Samuel M. Peterson

Extensive Genetic Diversity and Substructuring Among Zebrafish Strains Revealed through Copy Number Variant Analysis

Copy number variants (CNVs) represent a substantial source of genomic variation in vertebrates and have been associated with numerous human diseases. Despite this, the extent of CNVs in the zebrafish, an important model for human disease, remains unknown. Using 80 zebrafish genomes, representing three commonly used laboratory strains and one native population, we constructed a genome-wide, high-resolution CNV map for the zebrafish comprising 6,080 CNV elements and encompassing 14.6% of the zebrafish reference genome. This amount of copy number variation is four times that previously observed in other vertebrates, including humans. Moreover, 69% of the CNV elements exhibited strain specificity, with the highest number observed for Tubingen. This variation likely arose, in part, from Tubingens large founding size and composite population origin. Additional population genetic studies also provided important insight into the origins and substructure of these commonly used laboratory strains. This extensive variation among and within zebrafish strains may have functional effects that impact phenotype and, if not properly addressed, such extensive levels of germ-line variation and population substructure in this commonly used model organism can potentially confound studies intended for translation to human diseases.

Transcriptome alterations following developmental atrazine exposure in zebrafish are associated with disruption of neuroendocrine and reproductive system function, cell cycle, and carcinogenesis

Atrazine, a herbicide commonly applied to agricultural areas and a common contaminant of potable water supplies, is implicated as an endocrine-disrupting chemical (EDC) and potential carcinogen. Studies show that EDCs can cause irreversible changes in tissue formation, decreased reproductive potential, obesity, and cancer. The U.S. Environmental Protection Agency considers an atrazine concentration of ≤ 3 ppb in drinking water safe for consumption. The specific adverse human health effects associated with a developmental atrazine exposure and the underlying genetic mechanisms of these effects are not well defined. In this study, zebrafish embryos were exposed to a range of atrazine concentrations to establish toxicity. Morphological, transcriptomic, and protein alterations were then assessed at 72h postfertilization following developmental atrazine exposure at 0, 0.3, 3, or 30 ppb. A significant increase in head length was observed in all three atrazine treatments. Transcriptomic profiles revealed 21, 62, and 64 genes with altered expression in the 0.3, 3, and 30 ppb atrazine treatments, respectively. Altered genes were associated with neuroendocrine and reproductive system development, function, and disease; cell cycle control; and carcinogenesis. There was a significant overlap (42 genes) between the 3 and 30 ppb differentially expressed gene lists, with two of these genes (CYP17A1 and SAMHD1) present in all three atrazine treatments. Increased transcript levels were translated to significant upregulation in protein expression. Overall, this study identifies genetic and molecular targets altered in response to a developmental atrazine exposure to further define the biological pathways and mechanisms of toxicity.

Global gene expression analysis reveals dynamic and developmental stage-dependent enrichment of lead-induced neurological gene alterations.

Background The underlying genetic mechanisms specific to subtle neurological alterations associated with environmental lead (Pb) exposures have not been clearly elucidated. Objectives The goal of this study was to identify novel gene targets and the underlying genetic mechanisms associated with developmental Pb neurotoxicity. Methods We first exposed zebrafish embryos to a range of Pb concentrations throughout early development to establish relative toxicity. Using the data from that experiment, we exposed another group of zebrafish embryos to a sublethal dose of Pb (100 ppb) immediately after fertilization through 72 or 120 hr postfertilization (hpf). Global gene expression was then analyzed for molecular pathways and gene ontology enrichment, and Western blot analysis was performed to investigate the translation of gene expression changes to protein levels. Results After 72 hpf, we identified 231 probes representing 90 nonredundant genes with well-established function or orthology to human genes as being altered by Pb exposure. This gene set was both confirmatory and novel in nature and was highly enriched for neurological development, function, and disease. Moreover, gene changes at this time point were correlated to altered protein levels. Alternatively, the gene set at 120 hpf did not share association with neurological development. Conclusions Global gene expression alterations associated with developmental Pb exposure were dynamic and dependent on developmental stage. Gene expression alterations at the 72-hpf time point were highly enriched with genes and molecular pathways associated with neurological development and disease. Moreover, we identified a number of novel targets for future exploration into their role in the genetic mechanisms underlying Pb-induced neurological alterations.

RNA isolation from embryonic zebrafish and cDNA synthesis for gene expression analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA. A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications. It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample. Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated. The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.

Decreased axonal density and altered expression profiles of axonal guidance genes underlying lead (Pb) neurodevelopmental toxicity at early embryonic stages in the zebrafish

Previous studies have reported that environmental lead (Pb) exposure can result in neurological alterations in children leading to reduced IQ, attention deficit hyperactivity disorder, and diminished reading and learning abilities. However, the specific alterations in neurodevelopmental morphology and the underlying genetic mechanisms of these alterations have not yet been thoroughly defined. To investigate alterations in neurologic morphology and test the hypothesis that developmental Pb neurotoxicity is partially mediated through alterations in neuronal growth and transport function of axons, the changes of specific axon tracts in the embryonic zebrafish brain were observed with anti-acetylated α-tubulin staining at several developmental time points through 36hours post fertilization (hpf). In addition, the role of a subset of axonogenesis-related genes including shha, epha4b, netrin1b, netrin2, and noiwas investigated with real-time quantitative PCR (qPCR). Pb treatment resulted in decreased axonal density at 18, 20, and 24hpf for specific axon tracts in the midbrain and forebrain. These observations corresponded to an observed down-regulation of shha and epha4b at 14 and 16hpf, respectively. The axonal density in Pb exposed individuals at later stages (30 and 36hpf) was not significantly different from controls. An overexpression of netrin2 at these two developmental stages suggests a novel role for this gene in regulating axonal density specific to Pb neurotoxicity. Although no significant differences in axonal density was observed in the two later developmental stages, further studies are needed to determine if the morphologic alterations observed at the earlier stages will have lasting functional impacts.

Directional and color preference in adult zebrafish: Implications in behavioral and learning assays in neurotoxicology studies

The zebrafish (Danio rerio) is a useful vertebrate model organism for neurological studies. While a number of behavior and learning assays are recently reported in the literature for zebrafish, many of these assays are still being refined. The initial purpose of this study was to apply a published T‐maze assay for adult zebrafish that measures how quickly an organism can discriminate between different color stimuli after receiving reinforcement to measure learning in a study investigating the later life impacts of developmental Pb exposure. The original results were inconclusive as the control group showed a directional and color preference. To assess directional preference further, a three‐chambered testing apparatus was constructed and rotated in several directions. The directional preference observed in males was alleviated by rotating the arms pointing west and east. In addition, color preference was investigated using all combinations of five different colors (orange, yellow, green, blue and purple). With directional preference alleviated results showed that both male and female zebrafish preferred colors of shorter wavelengths. An additional experiment tested changes in color preference due to developmental exposure to Pb in adult male zebrafish. Results revealed that Pb‐exposed males gained and lost certain color preferences compared to control males and the preference for short wavelengths was decreased. Overall, these results show that consideration and pretesting should be completed before applying behavioral and learning assays involving adult zebrafish to avoid innate preferences and confounding changes in neurotoxicology studies and that developmental Pb exposure alters color preferences in adult male zebrafish. Copyright

Developmental reelin expression and time point-specific alterations from lead exposure in zebrafish.

Reelin (RELN) is a large glycoprotein that plays critical roles during and after neuronal development. In a previous study utilizing developing zebrafish, the RELN ortholog was in a subset of genes associated with neuronal function that was altered following exposure to 100ppb Pb at 72h post-fertilization (hpf). Although RELN is associated with neurodevelopment and neurological diseases, it represents a previously uncharacterized target for developmental Pb neurotoxicity. To further investigate Pb-induced expression decrease of RELN, a more detailed analysis was done using in situ hybridization and quantitative PCR (qPCR) on developing zebrafish at multiple developmental time points and at different exposure concentrations. Time points included in the analysis were 24, 36, 48, 60, 72 and 96hpf. The four exposure conditions tested were 10, 50 or 100ppb Pb as well as a control. In situ hybridization demonstrated concentrated expression of the gene in the telencephalon and in midbrain, hindbrain and spinal cord. Expression was less spatially restricted early in development but became isolated to specific cell populations in the central nervous system by 48hpf. The telencephalon demonstrated a high degree of expression throughout development. Pb exposure did not affect the observed location of expression at any of the time points tested. However, qPCR analysis demonstrated that reln expression statistically decreased in embryos exposed to the highest concentration compared to controls at 60hpf. No changes in brain morphology as assessed by measuring brain length and no significant increase in the number of apoptotic cells in the brain were observed at the highest exposure concentration. These data demonstrate that a decrease in reln expression occurs following Pb exposure at a concentration below one that results in changes in brain length or in the number of apoptotic cells in the zebrafish model system during a short window and is likely linked to concurrent developmental processes.

Embryonic ionizing radiation exposure results in expression alterations of genes associated with cardiovascular and neurological development, function, and disease and modified cardiovascular function in zebrafish

The relationship between ionizing radiation (IR) and carcinogenesis is long established, but recently the association between IR and other diseases is starting to be recognized. Currently, there is limited information on the genetic mechanisms governing the role of IR in non-cancer related adverse health effects and in regards to an early developmental exposure. In this study, zebrafish embryos were exposed to a range of IR doses (0, 1, 2, 5, 10 Gy) at 26 h post fertilization (hpf). No significant increase in mortality or hatching rate was observed, but a significant decrease in total larval length, head length, and eye diameter was observed in the 10 Gy dose. Transcriptomic analysis was conducted at 120 hpf to compare gene expression profiles between the control and highest IR dose at which no significant differences were observed in morphological measurements (5 Gy). 253 genes with well-established function or orthology to human genes were significantly altered. Gene ontology and molecular network analysis revealed enrichment of genes associated with cardiovascular and neurological development, function, and disease. Expression of a subset of genetic targets with an emphasis on those associated with the cardiovascular system was assessed using Quantitative PCR (qPCR) to confirm altered expression at 5 Gy and then to investigate alterations at lower doses (1 and 2 Gy). Strong correlation between microarray and qPCR expression values was observed, but zebrafish exposed to 1 or 2 Gy resulted in a significant expression alteration in only one of these genes (LIN7B). Moreover, heart rate was analyzed through 120 hpf following IR dosing at 26 hpf. A significant decrease in heart rate was observed at 10 Gy, while a significant increase in heart rate was observed at 1, 2, and 5 Gy. Overall these findings indicate IR exposure at doses below those that induce gross morphological changes alters heart rate and expression of genes associated with cardiovascular and neurological functions.

Cancer cytogenetics in the zebrafish.

The zebrafish system has been established as a useful model for the study of carcinogenesis. The cytogenetic characterization of the genome is vital for furthering our understanding of the progression of the disease. Establishing a basic description of the zebrafish chromosomal karyotype and markers for each specific chromosome permitted the first cytogenetic characterization of the reference genome and the genome of cancer models. As the field of cancer cytogenetics is highly dependent on technology, each advance in technique and methodology has resulted in a corresponding wave of discoveries. We have witnessed great improvement in the resolution of the assays allowing for more detailed characterization of cytogenetic abnormalities, including the efficient and accurate identification of DNA copy number alterations of specific chromosomal regions. Herein, we will discuss major advancements in the field of cytogenetics, along with examples of how these technologies have been utilized in studies to characterize zebrafish cancer disease models. Finally, we will discuss the current state of the field and how microarray technology are being implemented to scan the whole genome at high resolution for DNA copy number alterations observed in various cancer types throughout the progression of the disease.

Sex-specific characterization and evaluation of the Alzheimer's disease genetic risk factor sorl1 in zebrafish during aging and in the adult brain following a 100 ppb embryonic lead exposure

Developmental lead (Pb) exposure is suggested in laboratory studies to be a trigger for neurodegenerative diseases such as Alzheimers disease (AD). Sortilin‐related receptor, L (DLR class) A repeats‐containing (SORL1) is a recently identified AD genetic risk factor. SORL1 has limited characterization in vertebrate models in comparison to other AD genetic risk factors. To characterize SORL1 further, protein sequence homology between humans, mice and zebrafish was analyzed and showed conservation of functional repeats and domain orientation. Next, spatial expression of sorl1 in zebrafish larvae was completed and diffuse expression in neural tissue that was not restricted to the brain was observed. Influences of sex and age on quantitative expression of sorl1 in the brain of adult zebrafish were then assessed. Sex‐specific alteration of sorl1 expression transpired during the aging process in females. The zebrafish was then utilized to investigate the impacts of a 100 ppb embryonic Pb exposure on sorl1 expression and other known AD genetic risk factors. Sex‐specific quantitative gene expression analysis was completed with adult zebrafish brain to compare those developmentally exposed to Pb or a control treatment, but no significant difference in sorl1 expression or other AD genetic risk factors was observed. Overall, this study provided characterization of sorl1 with changes in brain expression during aging being female‐specific. This finding is in agreement with females being more prone to the onset of AD, but analysis of additional AD genetic risk factors is needed to facilitate our understanding of the impact of a 100 ppb embryonic Pb exposure. Copyright