scholarly journals Differential localization of distinct keratin mRNA-species in mouse tongue epithelium by in situ hybridization with specific cDNA probes.

1986 ◽  
Vol 103 (6) ◽  
pp. 2583-2591 ◽  
Author(s):  
M Rentrop ◽  
B Knapp ◽  
H Winter ◽  
J Schweizer
Keyword(s):  
In Situ Hybridization ◽  
Basal Layer ◽  
Hybridization Technique ◽  
Type I ◽  
Type Ii ◽  
Basal Cells ◽  
Fungiform Papillae ◽  
Filiform Papillae ◽  
Tongue Epithelium

The tongue of the adult mouse is covered by a multilayered squamous epithelium which is continuous on the ventral surface, however interrupted on the dorsal surface by many filiform and few fungiform papillae. The filiform papillae themselves are subdivided into an anterior and posterior unit exhibiting different forms of keratinization. Thus, the entire epithelium shows a pronounced morphological diversity of well recognizable tissue units. We have used a highly sensitive in situ hybridization technique to investigate the differential expression of keratin mRNAs in the tongue epithelium. The hybridization probes used were cDNA restriction fragments complementary to the most specific 3'-regions of any given keratin mRNA. We could show that independent of the morphologically different tongue regions, all basal cells uniformly express the mRNA of a type I 52-kD keratin, typical also for basal cells of the epidermis. Immediately above the homogenous basal layer a vertically oriented specialization of the keratin expression occurs within the morphological tissue units. Thus the dorsal interpapillary and ventral epithelium express the mRNAs of a type II 57-kD and a type I 47-kD keratin pair. In contrast, in the anterior unit of the filiform papillae, only the 47-kD mRNA is present, indicating that this keratin may be coexpressed in tongue epithelium with different type II partners. In suprabasal cells of both, the fungiform papillae and the posterior unit of the filiform papillae, a mRNA of a type I 59-kD keratin could be detected; however, its type II 67-kD epidermal counterpart seems not to be present in these cells. Most surprisingly, in distinct cells of both types of papillae, a type I 50-kD keratin mRNA could be localized which usually is associated with epidermal hyperproliferation. In conclusion, the in situ hybridization technique applied has been proved to be a powerful method for detailed studies of differentiation processes, especially in morphologically complex epithelia.

Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia

1991 ◽  
Vol 100 (1) ◽  
pp. 23-33 ◽  
Author(s):  
K.K. Svoboda
Keyword(s):  
Endoplasmic Reticulum ◽  
In Situ Hybridization ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Confocal Laser ◽  
Type I ◽  
Basal Cells ◽  
Double Labeling ◽  
Collagen Mrna

The intracellular distribution of endoplasmic reticulum (ER) and types I and II collagen mRNA was analyzed in whole-mount preparations of freshly isolated corneal epithelia using in situ hybridization combined with confocal laser scanning analysis. The ER stained with DiOC6 (3) was prominent in both the periderm and basal cells. The basal cell ER distribution was perinuclear in the center of the cells, but below the nucleus the ER occupied nearly all of the cytoplasm in a reticular pattern similar to that seen with TEM cross-sections. Initial single label in situ hybridization studies showed that both the periderm and basal cells were positive for both types I and II collagen mRNA. The collagen cDNA probes appeared perinuclear in the center of the basal cells, similar to the DiOC6(3) staining pattern. In double-labeling experiments, the two mRNAs that translate chains of type I collagen, alpha 1 and alpha 2, colocalized within the same cell. However, the hybridization of probes specific for type I and II collagen mRNAs had separate, but overlapping, distributions within the same cell.

In situ hybridization reveals differential spatial distribution of mRNAs for type I and type II collagen in the chick limb bud

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 111-118 ◽  
Author(s):  
C.J. Devlin ◽  
P.M. Brickell ◽  
E.R. Taylor ◽  
A. Hornbruch ◽  
R.K. Craig ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Limb Development ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Limb Bud ◽  
Type I ◽  
Type Ii ◽  
Mrna Accumulation ◽  
Rna Probes

During limb development, type I collagen disappears from the region where cartilage develops and synthesis of type II collagen, which is characteristic of cartilage, begins. In situ hybridization using antisense RNA probes was used to investigate the spatial localization of type I and type II collagen mRNAs. The distribution of the mRNA for type II collagen corresponded well with the pattern of type II collagen synthesis, suggesting control at the level of transcription and mRNA accumulation. In contrast, the pattern of mRNA for type I collagen remained more or less uniform and did not correspond with the synthesis of the protein, suggesting control primarily at the level of translation or of RNA processing.

Sequence and expression of a type II keratin, K5, in human epidermal cells

1988 ◽  
Vol 8 (1) ◽  
pp. 486-493
Author(s):  
R Lersch ◽  
E Fuchs
Keyword(s):  
Human Gene ◽  
Basal Layer ◽  
Epidermal Cells ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Human Epidermal Cells ◽  
Rna Blot Analysis

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.

scholarly journals Sequence and expression of a type II keratin, K5, in human epidermal cells.

1988 ◽  
Vol 8 (1) ◽  
pp. 486-493 ◽  
Author(s):  
R Lersch ◽  
E Fuchs
Keyword(s):  
Human Gene ◽  
Basal Layer ◽  
Epidermal Cells ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Human Epidermal Cells ◽  
Rna Blot Analysis

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.

Secretion of collagen types I and II by epithelial and endothelial cells in the developing chick cornea demonstrated by in situ hybridization and immunohistochemistry

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 27-36 ◽  
Author(s):  
M. Hayashi ◽  
Y. Ninomiya ◽  
K. Hayashi ◽  
T.F. Linsenmayer ◽  
B.R. Olsen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Epithelial Cells ◽  
Type I Collagen ◽  
Type I ◽  
Basal Cells ◽  
Collagen Types ◽  
Electron Microscopic ◽  
Specific Mrnas

Cells involved in the synthesis of collagen types I and II in the cornea of developing chick embryos have been studied by using in situ hybridization and immunohistochemistry. Corneas processed for in situ hybridization with the type I and II collagen probes demonstrated specific mRNAs in the epithelium of embryos at stage 18 with an increase at stages between 26 and 31, and then gradual decrease to the background level in the next several days. In the endothelium, a small amount of specific mRNA was recognized through these stages. In the stroma, only sections hybridized with the type I probe demonstrated mRNA in fibroblasts. Immunostaining demonstrated specific collagen types in the stroma at sites which were closely associated with cells containing specific mRNAs. Both collagens type I and II were present beneath the epithelium as narrow bands at stage 18; as the thicker primary stroma at stages 20 and 26; and as subepithelial, subendothelial and stromal staining at stage 31. Thereafter, type I collagen was increased in the stroma but it was also noted in the subepithelial and, to a lesser degree, subendothelial regions, whereas type II collagen was gradually confined to the subendothelial matrix. Electron microscopic examination of sections from 5-day-old (stage-27) embryo corneas using antibodies against the carboxyl propeptides of type I and II procollagens revealed the presence of these procollagens within the cisternae of the endoplasmic reticulum and Golgi vesicles in both epithelial and endothelial cells. In the epithelial cells both the periderm and basal cells contained these procollagens within the cytoplasmic organelles. These results indicate that not only the epithelial cells, but also the endothelial cells secrete collagen types I and II during the formation of the primary corneal stroma and for several days after invasion of fibroblasts.

scholarly journals Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

2001 ◽  
Vol 67 (10) ◽  
pp. 4850-4857 ◽  
Author(s):  
Svetlana N. Dedysh ◽  
Manigee Derakshani ◽  
Werner Liesack
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Fluorescence Signal ◽  
Type I ◽  
Oligonucleotide Probes ◽  
Type Ii ◽  
Cell Counts ◽  
Wet Weight

ABSTRACT Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or theMethylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in aSphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 106 cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 104 type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 106 type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus andMethylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion ofMethylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 106 cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population ofMethylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.

scholarly journals Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes.

1986 ◽  
Vol 102 (6) ◽  
pp. 2302-2309 ◽  
Author(s):  
M Hayashi ◽  
Y Ninomiya ◽  
J Parsons ◽  
K Hayashi ◽  
B R Olsen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Epithelial Cells ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Collagen Types ◽  
Paraffin Sections ◽  
Extracellular Matrix Components

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.

scholarly journals Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

1987 ◽  
Vol 104 (4) ◽  
pp. 1077-1084 ◽  
Author(s):  
M Sandberg ◽  
E Vuorio
Keyword(s):  
In Situ Hybridization ◽  
Type I Collagen ◽  
Cdna Probe ◽  
Type Ii Collagen ◽  
Northern Hybridization ◽  
Cdna Clones ◽  
Sequence Information ◽  
Type I ◽  
Type Ii

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.

scholarly journals Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent in situ Hybridization (FISH)

2010 ◽  
Vol 7 (5) ◽  
pp. 7475-7503 ◽  
Author(s):  
C. Borrelli ◽  
A. Sabbatini ◽  
G. M. Luna ◽  
C. Morigi ◽  
R. Danovaro ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Rose Bengal ◽  
Benthic Foraminifera ◽  
Fluorescent In Situ Hybridization ◽  
Physiological Status ◽  
Hybridization Technique ◽  
Fish Analysis ◽  
Type I ◽  
Active Metabolism

Abstract. Benthic foraminifera are an important component of the marine living biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but this approach does not allow discriminating between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a potentially useful approach identifying living cells with active metabolism cells. In this work, we tested for the first time the suitability of the FISH technique based on fluorescent probes targeting the 18S rRNA, to detect these live benthic protists. The protocol was applied on the genus Ammonia, on the Miliolidae group and an attempt was made also with agglutinated species (i.e., Leptohalysis scottii and Eggerella scabra). In addition microscopic analysis of the cytoplasm colour, presence of pigments and, sometimes, those of pseudopodial activity where conducted. The results of the present study indicate that FISH targeted only live and metabolically active foraminifera. These results allowed to identify as "live", cells improperly classified as "dead" by means of the classical technique (Type I error) and vice versa to identify as dead the foraminifera without rRNA, but stained using Rose Bengal (Type II error). In addition, the comparative FISH analysis of starved and actively growing cells demonstrated that individuals with active metabolism were stained more intensively than starved cells. This finding supports the hypothesis that the physiological status of cells can be directly related with the intensity of the fluorescent signal emitted by the fluorescent probe. We conclude that the use of molecular approaches could represent a key tool for acquiring crucial information on living foraminifera specimens and for investigating their ecological role in marine sediments.

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