Ontogenetic expression of thyroid hormone signaling genes: An in vitro and in vivo species comparison.

Thyroid hormone (TH) is essential for brain development. While disruption of TH signaling by environmental chemicals has been discussed as a mechanism of developmental neurotoxicity (DNT) for more than a decade, there remains a paucity of information linking specific TH disrupting chemicals to adverse neurodevelopmental outcomes. This data gap reflects, in part, the fact that the molecular machinery of TH signaling is complex and varies according to cell type and developmental time. Thus, establishing a baseline of the ontogenetic profile of expression of TH signaling molecules in relevant cell types is critical for developing in vitro and alternative systems-based models for screening TH disrupting chemicals for DNT. Here, we characterize the transcriptomic profile of molecules critical to TH signaling across three species-human, rat, and zebrafish-in vitro and in vivo across different stages of neurodevelopment. Our data indicate that while cultured human and rat neural progenitor cells, primary cultures of rat cortical cells, and larval zebrafish all express a fairly comprehensive transcriptome of TH signaling molecules, the spatiotemporal expression profiles as well as the responses to TH vary across species and developmental stages. The data presented here provides a roadmap for identifying appropriate in vitro and in simpler systems-based models for mechanistic studies and screening of chemicals that alter neurodevelopment via interference with TH action.

Deficiency of the clock gene Bmal1 affects neural progenitor cell migration.

We demonstrate the impact of a disrupted molecular clock in Bmal1-deficient (Bmal1-/-) mice on migration of neural progenitor cells (NPCs). Proliferation of NPCs in rostral migratory stream (RMS) was reduced in Bmal1-/- mice, consistent with our earlier studies on adult neurogenesis in hippocampus. However, a significantly higher number of NPCs from Bmal1-/- mice reached the olfactory bulb as compared to wild-type littermates (Bmal1+/+ mice), indicating a higher migration velocity in Bmal1-/- mice. In isolated NPCs from Bmal1-/- mice, not only migration velocity and expression pattern of genes involved in detoxification of reactive oxygen species were affected, but also RNA oxidation of catalase was increased and catalase protein levels were decreased. Bmal1+/+ migration phenotype could be restored by treatment with catalase, while treatment of NPCs from Bmal1+/+ mice with hydrogen peroxide mimicked Bmal1-/- migration phenotype. Thus, we conclude that Bmal1 deficiency affects NPC migration as a consequence of dysregulated detoxification of reactive oxygen species.

Teratological and Behavioral Screening of the National Toxicology Program 91-Compound Library in Zebrafish (Danio rerio).

To screen the tens of thousands of chemicals for which no toxicity data currently exists, it is necessary to move from in vivo rodent models to alternative models, such as zebrafish. Here, we used dechorionated Tropical 5D wild-type zebrafish embryos to screen a 91-compound library provided by the National Toxicology Program (NTP) for developmental toxicity. This library contained 86 unique chemicals that included negative controls, flame retardants, polycyclic aromatic hydrocarbons (PAHs), drugs, industrial chemicals, and pesticides. Fish were exposed to 5 concentrations of each chemical or an equal amount of vehicle (0.5% DMSO) in embryo medium from 6 h post-fertilization (hpf) to 5 days post-fertilization (dpf). Fish were examined daily for mortality and teratogenic effects and photomotor behavior was assessed at 4 and 5 dpf. Of the 5 negative control compounds in the library, none caused mortality/teratogenesis, but two altered behavior. Chemicals provided in duplicate produced similar outcomes. Overall, 13 compounds caused mortality/teratology but not behavioral abnormalities, 24 only affected behavior, and 18 altered both endpoints, with behavior affected at concentrations that did not cause mortality/teratology (55/86 hits). Of the compounds that affected behavior, 52% caused behavioral abnormalities at either 4 or 5 dpf. Compounds within the same functional group caused different behavioral abnormalities, while similar behavioral patterns were caused by compounds from different groups. Our data suggest that behavior is a sensitive endpoint for developmental toxicity screening that integrates multiple modes of toxic action and is influenced by the age of the larval fish at the time of testing.

A transcriptome comparison of time-matched developing human, mouse and rat neural progenitor cells reveals human uniqueness.

It is widely accepted that human brain development has unique features that cannot be represented by rodents. Obvious reasons are the evolutionary distance and divergent physiology. This might lead to false predictions when rodents are used for safety or pharmacological efficacy studies. For a better translation of animal-based research to the human situation, human in vitro systems might be useful. In this study, we characterize developing neural progenitor cells from prenatal human and time-matched rat and mouse brains by analyzing the changes in their transcriptome profile during neural differentiation. Moreover, we identify hub molecules that regulate neurodevelopmental processes like migration and differentiation. Consequences of modulation of three of those hubs on these processes were studied in a species-specific context. We found that although the gene expression profiles of the three species largely differ qualitatively and quantitatively, they cluster in similar GO terms like cell migration, gliogenesis, neurogenesis or development of multicellular organism. Pharmacological modulation of the identified hub molecules triggered species-specific cellular responses. This study underlines the importance of understanding species differences on the molecular level and advocates the use of human based in vitro models for pharmacological and toxicological research.

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.

Omnisphero: a high-content image analysis (HCA) approach for phenotypic developmental neurotoxicity (DNT) screenings of organoid neurosphere cultures in vitro.

Current developmental neurotoxicity (DNT) testing in animals faces major limitations, such as high cost and time demands as well as uncertainties in their methodology, evaluation and regulation. Therefore, the use of human-based 3D in vitro systems in combination with high-content image analysis (HCA) might contribute to DNT testing with lower costs, increased throughput and enhanced predictivity for human hazard identification. Human neural progenitor cells (hNPCs) grown as 3D neurospheres mimic basic processes of brain development including hNPC migration and differentiation and are therefore useful for DNT hazard identification. HCA of migrated neurospheres creates new challenges for automated evaluations because it encompasses variable cell densities, inconsistent z-layers and heterogeneous cell populations. We tackle those challenges with our Omnisphero software, which assesses multiple endpoints of the 'Neurosphere Assay.' For neuronal identification, Omnisphero reaches a true positive rate (TPR) of 83.8 % and a false discovery rate (FDR) of 11.4 %, thus being comparable to the interindividual difference among two researchers (TPR = 94.3, FDR = 11.0 %) and largely improving the results obtained by an existing HCA approach, whose TPR does not exceed 50 % at a FDR above 50 %. The high FDR of existing methods results in incorrect measurements of neuronal morphological features accompanied by an overestimation of compound effects. Omnisphero additionally includes novel algorithms to assess 'neurosphere-specific' endpoints like radial migration and neuronal density distribution within the migration area. Furthermore, a user-assisted parameter optimization procedure makes Omnisphero accessible to non-expert end users.