75 are factors involved in the cyclic transcription of Td'cyc in the firebrat. The RNAi experiment showed that Td'E75 may be a repressor of Td'cyc transcription, similar to mammalian REV-ERBα [15], because its RNAi resulted in substantial upregulation of Td'cyc mRNA levels (Fig. 4A). However, Td'HR3 may act as a phase regulator of Td'cyc since its RNAi resulted in a phase reversal in Td'cyc oscillation with an increase of mRNA levels during the night (Fig. 4A). The results also imply that this may occur through another unknown factor(s) that activates the transcription of Td'cyc during the night.
In addition, Td'HR3 might also participate in the regulation of Td'Clk expression, since its RNAi induced a circadian oscillation and an upregulation of Td'Clk mRNA levels (Fig. 4C). Interestingly, Td'cyc, Td'Clk, and Td'tim oscillated in phase to peak around mid–late night in the Td'HR3 RNAi firebrats (Fig. 4A–C). However, in the Td'E75 RNAi firebrats, Td'cyc and Td'tim showed an in-phase oscillation that peaked during the day (Fig. 4A–B), suggesting that Td'E75 regulates the phase of Td'tim. The phase coherence was never observed in intact firebrats; Td'cyc and Td'tim oscillated with differential peaks phased during the day and late day to early night, respectively. Their in-phase oscillation in RNAi firebrats suggests that their transcriptions are regulated by a common factor, and Td'HR3 and Td'E75 play a role as phase regulators to directly or indirectly set the clock gene oscillations in a proper phase relationship. A similar phase coherence was observed in the sandfly Lutzomyia longipalpis, in which Clk and cyc transcripts peaked simultaneously with or slightly earlier than per and tim[21], [22], and in some mammalian peripheral tissues where Bmal1 and Per were expressed almost in phase [23], [24]. This feature is in opposition to the hypotheses of the Drosophila and mammalian central clocks, where peaks of per/tim (Per/Cry) and Clk/cyc (Clk/Bmal1) transcriptions are about 12 h apart from each other [25]. The reason and mechanism underlying the phase coherence should be addressed in future studies.
The behavioral analysis of Td'HR3 RNAi and Td'E75 RNAi firebrats revealed that both genes have two roles. First, they are important for the persistence of locomotor activity rhythms in DD (Fig. 3A–B; Table 3), which is also seen in mice where RORα and REV-ERBα are required for normal daily locomotor activity [15], [16], [26]. The Td'HR3 RNAi or Td'E75 RNAi firebrats showed in-phase oscillations of Td'cyc, Td'tim, and Td'Clk or Td'cyc and Td'tim, respectively (Fig. 4A–C). Nevertheless, they lost the locomotor rhythm in DD (Fig. 3A–B). This result suggests that the persistence of the locomotor rhythm requires a temporally coordinated and/or properly leveled expression of those clock genes in the firebrat. Thus, HR3 and E75 might have been involved as essential components in the ancestral insect clock. In addition, they are known to play important roles within the ecdysone-signaling pathway in Drosophila[20], and this may be true in the firebrat because they were upregulated by 20E (Fig. 6). This hypothesis also explains why a majority of firebrats with Td'HR3 and Td'E75 RNAi died from ecdysis failure (Fig. 2E), although no such failure was observed in the RNAi of other clock- and clock-related genes [9], [10]. Thus, they may couple the developmental processes and circadian rhythms in the firebrat.
The circadian clock machinery has been substantially clarified in Drosophila and mammals. In the current Drosophila clock model, per and tim are rhythmically expressed by the core-feedback loop consisting of transcriptional repressors, PER and TIM, and activators, CYC and CLK [27]. Clk is also rhythmically expressed by a loop, including vri and Pdp1ε[6], [8]. It seems likely that the firebrat's core loop might be operating in a similar way. Td'TIM may have two roles, i.e., repression of the transcriptional activity of Td'CYC and activation of the Td'cyc transcription through some unknown mechanism, because our previous results have shown that Td'tim RNAi downregulates the Td'cyc transcripts [10]. This scheme also explains why Td'tim RNAi downregulated both Td'HR3 and Td'E75 (Fig. 5). In contrast to Drosophila, Td'cyc is rhythmically expressed in the firebrat [9], [10], and our results revealed that the oscillatory mechanism involved Td'HR3 and Td'E75 although the details have yet to be clarified. This property resembles the mammalian clock.
Unexpectedly, Td'HR3 RNAi revealed the rhythmic expression of Td'Clk, suggesting the coexistence of an oscillatory mechanism for Td'Clk. In comparison to the Drosophila circadian clock, we currently hypothesize that the rhythmic transcription of Td'Clk is regulated by VRI and PDP1ε. In many insects, cyc but not Clk, has been shown to be rhythmically expressed. However, our data suggest that, even in those species, the mechanism for the cyclic expression of Clk might be retained but concealed in normal physiological conditions. In fact, we have recently shown that Clk is rhythmically expressed when cyc is knocked down in the cricket G. bimaculatus[12]. Interestingly, both Clk and cyc are expressed in a rhythmic manner in the sandflies [21], [22]. Together with these facts, our results suggest that an ancestral insect clock has mechanisms for the rhythmic expression of both cyc and Clk but have diversified to express either or both of them during the course of adaptation to specific environments. Further comparative studies are necessary to test this view.