BDE-99 impairs differentiation of human and mouse NPCs into the oligodendroglial lineage by species-specific modes of action

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants causing developmental neurotoxicity (DNT) in humans and rodents. Their DNT effects are suspected to involve thyroid hormone (TH) signaling disruption. Here, we tested the hypothesis whether disturbance of neural progenitor cell (NPC) differentiation into the oligodendrocyte lineage (O4+ cells) by BDE-99 involves disruption of TH action in human and mouse (h,m)NPCs. Therefore, we quantified differentiation of NPCs into O4+ cells and measured their maturation via expression of myelin-associated genes (hMBP, mMog) in presence and absence of TH and/or BDE-99. T3 promoted O4+ cell differentiation in mouse, but not hNPCs, and induced hMBP/mMog gene expression in both species. BDE-99 reduced generation of human and mouse O4+ cells, but there is no indication for BDE-99 interfering with cellular TH signaling during O4+ cell formation. BDE-99 reduced hMBP expression due to oligodendrocyte reduction, but concentrations that did not affect the number of mouse O4+ cells inhibited TH-induced mMog transcription by a yet unknown mechanism. In addition, ascorbic acid antagonized only the BDE-99-dependent loss of human, not mouse, O4+ cells by a mechanism probably independent of reactive oxygen species. These data point to species-specific modes of action of BDE-99 on h/mNPC development into the oligodendrocyte lineage.

Scientific RepoRts | 7:44861 | DOI: 10.1038/srep44861 To shed light on this question, this study aimed to elucidate if disturbance of TH signaling by BDE-99 interferes with the neurodevelopmental process of oligodendrogenesis in vitro and thus might be a potential mode of action (MoA) of PBDE-induced DNT. First, we characterized the effects of BDE-99 on the formation of O4 + cells and their maturation in human and mouse NPCs (h,mNPCs). Second, we demonstrated the functionality of TH signaling in NPCs and assessed the species-specific contribution of TH to oligodendrocyte differentiation. Third, we elucidated the species-specific MoA of BDE-99 on the formation of O4 + cells and their maturation in hNPCs and mNPCs.

BDE-99 reduces NPC differentiation to the oligodendrocyte lineage.
Human and mouse NPCs grown as neurospheres migrate radially out of the sphere upon plating onto a poly-D-lysin/laminin matrix and form on average 6-8% O4 + cells/nuclei after 5 days of migration and simultaneous differentiation 33,34 . These percentages are similar to the oligodendrocyte fractions in human 35,36 and mouse 37,38 cortex in vivo supporting the physiological relevance of our findings. BDE-99 reduced differentiated O4 + cells/nuclei of both species concentration-dependently after 5 days without reducing viability (Fig. 1A,C). Human O4 + cell formation was found to be 7 times more sensitive towards the BDE-99 treatment than the murine process (IC 50 : 1.9 μ M and 13.6 μ M BDE-99 and IC 20 : 0.9 μ M and 6.9 μ M for hNPCs and mNPCs, respectively) showing significance in the nanomolar range (LOEAC 900 nM). Fluorescence microscope images illustrate the BDE-99-mediated reduction in O4 + cells after 5 days of differentiation (Fig. 1B,D) with the human IC 50 concentration of 2 μ M not reducing number of mouse O4 + cells/nuclei ( Supplementary Fig. S1).

TH signaling is functional in NPCs.
Human and mouse NPCs, as also previously described by others 39,40 , express genes encoding for TH receptors (TRs) α and β ( Supplementary Fig. S2A-D). Although the expression of the gene encoding for human TRβ 1 (THRB1) was significantly induced by the receptor active thyroid hormone L-triiodothyronine (T3), the low copy number change from 3 to 7 copies/10,000 β-actin makes a biological significance of this increase highly unlikely. Compared to the approximately 20-fold higher expression of TRα 1 in both species it is assumed that this receptor is more important at this stage of brain development. In vivo TRα 1 plays a role during early brain development and TRβ 1expression strongly increases postnatally in the rodent brain 41 . Functionality of TRs was demonstrated by T3-induced hairless mRNA expression ( Supplementary Fig. S2E,F). Hairless is a TH-inducible gene in fetal brains in vivo 42,43 that is TRα -dependent 44 . Furthermore, T3 induced its own metabolism by stimulating deiodinase 3 mRNA expression ( Supplementary Fig. S2G,H). TH effects on basal and BDE-99-reduced mouse and human O4 + cell formation. T3 and T4 only induced formation of O4 + cells/nuclei in mNPCs, but not in hNPCs after 5 days of differentiation ( Fig. 2A,D). A T3 concentration of 3 nM was used for all further experiments because it is close to the total T3 concentration in human serum [45][46][47][48] and produced the highest percentage of O4 + cells in mNPCs (Fig. 2D). After 5 days, 3 nM O4 + cells were immunocytochemically stained with an antibody against O4 and nuclei were counterstained with Hoechst 33258. Viability was measured with the Alamar-Blue assay two hours prior fixation. (A,C) Data for viability and the percentage of O4 + cells/nuclei in the migration area were normalized to the solvent control (mean ± SEM, n ≥ 3). A sigmoidal curve fit was applied and for oligodendrogenesis IC 20  T3 or T4 increased the percentage of O4 + cells/nuclei in control as well as in BDE-99 treated mNPCs, but not in hNPCs (Fig. 2B,E). Time-course experiments revealed that human O4 + cells form during the first 4 days of differentiation. Treatment of the cells with T3 accelerated, but did not increase the percentage of human O4 + cells after 5 days of differentiation. Moreover, T3 did not antagonize the inhibitory BDE-99 effect on human O4 + cell formation at any time point (Fig. 2C). In mouse cultures, O4 + cells formed within the first 1-2 days and T3 induced their formation in control as well as in BDE-99-treated NPCs at all timepoints with a similar potency (Fig. 2F).

TH effects on basal and BDE-99-reduced mouse and human oligodendrocyte maturation. For
investigating oligodendrocyte maturation, changes in expression of the oligodendrocyte-specific markers human myelin basic protein (hMBP) and murine myelin oligodendrocyte glycoprotein (mMog) were analyzed in the total neurosphere cell population over a time-course of 5 days (Fig. 3A,D). These different markers were used, because human oligodendrocytes do not express the late marker MOG after 5 days of differentiation due to their immaturity, while for murine oligodendrocytes -due to their high maturation stage -Mbp was already highly expressed on day 1 of differentiation and did not increase over differentiation time (data not shown). We also confirmed this species-dependent difference in oligodendrocyte maturation stage by morphological analysis as branched, sheet-forming O4 + cells found in murine culture represent phenotypically a more mature state than the more linear O4 + cells observed in human cultures (Fig. 3C). Both, hMBP and mMog expression increased significantly during the 5 days of differentiation indicating oligodendrocyte maturation (Fig. 3A,D). During this time period, T3 significantly induced gene copy numbers of NPCs' hMBP and mMog, while BDE-99 reduced T3-induced hMBP as well as mMog expression. To evaluate if this reduced hMBP and mMog expression is a consequence of reduced O4 + cell formation or rather a consequence of compromised oligodendrocyte maturation, hMBP and mMog expression after 5 days of differentiation were normalized to the percentage of O4 + cells in the respective cultures (6-8% O4 + cells;

TR involvement in TH and BDE-99 effects on mNPCs' differentiation to O4 + cells and mMog gene expression.
The involvement of TRs in T3 and BDE-99 effects on mNPCs' differentiation to O4 + cells and mMog gene expression was studied using neurosphere cultures prepared from PND1 TRα and β knockout mouse brains. Number of O4 + cells/nuclei as well as mMog gene expression/% O4 + cells (Q M ) were normalized to the respective genotype control because raw values did not differ significantly between genotype controls ( Supplementary Fig. S5). T3 significantly induced number of O4 + cells/nuclei (Fig. 4A) and their maturation ( Fig. 4B) in wildtype and TRβ , but not in TRα knockout neurospheres, confirming the specific involvement of TRα in T3-induced O4 + cell formation and maturation. On the contrary, BDE-99 effects on mouse O4 + cell formation and oligodendrocyte maturation were not mediated by TRs, because 10 μ M BDE-99 reduced the number of differentiated O4 + cells as well as Mog expression/% O4 + cells in TR knockout neurospheres comparable with wildtype neurospheres (Fig. 4A,B).
To confirm that 10 μ M BDE-99 indeed does not interfere with TR-mediated TH signaling, expression of TRs, deiodinase 3 and hairless, the latter TR-dependent gene products, was evaluated. BDE-99 did not influence TR expression ( Supplementary Fig. S2A-D) or TRα -mediated transcription of hairless alone or in combination with T3 ( Supplementary Fig. S2E,F). Furthermore, BDE-99 did not alter basal or T3-induced deiodinase 3 expression ( Supplementary Fig. S2G,H).

Reactive Oxygen Species (ROS) do not seem to be involved in BDE-99-dependent reduction of O4 + cells.
Since our studies revealed that TH disruption was not the mechanism by which BDE-99 compromised formation of human O4 + cells (Fig. 2B,C), we considered an alternative BDE-99 MoA, i.e. ROS formation. We hypothesized that the production of ROS might be involved in BDE-99-induced toxicity on O4 + cell formation. One indicator of ROS production is an adaptive induction of antioxidative defense gene expression like heme oxygenase 1, catalase, superoxide dismutase and glutathione peroxidase. 2 μ M BDE-99 did not alter the expression of any of these genes after 24 hours of BDE-99 exposure suggesting that BDE-99 is not producing an excess of ROS in hNPCs (Fig. 5A). However, application of ascorbic acid (50 and 100 μ M, not inducing O4 + cell formation per se) antagonized the BDE-99-dependent reduction of human O4 + cell formation (Fig. 5B). As Trolox, a water-soluble derivative of vitamin E and effective ROS-scavenger, did not exert comparable antagonizing effects in presence of BDE-99 (Fig. 5C), it is likely that ascorbic acid rescues BDE-99-reduced O4 + cell formation by a mechanism other than ROS scavenging. In contrast to human O4 + cell formation, mouse O4 + cell formation was not rescued by ascorbic acid against BDE-99 (Fig. 5E). Also Trolox did not rescue murine O4 + cells against BDE-99-induced toxicity (Fig. 5F). It is to note, that for scavenging BDE-99 effects in mNPCs, lower Trolox concentrations (10 and 20 μ M) were used than for hNPCs since in mNPCs, 50 and 100 μ M Trolox reduced the culture viability and O4 + cell formation (data not shown).

Discussion
PBDEs were recently classified as human DNT compounds 10 . The molecular mechanisms of PBDE-induced DNT, however, remain elusive. PBDEs alter TH homeostasis in rodents and interfere with TH signaling in vitro 49 . In addition, BDE-99 reduced oligodendrocyte differentiation of human NPCs 32 . Because TH is crucial for oligodendrogenesis and/or myelin production in vivo [17][18][19][50][51][52] , we investigated in this study if disruption of cellular TH signaling in hNPCs and mNPCs might be an underlying molecular mechanism for BDE-99-mediated reduction of O4 + cell formation and oligodendrocyte maturation.
BDE-99 reduced human and murine O4 + cell formation in a concentration-dependent manner (IC 50 1.9 μ M and 13.6 μ M, respectively and IC 20 0.9 μ M and 6.9 μ M, respectively) with human NPCs being 7-times more sensitive towards BDE-99 exposure than their murine counterparts. These data are in accordance with two other publications dealing with PBDE effects on oligodendrogenesis: our previous study with an IC 50 for inhibiting human O4 + cell formation around 1 μ M BDE-99 32 and inhibition of murine NSC differentiation into O4 + cells with an IC 25 for BDE-47 of 10 μ M 53 .
The IC 20 value for inhibiting human O4 + cell formation identified in this study lies in the upper nanomolar range (900 nM; approximately 450 ng/ml). Breast milk fed children are assumed to reach a daily PBDE exposure up to 306 ng/kg/day 54,55 and the serum concentration of the five most abundant PBDEs reached 480 ng/g lipid weight (lw; approx. 960 nM in serum lipids) for Californian toddlers 7 . In vivo, up to a 1.5-fold accumulation of PBDEs in brain lipids compared to the corresponding serum lipid samples in birds 56 and a 5.5 fold accumulation of BDE-47 in the rat brain versus serum 57 were observed indicating that PBDEs pass the blood-brain barrier and accumulate in the brain. Assuming similar accumulation in human brain lipids, our rough kinetic estimation suggests that toddler PBDE brain lipid concentrations could exceed 720 ng/g lw (approx. 1440 nM in brain lipids), which is higher than the observed IC 20 (900 nM) for human NPC differentiation in this study.
To test the hypothesis if TH disruption might be the underlying molecular mechanism for BDE-99-dependent inhibition of O4 + cell differentiation, we first characterized TH effects on human and mouse oligodendrogenesis. TH alone accelerated, but did not induce O4 + cell formation from hNPCs, while it induced the number of O4 + cells differentiated from mNPCs. In addition, TH enhanced human and murine oligodendrocyte maturation. Accelerated oligodendrocyte maturation due to TH treatment was also observed in mouse and human oligodendrocyte precursor cells (OPCs) 58 and in embryonic hNPCs differentiating into O4 + cells in vitro 59 supporting our findings. Moreover, similar to the rodent in vitro findings in this study, TH determines timing and amount of oligodendrocyte formation in rodents 52,60-62 in a TRα -dependent manner 63,64 , and hypothyroid mice express lower levels of Mbp than euthyroid mice, which might be due to less oligodendrocyte formation, their reduced maturation or a combination of both 52 . Neurospheres prepared from TRα and TRβ knockout mice indicated that TH-dependent induction of murine O4 + cell formation and oligodendrocyte maturation from mNPCs in vitro was also TRα -, not TRβ -dependent. Human patients with hypothyroidism during brain development also show delayed myelination 15,16,65 due to less oligodendrocyte formation or maturation or a combination of both 66 . Our species-comparative in vitro study suggests that in contrast to mNPCs, TH mainly guides hNPC-derived oligodendrocyte maturation as supported by others 58,59 . Concerning the speed of O4 + cell formation and oligodendrocyte maturation we found that these processes happen much faster in mouse than in human NPC cultures. Also in vivo oligodendrocytes form and mature much faster in rodents than in humans 67 . Thus, the closeness of the in vitro systems mirroring species-specific maturation speed strengthens the physiological relevance of our cell systems.
BDE-99 reduced NPC differentiation into O4 + cells in both species independent of TH as (i) human O4 + cell formation is not affected by TH and the TR antagonist NH-3 does not affect human NPC differentiation into O4 + cells (Supplementary Fig. S4) and (ii) BDE-99 reduces number of mouse O4 + cells in TH-induced neurospheres with the same magnitude than in untreated cells. Experiments with TRα and TRβ knockout mouse neurospheres support this data and add the information that TRs are not involved in BDE-99 action on O4 + cell formation. In accordance, BDE-99 did not inhibit T3-induced expression of hairless, a gene that is regulated by TRα in mice 44 and did not alter TR or T3-induced deiodinase 3 mRNA expression in either species.
As another possible MoA we postulated that the formation of ROS might be involved in BDE-99 toxicity on oligodendrogenesis. ROS formation by PBDEs is discussed controversially in the literature depending on the congener, cell type and method used 49 . BDE-99 did not up-regulate ROS-related genes in hNPCs and mNPCs and sub-cytotoxic concentrations of the antioxidant Trolox did not antagonize the BDE-99 effect on O4 + cell formation. Interestingly, ascorbic acid (50-100 μ M) rescued BDE-99-reduced O4 + cell formation in human, but not in mouse NPCs at concentrations not inducing oligodendrocytes in the cultures per se. A high ascorbic acid concentration (500 μ M) strongly induced formation of human, but not mouse O4 + cells (Supplementary Fig. S6). Ascorbic acid enables hydroxylation of proline to hydroxyproline 68 , a major component of the extracellular matrix protein collagen promoting rodent myelination of dorsal root ganglion neurons by Schwann cells in the peripheral nervous system [69][70][71] . The effects of ascorbic acid on central nervous system oligodendrogenesis are not known. However, the mechanism by which ascorbic acid antagonizes BDE-99-dependent reduction in human O4 + cell formation might involve collagen production. Interference of BDE-99 with mouse O4 + cell formation is clearly mediated by a different, yet unknown, MoA, as neither ascorbic acid nor Trolox antagonize this adverse effect. The maturation quotient Q M demonstrated that BDE-99 at the IC 50 concentration for O4 + cell formation did not affect human, but inhibited mouse gene expression related to oligodendrocyte maturation. These BDE-99 effects on mMog expression were also independent of TH signaling as TH did not antagonize the effect and TR knockout neurospheres were not protected against BDE-99-dependent loss of O4 + cells. Moreover, ascorbic acid did not antagonize reduction of BDE-99-dependent mMog expression (Supplementary Fig. S7) pointing to a mechanism distinct from the MoA in hNPCs. BDE-99 concentrations, which did not interfere with basal O4 + cell differentiation or mMog expression, however, prohibited T3 induction of mMog by a yet unknown mechanism.
Another possibility that was not addressed within this paper but is worth exploring in the future is that BDE-99 does not directly act on oligodendrocytes, but exerts its actions indirectly e.g. via astrocyte signaling. Astrocytes play a major role in growth factor release like PDGF, FGF2, LIF, CNTF, IGF-1, NT-3, extracellular matrix-related molecules or BMPs, which obtain known functions in oligodendrogenesis 19,[72][73][74] . These soluble factors have the ability to influence oligodendrocyte differentiation e.g. by epigenetic mechanisms and thus might provide a speculative rationale for BDE-99-dependent effects on oligodendrocyte differentiation via suppressed gene expression in cells of the oligodendrocyte lineage 72 . Unique features of the 'Neurosphere Assay' allowing investigations of such paracrine aspects in the future are its multicellularity, and the possibility to study multiple species in equivalent cell systems. However, in case of e.g. RT-PCR evaluation of cell type-specific markers the desired cell population might only be a fraction of the total cell number like in this study, where we find 6-8% oligodendrocytes after 5 days of differentiation 34 . With regards to the species comparison, the timing aspect has to be carefully considered as speed of developmental processes observed in neurospheres seem to reflect at least to some extent the pace of the species of cell origin, which is very different between humans and rodents 33 .

Conclusions
We developed a human and mouse NPC-based test system to identify compounds with adverse effects on oligodendrogenesis including TH signaling disruptors. With this test we identified BDE-99 as an inhibitor of O4 + cell differentiation in both species and a blocker of oligodendrocyte maturation-related gene expression (mMog) only in mouse cultures. BDE-99 effects follow unknown MoAs in both species, but ascorbic acid rescued inhibited O4 + cell differentiation only in human cultures, while it exerted no effect on any of the endpoints studied in mouse neurospheres. This study indicates the need to consider species differences in susceptibility and MoA analyses of chemicals for their DNT potential and thus suggests the necessity of using human cells for human hazard characterization of compounds that might be toxic for the developing brain. Neurosphere Culture. Human neural progenitor cells (gestational week (GW) [16][17][18][19][20] were purchased from Lonza Verviers SPRL (Belgium). Developmental time-matched 76 mouse NPCs from postnatal day (PND) 1 were prepared as described previously for rat NPCs 77 with the modification that only the mouse forebrain was used and digested with papain for 7 min. Experiments with human NPCs purchased from Lonza Verviers SPRL (Belgium) are ethical approved by the ethics committee of the Heinrich-Heine-University, Düsseldorf. Preparation of NPCs from mouse brain tissue and experiments with mouse NPCs are in accordance with German regulations and the experimental guidelines of the State Agency for Nature, Environment and Consumer Protection in North Rhine-Westphalia in Germany (LANUV) and are approved by the LANUV (license number: 84-02.05.40.14.140). To study the involvement of TH receptors (TRs) in oligodendrogenesis, NPCs from TRα and TRβ knockout mice were prepared. Human and mouse NPCs were cultured as neurospheres in proliferation medium consisting of DMEM (Life Technologies) and Hams F12 (Life Technologies) (3:1) supplemented with 2% B27 (Life Technologies), 20 ng/ml epidermal growth factor (EGF, Life Technologies), 20 ng/mL (hNPCs) or 10 ng/ml (mNPCs) recombinant human fibroblast growth factor (FGF, R&D Systems), and 1% penicillin and streptomycin (Pan-Biotech). Neurospheres were maintained at 37 °C with 5% CO 2 and passaged mechanically with a tissue chopper to 0.2 mm once a week. Half of the medium was replaced thrice a week.

Differentiation of NPCs.
NPCs were chopped to 0.2 mm 1-3 days (hNPCs) or 3-5 days (mNPCs) before plating. The longer time between chopping and plating is due to the fact that mouse neurospheres need longer to regenerate and form even, round spheres again. Human or mouse NPCs were differentiated in differentiation medium [DMEM (Life Technologies), Hams F12 (Life Technologies) 3:1 supplemented with 1% of N2 (Life Technologies) and 1% penicillin and streptomycin (Pan-Biotech) and only for the mouse 1% hormone-free fetal calf serum (FCS; Biochrom) to prevent apoptosis was added] containing the respective exposure(s) or solvent(s). Half of the exposure/solvent medium was refreshed at day 2 of differentiation. Under differentiating conditions NPCs radially migrate out of the neurosphere core (migration area) and differentiate into the three major cell types of the brain: neurons, oligodendrocytes and astrocytes 78,79 .
Scientific RepoRts | 7:44861 | DOI: 10.1038/srep44861 Differentiation of NPCs for viability measurement and staining of O4 + cells. Neurospheres of 0.3 mm diameter were washed in differentiation medium and plated into poly-D-lysine (PDL)/laminin (Sigma Aldrich) coated 8-chamber glass cover slides (LMS Consult). In each chamber, five neurospheres were plated in 500 μ l differentiation medium containing the respective exposure(s) or solvent(s). Neurospheres were differentiated for 5 days or in the time course experiment for 1, 2, 3, 4 and 5 days, where 5 slides were plated on the same day and one was fixed each day. Each experiment was repeated at least 3 times. Mean and SEM were calculated from those repeated experiments.
Differentiation of NPCs for RT-PCR. Neurospheres were differentiated in PDL/laminin coated wells of 24 well plates (Sarstedt) in 1 ml differentiation medium containing the respective exposure(s)/solvent(s). Three wells with ten 0.3 mm neurospheres each were used per exposure condition. Neurospheres were differentiated for 5 days or in time course experiments for 1, 3 and 5 days, where 3 plates were plated at the same day and cells in one plate were lysed on each day 1, 3 or 5 of differentiation. Each experiment was repeated at least 3 times. Mean and SEM were calculated from those repeated experiments. Viability assay. Cell viability was assessed using Alamar Blue reagent (CellTiter-Blue assay, Promega) by measuring mitochondrial reductase activity after 5 days of differentiation in the same slides which cells were later fixed and immunocytochemically stained for O4 + cells. The reagent was pre-diluted 1:3 in differentiation medium and added to the chambers (1:4) two hours prior to fixation. The neurospheres were incubated for 2 h at 37 °C and 5% CO 2 . The incubated reagent was pipetted into a 96-well plate in technical duplicates of 100 μ l to determine fluorescence with the Tecan infinite M200 Pro reader (ex: 540 nm; em: 590 nm). Background of only medium without cells was subtracted from the sample RFU values. Duplicate RFU values from each chamber were pooled. Each viability experiment was repeated at least three times independently. Mean and SEM were calculated from those repeated experiments. Viability data which are not included in the main manuscript are shown in the Supplementary Fig. S3. Fluorescence microscopy and quantification of O4 + cells. In each experiment five neurospheres/treatment and two fluorescence images/neurosphere, taken at defined, opposite sides of the neurosphere core (in the same distance to the neurosphere core for each differentiation day), were evaluated resulting in 10 image evaluations/ treatment/experiment. Images were taken using two channels of a fluorescent microscope (Zeiss, Axio Observer D1): ex: 359 nm; em: 461 nm for visualizing Hoechst staining and ex: 495 nm; em: 519 nm for O4 evaluation. Images were converted to 8-bit, binarized and watershed transformed for quantification of the number of nuclei by using an ImageJ macro. Only segments satisfying suitable size constraints (x-y) were considered as valid nuclei. O4 + cells were counted manually. Then the percentage of O4 + cells/nuclei was calculated for each picture and the percentages were pooled between the two pictures of the same neurosphere. Afterwards the mean and standard deviation between the five neurospheres was calculated for each treatment. The solvent control was set to 100% (except for the time-course experiment showing formation of O4 + cells over time). Each figure contains data from at least three independent experiments and for each condition mean and SEM were calculated from those independent experiments.

Immunocytochemistry.
On average for all O4 + cell formation experiments included in the manuscript, solvent controls of hNPCs contained 620 nuclei and 38 O4 + cells per image (6% O4 + cells) and of mNPCs with a less dense migration area 310 nuclei and 18 O4 + cells (6% O4 + cells) per image. For ten microscope pictures per solvent control this ends up in approximately 380 O4 + cells/6200 nuclei for hNPCs and 180 O4 + cells/3100 nuclei for mNPCs per n. Substance treatment does not change the amount of nuclei per picture, but only the amount of O4 + cells.
Quantitative RT-PCR (qRT-PCR). RNA of untreated proliferating (triplicated samples of 50 neurospheres with a 0.3 mm diameter were collected 2 (hNPCs) or 3 (mNPCs) days after chopping) or 1,3 or 5 days under exposures/solvents differentiated NPCs (as described above) was isolated (RNeasy Mini Kit, Qiagen, Hilden, Germany) and cDNA was transcribed (Quantitect Reverse Transcription Kit, Qiagen) according to the manufacturer instructions. qRT-PCR was performed using QuantiFast SYBR Green PCR Kit (Qiagen) in the Rotor Gene Q cycler (Qiagen). Copy numbers of the gene of interest were normalized to β-actin expression by using standards or expression was evaluated by the ddCT method normalizing to β-actin expression in the solvent control samples. Each PCR experiment was performed at least three times independently. Means and SEMs were calculated from those independent experiments. The detailed PCR protocol and details for evaluation as well as the used primer sequences are given in the Supplementary Material.

Statistics.
For dose-response curves a sigmoidal curve fit (variable slope) was applied using Graphpad Prism 6.0. Data was analyzed with the same software using OneWay ANOVA for dose-response experiments, TwoWay ANOVA for co-exposure and time-course experiments (non-repeated measures) or a student's t-test for comparison of wildtype and knockout mice within the same treatment. As post-hoc tests Bonferroni's (OneWay ANOVA) or Tukey's (TwoWay ANOVA) multiple comparison test were used. T-tests were performed with Welsh's correction assuming different standard deviations for the treatments. Significance threshold was established at p < 0.05.