The role of Drosophila cytidine monophosphate-sialic acid synthetase in the nervous system.

While sialylation plays important functions in the nervous system, the complexity of glycosylation pathways and limitations of genetic approaches preclude the efficient analysis of these functions in mammalian organisms. Drosophila has recently emerged as a promising model for studying neural sialylation. Drosophila sialyltransferase, DSiaT, was shown to be involved in the regulation of neural transmission. However, the sialylation pathway was not investigated in Drosophila beyond the DSiaT-mediated step. Here we focused on the function of Drosophila cytidine monophosphate-sialic acid synthetase (CSAS), the enzyme providing a sugar donor for DSiaT. Our results revealed that the expression of CSAS is tightly regulated and restricted to the CNS throughout development and in adult flies. We generated CSAS mutants and analyzed their phenotypes using behavioral and physiological approaches. Our experiments demonstrated that mutant phenotypes of CSAS are similar to those of DSiaT, including decreased longevity, temperature-induced paralysis, locomotor abnormalities, and defects of neural transmission at neuromuscular junctions. Genetic interactions between CSAS, DSiaT, and voltage-gated channel genes paralytic and seizure were consistent with the hypothesis that CSAS and DSiaT function within the same pathway regulating neural excitability. Intriguingly, these interactions also suggested that CSAS and DSiaT have some additional, independent functions. Moreover, unlike its mammalian counterparts that work in the nucleus, Drosophila CSAS was found to be a glycoprotein-bearing N-glycans and predominantly localized in vivo to the Golgi compartment. Our work provides the first systematic analysis of in vivo functions of a eukaryotic CSAS gene and sheds light on evolutionary relationships among metazoan CSAS proteins.

Alcohol-induced epigenetic alterations to developmentally crucial genes regulating neural stemness and differentiation.

BACKGROUND: From studies using a diverse range of model organisms, we now acknowledge that epigenetic changes to chromatin structure provide a plausible link between environmental teratogens and alterations in gene expression leading to disease. Observations from a number of independent laboratories indicate that ethanol (EtOH) has the capacity to act as a powerful epigenetic disruptor and potentially derail the coordinated processes of cellular differentiation. In this study, we sought to examine whether primary neurospheres cultured under conditions maintaining stemness were susceptible to alcohol-induced alterations in the histone code. We focused our studies on trimethylated histone 3 lysine 4 and trimethylated histone 3 lysine 27, as these are 2 of the most prominent posttranslational histone modifications regulating stem cell maintenance and neural differentiation. METHODS: Primary neurosphere cultures were maintained under conditions promoting the stem cell state and treated with EtOH for 5 days. Control and EtOH-treated cellular extracts were examined using a combination of quantitative RT-PCR and chromatin immunoprecipitation techniques. RESULTS: We find that the regulatory regions of genes controlling both neural precursor cell identity and processes of differentiation exhibited significant declines in the enrichment of the chromatin marks examined. Despite these widespread changes in chromatin structure, only a small subset of genes including Dlx2, Fabp7, Nestin, Olig2, and Pax6 displayed EtOH-induced alterations in transcription. Unexpectedly, the majority of chromatin-modifying enzymes examined including members of the Polycomb Repressive Complex displayed minimal changes in expression and localization. Only transcripts encoding Dnmt1, Uhrf1, Ehmt1, Ash2 l, Wdr5, and Kdm1b exhibited significant differences. CONCLUSIONS: Our results indicate that primary neurospheres maintained as stem cells in vitro are susceptible to alcohol-induced perturbation of the histone code and errors in the epigenetic program. These observations indicate that alterations to chromatin structure may represent a crucial component of alcohol teratogenesis and progress toward a better understanding of the developmental origins of fetal alcohol spectrum disorders.

Identification of cell-specific patterns of reference gene stability in quantitative reverse-transcriptase polymerase chain reaction studies of embryonic, placental and neural stem models of prenatal ethanol exposure.

Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean C(T) values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens.