A bioinformatics framework for targeted gene expression assay design: Application to in vitro developmental neurotoxicity screening in a rat model.

Brain development involves a series of intricately choreographed neuronal differentiation and maturation steps that are acutely vulnerable to interferences from chemical exposures. Many genes involved in neurodevelopmental processes show evolutionarily conserved expression patterns in mammals and may constitute useful indicators/biomarkers for the evaluation of potential developmental neurotoxicity. Based on these premises, this study developed a bioinformatics framework to guide the design of a gene expression-based in vitro developmental neurotoxicity assay targeting evolutionary conserved genes associated with neuronal differentiation and maturation in rat cerebellar granule cells (CGCs). Rat, mouse and human genes involved in neurodevelopment and presenting one-to-one orthology were selected and orthologous exons within these genes were identified. PCR primer sets were designed within these orthologous exons and their specificity was evaluated in silico. The performance and specificity of rat, mouse and human PCR primer sets were then confirmed experimentally. Finally, RT-qPCR analyses in CGCs exposed in vitro to well-known neurotoxicants (Chlorpyrifos and Chlorpyrifos oxon) uncovered perturbations of expression levels for most of the selected genes. This bioinformatics framework for gene and target sequence selection may facilitate the identification of transcriptional biomarkers for developmental neurotoxicity assays and the comparison of gene expression data across experimental models from different mammalian species.

Characterization of the rat Acetylcholinesterase readthrough (AChE-R) splice variant: Implications for toxicological studies.

Exposure to chemicals and other environmental stressors can differentially impact the expression of Acetylcholinesterase (AChE) splice variants. Surprisingly, despite the widespread use of the rat model in toxicological studies and the wealth of literature on this important biomarker of neurotoxicity, AChE coding exons and splice variants are not yet fully annotated in this species. To address this knowledge gap, a short problematic region of the rat AChE genomic DNA present in GenBank was first re-sequenced. This revised genomic sequence was then aligned to rat AChE RefSeq mRNA and compared to orthologous mammalian sequences, in order to map the coding exon and intron boundaries of the rat AChE gene. Based on these bioinformatics analyses, a sequence was predicted for the yet-unannotated rat Acetylcholinesterase readthrough (AChE-R) splice variant. PCR primers designed to specifically amplify rat AChE-R were used to confirm its expression in rat PC12 cells. Compared to the canonical AChE-S splice variant, AChE-R was expressed at much lower levels but presented distinct regulation patterns in PC12 cells and rat primary cerebral granule cells (CGCs) following exposure to Chlorpyrifos (a well-known neurotoxic organophosphate pesticide). Taken together, these observations point to the evolutionary conservation of the AChE-R splicing event between rodents and human and to the distinct regulation of AChE splice variants in response to toxicological challenges.

A bioinformatics workflow for the evaluation of RT-qPCR primer specificity: Application for the assessment of gene expression data reliability in toxicological studies.

The reliability of Reverse Transcription quantitative real-time PCR (RT-qPCR) gene expression data depends on proper primer design and RNA quality controls. Despite freely available genomic databases and bioinformatics tools, primer design deficiencies can be found across life science publications. In order to assess the prevalence of such deficiencies in the toxicological literature, 504 primer sets extracted from a random selection of 70 recent rat toxicological studies were evaluated. The specificity of each primer set was systematically analysed using a bioinformatics workflow developed from publicly available resources (NCBI Primer BLAST, in silico PCR in UCSC genome browser, Ensembl DNA database). Potential mismatches (9%), cross-matches (13.5%), co-amplification of multiple gene splice variants (9%) and sub-optimal amplicon sizes (25%) were identified for a significant proportion of the primer sets assessed in silico. Quality controls for gDNA contamination of RNA samples were infrequently reported in the surveyed manuscripts. Hence, the impacts of gDNA contamination on RT-qPCR data were further investigated, revealing that lowly expressed genes presented higher susceptibility to contaminating gDNA. In addition to the retrospective identification of potential primer design issues presented in this study, the described bioinformatics workflow can also be used prospectively to select candidate primer sets for experimental validation.

A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples.

Reverse Transcription quantitative real-time PCR (RT-qPCR) is applied to quantify gene transcript levels in a wide range of investigations. Proper assessment of RNA integrity is essential for reliable assessment of gene expression levels, as RNA molecules are acutely vulnerable to degradation. However, RNA quality control measures are still infrequently reported in rat toxicological studies, which impede proper evaluation of gene expression data reliability. The high operational cost of microfluidic capillary electrophoresis systems along with paucity of alternative methods for the quantitative assessment of rat RNA integrity constitute potential hurdles to the systematic implementation and reporting of RNA integrity assessment in rat studies. This manuscript describes the adaptation of an alternative RT-qPCR-based 3':5' assay as an additional option for the quantitative assessment of rat RNA integrity. Two PCR primer sets were designed on the 3' and 5' regions of a rat housekeeping gene to evaluate RNA integrity by measuring the relative expression (3':5' ratio) of these amplicons. The 3':5' ratios were then compared to Agilent Bioanalyzer's RNA integrity number (RIN) for a wide range of RNA samples originating from different tissues, cultured cell lines and rat strains that were prepared freshly, stored for years at -80 °C, purchased commercially or intentionally degraded. The 3':5' ratios and RIN values presented similar assessment of RNA integrity status from intact to heavily degraded samples. Based on the LOWESS regression of this large comparison dataset, 3':5' ratio threshold criteria equivalent to RIN cut-off values can be proposed for the selection of RNA samples for RT-qPCR analyses. This qPCR-based assay is easy to implement, cost-effective, and provides a reliable quantification of RNA integrity to assist in the selection of rat RNA samples suitable for downstream RT-qPCR gene expression analyses.

A PCR-based approach to assess genomic DNA contamination in RNA: Application to rat RNA samples.

Genomic DNA (gDNA) contamination of RNA samples can lead to inaccurate measurement of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR). We describe an easily adoptable PCR-based method where gDNA contamination in RNA samples is assessed by comparing the amplification of intronic and exonic sequences from a housekeeping gene. Although this alternative assay was developed for rat RNA samples, it could be easily adapted to other species. As a proof of concept, we assessed the effects of detectable gDNA contamination levels on the expression of a few genes that illustrate the importance of RNA quality in acquiring reliable data.

Perinatal methylmercury exposure perturbs the expression of Plp1 and Cnp splice variants in cerebellum of rat pups.

Early life exposure to environmental chemicals can interfere with myelin formation in the developing brain, leading to neurological disorders. The Proteolipid Protein 1 (Plp1), Myelin Basic Protein (Mbp) and 2',3'-Cyclic Nucleotide 3'Phosphodiesterase (Cnp) genes expressed in oligodendrocytes and involved in myelination processes can be useful biomarkers of potential developmental neurotoxicity. In an earlier study, we concluded that the reduction in the expression levels of Mbp splice variants in juvenile rat cerebellum following perinatal methylmercury (MeHg) exposure were compatible with an overall reduction of mature oligodendrocytes population. This observation prompted us to analyze the expression of Plp1 and Cnp in developing rat cerebellum to further confirm and investigate the toxic effects of MeHg on vulnerable oligodendrocytes. Splice variants of Plp1 in human and of Cnp in mouse are curated in NCBI RefSeq database, but not for rat. Lack of annotation of splice variants can pose significant challenge for the reliable quantification of gene expression levels in toxicological studies. Therefore, we applied a "comparative sequence analysis" approach, relying on annotated splice variants in human/mouse and on evolutionary conservation of intron-exon structures, to identify additional splice variants of Plp1 and Cnp in rat. Then, we confirmed their identity by nucleotide sequencing and characterized their temporal expression patterns during brain development by RT-PCR. The measurement of total transcripts and individual splice variants of Plp1 and Cnp in the cerebellum of MeHg-exposed rat pups revealed a relatively similar level of reduction in their expression levels. This study further confirms that perinatal exposure to MeHg can impact oligodendrocytes in pups. Based on these observations, we conclude that monitoring the expression of these oligodendrocyte-enriched genes can be useful to identify toxic chemicals affecting myelination.

Cyclooxygenase 1 mRNA expression is undetectable in Madin Darby Canine Kidney cells.

BACKGROUND: Madin Darby Canine Kidney (MDCK) cells form polarized epithelium in vitro and are routinely used in research fields ranging from protein trafficking to influenza. However, the canine origin of these cells also means that compared to man or mouse, genomic resources are more limited and performance of commercially available antibodies often untested. The synthesis of pro-inflammatory prostaglandins in the kidney is mediated by the constitutively expressed cyclooxygenase 1 and the inducible cyclooxygenase 2 (COX-1 and COX-2, respectively). There are conflicting reports on the expression of COX-1 and COX-2 in MDCK cells and this lingering uncertainty about such important pharmacological targets may affect the interpretation of results obtained from this cell line. RESULTS: In order to definitively settle the issue of cyclooxygenase expression in MDCK cells, we designed PCR primers based on dog genomic sequences to probe COX-1 and COX-2 mRNA expression in MDCK cells and dog kidney. We report that while COX-1 and COX-2 genes are both expressed in dog kidney, COX-1 expression is undetectable in MDCK cells. CONCLUSIONS: By improving the characterization of cyclooxygenase expression in MDCK cells, this study will contribute to a better understanding of the properties of this cell line and lead to improved experimental designs and data interpretations.

Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil.

New health safety concerns may arise from the increasing production and use of Jatropha oil, a biodiesel feedstock that also contains toxic, pro-inflammatory, and co-carcinogenic phorbol esters. Based on the exceptional sensitivity of Madin-Darby canine kidney (MDCK) cells to the model phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a robust bioassay was developed to quantify the biological activity of Jatropha phorbol esters directly in oil, without sample extraction. We first verified that the characteristic response of MDCK cells to TPA was also observed following direct exposure to phorbol esters in Jatropha oil. We further confirmed that similarly to TPA, Jatropha oil's phorbol esters can activate protein kinase C (PKC). We then assessed the transcriptional response of MDCK cells to Jatropha oil exposure by measuring the expression of cyclooxygenase-2 (COX-2), a gene involved in inflammatory processes which is strongly upregulated following PKC activation. Based on the parameterization of a TPA dose-response curve, the transcriptional response of MDCK cells to Jatropha oil exposure was expressed in term of TPA toxic equivalent (TEQ), a convenient metric to report the inflammatory potential of complex mixtures. The sensitive bioassay described in this manuscript may prove useful for risk assessment, as it provides a quantitative method and a convenient metric to report the inflammatory potential of phorbol esters in Jatropha oil. This bioassay may also be adapted for the detection of bioactive phorbol esters in other matrices.

Expression pattern of an evolutionarily conserved splice variant in the rat Tacc2 gene.

The transforming acidic coiled-coil containing protein 2 (Tacc2) gene and its paralogs, Tacc1 and Tacc3 encode proteins that are associated with the centrosome and involved in microtubule assembly during the cell cycle. Tacc2 produces several splice variants, which are poorly characterized, especially in the rat. Characterization of the temporal/spatial expression patterns of these isoforms would be useful in understanding their distinct and overlapping functions. By comparative sequence analyses of Tacc2 in multiple species, we identified a third splice variant in rat, which is much shorter in size (1,021 aa) than the longest isoform (2,834 aa). This newly identified Tacc2 splice variant (isoform 3) uses a distinct first exon and generates a different open reading frame. Although Isoform 3 is expressed predominantly during developmental stages, the long Tacc2 isoform (isoform 1) is distributed mainly in adult tissues. Multiple protein sequence analyses revealed that Tacc2 Isoform 3 could be the ancient form, as it is conserved in mammals, birds, and amphibians; whereas the long Tacc2 isoforms may have evolved in the mammalian lineage by adding exons toward the 5' region of the ancient isoform.

Perturbation of myelin basic protein (Mbp) splice variant expression in developing rat cerebellum following perinatal exposure to methylmercury.

Myelin sheaths surrounding axons are essential for saltatory conduction of nerve impulse in the central nervous system. A major protein constituent of myelin sheaths is produced by the myelin basic protein (Mbp) gene, whose expression in oligodendrocytes is conserved across vertebrates. In rat, five Mbp splice variants resulting from alternative splicing of exons 2, 5 and/or 6 are characterized. We developed a PCR-based strategy to quantify individual Mbp splice variants and characterized a sixth Mbp splice variant lacking only exon 5. This newly identified splice variant is predominantly expressed in developing rat brain and has orthologs in mouse and human. Many neurotoxic chemicals can perturb myelination and Mbp gene expression. Regulation of Mbp gene expression at the post-transcriptional level was assessed following perinatal exposure to neurotoxic methylmercury (2 mg/kg b.w./day). Similar reductions in total and individual Mbp splice variant mRNA levels suggest that methylmercury-induced perturbation in Mbp gene expression occurred as a consequence of decreased oligodendrocyte cell population in absence of a significant impact on its post-transcriptional regulation.

Loss of fish actinotrichia proteins and the fin-to-limb transition.

The early development of teleost paired fins is strikingly similar to that of tetrapod limb buds and is controlled by similar mechanisms. One early morphological divergence between pectoral fins and limbs is in the fate of the apical ectodermal ridge (AER), the distal epidermis that rims the bud. Whereas the AER of tetrapods regresses after specification of the skeletal progenitors, the AER of teleost fishes forms a fold that elongates. Formation of the fin fold is accompanied by the synthesis of two rows of rigid, unmineralized fibrils called actinotrichia, which keep the fold straight and guide the migration of mesenchymal cells within the fold. The actinotrichia are made of elastoidin, the components of which, apart from collagen, are unknown. Here we show that two zebrafish proteins, which we name actinodin 1 and 2 (And1 and And2), are essential structural components of elastoidin. The presence of actinodin sequences in several teleost fishes and in the elephant shark (Callorhinchus milii, which occupies a basal phylogenetic position), but not in tetrapods, suggests that these genes have been lost during tetrapod species evolution. Double gene knockdown of and1 and and2 in zebrafish embryos results in the absence of actinotrichia and impaired fin folds. Gene expression profiles in embryos lacking and1 and and2 function are consistent with pectoral fin truncation and may offer a potential explanation for the polydactyly observed in early tetrapod fossils. We propose that the loss of both actinodins and actinotrichia during evolution may have led to the loss of lepidotrichia and may have contributed to the fin-to-limb transition.

Screen for genes differentially expressed during regeneration of the zebrafish caudal fin.

The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. The cascade of genes induced after amputation or injury, leading to restoration of the lost fin structures, include those responsible for wound healing, blastema formation, tissue outgrowth, and patterning. We carried out a systematic study to identify genes that are up-regulated during "initiation" (1 day) and "outgrowth and differentiation" (4 days) of fin regeneration by using two complementary methods, suppression subtraction hybridization (SSH) and differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). We obtained 298 distinct genes/sequences from SSH libraries and 24 distinct genes/sequences by DDRT-PCR. We determined the expression of 54 of these genes using in situ hybridization. In parallel, gene expression analyses were done in zebrafish embryos and early larvae. The information gathered from the present study provides resources for further investigations into the molecular mechanisms of fin development and regeneration.