Single-cell mRNA Research Articles

In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert.

To investigate the cellular mechanisms and cell-cell heterogeneity of the actions of insulin-like growth factor-1 (IGF-1) and follicle-stimulating hormone (FSH) exerted alone and in combination on ovarian cholesterol side-chain cleavage gene expression (P450scc mRNA) in (pig) granulosa cells, we implemented semiquantitative in situ molecular hybridization at the single target-cell level. To this end, a 1-kb cDNA specific to the catalytic region of porcine p450scc gene was subcloned into pGEM-3 and directionally transcribed in vitro in the presence of 35S-dUTP to yield radiolabeled antisense (and sense, negative control) cRNA hybridization probes. Swine granulosa cells harvested nonenzymatically from immature (1-5 mm) Graafian follicles were anchored on eight-chamber multiwell slides and treated with control solvent, human recombinant IGF-1 (10nM), ovine FSH (10nM), or both hormones, for 48 h to stimulate progestin biosynthesis maximally. After appropriate cellular permeabilization, cRNA hybridization, and solvent washes, granulosa cells were exposed to Kodak NTB-2 emulsion for 6 wk. Semiquantitative automated image analysis software (NIH IMAGE 1.5) was used to evaluate the number of silver grains deposited/20,000 square pixels. Specificity controls included labeled sense riboprobe, pretreatment with RNase, and 100-fold molar excess unlabeled cRNA. Grain counts and their distributions were examined by ANOVA and the Wilcoxon nonparametric test. The mean number of silver grains deposited per granulosa cell increased over control (reflecting specific P450scc mRNA expression) in granulosa cells pretreated with IGF-1, FSH, or IGF-1 + FSH (p < 0.05 by ANOVA). The rank order of abundance of expression of P450scc mRNA (grains/ovarian cell) was (IGF-1 + FSH) > FSH > IGF-1 > control treatment. Distributional analysis showed that each treatment introduced skewed distributions toward granulosa cells expressing more P450scc per cell than controls (p < 0.01). The median grain count of granulosa cells treated with FSH was significantly increased over that of IGF-1 treatment (p < 0.05). Treatment with both IGF-1 and FSH further shifted the grain count distribution per cell to favor granulosa cells expressing more P450scc mRNA compared to IGF-1 or FSH treatment alone (p < 0.05). Accordingly, a demonstrable mechanism of IGF-1 and FSH's regulation of specific P450scc gene expression at the single granulosa cell level is amplification in the number of target ovarian cells expressing this enzymatically rate-determining gene transcript. Interestingly, the induction of P450scc mRNA is not sufficient to explain fully the synergistic increases in progesterone accumulation driven by combined treatment with IGF-1 and FSH, thus suggesting that other steroidogenic control points are also targets of IGF-1/FSH action.

Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons.

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma-aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD protein or GABA. A similar fraction hybridize with RNA probes for GAD65 and GAD67. Hippocampal CA1 pyramidal neurons in slice preparations, which are traditionally thought to be excitatory, also contain mRNA for GAD65 and GAD67. Hippocampal neurons in culture did not contain mRNA for two other neurotransmitter synthesizing enzymes, tyrosine hydroxylase, and choline acetyl transferase. These data suggest that in some neurons, presumably the excitatory neurons, GAD mRNA is selectively regulated at the level of translation. We propose that neurotransmitter phenotype may be posttranscriptionally regulated and neurons may exhibit transient phenotypic plasticity in response to environmental influences.

Chronic estrogen treatment in male rats reveals mammosomatotropes and allows inhibition of prolactin secretion by somatostatin.

Previous in vivo studies demonstrated that estrogen treatment of male rats allows somatostatin (SRIF) to inhibit PRL release. The objective of this study was to determine whether chronic estrogen (E2) treatment of male rats can induce the conversion of somatotropes to mammosomatotropes. In situ hybridization and reverse hemolytic plaque assay were used to evaluate the effects of E2 treatment on GH and PRL messenger RNA (mRNA) content and hormone secretion in individual pituitary cells. Male rats were implanted for 2-6 weeks with placebo or estradiol-containing pellets (5mg/90-day release). Pituitaries were removed and prepared for reverse haemolytic plaque assay to determine PRL and GH secretion. This was followed by in situ hybridization using 35S-labeled riboprobes for PRL and GH mRNA. Chronic E2 treatment increased both the percentage of pituitary cells that secreted PRL and the amount of PRL secreted per cell. Concomitantly, there was a decrease in both the percentage of GH-secreting cells and that amount of GH secreted per cell. In situ hybridization demonstrated that E2 treatment increased PRL mRNA while decreasing GH mRNA in single pituitary cells. Significantly, in control male rat pituitary cell cultures, no PRL-secreting cells were positive for GH mRNA. In contrast, after chronic E2 treatment, 10% of PRL-secreting cells contained GH mRNA. In the control pituitary cell cultures, SRIF had no effect on PRL release, but SRIF significantly inhibited PRL release from pituitary cell cultures prepared from E2-treated male rats. These studies demonstrate that the adult pituitary preserves plasticity and, under the appropriate steroid milieu, allows conversion of somatotropes to mammosomatotropes.

Review : Single-Cell mRNA Amplification: Implications for Basic and Clinical Neuroscience

The application of molecular biological techniques to the study of single cells has provided a unique window for exploring the mechanisms that underlie integrated cellular function. Analysis of gene expression in indi vidual cells of the central nervous system is critical to understanding how distinct cell populations with differing chemical and anatomic phenotypes respond to pharmacological agents or are altered in disease states. Quantification of mRNA by single-cell analysis gives a high-resolution picture of changes in gene expression within individual cells, whereas more conventional types of mRNA analysis may obscure subtle changes in gene expression because of a lack of change in surrounding cells that are included in the mRNA sample. In addition, the sensitivity for detecting low levels of mRNA is enhanced when individual versus groups of cells are analyzed. With the advent of various mRNA amplification strategies, it is now possible to determine the mRNA composition or "expression profile" of individual cells. Information about relative levels of different mRNAs, the subcellular localization of mRNAs, and insight into cell-specific RNA splicing and RNA editing can be obtained. When these molecular data are combined with electrophysiological, morpho logical, immunohistochemical, and anatomical analyses, a detailed portrait of neuronal functioning can be obtained. Moreover, alterations in cellular functioning induced by physiological manipulation, drug adminis tration, or disease state can be monitored by combining these approaches. This precise cellular information may be useful in developing pharmaceuticals designed to alter mRNA levels or protein levels in a predictable manner (transcript-aided drug design) to elicit specific physiological states. The Neuroscientist 1:200-211, 1995

In situ transcription with Tth DNA polymerase and fluorescent nucleotides

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, bu the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95°C, and 5–10 μl of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 μl of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl 2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95°C, 1 min at 50°C and 10 min at 72°C, the slides were washed in 0.5 × phosphate-buffered saline, pH 7.0, at 42°C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.

Analysis of Multiple Heterogeneous mRNAs in Single Cells

We describe a two-stage amplification procedure designed to analyze multiple heterogeneous mRNA from single cells. Using an oligo(dT) primer with an attached phage promoter, the whole mRNA pool from a single cell was first transcriptionally amplified. This step brings the weak signal from one cell to a range in which it can be reliably picked up by the polymerase chain reaction (PCR) procedure. The cDNA was then divided for separate PCR amplification to obtain an unambiguous signal for each gene product. The linear amplification of phage transcription increased the convenience and reliability of detecting multiple messengers in the second stage, The procedure is extremely sensitive because it combines the amplification generated by both phage transcription and PCR. Using a pAW109 artificial RNA, we demonstrated that this procedure detects 10 copies of pAW109 RNA per original sample with 90% confidence and 50 copies per sample with >95% confidence. This procedure of multiple mRNA analysis allows "phenotyping" of any cell for its mRNA composition. Examples involving several immediate early genes and subunits of the γ-aminobutyric acidA receptor genes are given. The method should greatly facilitate the analysis of combinatorial expression of various regulatory or channel molecules in their native environments. The procedure should also provide a direct and efficient way of decoding the developmental instruction coded through combinatorial transcriptional regulation.

Differences in Ca 2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression

Fast excitatory synaptic transmission in the CNS is mediated by AMPA-type glutamate receptor (GIuR) channels. Heterologous expression suggested that the Ca 2+ permeability of these receptors critically depends on the sub-unit composition. Using patch-clamp techniques in brain slices, we found that the Ca 2+ permeability of native AMPA-type GluRs was markedly higher in nonpyramidal ( P Ca P K ≈ 0.63 ) than in pyramidal ( P CaG P K ≈ 0.05 ) neurons of rat neocortex. Analysis of mRNA in single cells indicated that the relative abundance of GIuR-B-specific mRNA was significantly lower in nonpyramidal ( GluR-B GluR-non-B ≈ 0.3 ) than in pyramidal ( sol GluR-B GluRnon-B ≈ 3 ) cells. This suggests that differences in relative abundance of GIuR-B-specific mRNA generate functional diversity of AMPA-type GIuRs in neurons with respect to Ca 2+ permeability.

Are neostriatal dopamine receptors co-localized?

The postsynaptic effects of dopamine in the neostriatum are mediated by five G-protein-coupled receptors. The extent to which these receptors are co-localized in neostriatal neurons has become controversial. This debate has far-reaching implications for treatment strategies in disorders of dopaminergic signaling, such as Parkinson's disease and schizophrenia. This review examines the molecular and cellular evidence for and against co-localization, including new information derived from single-cell mRNA amplification and patch-clamping of isolated neurons. It is concluded that this evidence is largely consistent with co-localization of functionally significant receptors of the D1 and D2 families in the majority of neostriatal efferent neurons. This conclusion has important implications for parallel processing models of the neostriatum.

Colocalization of fibroblast growth factor receptor mRNA and choline acetyltransferase immunoreactivity in single cells in laterodorsal tegmental nucleus in the rat

Colocalization of fibroblast growth factor receptor mRNA and choline acetyltransferase immunoreactivity in single cells in laterodorsal tegmental nucleus in the rat

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.

Detection of colony‐stimulating factor messenger RNA in single T cells by in situ hybridization

In situ hybridization has been used to study the accumulation of colony-stimulating factor (CSF) mRNA in single cells of a T lymphocyte clone (E9.D4) following antibody-mediated (F23.1) activation via the Ti-T3 complex in filler-independent bulk cultures. The specificity of hybridization for cellular RNA was demonstrated by pretreating the cells with the Ca2+-dependent enzyme micrococcal nuclease by using a novel protocol developed for use with riboprobes. Maximal levels of granulocyte-macrophage (GM) and multipotential-CSF (interleukin 3) mRNA were detected after 8-10 h, with GM-CSF mRNA being detected earlier and at a lower concentration of stimulus. The rise in intracellular mRNA was accompanied by an increase in the corresponding CSF bioactivity in the supernatant. In situ hybridization was of comparable sensitivity to Northern blot analysis and revealed significant heterogeneity in the accumulation of CSF mRNA within individual cells of the clone following stimulation with F23.1. This could account for the corresponding heterogeneity in CSF production by single cells. Under optimal conditions at least 25% of cells contained both transcripts. The method has been used to examine CSF production by normal spleen cells and should be useful in the further analysis of lymphokine gene regulation in single T cells.

Novel method for studying mRNA phenotypes in single or small numbers of cells

Biological processes, such as growth control, are often governed by biochemical steps involving mRNA transcripts that are short-lived and have a low copy number. Furthermore, the cells involved in these processes are often available in low numbers from in vivo sources. We now report a method that is superior to in situ hybridization, RNA blot analysis, and the nuclease protection assay for the study of short-lived, low-copy-number mRNA transcripts. The method consists of a microprocedure for isolating RNA from one to a few thousand cells and two coupled enzymatic steps: reverse transcription of whole cellular RNA, followed by amplification of the cDNA by a specifically primed polymerase chain reaction to give specific cDNA fragments that can be visualized on agarose gels by ethidium bromide staining. With this method we have detected actin mRNA from a single cell, or less than 100 cRNA molecules, and have quantified differences in RNA concentrations of less than threefold. The reverse transcription reaction products can be divided for the polymerase chain reaction, and several mRNA species can be assayed simultaneously. Therefore, we call the method single-cell mRNA phenotyping. This technique is applicable to the analysis of low-copy-number growth factor transcripts in cells in culture and in vivo.

Biosynthesis of calcitonin by human lung cancer cells.

The ectopic secretion of calcitonin (CT) by a wide variety of nonthyroidal human tumors has been studied by CT RIA, but little information is available concerning the biosynthesis of CT in these tumors. In the present study, a human lung cancer cell line (BEN), secreting high mol wt forms of CT was investigated to characterize the CT gene products synthesized. When conditioned medium from BEN cells was chromatographed through a Bio-Gel P-30 column, larger species of immunoreactive CT were detected with mol wt of approximately 8,000 and 18,000. Little, if any, CT of 3,500 mol wt was detected. To examine CT gene products produced in BEN cells, poly A+ RNA was isolated from BEN cells and subjected to cell-free translation assays and DNA/RNA hybridization assays. In the wheat germ cell-free translation assay, a single BEN cell product which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent mol wt of 17,000 could be specifically immunoprecipitated with CT antisera. A similar sized CT-related translation product is produced in wheat germ assays programmed by mRNA prepared from human medullary thyroid carcinomas. In DNA/RNA hybridization assays, a single BEN cell mRNA species of 1,000 base pairs, identical in size to human thyroidal CT mRNA, hybridized to a radiolabeled CT cDNA probe. Hybridization of the CT cDNA probe with BEN cell mRNA was confirmed by RNA dot blot hybridization and cytoplasmic RNA blotting procedures. These results indicate that larger mol wt forms of CT secreted by BEN cells are derived from a translation product and a mRNA which are of similar, if not identical, size as CT gene products produced in human thyroidal tissues. The inability of lung tumor cells to process the CT precursor to calcitonin of 3,500 mol wt may reflect a lack of specific prohormone processing enzymes in these tumor cells or may be due to structural polymorphism in the CT precursor expressed in the lung cells.

The use of Xenopus egg cells to assay the mRNA of single cells

TRANSLATION of mRNA in egg cells and oocytes of Xenopus laevis Daudin has been shown to be very efficient for mRN As from a variety of cells1–7 and has uses in developmental biology, molecular biology and immunology8. We have also reported its use in cancer research9. One of the main disadvantages of this tool, however, is the fact that fairly extensive RNA extraction and purification procedures are required. In particular, in cases when different cell types are mixed, as in the case with immunologically active cells, it is almost impossible to obtain information about the activity of the separate cell types. Studies in this field are always dealing with, at best, highly enriched cell populations. Therefore we have looked for a method which avoids RNA extraction and purification, with the aim of being able to work with separate cells. We have tried to inject cell homogenates directly into egg cells of Xenopus, and in the experiments reported here, we chose myeloma cells (LOU/M/WS1 rats with myeloma IR2 synthesising immunoglobulin E), because we could analyse the translation products easily and we could start with a relatively homogeneous cell population. Our results suggest that it is possible to study the translation products of single myeloma cells using this technique.