BlockSupplementalMaterial – Supplemental material for Early Gender Differences in Core Values Predict Anticipated Family Versus Career Orientation

<p>Supplemental material, BlockSupplementalMaterial for Early Gender Differences in Core Values Predict Anticipated Family Versus Career Orientation by Katharina Block, Antonya Marie Gonzalez, Toni Schmader, and Andrew Scott Baron in Psychological Science</p

BlockOpenPracticesDisclosure – Supplemental material for Early Gender Differences in Core Values Predict Anticipated Family Versus Career Orientation

<p>Supplemental material, BlockOpenPracticesDisclosure for Early Gender Differences in Core Values Predict Anticipated Family Versus Career Orientation by Katharina Block, Antonya Marie Gonzalez, Toni Schmader, and Andrew Scott Baron in Psychological Science</p

MicroRNA Profiling as Tool for <i>In Vitro</i> Developmental Neurotoxicity Testing: The Case of Sodium Valproate

<div><p>Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative <i>in vitro</i> testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs <i>in vitro</i> allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, <i>mir-206, mir-133a</i> and <i>mir-10a</i>, and downregulation of neural-specific <i>mir-124a, mir-128</i> and <i>mir-137</i>). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (<i>Actc1, calponin</i>, <i>myosin light chain, asporin, decorin</i>) were found elevated, while genes involved in neurogenesis (e.g. <i>Otx1</i>, <i>2, and Zic3, 4, 5</i>) were repressed. These results were specific for valproate treatment and―based on the following two observations―most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant <i>mir-206</i> and <i>mir-10a</i> was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA.</p></div

Valproate effects on viability and expression of β-III-Tubulin.

<p>The cells were induced to differentiate into neurons for 16 days under continuous substance exposure. Cell viability was estimated using CellTiterBlue assay and is shown as a percentage of solvent control (A), expression of β-III-tubulin was analyzed by flow cytometry and is shown as a percentage of solvent control for each concentration tested (B). Results represent a mean of three independent differentiation experiments ± SEM.</p

Volcano plots comparing VPA and arsenite miRNA signatures 16 days after neural differentiation of mESCs.

<p>The –log₁₀ of P-values for each miRNA are plotted against log₂ mean ratio (three replicates) of the normalized miRNA signals of treated samples compared to solvent control. A. VPA induced changes in miRNA profile of neural-differentiated mESCs. B. Arsenite effects on miRNA expression in neural-induced mESCs. miRNAs which were included in further qPCR analysis or were regulated in opposite direction by both substances are marked in red.</p

Expression of neuron- and myocyte-markers under VPA exposure.

<p>Neural-differentiated ES cells were immuno-stained with neuron specific marker β-III-tubulin (red) and muscle specific marker α-actinin (green) after VPA (A) or PBS (B) treatment.</p

Gene expression under VPA treatment.

<p>A. RT-PCR verification of Affymetrix whole genome array data. The graph demonstrates mean of log₂ fold change in three independent differentiation processes (VPA vs. solvent control) ± SEM for nine upregulated and four downregulated mRNAs. (n = 3 independent biological replicates, t-test, *p<0.05, **p<0.01, ***p<0.001). B. Induction of expression of myogenic regulation factors (MRFs) by VPA in neural-differentiated ES cells. The graph demonstrates mean of log₂ fold change (VPA vs. solvent control) ± SEM. (n = 3 independent biological replicates, t-test, *p<0.05, **p<0.01, ***p<0.001).</p

TSA induction of <i>mir-206</i> and <i>mir-10a</i> during neural differentiation of mES cells.

<p>The graph demonstrates mean of log₂ fold change (TSA vs. solvent control) in two independent biological replicates ± SEM.</p

List of biological functions and canonical pathways affected by VPA according to IPA analysis.

<p>*Arrows show VPA induced up or down regulation of genes involved in the giving pathway.</p

Hierarchical cluster analysis (HCA) of miRNA expression in treated and control samples.

<p>HCA was carried out using Euclidian algorithm to build the cluster tree of the average significantly altered miRNAs by VPA or arsenite in comparison to negative/solvent control (NC) in neural- differentiated ESCs. The miRNA expression intensities of all probe set IDs are scaled as a Z-score (all microarray experiments were done in triplicates). Red color denotes upregulated miRNAs, green color – down regulated miRNAs. A. HCA of miRNAs responding to VPA treatment. B. HCA of miRNAs responding to arsenite treatment.</p