Identification of class I MHC mRNA in human first trimester trophoblast cells by in situ hybridization

Special gestation-related regulatory mechanisms for the expression of class I Ag by trophoblast cells directly exposed to maternal blood and tissues may be required for semiallogeneic pregnancy to be successful. Analysis of class I MHC mRNA by in situ hybridization and class I MHC Ag by immunohistology has revealed two phenotypically distinct subpopulations of trophoblast cells in term placentas and extraplacental membranes. Trophoblast cells external to the placenta are mRNA +/Ag+. They contain class I mRNA and express class I Ag that differ serologically from HLA-A,B,C. In contrast, trophoblast cells forming the syncytial layer of placental villi are mRNA-/Ag-. By immunohistology, trophoblast cells in 1st trimester placental tissues are similar to those in term tissues. In our study, in situ hybridization was used to determine if patterns of trophoblast cell class I mRNA were the same or different. Trophoblast cells external to the placental villi in 1st trimester tissues contained class I mRNA as would be predicted from the results with term tissues. Unexpectedly, class I mRNA was found in villous trophoblast cells. Thus, these studies identified an mRNA+/Ag- trophoblast cell subpopulation. The results suggest that tissue-specific mechanisms may interfere with translation of class I mRNA in 1st trimester villous trophoblast cells and/or that the protein products of the mRNA are not identified by available mAb.     

Cellular localization of induced human interferon-beta mRNA by non-radioactive in situ hybridization

Induced interferon-beta (IFN-beta) mRNA was localized in human FS-4 fibroblasts by in situ hybridization using biotinylated probes. The hybridization sites were detected by incubation with a nick-translated genomic DNA probe (1.8 kb) via streptavidin-colloidal gold followed by silver contrast enhancement. The positive signals were observed by reflection-contrast light microscopy. IFN-beta mRNA was transiently induced by poly r(I): r(C) in fibroblasts 2-4 h after induction. Induction in the presence of cycloheximide and actinomycin D (superinduction conditions) exhibited an enhanced level of IFN-beta mRNA with a maximum at 4-8 h. The kinetics of the IFN-beta mRNA expression in the cytoplasm as revealed by in situ hybridization proved to be compatible with the results of Northern blotting experiments of total cellular RNA.     

Arginine vasotocin mRNA revealed by in situ hybridization in bovine pineal gland cells

Arginine vasopressin (AVP) is the main antidiuretic hormone in mammals and arginine vasotocin (AVT) in submammalian vertebrates. The possibility that the genetic material encoding AVT is maintained in mammals is controversial. In this study, we investigated by radioactive in situ hybridization the possible presence of the mRNA encoding AVP and AVT, and using immunocytochemistry the presence of structures immunoreactive for AVP and AVT in the bovine pineal gland. In situ hybridization was performed by use of ³⁵S-labelled oligoprobes. Immunocytochemistry was performed using specific polyclonal rabbit antibodies and the avidin-biotin-complex method. In situ hybridization revealed positive signals for both AVP mRNA and AVT mRNA in a few cells scattered throughout the pineal body. Immunocytochemistry revealed thin AVP-immunoreactive fibres in the pineal stalk and the pineal gland. It also revealed staining of several AVT-immunoreactive nerve fibres in both the pineal stalk and the gland. In addition, polyhedral, neuron-like cell bodies from which two to three processes emerged were also AVT-immunoreactive. Thus, our investigation shows the presence of AVP/AVT-immunoreactive cellular structures in the bovine pineal gland. Our data further show the presence of mRNAs encoding both AVT and AVP. We therefore suggest that AVT mRNA is translated into an AVT-like peptide in the bovine pineal.     

In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels

The localization of mRNA encoding preproatrial natriuretic peptide (ANP) was investigated in cultured human umbilical vein endothelial cells (HUVEC) and tissue preparations of umbilical vein and artery. The techniques used were in situ hybridization and in situ hybridization combined with immunocytochemistry, using ³²P-radiolabelled and non-radioactive digoxigenin labelled complementary RNA probes. Human ANP mRNAs are mainly localized in the endothelial cells of the umbilical vein and, to a lesser extent, in the endothelial cells of the umbilical artery. The autoradiographic labelling and the intensity of digoxigenin staining were significantly reduced by treatment with RNase before in situ hybridization. This study provides unequivocal evidence for the expression of the ANP gene in the endothelial cells of human umbilical vessels, confirming that these endothelial cells have the ability to synthesize this peptide. The functional significance of the presence of the ANP mRNA in the endothelial cells of human umbilical vessels is discussed.     

Analysis of prion protein mRNA by in situ hybridization in brain and placenta of sheep

In this study, prion protein (PrP) mRNA was focally detected in brain and placenta of pregnant sheep by Northern blot analysis. In addition, host-encoded cellular prion protein (PrP(C)) was observed in brain and placenta of the ruminant by Western blot analysis as well. Localization of PrP mRNA in pregnant sheep tissues was rendered possible with in situ hybridization. In sheep brain, PrP mRNA was predominantly localized within large neocortical neurons in the cerebrum, Purkinje cells and neurons of the molecular and granule cell layers in the cerebellum. In the placenta, signals were observed in the myometrium, including stratum longitudinale tunicae muscles and circular layers of muscular tunics. In the caruncle and placentome, signals were stronger by in situ hybridization. Since accumulation of the scrapie isoform PrP (PrP(Sc)) is required to PrP(C), these results suggest that brain and placenta of sheep may be important organs and sites for the conversion of PrP(C) to PrP(Sc).     

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/ VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22–79, PHM, prepro-VIP 111–122, VIP and prepro-VIP 156–170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single-or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.     

Probing VH gene-family utilization in human peripheral B cells by in situ hybridization

Recent studies on the genomic organization of the human Ig VH locus have revealed the presence of an important proportion of VH pseudogenes and a high degree of interspersion among VH gene-family members. The mechanisms of selection of VH genes expressed by differentiating B cells remain to be elucidated. We have made use of RNA-RNA in situ hybridization to probe the repertoire of VH gene-families assembled in peripheral B cells of normal adults. Cells were in vitro activated with mitogens and then hybridized to [35S]-labeled anti-sense RNA probes specific for C mu and C gamma genes, and for the six known human VH gene-families. We found that the numbers of cells expressing C mu and C gamma mRNA were similar to the total numbers of cells expressing members of the six VH gene-families. Therefore, the six known VH gene-families represent essentially the human VH locus. We also found that expression of VH gene-families does not closely correlate with their relative genomic complexity. This apparently biased expression may suggest that in some VH gene-families the ratio of pseudogenes/functional genes is particularly high, and/or that regulatory mechanisms play a major role in shaping the available VH gene repertoire in differentiating B cells.     

Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization

Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.     

Detection of CD1 mRNA in Paneth cells of the mouse intestine by in situ hybridization.

Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Although their function(s) remains unknown, it has been proposed that CD1 may present antigens to specific subsets of peripheral T-cells. We now provide evidence in support of this hypothesis through the demonstration by in situ hybridization that Paneth cells of the mouse intestine express CD1 mRNA. These cells are thought to be involved in the immunological regulation of intestinal flora and could accomplish this task through interactions with intestinal intraepithelial lymphocytes. The expression and localization of CD1 mRNA was confirmed by both autoradiographic and non-isotopic techniques. The relevance of these results to CD1 function as well as to Paneth cell biology is discussed.     

Regulation of tyrosine hydroxylase mRNA in catecholaminergic cells of embryonic rat: analysis by in situ hybridization

In situ hybridization was used to examine the appearance of mRNA specific for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (CA) biosynthesis, in neural crest derivatives of the rat embryo. These derivatives include sympathetic ganglia and transient catecholaminergic cells of embryonic intestine. Messenger RNA is first detected in sympathetic ganglia at E11.5, the age corresponding to the initial immunocytochemical expression of TH protein. In older embryos increased accumulation of TH-specific mRNA in sympathetic ganglia parallels the increase in TH immunoreactivity. By contrast, mRNA for TH is difficult to detect in embryonic intestines at E11.5 but is found instead in cells clustered at the dorsal boundaries of the pharynx and foregut. Cells expressing TH mRNA are infrequently found in embryonic intestines at any age, even though TH protein is immunohistochemically apparent. Treatment of pregnant rats with doses of reserpine, known to increase circulating levels of glucocorticoid hormones and prolong the expression of TH protein in embryonic gut cells, dramatically but transiently increases the number of gut cells at E12.5 with detectable TH mRNA. After E13.5 TH mRNA is undetectable even in reserpine-treated guts. Reserpine treatment also increases the labeling density in sympathetic ganglia. Taken together, these data are consistent with the hypothesis that the microenvironment of the embryonic intestine affects gene expression directly to alter phenotype. Moreover, although reserpine administration briefly increases TH mRNA levels, the effect is short-lived and does not alter neurotransmitter phenotypic conversion.     

Analysis of whole-arm translocations in malignant blood cells by nonisotopic in situ hybridization

Whole-arm translocations in five leukemic patients were studied using nonisotopic in situ hybridization with alpha satellite DNA and simple satellite chromosome-specific DNA probes to detect the target sequences. The results show that this technique provides additional information on the involvement of these DNA sequences in whole-arm translocations.