Transcriptomic profiling of mTOR and ryanodine receptor signaling molecules in developing zebrafish in the absence and presence of PCB 95

Chemicals

2,2′,3,5′,6-Pentachlorobiphenyl (PCB 95; 99.7% purity) was purchased from AccuStandard (New Haven, CT, USA).

Fish husbandry and spawning

Fish husbandry, spawning and all research involving zebrafish were performed in accordance with UC Davis Institutional Animal Care and Use Committee protocol #17645. Adult wild-type, tropical 5D zebrafish (D. rerio) were kept in 2 L tanks at a density of 10–14 fish at 28.5 ± 0.5 °C under a 14 h light:10 h dark cycle. Culture water pH was kept in the range of 7.2–7.8, and electric conductivity between 600 and 800 μS cm⁻¹. Adult fish were fed twice a day with Artemia nauplii (INVE Aquaculture, Inc., Salt Lake City, UT, USA) and commercial flake (a combination of Zebrafish Select Diet, Aquaneering, San Diego, CA, USA and Golden Pearls, Artemia International LLC, Fairview, TX, USA). Embryos were obtained by spawning groups of 8–12 fish in a 1:2 female/male ratio. Spawning time was coordinated using a barrier to separate male and female fish, which was removed in the morning after the lights turned on, thus producing age-matched fertilized eggs, which were collected within 20 min of spawning.

Quantitative polymerase chain reaction

For the initial studies of the normal ontogenetic profiles of transcription of mTOR and RyR signaling molecules, embryos were directly transferred into 100 × 20 mm polystyrene tissue culture dishes (Corning Inc., Corning, NY, USA) containing 60 mL standardized Embryo Medium (Westerfield, 2007) and placed into an incubator at a constant temperature of 28.5 ± 1 °C, with a 14 h light:10 h dark photoperiod. Fish were maintained at a density of 50 individuals per petri dish, and RNA was extracted at three different time points (24, 72 and 120 hpf). Fish from three independent spawns were used to obtain three biological replicates at each time point. To minimize variability due to differences in spawning time, all embryos were collected within a 20 min spawning window. Fish within each spawn were pooled into batches of 350 or 500 to extract sufficient amounts of RNA for quantitative polymerase chain reaction (qPCR) analyses. The first biological replicate represented a pooled sample of 500 embryos in order to collect a sufficient amount of RNA for method validation. Embryo numbers were chosen using a conservative approach of assessing average population transcription of target genes, rather than gene expression from single individuals. We quantified baseline transcription of 36 genes, 23 associated with the mTOR signaling pathway (Table S1) and 13 associated with RyR-dependent Ca²⁺ signaling (Table S2).

Transcript levels of target genes were assessed by qPCR using gene-specific primers derived from mRNA sequences obtained from the Zebrafish Model Organism Database (http://zfin.org/). Primers were designed using NCBI Primer Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), and obtained from Integrated DNA Technology (Integrated DNA Technologies, Inc., Coralville, IA, USA).

RNA extractions were performed with a Qiagen Qiacube robotic workstation using RNeasy Mini Kit spin columns (Qiagen, Valencia, CA, USA) as per the manufacturer’s directions. Total RNA integrity was verified using an RNA 6000 Nano Kit on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) (Fig. S3). The RNA Integrity Ratio (RIN) scores were calculated, and RNA was deemed to be of good quality with RIN scores of over 8 (Table S3). RNA concentration was determined using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).

Complementary DNA (cDNA) was synthesized using 1 μg total RNA in a reaction with 4 μL Superscript Vilo Mastermix (SuperScript® VILO™ MasterMix, Invitrogen, Carlsbad, CA, USA) according to the user’s manual. Reactions were incubated for 10 min at 25 °C, 60 min at 42 °C followed by a 5 min denaturation step at 85 °C. Samples were then diluted with nuclease-free water in a 1:5 ratio to produce acceptable concentrations for quantitative PCR evaluations. Success of the cDNA-synthesis was tested using beta-actin primers with a polymerase chain reaction (5 min at 95 °C; 30 s at 95 °C, 30 s at 60 °C, 45 s at 72 °C, in 35 cycles; 10 min at 72 °C) visualized through gel electrophoresis.

Quantitative polymerase chain reaction was conducted using Power SYBR Green PCR Master Mix (Life-technologies, Carlsbad, CA, USA). Primer validation was performed using a seven point standard curve with three replicates; amplification efficiencies ranged between 90.1% and 108.7%. Cycling conditions were 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, followed by a thermal ramping stage for dissociation evaluation. Amplification data were analyzed using Sequence Detection Systems software (SDS v2.4.1, Applied Biosciences). Relative gene expression was calculated using the Log2^−ΔΔCT method (Livak & Schmittgen, 2001) relative to the reference genes elongation factor 1 alpha (eef1a1) and beta actin 2 (actb2) (Table S2), which sustained best scores in GeNorme (Vandesompele et al., 2002). All data were normalized to 24 hpf samples. To verify primer quality, sequences obtained from NCBI were checked additionally in Ensembl genome browser (http://www.ensembl.org/index.html) to identify chromosome and exon location (Tables S1 and S2).

Whole mount in situ hybridization

Digoxigenin-labeled probes were prepared from 24 hpf embryonic cDNA for ryr3. We chose ryr3 because it experiences a highly significant increase in transcription between 24 and 72 hpf as well as between 24 and 120 hpf, and because of discrepancies in the published data, which describe ryr3 transcription in zebrafish prior to 24 hpf in skeletal muscle only, or predominantly in hindbrain regions at later time points (Thisse & Thisse, 2005; Wu, Brennan & Ashworth, 2011). Specifically, we designed primers (Table S2) containing a T3 RNA polymerase promoter on the 5′-end of the reverse primer, thereby allowing antisense probe transcription. Procedures for embryo and larval preparation and in situ hybridization assay were performed as described previously (Thisse & Thisse, 2014), using BM purple (Roche, Basel, Switzerland) as labeling solution.

PCB 95 exposures

Embryos collected from spawning tanks were transferred to 100 × 20 mm polystyrene tissue culture dishes (Corning Inc., Corning, NY, USA) and maintained at a constant temperature of 28.5 ± 1 °C. At 4 hpf, embryos were enzymatically dechorionated using 50 μL of 41 mg/mL pronase (Sigma-Aldrich, St. Louis, MO, USA) in 25 mL of culture water for a maximum of 6 min (Truong, Harper & Tanguay, 2011). Embryos were then allowed to recover for 2 h in culture water before being transferred one embryo per well into a 96-well plate (Falcon™; Corning Inc., Corning, NY, USA) containing 100 μL of standardized Embryo Medium (Westerfield, 2007). At 6 hpf, 100 μL of 2× PCB 95 solution was added directly into each well to yield final concentrations of 0.1, 0.3, 1.0, 3.0 or 10.0 μM PCB 95; control embryos were exposed to vehicle (0.2% DMSO). Plates were covered with Parafilm M (Bemis NA, Neenah, WI, USA) to reduce evaporation, and were then placed into an incubator at a constant temperature of 28.5 ± 1 °C and a 14 h light:10 h dark cycle until fish (n = 16 per treatment in each plate) were harvested for transcriptomic assessments at 72 and 120 hpf. Embryos or larvae were pooled into batches of 12–16 individuals to generate sufficient RNA for qPCR analyses. Fish from three independent spawns were used to obtain three biological replicates at each time point.

Statistical analysis

Significant differences in gene transcription at 72 and 120 hpf relative to transcription at 24 hpf were identified using one-way ANOVA with significance set at P < 0.05 followed by a Tukey’s post hoc test. If data did not fit the ANOVA assumptions of normality, a Kruskal–Wallace test was applied (P < 0.05), followed by the Nemenyi–Damico–Wolfe–Dunn post hoc test. Shapiro–Wilk normality and Bartlett tests were used to determine which algorithms are appropriate for determining significant differences between time points. R-packages “stats” (Team, 2014), “PMCMR”; pairwise multiple comparisons of mean rank sums (Pohlert, 2014) and “multcomp”; multiple components (Hothorn et al., 2008) were used to perform statistical analyses. Data from the PCB 95 exposure studies were similarly analyzed, with the different PCB exposures normalized to the solvent control.

Ontogenetic profile of transcripts encoding mTOR signaling pathway genes

Overall, there was increased transcription of genes encoding signaling molecules upstream of mTOR between 24 and 120 hpf (Figs. 1A and 1B). Ribosomal protein S6 kinase polypeptide 1 (rps6ka1) was significantly upregulated (P < 0.05) at both 72 and 120 hpf relative to 24 hpf, while tuberous sclerosis 1a (tsc1a) and mitogen-activated protein kinase 1 (mapk1) were significantly upregulated (P < 0.05) at 120 hpf relative to 24 hpf. Mitogen-activated protein kinase 3 (mapk3) and insulin receptor substrate 1 (irs1) remained constant across all time points, and V-akt murine thymoma viral oncogene homolog 1 (akt1) showed a declining trend in relative expression over time. All other upstream targets of the mTOR complex exhibited a general increased transcription from 24 to 120 hpf that was not statistically significant.

There were no significant differences in transcription of genes within the mTORC1 complex from 24 to 72 or 120 hpf (Fig. 1C). However, relative to transcript levels at 24 hpf, transcription of raptor-independent companion of mTOR (rictora) decreased at 72 hpf but was significantly upregulated from 72 to 120 hpf. Transcription of genes encoding signaling molecules downstream of mTOR generally decreased with increasing hpf, but none of these changes were statistically significant (Figs. 1D–1F). The one exception was the p70 ribosomal S6 kinase a (rps6kb1a), which showed the tendency of increased transcription at 72 hpf, although this change was not statistically significant.

Baseline transcription of genes involved in RyR-dependent signaling

Zebrafish express five RyR-paralogs (Wu, Brennan & Ashworth, 2011): ryr1a and ryr1b are transcribed predominantly in slow and fast twitch muscle tissue, respectively (Hirata et al., 2007); ryr2a is predominantly expressed in the central nervous system (CNS), ryr2b, in the heart of developing zebrafish (Wu, Brennan & Ashworth, 2011); and ryr3 has been detected in zebrafish skeletal muscle prior to 24 hpf (Wu, Brennan & Ashworth, 2011) but is reported to be expressed predominantly in hindbrain regions at later developmental stages (Thisse & Thisse, 2005). Transcripts of ryr2a and ryr3 were significantly elevated at 72 and 120 hpf, whereas relative expression of ryr1a, ryr1b and ryr2b did not change significantly over the 120 hpf assessment period (Fig. 2A). Transcription of wnt2ba increased in a time-dependent manner with a significant increase noted at 120 hpf, whereas wnt2bb did not change significantly over the 120 hpf assessment (Fig. 2B).

Significant increases in gene transcription were observed for Homer homolog 1b (homer 1b) and the alpha 1c subunit of the voltage-dependent L type calcium channel (cacna1c) at 72 and 120 hpf. Transcript levels of the alpha 1S subunit 1 of the voltage-dependent L type calcium channel (cacna1sa) were significantly increased at 72 hpf (Fig. 2C). Transcription of psen1 and psen2 was elevated at 72 hpf, but this change was not significant (Fig. 2C), whereas genes involved in regulating RyR-dependent signaling (like trdn) remained relatively consistent across the three time points evaluated in this study.

In-situ hybridization of ryr3

Since ryr3 is expressed in both the skeletal muscle and brain at different developmental stages (Thisse & Thisse, 2005; Wu, Brennan & Ashworth, 2011), to determine whether increased transcripts of ryr3 detected by qPCR reflect ryr3 upregulation in the CNS, we examined its expression by in situ hybridization. At 24 hpf, ryr3 transcription was observed only in skeletal muscle (Fig. 3A). However, by 26 hpf, ryr3 transcripts were detected in brain tissue (Figs. 3B and 3C). By 72 hpf, ryr3 was abundantly expressed in the brain (Fig. 3D).

Transcriptional effects of PCB 95

The influence of developmental PCB 95 exposure on transcription of NDD-relevant genes was tested on six genes associated with the mTOR signaling pathway (Table S1; genes in bold font) and six associated with RyR-dependent Ca²⁺ signaling (Table S2; gene in bold font) (Fig. 4). The mTORC1 member, rptor, was significantly downregulated in PCB 95-exposed fish at both 72 and 120 hpf (Fig. 4B). At both time points, the effect of PCB 95 was concentration-dependent. At 72 hpf, the fold-change progressively decreased with increasing concentrations of PCB 95, with statistical significance reached at the highest PCB 95 concentration of 10 μM. At 120 hpf, statistically significant downregulation was observed at 3.0 and 10.0 μM. The other five genes of the mTOR signaling pathway that we examined were not significantly altered at 72 or 120 hpf in fish exposed to PCB 95 at any of the concentrations tested in this study (Figs. 4A, 4C–4F). Within the subset of genes relevant to RyR signaling that we examined, two were found to be significantly upregulated by PCB 95 exposure but interestingly, only at 72 hpf (Figs. 4K and 4L). Transcription of ryr2b and wnt2ba showed a concentration-dependent increase, which reached statistical significance at 10 μM PCB 95.