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Summary: Prenatal alcohol exposure (PAE) results in cerebral cortical dysgenesis. Single-cell RNA sequencing was performed on murine fetal cerebral cortical cells from six timed pregnancies, to decipher persistent cell- and sex-specific effects of an episode of PAE during early neurogenesis. We found, in an analysis of 38 distinct neural subpopulations across 8 lineage subtypes, that PAE altered neural maturation and cell cycle and disrupted gene co-expression networks. Whereas most differentially regulated genes were inhibited, particularly in females, PAE also induced sex-independent neural expression of fetal hemoglobin, a presumptive epigenetic stress adaptation. PAE inhibited Bcl11a, Htt, Ctnnb1, and other upstream regulators of differentially expressed genes and inhibited several autism-linked genes, suggesting that neurodevelopmental disorders share underlying mechanisms. PAE females exhibited neural loss of X-inactivation, with correlated activation of autosomal genes and evidence for spliceosome dysfunction. Thus, episodic PAE persistently alters the developing neural transcriptome, contributing to sex- and cell-type-specific teratology.

Prenatal ethanol exposure can result in loss of neural stem cells (NSCs) and decreased brain growth. Here, we assessed whether a noncoding RNA (ncRNA) related to the NSC self-renewal factor Oct4/Pou5f1, and transcribed from a processed pseudogene locus on mouse chromosome 9 (mOct4pg9), contributed to the loss of NSCs due to ethanol. Mouse fetal cortical-derived NSCs, cultured ex vivo to mimic the early neurogenic environment of the fetal telencephalon, expressed mOct4pg9 ncRNA at significantly higher levels than the parent Oct4/Pou5f1 mRNA. Ethanol exposure increased expression of mOct4pg9 ncRNA, but decreased expression of OCT4/POU5F1. Gain- and loss-of-function analyses indicated that mOct4pg9 overexpression generally mimicked effects of ethanol exposure, resulting in increased proliferation and expression of transcripts associated with neural maturation. Moreover, mOct4pg9 associated with Ago2 and with miRNAs, including the anti-proliferative miR-328-3p, whose levels were reduced following mOct4pg9 overexpression. Finally, mOct4pg9 inhibited Oct4/Pou5f1-3’UTR-dependent protein translation. Consistent with these observations, data from single-cell transcriptome analysis showed that mOct4pg9-expressing progenitors lack Oct4/Pou5f1, but instead overexpress transcripts for increased mitosis, suggesting initiation of transit amplification. Collectively, these data suggest that the inhibitory effects of ethanol on brain development are explained, in part, by a novel ncRNA which promotes loss of NSC identity and maturation.

Early developmental exposure to ethanol, a known teratogen, can result in a range of neurodevelopmental disorders, collectively referred to as Fetal Alcohol Spectrum Disorders (FASDs). Changes in the environment, including exposure to teratogens, can result in long term alterations to the epigenetic landscape of a cell, thereby altering gene expression. Noncoding RNAs (ncRNAs) can affect transcription and translation of networks of genes. ncRNAs are dynamically expressed during development and have been identified as a target of alcohol. ncRNAs therefore make for attractive targets for novel therapeutics to address the developmental deficits associated with FASDs.

• Epigenetic modifications change global gene expression in response to environmental factors.

Poverty, conflict, chemical contaminants, and substance abuse are examples of environmental conditions that are associated with malnutrition, stress, vulnerability to obesity, cancer, psychiatric and other diseases, and diminished life expectancy. We have come to expect that conditions such as these result in deleterious consequences for target populations. However, what if the effects of these conditions transcend generations? What if grandparents’ adverse life experiences influence health and disease susceptibility of their grandchildren or even great-grandchildren? What are the implications for the practice of medicine and social justice? Evidence from human and animal studies in the rapidly growing field of transgenerational epigenetics suggests that the inherited consequences of adverse ancestral life experiences are real. Biology can be programmed in one generation to produce long-lasting, multigenerational maladaptation. Periods of embryonic development and epigenetic programing, which modify the output of genes, are specifically vulnerable to environmental perturbations. This chapter discusses the somewhat checkered history of enquiry into the heritability of acquired traits, along with more recent experimental and epidemiological data that validates the phenomenon of transgenerational epigenetics. Last, we focus on specific examples of environmental perturbations, diet, exposure to toxic chemicals, and addiction that can be modeled in the laboratory, to show how transgenerational epigenetic mechanisms may underlie human susceptibility to disease.

Within neurons of several regions of the CNS, mature dendrite architecture is attained via extensive reorganization of arbor during the developmental period. Since dendrite morphology determines the firing patterns of the neuron, morphological refinement of dendritic arbor may have important implications for mature network activity. In the neocortex, a region of brain that is sensitive to activity-dependent structural rearrangement of dendritic arbor, the proportion of AMPA receptors increases over the developmental period. However, it is unclear whether changes in AMPA receptor expression contribute to maturation of dendritic architecture. To determine the effects of increasing AMPA receptor expression on dendrite morphology and connectivity within the neocortex, and to determine whether these effects are dependent on specific AMPA receptor subunits, we overexpressed the AMPA glutamate receptor subunit 1 (GluR1) and glutamate receptor subunit 2 (GluR2) in cultured rat neocortical neurons at the time that AMPA receptors would normally be incorporated into synapses. Following expression of GluR1 or GluR2 we observed increases in the length and complexity of dendritic arbor of cortical neurons, and a concurrent reduction in motility of spines. In addition, expression of either subunit was associated with an increased density of excitatory postsynaptic puncta. These results suggest that AMPA receptor expression is an important determinant of dendrite morphology and connectivity in neocortical neurons, and further, that contrary to other regions of the CNS, the effects of AMPA receptors on dendrite morphology are not subunit-specific.

The level and integrity of glutamate transmission during critical periods of postnatal development plays an important role in the refinement of pyramidal neuron dendritic arbor, synaptic plasticity, and cognition. Presently, it is not clear how excitatory transmission via the two predominant isoforms of the vesicular glutamate transporter (VGLUT1 and VGLUT2) participate in this process. To assess a neurodevelopmental role for VGLUT2 in pyramidal neuron maturation, we generated recombinant VGLUT2 knock-out mice and inactivated VGLUT2 throughout development using Emx1-Cre+/+knock-in mice. We show that VGLUT2 deficiency in corticolimbic circuits results in reduced evoked glutamate transmission, release probability, and LTD at hippocampal CA3-CA1 synapses during a formative developmental period (postnatal days 11–14). In adults, we find a marked reduction in the amount of dendritic arbor across the span of the dendritic tree of CA1 pyramidal neurons and reduced long-term potentiation and levels of synaptic markers spinophilin and VGLUT1. Loss of dendritic arbor is accompanied by corresponding reductions in the number of dendritic spines, suggesting widespread alterations in synaptic connectivity. Conditional VGLUT2 knock-out mice exhibit increased open-field exploratory activity yet impaired spatial learning and memory, endophenotypes similar to those of NMDA receptor knock-down mice. Remarkably, the impairment in learning can be partially restored by selectively increasing NMDA receptor-mediated glutamate transmission in adult mice by prolonged treatment withd-serine and ad-amino acid oxidase inhibitor. Our data indicate that VGLUT2 expression is pivotal to the proper development of mature pyramidal neuronal architecture and plasticity, and that such glutamatergic deficiency leads to cognitive malfunction as observed in several neurodevelopmental psychiatric disorders.