Differential expression of alpha-tubulin mRNA in rat cerebellum as revealed by in situ hybridization

Nucleotide sequence analysis of two rat alpha-tubulin cDNA clones showed a marked divergence in their 3'-untranslated regions. However, each of the alpha-tubulin isotypes shows a high interspecies homology in this region, when compared with an isotubulin sequence from human and Chinese hamster. In situ hybridization of rat cerebellum with alpha-tubulin cDNA revealed differential expression in various cell layers. The mitotically active cells in the external granular layer show the highest level of alpha-tubulin mRNA, while lower levels are observed in the migrating cells in the molecular layer and in the differentiating cells in the internal granular layer. Very low levels of the mRNA are observed in the prenatally differentiated Purkinje cells.     

Hydroxyproline-rich glycoprotein mRNA accumulation in maize root cells colonized by an arbuscular mycorrhizal fungus as revealed by in situ hybridization

Summary To determine whether the expression of cell wall related genes changes during the establishment of an arbuscular mycorrhizal symbiosis (AM), we studied the expression of a maize hydroxyproline-rich glycoprotein (HRGP) gene. In situ hybridization showed that, in differentiated cells of maize roots, mRNA accumulation corresponding to the gene encoding for HRGP was only found when the cells were colonized by the endomycorrhizal fungusGlomus versiforme.     

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.     

Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization

Summary An antiserum raised against N-amino-3-propyl melatonin bound to a protein carrier was used to visualize melatonin by immunohistochemistry and to measure melatonin concentration by radioimmunoassay in the pineal gland of intact mink females killed throughout the 24 h cycle and females killed after a bilateral ablation of the cervical superior ganglion. Melatonin immunoreactivity revealed by immunofluorescence or by the peroxidase-antiperoxidase complex was observed in the cytoplasm of presumed pinealocytes of all the females. Circadian changes in pineal melatonin content were not visualized by immunohistochemistry; furthermore, immunoreactivity was also present in the pineal gland of the ganglionectomized females. However, the melatonin content measured by radioimmunoassay was significantly higher in the pineal gland from intact females killed during the night compared with that of intact females killed during the day or of ganglionectomized females. The discrepancy between the results obtained using the two methods may arise because immunohistochemistry can detect very small amounts of melatonin.     

Estrogen mercapturic acids in the adult male rat

N-Acetylcysteine derivatives of catechol estrogens have been isolated from the urine of adult male hooded rats with ligated bile ducts, following injection of [4-¹⁴C]2-hydroxyestradiol-17β and of [4-¹⁴C]estradiol-17β-By application of double isotope methods previously described, it was shown that 2-hydroxyestradiol-17β was converted into mercapturic acids in a yield of 6–8%, confirming two previous experiments with bile duct cannulated rats, and that estradiol-17β was converted into mercapturic acids to the extent of 3–6%. Since these figures are small, and since it has been shown that in two women estrogen mercapturic acids were not formed, it appears that this class of compound will not provide an answer to the problem of unidentified water-soluble metabolites of the estrogens.     

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.     

Expression of myelin basic protein gene in the developing rat brain as revealed by in situ hybridization

The developmental program controlling the expression of myelin basic protein (MBP) gene was studied in the rat using the technique of in situ hybridization. A 35S-labeled cDNA clone of mouse MBP encoding an amino acid sequence present in all four of the major forms of rodent MBP was used. The probe hybridized to the tracts of white matter with different intensities, depending on the age of the animal and the region of the brain examined. In the medulla oblongata, maximal hybridization was found in 5- and 7-day-old rats and was confined to the tectospinal tracts, fibers of the seventh cranial nerve, and the spinocerebellar tracts. By 12 days the amount of MBP mRNA had decreased in these areas. In the cerebrum, the greatest amount of MBP mRNA was observed in 17-day-old rats in the radiations of the corpus callosum. Thereafter, the levels decreased but could still be observed in the adult animals. Thus, using this technique, we have been able to demonstrate that the level of MBP-specific mRNA correlates closely with the development of myelin in different regions of the brain.     

Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary

Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.     

The regulated expression of mRNA for Clara cell protein in the developing airways of the rat, as revealed by tissue in situ hybridization.

Tissue in situ hybridization has been used on sections of developing rat lung to follow the cellular sites of mRNA expression for a protein identified only in bronchiolar Clara cells. The mRNA for this Clara cell protein (CCP) was first detected on gestational day 16 in only one of the two types of tubules existing in the lung at this developmental stage. During the next 2 days CCP mRNA expression increased uniformly only in the epithelium lining the respiratory tubules. By gestational day 19, CCP mRNA expression became limited to secretory epithelial cells lining the bronchi, and terminal bronchioles. By neonatal day 1, an intense hybridization signal was observed along all of the conducting airways, but it was irregular due to the fact that expression of the CCP gene was limited to the secretory epithelial cells. In adult rats, CCP mRNA was expressed not only in secretory cells of the intrapulmonary airways at all anatomical levels, but also in secretory epithelial cells lining the trachea and its glands, as well as in specific alveolar cells thought to be type II pneumocytes. These findings demonstrate that the regulation of the CCP gene during lung development is a complicated process and that the expression of CCP mRNA does not parallel exactly the sequential development of the airways.     

Differential expression of c-fos mRNA in the rat neocortex by in situ hybridization

The c-fos mRNA expression pattern in rat neocortex, was determined in the rat kept in a 12:12 light/dark cycle, in constant dark, or in constant light by in situ hybridization. At the beginning of the light period, c-fos mRNA was induced both in the neocortex and suprachiasmatic nucleus (SCN). Transiently increased c-fos mRNA expression was detected from 0830 to 0900 and soon declined to basal levels. Immediately prior to the beginning of the dark period, c-fos mRNA expression also increased and remained elevated in the neocortex following the dark period. In the constant dark group, c-fos mRNA expression showed no transient elevation at the beginning of the light period. On the other hand, c-fos mRNA expression in the constant light group increased during their subjective dark period as well as normal light/dark cycle. These results demonstrate a circadian pattern of c-fos mRNA expression in the neocortex which is similar to that observed previously in the inner and outer nuclear layers of the retina.     

Estrogen receptor mRNA in uteri of normal estrous cycling and ovariectomized rats by in situ hybridization.

Biological effects of estrogen are mediated via its binding to the estrogen receptor (ER), the contents of its protein and mRNA varying during the estrous cycle. In the present study, the ERalpha mRNA expression in different cell components of the uterus was investigated in normal estrous cycling rats using nonisotopic in situ hybridization. Additionally, ovariectomized (OVX) rats treated with 17beta-estradiol (E2: 5 microg/kg, sc injection daily) were also investigated to clarify the effects of exogenous E2. At proestrus and diestrus, and especially the former, the luminal and glandular epithelial (LE and GE) cells were strongly positive, along with stromal cells beneath the luminal epithelium. At estrus, the expression was slightly diminished in LE cells, but almost completely lacking in GE cells. At metestrus, positive signals appeared again in GE cells. In the myometrium, ER mRNA was demonstrated to be constantly positive in all estrous cycle stages. OVX rat uteri underwent marked atrophy, but ER mRNA still remained in all cell types. After 2 consecutive days of E2 treatment, markedly increased intensity was observed, especially in LE and GE cells. The uteri of OVX rats treated with E2 for 14 days, however, showed slightly diminished expression, whereas the serum concentration of E2 was comparable to that in rats after 2 days. These results provide evidence that cell-type specific patterns of ER mRNA expression characterize the uteri of both normal estrous cycling rats and OVX rats after estrogen treatment.     

Individual interphase chromosome domains revealed by in situ hybridization

Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.     

Androgens and glucocorticoids modulate heparin-binding growth factor I mRNA accumulation in DDT1 cells as analyzed by in situ hybridization

The ductus deferens smooth muscle tumor cell line (DDT1-MF-2) is very sensitive to steroids. Treatment with 10 nM testosterone accelerates the growth of DDT1 cells in the absence of serum. Glucocorticoids in the presence or absence of androgens inhibits growth. Stimulation of growth of DDT1 cells by testosterone can be replaced by the addition of heparin-binding growth factor I and II (HBGF). Addition of testosterone plus HBGF growth factors results in a further increase in cell number. By in situ hybridization, accumulation of HBGF-I mRNA is significantly increased by testosterone treatment of low density cultures. Testosterone treatment of high density cultures results in no stimulation of HBGF-I mRNA accumulation. Glucocorticoids alone, which block growth of DDT1 cells, have no effect of HBGF-I mRNA accumulation. However, the simultaneous addition of glucocorticoid and androgens to DDT1 cells results in a rapid accumulation of HBGF-I mRNA by 12 h, although growth is inhibited by the presence of both steroid analogs.     

Changes in rat pituitary POMC mRNA after exposure to cold or a novel environment, detected by in situ hybridization.

Plasma adrenocorticotropin (ACTH) levels increase after acute cold exposure. The purpose of this study was to determine if there were parallel changes in pituitary proopiomelanocortin (POMC) mRNA. Male rats were exposed to cold (3-5 degrees C) or a novel environment for 15 or 30 min. Others were unstressed. POMC mRNAs in frozen sections or dissociated cells were hybridized with a photobiotinylated oligonucleotide probe which was detected in situ by streptavidin-alkaline phosphatase. The percentage of area labeled for POMC mRNA was quantified by the Cue-3 color image analysis system. In frozen sections, 24-fold increases in the percentage of area labeled for POMC mRNA were evident in intermediate lobes (IL) 30 min after stress. No change was seen in anterior lobes (AL). If the ALs were dissociated, a 66-99% increase in percentage of labeled cells was detected 2-3 hr after the cold exposure. Fifteen min of cold stress (CS) also caused a 117% increase in the area of label for POMC mRNA per corticotrope. No change was seen after 30 min. Exposure to a novel environment caused a 73% increase in the percentage of area labeled for POMC mRNA per AL corticotrope and an 11-fold increase in the IL. These results indicate that both AL corticotropes and IL melanotropes are stimulated by acute exposure to cold and novel environments.     

N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization

The N-myc proto-oncogene is expressed during embryogenesis, suggesting that it plays a role in normal development. Since the myc-family oncogenes have been implicated in the control of cell growth, the embryonic expression may reflect rapid proliferation known to occur in development. Alternatively, N-myc expression may be involved in specific differentiation stages. In many embryonic tissues, early and late differentiation events occur in different locations. By in situ hybridization of tissue sections, we now demonstrate a restricted expression of N-myc mRNA to a few tissues and to areas where the first differentiation stages occur. N-myc expression was most strongly expressed in the developing kidney, hair follicles, and in various parts of the central nervous system. In these tissues, expression was restricted to a few cell lineages. In all lineages, expression was confined to early differentiation stages, and, at onset of overt differentiation, the level of expression decreased dramatically. Several rapidly proliferating tissues showed very little, if any, N-myc expression. In the brain, post-mitotic but not yet differentiated cells expressed high levels of N-myc mRNA. Therefore, N-myc expression is not a simple marker for proliferation in the embryo. Rather, N-myc expression seems to be a feature of early differentiation stages of some cell lineages in kidney, brain, and hair follicles, regardless of the proliferative status of the cell. The results raise the possibility that N-myc may participate in the control of these early differentiation events.     

Detection of CAIII mRNA in rat skeletal muscle and liver by in situ hybridization.

We carried out a variety of in situ methods of hybridization on rat liver and rat skeletal muscle using 35S-labeled or biotin-labeled rat carbonic anhydrase III (CAIII) cDNA clone. The methods were compared and evaluated. Use of the biotin system produced defined but nonspecific results which were shown not to be due to the biotinylated cDNA probe binding to the mRNA in the muscle sections. This artifact was shown to persist despite various attempts to eliminate it. Alternatively, using 35S-labeled cDNA gave reproducible results which were shown to be consistent with probe binding specifically to mRNA in the muscle section.     

Assignment of human satellite 1 DNA as revealed by fluorescent in situ hybridization with oligonucleotides

We have used a fluorescent in situ hybridization procedure to detect human satellite 1 DNA, the simple sequence family that constitutes the non-male-specific fraction of classical satellite 1 DNA. Satellite 1 appears to be located on pericentromeric regions of chromosomes 3, 4 and 13, and on satellites of each acrocentric chromosome. These results suggest a possible relationship between quinacrine fluorescence of heterochromatin and DNA composition. Furthermore, by means of multicolour in situ hybridization, we have spatially resolved satellite 1 sequences and centromeric -satellite within heterochromatic blocks.