Expression patterns of zebrafish nocturnin genes and the transcriptional activity of the frog nocturnin promoter in zebrafish rod photoreceptors

Zebrafish care

Adult AB zebrafish were raised at 28.5 °C on a 14 h:10 h light-dark cycle. Zebrafish embryos were collected and raised at 28.5 °C. All procedures for zebrafish care conformed to the University of Pittsburgh standards for care and use of animals in research (University of Pittsburgh Animal Protocol Number: 0909487B-3; Assurance Number: A3187–01). The study adhered to the ARVO Statement for Use of Animals in Research.

RT–PCR confirmation of the zebrafish nocturnin-b cDNA open reading frame

For RT–PCR analysis of nocturnin expression, total RNA molecules were either isolated at 8 AM, 12 PM, 4 PM, 8 PM, 12 AM, and 4 AM from adult fish eyes or isolated at 12 AM from 5-dpf larval eyes and 5-dpf whole larval fish. The samples were cut into small pieces and incubated with TRIzol (Thermo Fisher Scientific, Waltham, MA) for 5 min at room temperature. Then 0.2 ml of chloroform was added to every 1 ml of TRIzol tissue extraction, followed by gentle shaking for 15 s and 3 min of incubation at room temperature. The aqueous fractions of the lysates were retrieved and used for RNA precipitation with isopropyl alcohol with centrifugation. The RNA pellet was washed with 75% ethanol three times and redissolved in 100% formamide and stored at −80 °C until later usage. To make a cDNA library, the total RNA was first incubated at 65 °C for 5 min to remove their secondary structures and then reversely transcribed using an oligo dT primer and SuperScript III reverse transcriptase (Invitrogen). The expression of the zebrafish nocturnin-a and nocturnin-b genes in the eye was examined with RT–PCR reactions using the following primers (Figure 1C): noc-a F7 (5′-AGC CTT CAG AGT TCA CG ACA-3′); noc-a R7 (5′-GGC TCT GGT CAT GAT GAA CT-3′); noc-a F8 (5′-ATT TAG GAG TTC TTC ATT A-3′); noc-a R8 (5′-ACT GAA GCT GAG GTC ACA CA-3′); noc-b F4 (5′-ACA ATC TGA ATC TGG ATC AG-3′); noc-b R4 (5′-TCC CTC CAG GAC ATG TAC TT-3′); noc-b F5 (5′-GAA GAC GTC TAT AGG AAT TT-3′); noc-b R5 (5′- AAA GGT GAT CTG AAG GGT AG-3′); elf2a-F (5′-TTG AGA AGA AAA TCG GTG GTG CTG-3′); and elf2a-R (5′-GGA ACG GTG TGA TTG AGG GAA ATT C-3′). The PCR conditions were initial denaturation at 95 °C for 1 min, and 40 cycles of amplification and quantification (95 °C for 20 s, 55 °C for 20 s, and 72 °C 30 s). To confirm the sequence of the nocturnin-b open reading frame (ORF), we sequenced an RT–PCR nocturnin-b ORF DNA (cloned in a pDrive vector), which was amplified with a forward primer covering the start codon (5′-TCC GAA TTC CGT GTT CCA TGG GCA GCG GC-3′; the underlined sequence is an EcoR I site) and a reverse primer binding to its 3′ untranslated region (UTR) (5′-TCC AAG CTT CGT GAA TTC ATA CAA AAT TGC ACA G-3′; the underlined sequence is a Hind III site).

RACE identification of the full-length sequence of the zebrafish nocturnin-b cDNA

The total RNA of the adult AB wild-type zebrafish eyes was extracted with an RNeasy Mini Kit (Qiagen, Hilden, Germany). Briefly, small pieces of eye tissue (30 mg) were dissociated and homogenized gently in an RLT Buffer with a MACS Dissociator (Miltenyi Biotec., Teterow, Germany). An equal volume of 70% ethanol was added to the lysates to allow selective binding of RNA to the RNeasy membranes for purification. The total RNA was eluted in RNase-free water and stored at −80 °C until later usage.

To identify the full-length sequence of the zebrafish nocturnin-b cDNA sequence, 5′ and 3′ rapid amplification of cDNA ends (RACE) analyses were performed with the total RNA using a GeneRacer Kit (Invitrogen) following the manufacturer’s protocol. For 5′ RACE, nocturnin-b-specific reverse primer 1 (5′-TCC AAG CTT CGT GAA TTC ATA CAA AAT TGC ACA G-3′) and primer 2 (5′-CTA CAT CGA GAC ATG GAG ACC ATG G-3′) were used for the first-round PCR and then nested-PCR, respectively. For 3′ RACE, nocturnin-b-specific forward primer 1 (5′-TCC GAA TTC CGT GTT CCA TGG GCA GCG GC-3′) and primer 2 (5′-CGT GGA AGA TCC GAC CCA GTG GCG AGA TT-3′) were used for the first-round PCR and the subsequent nested-PCR, respectively. Platinum High Fidelity Taq DNA Polymerase was used for all PCR reactions. The PCR conditions were initial denaturation at 94 °C for 2 min, and 35 cycles of amplification and quantification (94 °C for 30 s, 55 °C for 30 s, and 68 °C 2 min). The amplified PCR fragments of the nocturnin-b cDNA were cloned with a TOPO TA cloning kit (Invitrogen) and then sequenced (Figure 1B).

qRT-PCR

Quantitative real-time PCR (qRT-PCR) analysis of nocturnin-b at four time points during the day was performed with SYBR Green/ROX qPCR Master Mix (Fermentas, Thermo Fisher Scientific, Waltham, MA) on a Roter-Gene 6000 real-time genetic analyzer (Corbett Life Science, Qiagen, Hilden, Germany) according to the manufacturer’s instructions; elf2a was used as an internal control. The following primers were used for qRT-PCR: noc-b F4, noc-b R4, elf2a-F, and elf2a-R. Each sample was replicated three times. The qRT-PCR was performed with the following conditions: initial denaturation at 95 °C for 2 min, 40 cycles of amplification and quantification (95 °C for 5 s, 55-57 °C for 30 s), and a melting curve program 55-95 °C with 0.5 °C increment each cycle).

Computational analyses of genes

We used Clustalw2 software to analyze the amino acid sequence similarities among various full-length nocturnin homologs of the following species: zebrafish (nocturnin-b, XP_697426; nocturnin-a, XP_700794); Xenopuslaevis (NM001085812), mouse (NM009834), and human (NM012118). The amino acid sequence of zebrafish nocturnin-b was predicted from the sequence of its cDNA that was prepared from the adult whole eye total RNA. The unrooted phylogenetic relationships between different nocturnin proteins were analyzed with VectorNTI and the MEGA4 program using the neighbor-joining method.

Transgenesis

A 417-bp DNA fragment of the frog nocturnin promoter [28] was amplified with PCR using Xenopus genomic DNA, a forward primer (5′-CTG CTC GAG CTA GTG AAA TGT CAC TGT G-3'; Xho I site, underlined), and a reverse primer (5′-ATA GAA TTC CTG TGC TTC CCT CTC AGC AC-3′; EcoR I site, underlined). The PCR conditions were initial denaturation at 94 °C for 2 min, and 35 cycles of amplification (94 °C for 30 s, 55 °C for 30 s, and 68 °C 2 min). The Tol2-based transgenesis system was used to generate a transgenic fish line that expresses green fluorescent protein (GFP) under the control of the frog nocturnin promoter using a standard protocol as described previously [29]. A stable zebrafish transgenic line, designated Tg(nocturnin^frog:GFP)^pt158, was identified through PCR genotyping of F1 generation and screening of GFP expression.

Immunohistochemistry

Immunohistochemical confocal microscopic analyses were performed to examine the GFP expression patterns in the Tg(nocturnin^frog:GFP)^pt158 transgenic fish using protocols as described previously [30]. Anti-GFP (Sigma-Aldrich, St. Louis, MO, G1544–100UG; 1:300), zpr1 antibody (ZFIN, 1:300), and zpr3 antibody (ZFIN, 1:300) were used to visualize GFP, green/red double cones, and rods, respectively.

Western blotting

The adult fish eyes were homogenized and extracted with a lysis buffer (1X PBS [137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.3], 1% Triton X-100, and 1X protease inhibitor cocktail [Roach Diagnostics, Indianapolis, IN]) for 30 min on ice. The supernatants of the lysates were mixed with a 6X SDS loading buffer and boiled for 5 min. Protein lysates was separated with 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed with standard western blotting. The signal intensities of the GFP expression were measured with ImageJ software (National Institutes of Health, Bethesda, MA), and their relativity to α-tubulin signal intensities were determined with the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA), with the relative intensity at 8 AM set as 1.

Nocturnin-b in situ hybridization

A zebrafish nocturnin-b cDNA fragment was amplified with RT–PCR with a forward primer (5′-TCC GAA TTC CGT GTT CCA TGG GCA GCG GC-3′; EcoR I site, underlined) and a reverse primer (5′-TCC AAG CTT ATA ACA ATC TGG CTG GAT CTT CGT-3′; Hind III site, underlined) and subsequently cloned in the pDrive vector. The resulting pDrive construct was linearized and transcribed with the T7/SP6 mMESSAGE mMACHINE Kit (Ambion, Thermo Fisher Scientific, Waltham, MA) to make the antisense and sense nocturnin-b Digoxigenin-RNA probes. After the RNA synthesis, the DNA templates were removed with DNase I digestion. The RNA probes were precipitated with ethanol and dissolved in 50% formamide and stored at −80 °C.

The adult zebrafish eyes were fixed with 4% paraformaldehyde overnight at 4 °C and cryosectioned at 300 μm thickness. The eye sections were dehydrated with a series of PBS/methanol solutions, followed by treatment with 100% acetone for 8 min at −20 °C. The samples were then rehydrated with a series of methanol/PBST (1% Triton X-100 in 1X PBS) washes and treated with 1 μg/ml proteinase K for 20 min. The proteinase K-treated sections were fixed again with 4% paraformaldehyde for 20 min at room temperature and washed with PBS. The retinal sections were then analyzed with in situ hybridization by using a previously published protocol [29]. The stained sections were then embedded in JB4 resin and cut at 4 μm thickness. The sections were stained with propidium iodide (PI; Invitrogen; 1:200) to visualize the cell nuclei. The slides were reviewed under an Axio Imager microscope (Carl Zeiss, Oberkochen, Germany).

Identification of the zebrafish nocturnin genes

To identify the zebrafish nocturnin genes, we searched the published zebrafish genome with the amino acid sequence of Xenopus nocturnin as a query [22]. The search identified two predicted zebrafish genes (XP_697426 and XP_700794) that show significant similarity to the frog nocturnin gene (Figure 1A,B). We designate the XP_700794 gene as the nocturnin-a gene and the XP_697426 gene as the zebrafish nocturnin-b gene. It is predicted that nocturnin-a encodes a polypeptide of 432 amino acids and nocturnin-b encodes a polypeptide of 371 amino acids. To determine which nocturnin gene is expressed in the eye, we next examined nocturnin-a and nocturnin-b mRNA levels in 5-dpf eyes, adult eyes, and 5-dpf whole fish with RT–PCR. We found that nocturnin-b was expressed in larval and adult eyes, whereas nocturnin-a was not, despite being expressed in the whole larval fish; these results suggest that the eye expresses nocturnin-b but not nocturnin-a, and that the two genes may promote mRNA depolyadenylation in a distinct tissue-specific fashion (Figure 1C). Furthermore, nocturnin-b expression in the eye is rhythmic, at higher levels at night than during the day (Figure 1D,E), echoing the rhythmic expression of nocturnin in the frog [22].

We next cloned and sequenced the entire cDNA of nocturnin-b to confirm the predicted amino acid sequence in the GenBank database. At the amino acid level, the two zebrafish nocturnin homologs show more than 60% similarity to their counterparts in the frog, human, and mouse (Figure 1B). In particular, the catalytic domains of the zebrafish nocturnin homologs are about 70% identical to their counterparts, suggesting a conserved catalytic function (Figure 1B, boxed area). Thus, the highly conserved nocturnin-b gene is expressed in the zebrafish eye, and the gene potentially regulates the mRNA turnover of the retinal cells in a circadian rhythm.

Expression patterns of nocturnin-b in zebrafish eyes

Previous studies showed that in the frog, the nocturnin gene is restrictively expressed in the retinal photoreceptors [22,28], whereas in the mouse, the gene is broadly expressed in various tissues, such as the liver, spleen, kidney, and heart [25]. This difference in expression pattern patterns prompted us to determine which cells express the nocturnin-b gene in the zebrafish retina. With in situ hybridization analysis, we found that the highest levels of the nocturnin-b gene were expressed in the photoreceptor layer (PRL), compared with the inner nuclear layer (INL) and the ganglion cell layer (GCL; Figure 2). The strongest expression of the nocturnin-b gene in zebrafish photoreceptors suggests that the nocturnin-b gene plays an important role in regulating the circadian turnover of mRNA in photoreceptors, a function that echoes the gene’s counterpart in the frog [22,28].

Transcriptional activity of the frog nocturnin promoter in zebrafish rods

Because transcription factors interact with cis-regulatory elements to drive transcription [31], the broader expression of the nocturnin-b gene in the zebrafish retina than the gene’s counterpart in the frog retina suggests mechanistic differences in activating nocturnin transcription. For example, the cis-regulatory elements of the frog nocturnin gene may be different from those of the nocturnin-b gene, and as a result, it can be activated only in photoreceptors [28]. Could it be possible that the cis-regulatory elements of the frog nocturnin gene are also functional specifically in the zebrafish photoreceptors?

To investigate this possibility, we next examined whether a 417-bp frog nocturnin promoter, which is sufficient to express a GFP reporter gene specifically in the frog photoreceptors [28], can drive photoreceptor expression of a GFP reporter in the zebrafish by generating a stable transgenic zebrafish line Tg(nocturnin^frog:GFP)^pt158 (Figure 3A). In Tg(nocturnin^frog:GFP) ^pt158, GFP was detected only in the outer nuclear layer (ONL) but not in other two retinal cellular layers. Interestingly, the GFP signals were restricted to rods: The GFP-positive cell nuclei, round and strongly TO-PRO-stained, resided in the basal half of the ONL, where rod nuclei localize (Figure 3B-E); furthermore, GFP immunostaining localized to cells that were positive for rod marker Zpr3 (Figure 3F-I). In contrast, GFP signals were not detected in the double cones and the ultraviolet (UV) cones (Figure 3E,J,M). In addition to rod-specific transcriptional activity, the frog nocturnin promoter in Tg(nocturnin^frog:GFP) ^pt158 was more than 1,000 times weaker than the rod opsin promoter in the Tg(−3.7rho:EGFP)^kj2 transgenic fish (Figure 3N) [32]. However, the GFP protein level did not show apparent rhythmic fluctuation, possibly due to the greater stability of the GFP protein (Figure 3O). Thus, the promoter of the frog nocturnin gene can be activated only in the rods in the zebrafish retina.

The frog nocturnin promoter contains an E-box-like nocturnin element, which functions as a cyclic AMP response element to recruit the phosphorylated CREB transcription factor for its activation [28]. The rod-specific expression of GFP in Tg(nocturnin^frog:GFP) ^pt158 suggests that the frog nocturnin promoter may carry sufficient cis-regulatory elements for its activation in the zebrafish rod photoreceptors but not necessarily for other zebrafish retinal cell types.

The characteristics of GFP expression in Tg(nocturnin^frog:GFP) ^pt158 make the frog nocturnin promoter desirable for moderate transgenic expression in the zebrafish rod photoreceptors. The zebrafish rod opsin promoters have been frequently used to express transgenes in rods thanks to the promoters’ superb rod photoreceptor specificity [32,33]. However, zebrafish rod opsin promoters can be problematic because their strong transcriptional activities may cause unwanted overexpression phenotypes in certain situations. Thus, the frog nocturnin promoter becomes particularly useful when low-level transgenic expression is desired in zebrafish rod photoreceptors.