The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.     

Detection of spatially regulated gene expression by hybridization histochemistry.

Steady state measurements of kidney androgen-regulated protein (KAP) mRNA suggested that KAP gene expression was unusually sensitive to low hormone-receptor levels. Two of the criteria used to reach this conclusion involved relative insensitivity to treatment with a competitive antiandrogen and a partial androgen response of the gene in Tfm/Y androgen receptor (AR) deficient mice. These data may indicate the ability of the KAP gene to respond to an extremely low level of androgen-AR complex or that the effect of androgens is, at least in part, indirect. Hybridization in situ revealed that KAP mRNA expression was restricted to proximal tubule epithelial cells in the juxtamedullary region of castrated animals rather than throughout the cortex as in intact males. Examination of sections of kidneys from Tfm/Y mice before and after testosterone (T) treatment revealed that only the juxtamedullary tubules expressed KAP mRNA and that T increased the level of KAP mRNA in these cells. Treatment of Tfm/Y animals with other steroids showed that beta-estradiol treatment mimicked the effect of T while dihydrotestosterone (DHT) had no effect. The facts that DHT and T both stimulate cortical expression of KAP mRNA in normal animals but DHT has no effect on the juxtamedullary cells in the Tfm/Y variant may indicate that the action of T is due to an estrogenic metabolite. Castrated, hypophysectomized males exhibited no KAP gene expression, while in the presence of T, expression was observed throughout the cortex as in intact males. These results clearly indicate the involvement of pituitary hormones in KAP gene expression in the juxtamedullary tubules. These studies have shown that the regulation of KAP gene expression in the mouse kidney is much more complex than originally believed. Future studies will further investigate the roles of estrogen and specific pituitary hormones in KAP gene expression.     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

Neuroendocrine gene expression in the hypothalamus:In situ hybridization histochemical studies

1. We have reviewed recent studies in which in situ hybridization histochemistry (ISHH) was used to investigate the regulation of expression of neurohypophysial peptides and hypothalamic releasing hormones. 2. ISHH is a technique in which the presence and quantity of a specific mRNA can be determined in tissue sections with a high degree of resolution and sensitivity. 3. ISHH has been used to measure changes in cellular levels of mRNAs encoding vasopressin, oxytocin, corticotropin-releasing factor, gonadotropin-releasing hormone, thyrotropin-releasing hormone and somatostatin in response to various physiological challenges. 4. A theme emerging from these studies is that changes in levels of mRNA encoding neuroendocrine peptides reflect changes in biosynthesis and secretion.     

Prealbumin gene expression during mouse development studied by in situ hybridization

Localization of prealbumin mRNA in tissues from mice at various stages of gestation was investigated using in situ hybridization procedures. Prealbumin mRNA was detected as early as the 10th day of gestation. It was specifically localized in endodermal cells of the visceral yolk sac, tela choroidea, and hepatocytes. In the adult mice, prealbumin mRNA was localized in the hepatocytes and choroid plexus epithelial cells. These observations indicate that synthesis of prealbumin mRNA is initiated in several different types of cells at early stages of fetal development.     

Visualization of gene expression in whole mouse retina by in situ hybridization

The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is useful to visualize the expression of specific genes in the entire vascular plexus and retina. However, in situ hybridization on whole-mount retinal preparations is problematic because isolated retinas have a tendency to curl up during hybridization and are difficult to stain. Here we provide a detailed protocol that overcomes these difficulties and visualizes the mRNA distribution of one or two genes in the context of the counterstained retinal vasculature. The protocol takes 3-4 d for single-probe stains, with an additional 2 d for immunohistochemistry co-labeling. In situ hybridization with two probes adds a further 3 d.     

High-throughput analysis of gene expression on tissue sections by in situ hybridization

Genome-scale sequencing projects, high-throughput RNAi screens, systematic gene targeting, and system-biology-based network predictions all depend on a validation of biological significance in order to understand the relevance of a particular finding. Such validation, for the most part, rests on low-throughput technologies. This article provides protocols that, in combination with suitable instrumentation, make possible a semi-automated analysis of gene expression on tissue sections by means of in situ hybridization. Knowledge of gene expression localization has the potential to aid, and thereby accelerate, the validation of gene functions.     

GEISHA: an in situ hybridization gene expression resource for the chicken embryo

An important and ongoing focus of biomedical and agricultural avian research is to understand gene function, which for a significant fraction of genes remains unknown. A first step is to determine when and where genes are expressed during development and in the adult. Whole mount in situ hybridization gives precise spatial and temporal resolution of gene expression throughout an embryo, and a comprehensive analysis and centralized repository of in situ hybridization information would provide a valuable research tool. The GEISHA project (gallus expression in situ hybridization analysis) was initiated to explore the utility of using high-throughput in situ hybridization as a means for gene discovery and annotation in chicken embryos, and to provide a unified repository for in situ hybridization information. This report describes the design and implementation of a new GEISHA database and user interface (www.geisha.arizona.edu), and illustrates its utility for researchers in the biomedical and poultry science communities. Results obtained from a high throughput screen of microRNA expression in chicken embryos are also presented.     

Detection of tissue-specific gene expression using an in situ hybridization method of histoblotting

A version of in situ hybridization on the histological sections that is used for screening specific mRNA in tissues in proposed. Sections of the frozen tissue samples are prepared on the cryostatic microtome, placed on nitrocellulose filters and hybridized with labelled DNA-probes under the conditions of RNA blot hybridization. The proposed method ("histoblotting") was used to study the distribution of actin and alpha-phetoprotein mRNA genes in tissues of 15 and 21-day rat embryos. The possibility of studying mRNA (by hibridizations) and protein (by immunoenzyme staining) and making histological analysis simultaneously by histoblotting is demonstrated.     

Erythropoietin gene expression in vitro and in vivo detected by in situ hybridization

Macrophages derived from unstimulated and unseparated mouse bone marrow cells have been shown to release erythropoietin into the extracellular fluid. Additional proof that macrophages can produce the hormone would be a demonstration that the gene is expressed and the mature protein released. In situ hybridization using a 1.2 kb biotinylated erythropoietin DNA probe demonstrates that both cultured macrophages and those present in normal mouse bone marrow express the gene. These results are discussed in terms of the role played by the macrophage in the hemopoietic cellular microenvironment and indicate that a subpopulation is responsible for this function and that cell interactions play an important role in hemopoietic differentiation.     

Quantitative analysis of in situ hybridization methods for the detection of actin gene expression.

We have implemented an efficient, quantitative approach for the optimization of in situ hybridization using double-stranded recombinant DNA probes. The model system studied was actin mRNA expression in chicken embryonic muscle cultures. Actin and control (pBR322) probes were nick-translated with p32 labeled nucleotides, hybridized to cells grown on coverslips, and quantitated in a scintillation counter. Cellular RNA retention was monitored via the incorporation of H3-Uridine into RNA prior to cell fixation. Over a thousand samples were analyzed, and among the technical variables examined were the fixation protocol, proteolytic cell pretreatment, the time course of hybridization, saturation kinetics, hybridization efficiency, and effect of probe size on hybridization and network formation. Results have allowed us to develop a reproducible in situ hybridization methodology which is simpler and less destructive to cellular RNA and morphology than other protocols. Moreover, this technique is highly sensitive and efficient in detection of cellular RNAs. Lastly, the rapid quantitative approach used for this analysis is valuable in itself as a potential alternative to filter or solution hybridizations.     

Survivin antiapoptotic gene expression as a prognostic factor in non-small cell lung cancer: in situ hybridization study

Survivin is an inhibitor of apoptosis that plays a significant role in cell cycle regulation and is important for survival prognosis in many neoplasms. Survivin expression was assessed by in situ hybridization (ISH) in 60 consecutive patients (54 males and 4 females) with NSCLC treated between 1993 and 1997. The examined patients had IIB and IIIA stage according to TNM system. In all cases the chemotherapy with cisplatin and etoposide (2 cycles) was administered prior the surgery; in patients responding to the therapy one more cycle was applied. Survivin gene overexpression was observed in 35 patients (58.3%). There was no correlation between survivin mRNA level and histological type of tumor, stage of cell differentiation, stage of disease according to TNM classification, performance status according to WHO and number of chemotherapy regimens administered (p > 0.05). However, the correlation between survivin gene expression and response to the chemotherapy was statistically significant (p = 0.04). Statistical analysis showed that median survival in patients with survivin gene overexpression was shorter (14.0 months) as compared to patients with no expression (60.0 months; p = 0.00002). In survival assessment by means of Kaplan-Meier test, 14.3% of five-year survival was achieved in the former group versus 60% in the latter (p = 0.00003). Univariate analysis (log-rank test) showed that significant independent prognostic factors in NSCLC included: stage of the disease according to TNM classification (p = 0.006), response to chemotherapy (p = 0.005) and pattern of survivin gene expression (p = 0.00003). Multivariate analysis utilizing Cox's model showed that for survival assessment the stage according to TNM, response to the chemotherapy and survivin expression estimated by means of ISH are of statistical significance (p=0.00001). The calculated predictive values showed that ISH technique was quite accurate in assessment of five-year survival. Our data show that survivin expression may be used as a prognostic factor and a target for therapy.     

Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells

In canine thyrocytes in primary culture, our previous studies have identified three mitogenic agents and pathways: thyrotropin (TSH) acting through cyclic AMP (cAMP), EGF and its receptor tyrosine protein kinase, and the phorbol esters that stimulate protein kinase C. TSH enhances, while EGF and phorbol esters inhibit, the expression of differentiation. Given that growth and differentiation expression are often considered as mutually exclusive activities of the cells, it was conceivable that the differentiating action of TSH was restricted to noncycling (Go) cells, while the inhibition of the differentiation expression by EGF and phorbol esters only concerned proliferating cells. Therefore, the capacity to express the thyroglobulin (Tg) gene, the most prominent marker of differentiation in thyrocytes, was studied in proliferative cells (with insulin) and in quiescent cells (without insulin). Using cRNA in situ hybridization, we observed that TSH (and, to a lesser extent, insulin and insulin-like growth factor I) restored or maintained the expression of the Tg gene. Without these hormones, the Tg mRNA content became undetectable in most of the cells. EGF and 12-0-tetradecanoyl phorbol-13-acetate (TPA) inhibited the Tg mRNA accumulation induced by TSH (and/or insulin). Most of the cells (up to 90%) responded to both TSH and EGF. Nevertheless, the range of individual response was quite variable. The effects of TSH and EGF on differentiation expression were not dependent on insulin and can therefore be dissociated from their mitogenic effects. Cell cycling did not affect the induction of Tg gene. Indeed, the same cell distribution of Tg mRNA content was observed in quiescent cells stimulated by TSH alone, or in cells approximately 50% of which had performed one mitotic cycle in response to TSH + insulin. Moreover, after proliferation in "dedifferentiating" conditions (EGF + serum + insulin), thyrocytes had acquired a fusiform fibroblast-like morphology, and responded to TSH by regaining a characteristic epithelial shape and high Tg mRNA content. 32 h after the replacement of EGF by TSH, cells in mitosis presented the same distribution of the Tg mRNA content as the rest of the cell population. This implies that cell cycling (at least 27 h, as previously shown) did not affect the induction of the Tg gene which is clearly detectable after a time lag of at least 24 h. The data unequivocally show that the reexpression of differentiation and proliferative activity are separate but fully compatible processes when induced by cAMP in thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Factors regulating the activity of striatal neurons: New perspectives fromin situ hybridization histochemistry

1. The basal ganglia contain a variety of putative peptide neurotransmitters. In situ hybridization allows changes in the levels of the mRNAs encoding these neuropeptides to be assessed at the cellular level of resolution. 2. Alterations in the activity of pathways within the basal ganglia of the rat produce distinct effects on the different neuropeptide mRNAs. 3. The evidence, where available, suggests that mRNA levels provide an index of peptide turnover. 4. This approach has consequently revealed much new information on the regulation of neuronal activity in the basal ganglia.     

Localization of the beta-globin gene by chromosomal in situ hybridization

A 3.7-kilobase (kb) genomic clone of the human beta-globin gene, including 1.5-kb upstream and approximately 0.5-kb downstream, was utilized in chromosomal in situ hybridization for precise mapping of the beta-globin locus on peripheral blood lymphocyte-derived metaphases from a normal male, and for further evaluation of a clonal t(7;11) (q22;p15) translocation on bone marrow-derived metaphases from a 46-year-old male with erythroleukemia. Analyses of 205 midmetaphases from a normal male hybridized with the tritium-labeled beta-globin probe and stained with quinacrine mustard dihydrochloride revealed approximately 12% of spreads to have silver-grain deposition over the p15 band of chromosome 11. Of the 365 silver grains observed to be located on or beside chromosomes, 25 (approximately 7%) grains were localized in band p15. Karyotype analysis of a bone marrow specimen from the patient with erythroleukemia revealed hypodiploidy with various unidentified marker chromosomes as well as a presumably balanced translocation between 7q and 11p . Chromosomal in situ hybridization showed localization of silver grains at the junction between chromosomes 7 and 11 as well as to the normal chromosome 11, indicating that the beta-globin locus had not been translocated in the chromosomal rearrangement. This case demonstrates the value of chromosomal in situ hybridization in the definition of chromosome rearrangements and provides further evidence for the localization of the beta-globin gene to 11p15 .     

Localization of nerve growth factor receptor mRNA in the rat basal forebrain within situ hybridization histochemistry

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.