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 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.

Expression of the type I collagen gene in rat periodontal ligament during tooth movement as revealed by in situ hybridization

1994 ◽  
Vol 39 (4) ◽  
pp. 289-294 ◽  
Author(s):  
Masahiro Nakagawa ◽  
Toshio Kukita ◽  
Akihiko Nakasima ◽  
Kojiro Kurisu
Keyword(s):  
In Situ Hybridization ◽  
Periodontal Ligament ◽  
Type I Collagen ◽  
Tooth Movement ◽  
Type I ◽  
Collagen Gene

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.

scholarly journals Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization.

1983 ◽  
Vol 97 (4) ◽  
pp. 986-992 ◽  
Author(s):  
M A Saber ◽  
D A Shafritz ◽  
M A Zern
Keyword(s):  
In Situ Hybridization ◽  
Schistosoma Mansoni ◽  
Liver Tissue ◽  
Molecular Mechanisms ◽  
Type I Collagen ◽  
Primary Source ◽  
Isolated Hepatocytes ◽  
Type I ◽  
Albumin Mrna

We have employed in situ hybridization to evaluate the molecular mechanisms responsible for hypoalbuminemia and increased liver collagen content in murine schistosomiasis. Results were compared using a simplified method of hybridizing isolated hepatocytes from Schistosoma mansoni-infected and normal mouse liver with mouse albumin (pmalb-2) and chick pro-alpha 2(l) collagen (pCg45) probes. Whereas hepatocytes from infected mice showed significantly less albumin mRNA than hepatocytes from control, there were more grains of procollagen mRNA in hepatocytes from infected as compared with control liver. Hybridization of infected liver tissue sections with the collagen probe showed more grains per field in granulomas than in liver regions, whereas with the albumin probe there was more hybridization in liver tissue than in granulomas. These results suggest that in murine schistosomiasis a reduction in albumin mRNA sequence content may be associated with decreased albumin synthesis and ultimately leads to hypoalbuminemia. In addition, although the granuloma seems to be the primary source of type I collagen synthesis, hepatocytes are also capable of synthesizing collagen, especially under fibrogenic stimulation.

scholarly journals Accumulation of worn-out GBM material substantially contributes to mesangial matrix expansion in diabetic nephropathy

2017 ◽  
Vol 312 (6) ◽  
pp. F1101-F1111 ◽  
Author(s):  
Wilhelm Kriz ◽  
Jana Löwen ◽  
Giuseppina Federico ◽  
Jacob van den Born ◽  
Elisabeth Gröne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Diabetic Nephropathy ◽  
In Situ Hybridization ◽  
Mesangial Cells ◽  
Collagen Iv ◽  
Mesangial Matrix ◽  
Electron Microscopic ◽  
Matrix Expansion ◽  
Production Mechanisms

Thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix are hallmarks of diabetic nephropathy (DN), generally considered to emerge from different sites of overproduction: GBM components from podocytes and mesangial matrix from mesangial cells. Reevaluation of 918 biopsies with DN revealed strong evidence that these mechanisms are connected to each other, wherein excess GBM components fail to undergo degradation and are deposited in the mesangium. These data do not exclude that mesangial cells also synthesize components that contribute to the accumulation of matrix in the mesangium. Light, electron microscopic, immunofluorescence, and in situ hybridization studies clearly show that the thickening of the GBM is due not only to overproduction of components of the mature GBM (α3 and α5 chains of collagen IV and agrin) by podocytes but also to resumed increased synthesis of the α1 chain of collagen IV and of perlecan by endothelial cells usually seen during embryonic development. We hypothesize that these abnormal production mechanisms are caused by different processes: overproduction of mature GBM-components by the diabetic milieu and regression of endothelial cells to an embryonic production mode by decreased availability of mediators from podocytes.

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 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.

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