<i>p21</i> is upregulated as a response to <i>Tra2b</i> depletion in the mouse brain and in neural stem cells.

<p>(<b>A,B</b>) Semi-quantitative RT-PCR using whole brain RNA of neuronal specific <i>Tra2b</i> KO mice and controls. <i>p21</i> is significantly upregulated by 1.5-fold in KO mice as compared to HET or control mice. <i>p21</i> expression is indifferent between controls and HET mice. <i>p21</i> expression was normalized to <i>Hprt</i>. (<b>C–G</b>) NSC34 neural stem cells were transfected with siRNAs specific to <i>Tra2b</i> or scrambled siRNAs. siRNA treatment but not scr-treatment effectively reduced Tra2b protein and mRNA levels after 24 h, 48 h and 72 h after transfection (<b>C–E</b>). Tra2b function was strongly reduced as the <i>Nasp</i> transcript showed a significantly lower inclusion of the T-exon at 24 h, 48 h and 72 hours after transfection (<b>F</b>). 48 hours after transfection <i>p21</i> expression was found slightly but significantly increased on RNA level (<b>G</b>) but not on protein level (<b>D</b>). 72 hours after transfection p21 was massively and highly significantly upregulated on RNA and protein level by +2.2-fold (<b>D,G</b>). a.u., arbitrary units; nt, non-treated; scr, scrambled siRNA; si, siRNA against <i>Tra2b</i>; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p

Brain malformations are initiated by massive apoptosis in the cortex.

<p>(<b>A</b>) Immunostaining for Caspase-3 on paraffin-embedded coronal sections indicates prominent apoptosis in the proximal cortical layers and in the thalamic area of 14.5 dpc and 15.5 dpc KO embryos (black arrowheads). Remaining cortical tissue does not show apoptosis at 16.5 dpc and later stages (light arrowhead). (<b>B</b>) Immunostaining for Ki-67 shows initial decrease of proliferation at 14.5 dpc which is fully lost at 16.5 dpc in KO animals (black arrowheads). Control and HET animals retain strong Ki-67 signals in the proximal cortical layers at all indicated developmental stages. Scale bar equals 400 µm; ctx, cortex; th, thalamus; cpt, caudoputamen; sn, septal nuclei; 3v, third ventricle; lv, lateral ventricle.</p

Mouse whole exon array analysis reveals <i>Tubd1</i> exon4 and <i>Sgol2</i> exon4 as <i>in vivo</i> targets of <i>Tra2b</i>.

<p>Whole brain RNA of 4 CTRL animals and 4 KO animals was analyzed on mouse exon array. (<b>A</b>) The inclusion ratio (PSI, percent splicing inclusion) of each identified exon is defined as [PSI_KO]/[PSI_CTRL]. PSI distribution reached from ∼0.2 until ∼4.0. (<b>B</b>) Initial filtering strategies comprised exclusion of PSIs between 0.66 and 1.5 (grey bars) as well as restriction to p-values smaller than 0.05 which yielded a total of 1,006 exons. Exons associated with transcripts identified as being transcriptionally up- or downregulated were excluded from analysis. Ranking of those was further refined using large PSI values and considering presence of putative Tra2b binding sites (AGAA-motifs). Thereby, exons had to contain at least a single AGAA-site and a AGAA-frequency higher than 1.5. (<b>C,D,G,H</b>) Semi-quantitative RT-PCT on whole brain RNA was carried out using isoform specific primers for <i>Sgol2</i> FL (<b>C</b>), <i>Sgol2</i> Δ4 (<b>D</b>), <i>Tubd1</i> FL (<b>G</b>) and <i>Tubd1</i> Δ4 (<b>H</b>) confirming splicing events identified on the microarray. All isoform expression levels were densitometrically measured and normalized against <i>Hprt</i> (<b>E,F,I</b>). The <i>Tubd1</i> Δ4 isoform could not be detected using whole brain RNA, as skipping of exon4 introduces numerous premature termination codons leading to nonsense-mediated decay of the transcript (<b>H</b>). Treatment of wt and <i>Tra2b</i>-depleted murine embryonic fibroblasts with emetine successfully inhibited NMD and the <i>Tubd1</i> Δ4 isoform was detectable in <i>Tra2b</i>-depleted cells only (<b>J</b>). FL, full length; Δ4, transcript lacking exon 4, (−) PCR negative control; a. u., arbitrary units; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p

Conditional ablation of <i>Tra2b</i> causes perinatal lethality and disturbed cortical patterning in mice.

<p>(<b>A</b>) Cross breading of <i>Tra2b^fl/fl</i> with <i>Tra2b^fl/+; Nestin-Cre^tg/0</i> mice allowed generation of neuronal-specific knock-out (KO) animals as well as controls (CRTL) and heterozygous knock-out animals (HET) in one litter. (<b>B</b>) KO animals are born alive and all possible genotypes were detected according to Mendelian law (N = 113). (<b>C</b>) General development of conditional knock-out mice is not impaired as there are no gross morphological differences in embryo appearance. (<b>D</b>) Hematoxylin/Eosin staining of paraffin-embedded coronal sections at indicated developmental stages. KO animals but not controls or HET animals show ventriculomegaly of the third and lateral ventricles starting at around 14.5 dpc. Cortical layers are largely distinguishable at 14.5 dpc but cortical patterning and the ependymal lining of the lateral ventricle appears highly disturbed (black arrowheads) at 16.5 dpc in knock-out brains. (<b>E</b>) Immunostaining of <i>Tra2b</i> on paraffin-embedded coronal sections shows efficient downregulation of <i>Tra2b</i> protein in knock-out brains compared to controls and heterozygote animals. Cells of the ventricular and subventricular zones of the cortex show strongest decrease in staining intensity (black arrowhead). Scale bar equals 400 µm; ctx, cortex; th, thalamus; cpt, caudoputamen; cp, cortical plate; iz, intermediate zone; svz, subventricular zone; vz, ventricular zone; sn, septal nuclei; 3v, third ventricle; lv, lateral ventricle; pc, choroid plexus.</p

Splicing of <i>Sgol2</i> and <i>Tubd1</i> is responsive to changes in Tra2b concentration.

<p>(<b>A</b>) The murine and human versions of the genomic regions comprising the identified exons of <i>Sgol2</i> and <i>Tubd1</i> were cloned into the pSPL3 exon trapping vector. (<b>B</b>) HEK293T cells were co-transfected with the pSPL3 minigene vector and siRNA specific for <i>Tra2b</i> or a <i>TRA2B-GFP</i> expression vector. Western Blot analysis shows efficiently reduced Tra2b protein levels and solid overexpression of TRA2B-GFP. (<b>C</b>) RNA was analyzed for exon inclusion after 48 h by semi-quantitative RT-PCR. (<b>D–G</b>) RT-PCR results were densitometrically quantified. Exon 4 of murine but not human <i>Sgol2</i> is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 43% to 89%. Knock-down of <i>Tra2b</i> is insufficient to reduce <i>Sgol2</i> exon4 splicing inclusion. Exon 4 of human but not murine <i>Tubd1</i> is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 79% to 94%. Knock-down of <i>Tra2b</i> decreased inclusion of exon 4 from 79% to 71%. Nt, non-treated; scr, scrambled siRNA; si, siRNA against <i>Tra2b</i>; n.s., non significant; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p

Enrichment of embryonic development associated genes in 14 days of hESC differentiation.

<p><b>A</b>, A schematic representation of the 14 days hESC differentiation. <b>B</b>, An unsupervised hierarchical clustering analysis of DEG in hESC differentiation showed two distinct clusters for up and down-regulated genes. <b>C</b>, The over expressed neuron development BP (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044228#pone.0044228.s008" target="_blank">Table S4</a>) encompasses 86 genes and their gene expression pattern showed in volcano plot. In x-axis represents fold change and y-axis showed FDR-controlled p-values (p≤0.05). <b>D</b>, The pluripotency markers were significantly downregulated (as shown by microarray) after 14 days of differentiation. <b>E</b>, The mass spectrometry analysis of regulated proteins unravelled the up-regulated spots for neuronal development related proteins and (<b>F</b>) the down-regulated spots for pluripotency regulators. The differentially regulated proteins (n = 3), (p≤0.01) are shown with their corresponding spot ID and UniProt ID (E (I), F (I)). Their expression patterns are represented as a percentage of volume (E (II), F (II)). The error bar represents SEM from 3 independent biological replicates.</p

Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

<div><p>Embryonic development can be partially recapitulated <em>in vitro</em> by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant <em>in vivo</em> and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many <em>in vivo</em> and <em>in vitro</em> platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.</p> </div

Dose-dependent gene expression response of thalidomide in hESC differentiation.

<p><b>A</b>, The experimental approach of hESC differentiation and thalidomide treatment. <b>B</b>, Selected thalidomide concentrations do not induce any cytotoxicity in hESC differentiation. The scale bar represents 250 µm. <b>C</b>, 3D PCA showed that thalidomide treatment induces the changes in the gene expression pattern with increasing concentration. The 3 biological replicates for each sample group are showed in single colour. <b>D</b>, The number of DEG progressively increased with thalidomide concentration. Black shows up-regulated and grey shows down-regulated transcripts. Fold change ≥2 or ≤−2, (p≤0.05). <b>E</b>, An unsupervised hierarchical clustering of DEG demonstrated a dose-dependent repression. The highly expressed genes in untreated control were repressed for thalidomide in a concentration dependent manner. Data represents from 3 biological replicates. <b>F</b>, To quantify the BP the average relative fold change values were calculated manually (for all transcripts present in each GO) and represented for 14 day old EBs and thalidomide treatment. The error bar represents SEM from fold change values for transcripts belong to each GO.</p

Thalidomide perturbed heart, limb development and WNT signalling associated genes.

<p><b>A</b>, The heat map shows the microarray expression pattern for the selected genes for heart, limb development (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044228#pone.0044228.s010" target="_blank">Table S6D, S6F</a>) and WNT signalling (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044228#pone.0044228.s013" target="_blank">Table S9</a>). Since multiple genes are involved in various developmental processes few redundant genes are present. The up-regulated genes in control were repressed in a dose dependent manner. <b>B</b>, The representative heart (I), limb development (II) associated genes were analysed using RT-qPCR analysis with an independent experiment. Thalidomide responsive genes were shown in I and II (*p-value≤0.01, thalidomide-treated vs untreated 14-days old EBs) and 14 days old EBs gene expression were shown in (III) *p-value≤0.01, 14 days old EBs vs ESCs . The error bars represents the SEM from 3 technical replicates. <b>C</b>, The mass spectrometry results showed down-regulation of DDAH2 gene (n = 3), (p≤0.01). Figure C (I) represents spot analysis of DDAH2, mRNA and protein fold change (FC) values. C (II) The immunoblotting analysis of representative genes for thalidomide treatment. <b>D</b>, The immunoflourescence results shows the localisation of β-catenin and the gradient suppression at 70 µM thalidomide treatment. The scale bar represents 20 µm. <b>E</b>, The figure representing limb bud patterning was adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044228#pone.0044228-Niswander1" target="_blank">[40]</a> and modified. The figure shows the molecular markers that SHH and ZPA (essential for limb development) were affected by thalidomide treatments. The green colour coded genes are more than −1.8 fold down-regulated.</p

Selected down-regulated significant (p≤0.01) GO categories after thalidomide treatment.

<p>GO categories were identified with DAVID analysis using DEG from the respective treatment concentration.</p