Browsing by Author "Zhou, Feng C."
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Item Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum(Plos, 2016-09) Zhou, Feng C.; Resendiz, Marisol; Lo, Chiao-Ling; Chen, Yuanyuan; Department of Anatomy & Cell Biology, IU School of MedicineGlobal DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology.Item Cis‐acting allele specific expression (ASE) differences induced by alcohol and impacted by sex as well as parental genotype of origin(Wiley, 2018) Lo, Chiao-Ling; Lumeng, Lawrence; Bell, Richard L.; Liang, Tiebing; Lossie, Amy C.; Muir, Williams M.; Zhou, Feng C.; Anatomy and Cell Biology, School of MedicineBackground Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high and low alcohol drinking (HAD and LAD) rat lines. Here we report allele specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. Methods The HAD2 and LAD2 rats which have been sequenced were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2; and, for the other 5 litters, the sire was a LAD2. From these 10 litters, two males and two females were picked from each F1 litter (N = 40 total). The F1‐pups were divided, with balancing for sex and direction of cross, into an alcohol (15%) vs a water control group. Alcohol‐drinking started in the middle of adolescence (~PND 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA‐sequencing and ASE analyses. Results Analyses revealed that adolescent ethanol drinking, individual ethanol drinking levels, parentage, and sex‐of‐animal affected ASEs of about 300 genes. The identified genes included those associated with ethanol metabolism (e.g., Aldh2); neuromodulatory function [e.g., Cckbr, Slc6a7, and Slc1a1]; ion channel activity (e.g., Kcnc3); as well as other synaptic and epigenetic function. Conclusion These data indicate that ethanol drinking differentially amplified paternal vs maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to ethanol‐induced changes in cis‐regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene‐by‐environment interactions mediating a genetic predisposition for, and/or the active development of, alcohol use disorders.Item Commonality in Down and Fetal Alcohol Syndromes(Wiley, 2013) Solzak, Jeffrey P.; Liang, Yun; Zhou, Feng C.; Roper, Randall J.; Biology, School of ScienceBackground: Down syndrome (DS) and Fetal Alcohol Syndrome (FAS) are two leading causes of birth defects with phenotypes ranging from craniofacial abnormalities to cognitive impairment. Despite different origins, we report that in addition to sharing many phenotypes, DS and FAS may have common underlying mechanisms of development. Methods: Literature was surveyed for DS and FAS as well as mouse models. Gene expression and apoptosis were compared in embryonic mouse models of DS and FAS by qPCR, immunohistochemical and immunoflurorescence analyses. The craniometry was examined using MicroCT at postnatal day 21. Results: A literature survey revealed over 20 comparable craniofacial and structural deficits in both humans with DS and FAS and corresponding mouse models. Similar phenotypes were experimentally found in pre- and postnatal craniofacial and neurological tissues of DS and FAS mice. Dysregulation of two genes, Dyrk1a and Rcan1, key to craniofacial and neurological precursors of DS, was shared in craniofacial precursors of DS and FAS embryos. Increased cleaved caspase 3 expression was also discovered in comparable regions of the craniofacial and brain precursors of DS and FAS embryos. Further mechanistic studies suggested overexpression of trisomic Ttc3 in DS embyros may influence nuclear pAkt localization and cell survival. Conclusions: This first and initial study indicates that DS and FAS share common dysmorphologies in humans and animal models. This work also suggests common mechanisms at cellular and molecular levels that are disrupted by trisomy or alcohol consumption during pregnancy and lead to craniofacial and neurological phenotypes associated with DS or FAS.Item Constructing a new nigrostriatal pathway in the Parkinsonian model with bridged neural transplantation in substantia nigra(Society for Neuroscience, 1996-11-01) Zhou, Feng C.; Chiang, Yung H.; Wang, Yun; Anatomy and Cell Biology, School of MedicineThe physical repair and restoration of a completely damaged pathway in the brain has not been achieved previously. In a previous study, using excitatory amino acid bridging and fetal neural transplantation, we demonstrated that a bridged mesencephalic transplant in the substantia nigra generated an artificial nerve pathway that reinnervated the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats. In the current study, we report that a bridged mesencephalic transplant can anatomically, neurochemically, and functionally reinstate the 6-OHDA-eradicated nigro-striatal pathway. An excitatory amino acid, kainic acid, laid down in a track during the transplant generated a trophic environment that effectively guided the robust growth of transplanted neuronal fibers in a bundle to innervate the distal striatum. Growth occurred at the remarkable speed of approximately 200 microm/d. Two separate and distinct types of dopamine (DA) innervation from the transplant have been achieved for the first time: (1) DA innervation of the striatum, and (2) DA innervation of the pars reticularis of the substantia nigra. In addition, neuronal tracing revealed that reciprocal connections were achieved. The grafted DA neurons in the SNr innervated the host's striatum, whereas the host's striatal neurons, in turn, innervated the graft within 3-8 weeks. Electrochemical volt- ammetry recording revealed the restoration of DA release and clearance in a broad striatal area associated with the DA reinnervation. Furthermore, the amphetamine-induced rotation was attenuated, which indicates that the artificial pathways were motor functional. This study provides additional evidences that our bridged transplantation technique is a potential means for the repair of a completely damaged neuronal pathway.Item Diversity of two forms of DNA methylation in the brain(Frontiers Media, 2014-03-10) Chen, Yuanyuan; Damayanti, Nur P.; Irudayaraj, Joseph; Dunn, Kenneth; Zhou, Feng C.; Anatomy, Cell Biology and Physiology, School of MedicineDNA methylation 5-methylcytosine (5mC) predicts a compacting chromatin inaccessible to transcription. The discovery of 5-hydroxymethylcytosine (5hmC), which is derived from 5mC, adds a new dimension to the mechanism and role of DNA methylation in epigenetics. Genomic evidence indicates that the 5hmC is located in the alternate regions to 5mC. However, the nature of 5hmC, as compared with classical 5mC remains unclear. Observing the mouse brain through embryonic development to the adult, first, we found that 5hmC is not merely an intermediate metabolite of demethylation, but is long lasting, chromatically distinct, and dynamically changing during neurodevelopment. Second, we found that 5hmC distinctly differs from 5mC in its chromatin affiliation during neural stem cell (NSC) development. Thirdly, we found both 5mC and 5hmC to be uniquely polarized and dynamic through the NSC development. 5mC was found to progressively polarize with MBD1 and MeCP2, and recruits H3K9me3 and H3K27me3; while 5hmC progressively co-localizes with MBD3 and recruits H3K4me2. Critical differential binding of 5mC with MBD1, and 5hmC with MBD3 was validated by Resonance Energy Transfer technique FLIM-FRET. This transition and polarization coincides with neuroprogenitor differentiation. Finally, at the time of synaptogenesis, 5mC gradually accumulates in the heterochromatin while 5hmC accumulates in the euchromatin, which is consistent with the co-localization of 5hmC with PolII, which mediates RNA transcription. Our data indicate that 5mC and 5hmC are diverse in their functional interactions with chromatin. This diversity is likely to contribute to the versatile epigenetic control of transcription mediating brain development and functional maintenance of adult brain.Item DNA methylation program during development(Frontiers Media, 2012) Zhou, Feng C.; Anatomy, Cell Biology and Physiology, School of MedicineDNA methylation is a key epigenetic mark when occurring in the promoter and enhancer regions regulates the accessibility of the binding protein and gene transcription. DNA methylation is inheritable and can be de novo-synthesized, erased and reinstated, making it arguably one of the most dynamic upstream regulators for gene expression and the most influential pacer for development. Recent progress has demonstrated that two forms of cytosine methylation and two pathways for demethylation constitute ample complexity for an instructional program for orchestrated gene expression and development. The forum of the current discussion and review are whether there is such a program, if so what the DNA methylation program entails, and what environment can change the DNA methylation program. The translational implication of the DNA methylation program is also proposed.Item DNA Methylation Program in Developing Hippocampus and Its Alteration by Alcohol(Public Library of Science, 2013) Chen, Yuanyuan; Ozturk, Nail Can; Zhou, Feng C.; Anatomy, Cell Biology and Physiology, School of MedicineDuring hippocampal development, the Cornus Ammonis (CA) and the dentate gyrus (DG) undergo waves of neurogenesis and neuronal migration and maturation independently. This stage is widely known to be vulnerable to environmental stresses, but its underlying mechanism is unclear. Alcohol exposure has been shown to alter the expression of genes that regulate the fate, survival, migration and differentiation of pyramidal and granule cells. Undermining this process might compromise hippocampal development underlying the learning and memory deficits known in Fetal Alcohol Spectrum Disorders (FASD). We have previously demonstrated that DNA methylation was programmed along with neural tube development. Here, we demonstrated that DNA methylation program (DMP) proceeded along with hippocampal neuronal differentiation and maturation, and how this DMP was affected by fetal alcohol exposure. C57BL/6 mice were treated with 4% v/v ethanol through a liquid diet along with pair-fed and chow-fed controls from gestation day (E) 7 to E16. We found that a characteristic DMP, including 5-methylcytidine (5mC), 5-hydroxylmethylcytidine (5hmC) and their binding proteins, led the hippocampal neuronal differentiation and maturation spatiotemporally as indicated by their phenotypic marks in the CA and DG pre- and post-natally. Alcohol hindered the acquisition and progression of methylation marks, and altered the chromatin translocation of these marks in the nucleus, which was correlated with developmental retardation.Item DNA Methylation program in normal and alcohol-induced thinning cortex(Elsevier, 2017-05) Öztürk, Nail Can; Resendiz, Marisol; Öztürk, Hakan; Zhou, Feng C.; Anatomy and Cell Biology, School of MedicineWhile cerebral underdevelopment is a hallmark of fetal alcohol spectrum disorders (FASD), the mechanism(s) guiding the broad cortical neurodevelopmental deficits are not clear. DNA methylation is known to regulate early development and tissue specification through gene regulation. Here, we examined DNA methylation in the onset of alcohol-induced cortical thinning in a mouse model of FASD. C57BL/6 (B6) mice were administered a 4% alcohol (v/v) liquid diet from embryonic (E) days 7–16, and their embryos were harvested at E17, along with isocaloric liquid diet and lab chow controls. Cortical neuroanatomy, neural phenotypes, and epigenetic markers of methylation were assessed using immunohistochemistry, Western blot, and methyl-DNA assays. We report that cortical thickness, neuroepithelial proliferation, and neuronal migration and maturity were found to be deterred by alcohol at E17. Simultaneously, DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxcylmethylcytosine (5hmC), which progresses as an intrinsic program guiding normal embryonic cortical development, was severely affected by in utero alcohol exposure. The intricate relationship between cortical thinning and this DNA methylation program disruption is detailed and illustrated. DNA methylation, dynamic across the multiple cortical layers during the late embryonic stage, is highly disrupted by fetal alcohol exposure; this disruption occurs in tandem with characteristic developmental abnormalities, ranging from structural to molecular. Finally, our findings point to a significant question for future exploration: whether epigenetics guides neurodevelopment or whether developmental conditions dictate epigenetic dynamics in the context of alcohol-induced cortical teratogenesis.Item Effect of Prenatal Alcohol Exposure on Bony Craniofacial Development: A Mouse MicroCT Study(Elsevier, 2013) Shen, Li; Ai, Huisi; Liang, Yun; Ren, Xiaowei; Anthony, Charles Bruce; Goodlett, Charles R.; Ward, Richard; Zhou, Feng C.; Radiology and Imaging Sciences, School of MedicineCraniofacial bone dysmorphology is an important but under-explored potential diagnostic feature of fetal alcohol spectrum disorders. This study used longitudinal MicroCT 3D imaging to examine the effect of prenatal alcohol exposure on craniofacial bone growth in a mouse model. C57BL/6J dams were divided into 3 groups: alcohol 4.2% v/v in PMI® liquid diet (ALC), 2 weeks prior to and during pregnancy from embryonic (E) days 7-E16; pair-fed controls (PF), isocalorically matched to the ALC group; chow controls (CHOW), given ad libitum chow and water. The MicroCT scans were performed on pups on postnatal days 7 (P7) and P21. The volumes of the neurocranium (volume encased by the frontal, parietal, and occipital bones) and the viscerocranium (volume encased by the mandible and nasal bone), along with total skull bone volume, head size, and head circumference were evaluated using general linear models and discriminant analyses. The pups in the alcohol-treated group, when compared to the chow-fed controls (ALC vs CHOW) and the isocaloric-fed controls (ALC vs PF), showed differences in head size and circumference at P7 and P21, the total skull volume and parietal bone volume at P7, and volume of all the tested bones except nasal at P21. There was a growth trend of ALC < CHOW and ALC < PF. While covarying for gender and head size or circumference, the treatment affected the total skull and mandible at P7 (ALC > CHOW), and the total skull, parietal bone, and occipital bone at P21 (ALC < CHOW, ALC < PF). While covarying for the P7 measures, the treatment affected only the 3 neurocranial bones at P21 (ALC < CHOW, ALC < PF). Discriminant analysis sensitively selected between ALC and CHOW (AUC = 0.967), between ALC and PF (AUC = 0.995), and between PF and CHOW (AUC = 0.805). These results supported our hypothesis that craniofacial bones might be a reliable and sensitive indicator for the diagnosis of prenatal alcohol exposure. Significantly, we found that the neurocranium (upper skull) was more sensitive to alcohol than the viscerocranium (face).Item Effects of Chronic Alcohol and Repeated Deprivations on Dopamine D1 and D2 Receptor Levels in the Extended Amygdala of Inbred Alcohol-Preferring Rats(Wiley Blackwell (Blackwell Publishing), 2006-01) Sari, Youssef; Bell, Richard L.; Zhou, Feng C.; Department of Anatomy & Cell Biology, IU School of MedicineBackground Dopaminergic (DA) activity in the extended amygdala (EA) has been known to play a pivotal role in mediating drug and alcohol addiction. Alterations of DA activity within the EA after chronic exposure to alcohol or substances of abuse are considered a major mechanism for the development of alcoholism and addiction. To date, it is not clear how different patterns of chronic alcohol drinking affect DA receptor levels. Therefore, the current studies investigated the effects of chronic ethanol consumption, with or without deprivations, on D1 and D2 receptor densities within the EA. Methods Inbred alcohol-preferring (iP) rats were divided into 3 groups with the following treatments: (1) water for 14 weeks; (2) continuous alcohol (C-Alc) for 14 weeks [24-hour concurrent access to 15 and 30% (v/v) ethanol]; or (3) repeatedly deprived of alcohol (RD-Alc) (24-hour concurrent access to 15 and 30% ethanol for 6 weeks, followed by 2 cycles of 2 weeks of deprivation of and 2 weeks of reexposure to ethanol access). At the end of 14 weeks, the rats were killed for autoradiographic labeling of D1 and D2 receptors. Results Compared with the water control group, both the C-Alc and the RD-Alc groups displayed increases in D1 receptor binding density in the anterior region of the Acb core, whereas the RD-Alc group displayed additional increases in D1 receptor binding density in anterior regions of the lateral and intercalated nuclei of the amygdala. Additionally, both C-Alc and RD-Alc rats displayed increases in D2 receptor binding density in anterior regions of the Acb shell and core, whereas RDAlc rats displayed additional increases in D2 receptor binding density in the dorsal striatum. Conclusion The results of this study indicate that 14-week extended alcohol drinking with continuous chronic or repeated deprivations increase binding sites of D1 and D2 receptors in specific regions of the EA with greater sensitivity in the anterior regions. The repeated deprivation has greater effect on altering D1 and D2 receptor binding sites in the Acb, dorsal striatum, and subamygdala regions. The current result indicates that the two drinking paradigms may have common as well as differential mechanisms on alteration of dopamine receptor–binding sites in specific regions of the EA.