Transcriptional and Behavioral Responses of Zebrafish Larvae to Microcystin-LR Exposure

Abstract

Microcystins are cyclic heptapeptides that constitute a diverse group of toxins produced by cyanobacteria. One of the most toxic variants of this family is microcystin-LR (MCLR) which is a potent inhibitor of protein phosphatase 2A (PP2A) and induces cytoskeleton alterations. In this study, zebrafish larvae exposed to 500 μg/L of MCLR for four days exhibited a 40% reduction of PP2A activity compared to the controls, indicating early effects of the toxin. Gene expression profiling of the MCLR-exposed larvae using microarray analysis revealed that keratin 96 (krt96) was the most downregulated gene, consistent with the well-documented effects of MCLR on cytoskeleton structure. In addition, our analysis revealed upregulation in all genes encoding for the enzymes of the retinal visual cycle, including rpe65a (retinal pigment epithelium-specific protein 65a), which is critical for the larval vision. Quantitative real-time PCR (qPCR) analysis confirmed the microarray data, showing that rpe65a was significantly upregulated at 50 μg/L and 500 μg/L MCLR in a dose-dependent manner. Consistent with the microarray data, MCLR-treated larvae displayed behavioral alterations such as weakening response to the sudden darkness and hypoactivity in the dark. Our work reveals new molecular targets for MCLR and provides further insights into the molecular mechanisms of MCLR toxicity during early development.

Towards the identification of causal genes for age-related macular degeneration

“…Considering the potential role of CNN2 and SARM1 in AMD pathophysiology, we sought to test the hypothesis whether visual impairment could be attributed to defects of vital photoreceptor or RPE genes. Thus, we conducted reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis to measure the expression levels of retinal signature genes: rhodopsin , four kinds of cone opsins ( red , green , blue , and uv ) and RPE-specific gene rpe65a 29,30 . For the cnn2 6.0 ng MO group and the sarm1 1.0 ng MO group, all these genes showed dramatic decreases compared to the standard MO control; rhodopsin decreased by 117.1-fold and 12.6-fold respectively, so as to cone opsin ( green opsin by 797.5-fold and 13.6-fold, blue opsin by 279.8-fold and 10.4-fold, red opsin by 107.4-fold and 8.0-fold, and uv opsin by 19.2-fold and 10.8-fold), and rpe65a (by 7.5-fold and 3.6-fold) ( Figure 4 ).…”

Effects of the Essential Oil from Pistacia lentiscus Var. chia on the Lateral Line System and the Gene Expression Profile of Zebrafish (Danio rerio)

“…The transcriptomic profile of control and mastic oil-treated zebrafish was analyzed by microarrays, using the Affymetrix Platform of the GeneCore Facility in EMBL (Heidelberg, Germany). RNA samples were processed, labeled, and hybridized to the Zebrafish Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA), according to standard Affymetrix protocols, as described [59]. Raw data were normalized, processed, and visualized with the GeneSpring software (Agilent Technologies) [59].…”

“…RNA samples were processed, labeled, and hybridized to the Zebrafish Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA), according to standard Affymetrix protocols, as described [59]. Raw data were normalized, processed, and visualized with the GeneSpring software (Agilent Technologies) [59]. The list of genes was generated from the final data by p -value sorting ≤ 0.05 and absolute differential expression ≥ 1.7 fold change.…”

Application of Benchmark Concentration (BMC) Analysis on Zebrafish Data: A New Perspective for Quantifying Toxicity in Alternative Animal Models

“…A Teratogenic Index (TI) was estimated as the ratio between the LC 50 and EC 50 values. Two TIs were calculated, one per developmental stage (Tzima et al, 2017;Van Voorhis et al, 2016).…”

“…The mean of the total distance moved by embryos in each group was measured in 2-min time bins and treated versus control groups were compared using the unpaired Student's t test. Compounds were considered active if significant differences were detected (p < .005) in more than one point at the same condition and tracking phase; the lowest tested concentration with a significant effect was then reported for the active compounds (ie, LOAEL) (Tzima et al, 2017;Van Voorhis et al, 2016).…”