DNA Methylation program in normal and alcohol-induced thinning cortex
Introduction
Children with Fetal Alcohol Spectrum Disorders (FASD) have been reported to suffer cognitive and neurological deficits, including learning disabilities, intellectual disabilities, and impairments of expressive and receptive language (Green, 2007, Jacobson et al., 2011, Jones et al., 2010, Lebel et al., 2012). Some of the underlying brain abnormalities of FASD include reduced brain volume (microcephaly), reduced grey matter (Nardelli, Lebel, Rasmussen, Andrew, & Beaulieu, 2011), and reduced corpus callosum (Yang, Phillips, et al., 2012). Cross-sectional neuroimaging studies have also recently revealed that children and adolescents suffering from FASD exhibit abnormalities in the thickness of different regions of the cerebral cortex, as compared to healthy controls (Robertson et al., 2016, Sowell et al., 2008, Yang et al., 2012b). While several observations have been made regarding the fundamental hindrance of alcohol on cortical development, e.g., apoptosis (Lebedeva et al., 2015), deficiency of neurotrophic factors, prevention of cell migration (Aronne et al., 2011, Chikhladze et al., 2011, Riar et al., 2016), and abnormal somatic morphologies of cortical neurons (Chikhladze et al., 2011, Lawrence et al., 2012), the mechanism underlying the structural abnormality systemically occurring in relationship to alcohol exposure is not clear.
Recently, alcohol has emerged as a key chemical player, which can reach nuclear chromatin and alter the core functions of DNA (for review see Resendiz, Lo, Badin, Chiu, & Zhou, 2016). We and other investigators (Perkins, Lehmann, Lawrence, & Kelly, 2013) have recently found that DNA methylation, an important regulator of gene expression, progresses in the developing nervous system as a program (Zhou, 2012), and is disturbed by alcohol in many aspects across neural tube (Zhou, Chen, & Love, 2011) and hippocampal development (Chen et al., 2013, Otero et al., 2012). Given the known intricacies of epigenetic mechanisms such as DNA methylation in gene regulation and cellular specification, we sought to characterize the epigenetic and phenotypic changes of chronic, moderate prenatal alcohol exposure in utero in the developing stages of the cortex. In this study, we report that beyond neural tube formation, the formation of the cortices adopt a systemic DNA methylation program (DMP) (including DNA methylation and its binding proteins), by which neuroepithelial cells (NEs) differentiate through the formation of cortical layers in a precise spatiotemporal manner. Aside from confirming cortical phenotypes of FASD, we also demonstrated novel deficiencies. These processes, together with global and cellular epigenetic mechanisms, may drive the consequential dysmorphology of the developing cortex. In this study, we demonstrate how alcohol interferes with the DMP, in parallel with cortical thinning and other abnormalities. Understanding the molecular drivers of alcohol-induced alteration of the highly ordered developmental cortical program is paramount toward uncovering how fetal environmental insults are established, maintained, and manifested into lasting cognitive and behavioral deficits.
Section snippets
Overview of experimental prenatal alcohol exposure
In this study, alcohol was administered via liquid diet according to the paradigm illustrated in Fig. 1A. The time course and types of analysis are summarized in Fig. 1B. Mice were conditioned to receive the liquid diet prior to mating. After conception, the liquid diet was re-introduced and alcohol was administered from E7–E16 (corresponding to brain development in the late first and second human trimester equivalent). The 4% alcohol liquid diet (v/v) administered in this paradigm has been
Results
First, we report the parallel development of the phenotypes and cellular features of normal corticogenesis alongside DNA methylation markers and their binding proteins layer-by-layer, revealing the cortical DNA methylation program of differentiating neuroepithelial cells into mature neurons. Subsequently, alcohol-induced aberrations of phenotypic and epigenetic features are demonstrably associated. Finally, a global analysis of the average cortical DNA methylation and MeCP2 protein are
Discussion
Fetal alcohol exposure has been associated with lasting cortical deficits through various molecular constructs and functional outcomes in human and rodent models of FASD (Abbott et al., 2016, El Shawa et al., 2013, Robertson et al., 2016, Zhou et al., 2011b). To date, however, the underpinnings of cortical thinning reported in human and animal models of FASD have not been clearly defined. We have previously demonstrated that an orderly progression of DNA methylation marks occurs parallel to the
Conclusion
Here we report that a dynamic DNA methylation program can be demonstrated throughout the distinct cortical laminae during development. 5mC and 5hmC, the two key methylation marks in the brain, demonstrated a differential distribution consistent with neural maturity. In the presence of fetal alcohol exposure, the DMP in the normal cortical condition was altered, globally, across individual cortical layers, and at a cellular level. More importantly, alcohol-induced alterations of the DMP
Funding sources
This work was supported by the National Institutes of Health AA024216, AA016698, and P50AA07611 to FCZ. Additional support was provided by the Indiana CTSI Cohort 11 core pilot grant to FCZ and BAP-SBEA 2011-5DR (Mersin University) to HÖ. NCÖ is supported in part by the Fulbright Visiting Researcher fellowship. MR is supported by National Institutes of Health training grant T32 AA007462.
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