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Genome-wide transcriptomic interrogation of organoids and 3D tissue models are increasingly used for characterizing drug, toxicity responses and neurodevelopmental disorders. We established here a neuro-toxicogenomic assay by utilizing “minibrain”, a humanin vitro3D brain model system and a low-cost, highly multiplexable RNA-seq methodology (BRB-seq) for screening the effect of trimethyltin chloride (TMT) induced neurotoxicity. We demonstrate that transcriptomic profiling is insightful to the cellular composition and regional identity of the minibrain. Further, we characterize the transcriptomic changes associated with the dose-time neurotoxic response of minibrain upon exposure to TMT. The distinct gene expression changes and molecular candidates identified with our pipeline provides insight to map the key events involved in the adverse outcome pathway of TMT associated neurotoxicity. We identify processes such as endoplasmic reticulum stress, dysregulation of synaptic genes and downregulation of neuron-morphology associated genes upon exposure to TMT. In response to TMT, we identify activation of an early response homeostatic mechanism in minibrain and an interplay of STAT pathways correlating with the dose severity. In this study, we present a neuro-toxicogenomic assay that demonstrates the power of a low-cost transcriptomic screening to study chemical induced neurotoxicity.

Neocortical neurons are born from progenitors situated in the ventricular zone (VZ) and migrate to the cortex to form circuits that underlie mammalian high skilled behavior. Genetic programs that broadly specify distinct subtypes of neurons within the neocortex are increasingly understood. But despite the mounting evidence on how different genes regulate different aspects of cortical neurogenesis and neuron differentiation, the genetic program that drives acquisition and maintenance of neuronal identity upon progenitor division is unknown. It is thus important to dissect the proper sequence of molecular events that occurs during acquisition of neuronal identity. A major limitation in studying this has been the inability to specifically identify, access and track specific cohort of neurons from their time of birth in vivo with the requisite temporal resolution. To overcome this obstacle, I have developed and standardized a novel technique “FlashTag” that enables precise in vivo tagging of isochronic cohorts of VZ-born neurons and progenitors from the time of mitosis. By combining this high temporal resolution “FlashTag” technology with single-cell RNA sequencing, we have identified and functionally characterized neuron-specific primordial transcriptional programs as they dynamically unfold in vivo during the first forty-eight hours following mitosis. Our results provide a detailed functional account of early neuronal life, including the discovery of successive “transcriptional waves” that organize the temporal sequence of cellular differentiation events. This high-resolution dynamic blueprint of neuronal specification can identify the transient molecular events that guide a neuron towards its final fate, and contribute to a roadmap for the reverse engineering of specific classes of cortical neurons from undifferentiated cells.

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self‐renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here, we generated knock‐in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates in undirected differentiation. Using ATAC‐seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation‐associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration‐dependent priming of differentiation‐associated enhancers.