Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.     

Gene expression profiling of cranial sensory ganglia that transmit food intake stimuli

Peripheral cranial sensory nerves projecting into the oral cavity receive food intake stimuli and transmit sensory signals to the central nervous system. They are derived from four cranial sensory ganglia, trigeminal, geniculate, petrosal, and nodose ganglia, each of which contains multiple kinds of sensory neurons with different cell morphologies and neuronal properties. We investigated the complex properties of these neurons from the viewpoint of gene expression using DNA microarrays. The 498 genes were selected from a total of 8,740 genes as showing tissue-dependent expression on the microarray by hierarchical cluster analysis, in which several genes known to be differentially expressed in cranial sensory ganglia are included. This suggests that DNA microarray cluster analysis revealed a number of characteristic genes for sensory neurons in these ganglia. Among the selected 498 genes, 44 genes are associated with neurotransmission, such as neuropeptides, their receptors, and vesicle transport, and 26 are ion channels regulating membrane potentials. The identification of a number of genes related directly to neural properties indicates that these sensory ganglia contain heterogeneous types of neurons with different neural properties.     

Astrocyte-specific genes are generally demethylated in neural precursor cells prior to astrocytic differentiation

Epigenetic changes are thought to lead to alterations in the property of cells, such as differentiation potential. Neural precursor cells (NPCs) differentiate only into neurons in the midgestational brain, yet they become able to generate astrocytes in the late stage of development. This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown. Here we analyzed DNA methylation changes in mouse NPCs between the mid-(E11.5) and late (E14.5) stage of development by a genome-wide DNA methylation profiling method using microarrays and found that many astrocytic genes are demethylated in late-stage NPCs, enabling the cell to become competent to express these genes. Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes. Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.     

Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation

Human NT2 cells, which differentiate into neurons and astrocytes, initially express and then permanently down-regulate Nanog and Oct-4 (POU5F1). We investigated the relationship between the expression of these genes and the methylation state of their 5'-flanking regions. Gene expression and DNA methylation were assayed with quantitative polymerase chain reaction and bisulfite genomic sequencing, respectively. Retinoic acid-induced differentiation of NT2 cells to neurons is accompanied by a sequential decrease in the expression of both genes, paralleled by sequential epigenetic modification of their upstream regions. This is the first report demonstrating changes in DNA methylation in the promoter regions of Nanog and Oct-4 in a human cell line.     

Rapid in vivo labeling of identified zebrafish neurons

We report a simple and rapid method to label individual neurons in live zebrafish embryos and to examine their gene expression profiles. Injection of plasmid DNA encoding an alpha-tubulin promotor driving GFP expression results in mosaic embryos containing a limited number of GFP-positive neurons. Labeled neurons express GFP in their soma and axon, providing the opportunity to analyze pathfinding behaviors of identified neurons in vivo. Moreover, the presence of only a small subset of GFP tagged neurons permits the rapid anatomical identification of these neurons based on soma position and axonal trajectory. Analysis of injected embryos reveals that most, if not all, spinal cord cell types and many other neuronal cell types elsewhere in the nervous system can be GFP tagged. Finally, by combining GFP labeling of individual neurons with fluorescent in situ hybridization, we demonstrate the potential of this method to elucidate gene expression patterns at single cell resolution.     

Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.     

Retention of cryptic genes in microbial populations

Cryptic genes are silenced genes that can still be reactivated by mutation. Since they can make no positive contribution to the fitness of their carriers, it is not clear why many cryptic genes in microbial populations have not degenerated into useless DNA sequences. Hall et al. (1983) have suggested that cryptic genes have persisted because of occasional strong environmental selection for reactivated genes. The present mathematical study supports their suggestion. It shows that a cryptic gene can be retained without having any selective advantage over a useless DNA sequence, if selection for the reactivated gene occasionally occurs for a substantially long time.      

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Retroviral-mediated gene transfer

There are now many examples of the successful expression of genes transduced by retroviruses in studies from outside the field of neuroscience. Retroviruses will undoubtedly also prove to be effective tools for neuro-scientists interested in expressing cloned neurotransmitter and receptor genes. There are also other less obvious applications of retroviruses, such as their insertional mutagenic effects, which may be useful in studies of the genetic factors and biochemical mechanisms involved in, for example, neurotoxicity. Strong cellular promoters have been identified by retroviral infection and subsequent rescue of the flanking genomic DNA. Retroviruses can be employed again to reintroduce these regulatory sequences back into cells. In this way the complexities of gene expression in the many subpopulations of neurons may be unraveled. Retroviruses can also serve as very useful genetic markers in studies of development and lineage relationships. Retroviruses may be used to efficiently transfer oncogenes into neuronal cells to create new cell lines. This application exploits one of the natural traits of retroviruses--oncogenesis--which led to their original discovery. Finally, there are neurotropic retroviruses that could serve as important vectors for delivering genes into neurons. Studying these retroviruses may lead to an understanding of how they cause neuropathologic changes in the CNS.     

Epigenetic regulation in psychiatric disorders

Many neurological and most psychiatric disorders are not due to mutations in a single gene; rather, they involve molecular disturbances entailing multiple genes and signals that control their expression. Recent research has demonstrated that complex 'epigenetic' mechanisms, which regulate gene activity without altering the DNA code, have long-lasting effects within mature neurons. This review summarizes recent evidence for the existence of sustained epigenetic mechanisms of gene regulation in neurons that have been implicated in the regulation of complex behaviour, including abnormalities in several psychiatric disorders such as depression, drug addiction and schizophrenia.     

The mammalian XRCC genes: their roles in DNA repair and genetic stability

Analysis of the XRCC genes has played an important part in understanding mammalian DNA repair processes, especially those involved in double-strand break (DSB) repair. Most of these genes were identified through their ability to correct DNA damage hypersensitivity in rodent cell lines, and they represent components of several different repair pathways including base-excision repair, non-homologous end joining, and homologous recombination. We document the phenotypic effects of mutation of the XRCC genes, and the current state of our knowledge of their functions. In addition to their continuing importance in discovering mechanisms of DNA repair, analysis of the XRCC genes is making a substantial contribution to the understanding of specific human disorders, including cancer.     

The cryptic HLA-DQA2 ("DX alpha") gene is expressed in human B cell lines.

Although there are two gene pairs in the HLA-DQ region, DQA1/B1 and DQA2/B2, the latter pair have never been found to be expressed. The DQA2 and DQB2 genes appear to be normal at the DNA sequence level, there being no indication that they should be pseudogenes, and normal splicing occurs when DQA2 is included in a retroviral vector. The putative amino acid sequences of DQA2 and DQB2 have been nearly invariant, raising the questions of how these genes have been maintained normal-appearing and nearly monomorphic if they are not expressed and, if they are expressed, in what cell types are they expressed. We present Northern blot, nuclease protection assay, and mRNA sequence data that demonstrate the existence of spliced DQA2 mRNA in human B lymphoblastoid cell lines. In contrast, we found no evidence of DQB2 mRNA in the same cell lines, nor of DQA2 mRNA in the monocyte line U937 or in activated T cells.     

Replication of the dihydrofolate reductase genes on double minute chromosomes in a murine cell line

The purpose of this study is to determine the kinetics of the replication of intrachromosomal versus extrachromosomal amplified dihydrofolate reductase (DHFR) genes. Previous studies reported that the DHFR gene, when carried intrachromosomally on a homogeneously staining region, replicates (as a unit) within the first 2 h of the S phase of the cell cycle. We wished to determine if the extrachromosomal location of the amplified genes carried on double minute chromosomes effects the timing of their replication. Equilibrium cesium chloride ultracentrifugation was used to separate newly replicated (BUdR-labeled) DNA from bulk DNA in a synchronized cell population. Hybridization with the cDNA for the DHFR gene allowed us to determine the period of time within the cell cycle in which the DHFR DNA sequences were replicated. We found that, in contrast to intrachromosomal dihydrofolate reductase genes that uniformly replicate as a unit at the beginning of the S phase of the cell cycle, dihydrofolate reductase genes carried on double minute chromosomes (DMs) replicate throughout the S phase of the cell cycle. These results suggest that control of replication of extrachromosomal DNA sequences may differ from intrachromosomal sequences.