Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Summary Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition,³⁵S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues usingin situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in ‘cement lines’. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results ofin situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites ofde novo bone formation. More moderate expression of BSP was observed in ‘older’ osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.     

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.     

Localization of the prostaglandin F2 alpha receptor in rat tissues

The localization of the prostaglandin F_2α (FP) receptor was examined in rat tissues by immunohistochemistry and in situ hybridization. Immunohistochemistry on paraffin sections was performed with a rabbit polyclonal antiserum raised against a synthetic peptide derived from the rat FP receptor sequence. In situ hybridization on cryosections was done with ³⁵S-labelled rat FP receptor antisense and sense riboprobes. The most intense FP receptor-like immunoreactivity was observed in granulosa luteal cells, muscle and epithelial cells, e.g. cardiac, skeletal and smooth muscle, and hepatocytes. Weaker immunoreactivity was found in connective tissue fibroblasts. In the eye, intense immunostaining was associated with the corneal and conjunctival epithelium and moderate staining with the ciliary body, retina, iris and connective tissues. In situ hybridization generally confirmed the results. The riboprobe hybridized weakly with the heart, skeletal muscle, uterus, liver, lung and corpus luteum. Thus, the prostaglandin FP receptor was found to be widely distributed in rat tissues.     

Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.     

Quantitation of beta-tubulin mRNA in mouse brain by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. Two types of riboprobes for quantitating mouse beta-tubulin mRNA were prepared; one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with the mouse brain RNA, yielding heat-stable hybrids. The truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of mice of 10 and 50 days old were quantitated.     

Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases

In the liver, neural cell adhesion molecule (NCAM) is a marker of immature cells committed to the biliary lineage and is expressed by reactive bile ductules in human liver diseases. We investigated the possible role of NCAM in the development of intrahepatic bile ducts and aimed at determining whether immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases. Therefore, we performed immunohistochemistry for NCAM and bile duct cell markers cytokeratin 7 and cytokeratin 19 on frozen sections of 85 liver specimens taken from 14 fetuses, 10 donor livers, 18 patients with congenital liver diseases characterized by ductal plate malformations (DPMs), and 43 cirrhotic explant livers. Duplicated ductal plates and incorporating bile ducts during development showed a patchy immunoreactivity for NCAM, while DPMs were continuously positive for NCAM. Bile ducts showing complete or patchy immunoreactivity for NCAM were found in cirrhotic livers, with higher frequency in biliary than in posthepatitic cirrhosis. Our results suggest that NCAM may have a function in the development of the intrahepatic bile ducts and that NCAM-positive immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases.     

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.     

A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines

A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.     

Analysis of Differentiation of Hepatocytes and Bile Duct Cells in Developing Mouse Liver by Albumin Immunofluorescence

Differentiation of endodermal cells in embryonic mouse liver was analyzed by study of their albumin immunofluorescence. Hepatocytes extending from the cranial diverticulum in 9.5-day embryos do not contain albumin. In 10.5-to 11.5-day embryos, hepatocytes are still immature, but they all show albumin immunofluorescence. Most precursor cells of intrahepatic bile-duct cells also give a positive reaction for albumin, like immature hepatocytes. The differentiation of intrahepatic bile-duct cells from immature hepatocytes is suggested.     

Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.

To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.     

In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.     

Ultrastructural distribution of myosin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment

Electron microscopy (EM) in situ hybridization provides the higher resolution necessary to determine the spatial relationship between a specific mRNA and the organelle containing the protein encoded by that message. EM in situ hybridization was used to determine the subcellular myosin heavy chain (MHC) mRNA distribution with respect to the myofibril in normal cardiac tissue. Sections of frozen or acrylic-embedded tissue were compared for ultrastructural integrity and content of endogenous mRNA. Papillary muscles dissected from hearts of normal rabbits were aldehyde-fixed and either frozen or embedded in LR White. EM in situ hybridization with no riboprobe, vector sequence, same-sense, and anti-sense biotinylated riboprobes was detected by indirect immunocytochemistry. Labeling density using an antisense probe was highest over the intermyofibrillar space, with an average signal five times that of background. Background labeling by nonspecific sense probe was consistently low but not random, also having the highest density of gold clusters over the intermyofibrillar space. Ultracryomicrotomy yielded a higher absolute number of gold clusters, but sections were fragmented and disrupted striated muscle morphology. LR White embedment maintained ultrastructural integrity but gave a lower absolute signal. Fortunately, MHC mRNA is an abundant message and can tolerate the decreased sensitivity of LR White.     

Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry.

Digoxigenin-labeled riboprobes and in situ hybridization of choline-O-acetyltransferase mRNA, both alone and in combination with immunohistochemical procedures for the synthetic enzyme of acetylcholine, were used to map the topography of putative cholinergic neurons in the rat central nervous system. Only the anti-sense riboprobe yielded specific labeling, which was absent in brain sections processed with sense riboprobe. Telencephalic neurons demonstrating the mRNA for choline-O-acetyltransferase and choline-O-acetyltransferase-like immunoreactivity were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, Islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis, as well as occasionally in the amygdala. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridization signal, even though some cells in those areas were observed to exhibit choline-O-acetyltransferase-like immunopositivity. Thalamic cells were devoid of hybrido- and immunoreactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few cell bodies labeled with riboprobe and co-localizing choline-O-acetyltransferase-like immunopositivity were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei evinced moderate hybridization signal, whereas cells of the parabigeminal nucleus were very weakly reactive. In contrast, motor neurons of the cranial nerve nuclei demonstrated high levels of choline-O-acetyltransferase mRNA and choline-O-acetyltransferase-like immunoreactivity. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord and around the central canal were also labeled with riboprobe. It is concluded that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons in most of the neural regions believed to contain them on the basis of acetylcholinesterase pharmacohistochemistry and choline-O-acetyltransferase immunocytochemistry, with the prominent exceptions of the cerebral cortex, hippocampus, olfactory bulb, anterior olfactory nucleus, and caudal raphe nuclei, which apparently do not possess neurons expressing detectable levels of the mRNA for the synthetic enzyme of acetylcholine.