Abstract:

MicroRNAs (miRs) are a family of small non-coding RNAs that regulate the expression of numerous downstream mRNAs. Cardiac dysfunction in early life stage mahi-mahi (Coryphaena hippurus) exposed to crude oil has been associated with upregulation of miR133b and subsequent downregulation of genes important for proper heart development, similar to mammals. To directly test the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish (Danio rerio) embryos were microinjected with a miR133b mimic. At 72 hours post-fertilization (hpf), miR133b-injected fish exhibited oil-like cardiovascular malformations relative to negative control miR-injected embryos. Phenotypic effects outside of the heart were not observed. Gene expression analysis showed that at 5 hpf miR133b decreased the abundance of cardiac-specific IKr kcnh6 and ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum. Together results demonstrate that miR133b may target cardiac tissues in teleost, similar to vertebrates, and contribute to cardiac perturbations in oil-exposed fish by targeting genes essential for proper heart development.

Suggested Citation:

Greer, Justin and Daniel Schlenk. 2021. Effects of microRNA 133 on developing zebrafish (Danio rerio) embryos. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/PNCJKYJ7

Purpose:

To further understand the molecular mechanisms of cardiotoxicity induced by oil.

Data Parameters and Units:

Data is organized by different data types obtained from microRNA133b injection in zebrafish (Danio rerio). Below are short descriptions of the data in each file and folder. Phenotypic_measurements = Heart rate, pericardial area, and eye area measurements obtained at 72 hours post fertilization --Treatment = Describes the treatment for each exposure group. Embryos were microinjected with 1-3 nanoliters of either a miR133b mimic or a negative control miR at a concentration of 75 uM. Embryos were maintained at a low density (~30 per dish) until 72 hpf, when phenotypes were measured. --Replicate = Biological replicate number within each exposure group. Each replicate number matches the images contained within the "Phenotype_images" folder. --Pericardial Area (mm) = measured pericardial area in millimeters, as determined in ImageJ. --Heart rate (beats per min) = Heart rate per minute, calculated by multiplying the number of heart beats in 10 sec * 6. Heart rate was not possible to measure in some replicates due to camera focus, in which case this field contains "n/a". --Eye area (in mm) = measured eye area in millimeters as determined in ImageJ. Folder: Phenotype_images - contains images obtained at 8x zoom for measurements of pericardial area and eye area. Images are labeled as Treatment_replicate. qPCR_data = Measured cycle threshold values from quantitative PCR (qPCR) performed on RNA extracted from whole embryos at 5 and 72 hpf. Genes important for calcium homeostasis and potassium cycling in cardiomyocytes were measured for alteration induced by miR133b or phenanthrene, a PAH commonly associated with cardiac disruption in early life stage fish. Data were analyzed using the delta delta Ct formula with EF1a as the reference gene. --Gene = Gene symbol for the gene tested --Treatment = Describes the treatment for each exposure group. For miR133 experiments embryos were microinjected with 1-3 nanoliters of either a miR133b mimic (miR133) or a negative control miR (miR_control) at a concentration of 75 uM. For phenanthrene experiments, embryos exposed to either 12 uM phenanthrene (phen 12uM) or a DMSO vehicle only control (phen_control). --Timepoint = Describes the number of hours post fertilization in which RNA was obtained and used for analysis. For miR133 experiments gene expression analysis was performed at both 5 and 72 hpf. For phenanthrene exposures gene expression was only performed at 72 hpf. --Replicate = Biological replicate number within each exposure group. Each reaction was performed in either technical duplicate or triplicate, hence each biological replicate consists of two or three values. --Ct = Threshold cycle value. The mean Ct value was calculated for technical replicates to perform downstream expression analysis.

Methods:

Adult wild-type zebrafish (Danio rerio) were maintained and bred using a recirculating system with a 14:10 light: dark cycle and conditioned reverse osmosis water maintained at 27-28 C. Cleavage-stage zebrafish embryos were microinjected with ~3 nL of either 75 uM negative control miR or 75 uM miR133b using a motorized Eppendorf Injectman NI2 and FemtoJet 4x. Lyophilized miR133b and nc-miR mimics were obtained from Thermo Fisher Scientific and reconstituted in nuclease free water. During the first several hours after injection embryos were monitored, and unhealthy embryos removed. Phenanthrene stock solutions were prepared by dissolving phenanthrene (98% purity, Sigma Aldrich) in 100% dimethyl sulfoxide (DMSO) and stored at 4 C. Exposure solutions were made with water collected from the recirculating system and vacuum-filtered through a 5-µm membrane. Filtered water was then spiked with DMSO or the phenanthrene stock to produce a DMSO vehicle control (0.08% DMSO) or 12 µM Phenanthrene exposure solutions. Exposures were conducted in 48-well polystyrene plates. Shield-stage zebrafish embryos (6 hpf) were randomly distributed into wells for exposure, with one embryo per well, and 75% (500 mL) renewal at 24 hpf and 48 hpf. At 72 hpf, embryos were collected in microcentrifuge tubes, snap frozen in liquid nitrogen, and stored at -80 C for subsequent RNA extraction. Live embryos were imaged at 72 hpf using transmitted light on a Leica MZ10 F stereomicroscope equipped with a DMC2900 camera. Images were assessed for eye area and pericardial area using ImageJ version 1.52a 44. To assess the effects of miR133 overabundance on the expression of cardiac K+ and Ca2+ genes, qPCR was performed on microinjected embryos at 5 and 72 hpf. Phenanthrene-exposed embryos were assayed only at the 72-hpf timepoint. At each timepoint, embryos were collected and snap frozen in liquid nitrogen. Total RNA was extracted from 12-15 embryos per replicate at 5 hpf and 7-10 embryos per replicate at 72 hpf. Frozen embryos were lysed in Buffer RLT (Qiagen) and homogenized using a Kontes Pellet Pestle Cordless Motor. Total RNA was extracted using RNeasy mini kit (Qiagen) following the manufacturer’s protocol. Quantity and quality of total RNA were assessed using a Nanodrop ND-1000 and Qubit fluorometer. 500 ng of total RNA then was used as input for cDNA synthesis using the Promega Reverse Transcription System (Promega, Madison, WI). Final 20 ul cDNA reactions were diluted to 100 ul using molecular-grade water to provide the final cDNA concentration. qPCR reactions consisted of 10 ul of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA), 0.2 uM forward and reverse primers, and 2 ul of cDNA in a 20 ul final volume. Each biological replicate was performed in triplicate on a CFX Connect Real-Time PCR Detection System with the following thermal cycler conditions: 95˚C for 10 minutes, followed by 40 cycles of 95˚C for 15 s, 58˚C for 30 s, 72˚C for 30 s. Relative expression was determined by the 2 dela-delta Ct method with elongation factor 1-alpha (ef1a) as the normalizing gene.

Individual Files: