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

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.104.4.1077 ◽

1987 ◽

Vol 104 (4) ◽

pp. 1077-1084 ◽

Cited By ~ 210

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.

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In Situ Hybridization Analysis of the Expression of the Type II Collagen Gene in the Developing Chicken Limb Bud

Collagen and Related Research ◽

10.1016/s0174-173x(88)80001-3 ◽

1988 ◽

Vol 8 (4) ◽

pp. 277-294 ◽

Cited By ~ 65

Author(s):

Hyun-Duck Nah ◽

Barbara J. Rodgers ◽

William M. Kulyk ◽

Barbara E. Kream ◽

Robert A. Kosher ◽

...

Keyword(s):

In Situ Hybridization ◽

Type Ii Collagen ◽

Hybridization Analysis ◽

Limb Bud ◽

Collagen Gene ◽

Type Ii

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Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis

Development ◽

10.1242/dev.57.1.51 ◽

1980 ◽

Vol 57 (1) ◽

pp. 51-60

Author(s):

W. Dessau ◽

H. Von Der Mark ◽

K. Von Der Mark ◽

S. Fischer

Keyword(s):

Type I Collagen ◽

Type Ii Collagen ◽

Limb Bud ◽

Condensation Process ◽

Type I ◽

Cellular Interactions ◽

Type Ii ◽

Cell Matrix ◽

Cartilage Differentiation ◽

Mesenchyme Cell

The distribution and sequence of appearance of fibronectin and of type-I and type-II collagen in the developing cartilage models of embryonic chick hind-limb buds was studied by immunofluorescence, using specific antibodies directed against these proteins. Fibronectin and type-I collagen are evenly distributed throughout the intercellular space ofthe mesenchyme prior to condensation of core mesenchyme of the limb anlage and formationof the cartilage blastema. With the onset of the condensation process fibronectin and type-I collagen appear to increase in the cartilage blastema compared to the surrounding loose mesenchyme, reaching a maximal density at the time of cartilage differentiation. The latter process is marked by the appearance of type-II collagen in the cartilage blastema. As cartilage differentiation progresses, type-I collagen is gradually replaced by type-II collagen; fibronectin disappears and is completely absent from mature cartilage. The transient appearance of type-I collagen and fibronectin suggests a temporal role in cell-matrix or cell-cell interactions in chondrogenesis, since it had been shown that(a) type-I collagen substrates stimulate cell proliferation and cartilage differentiation in limb-bud mesenchyme cell cultures; (b) fibronectin mediates attachment of cells to collagen substrates; and (c) fibronectin is directly involved in cellular interactions in chondrocyte cultures.

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

The Journal of Cell Biology ◽

10.1083/jcb.102.6.2302 ◽

1986 ◽

Vol 102 (6) ◽

pp. 2302-2309 ◽

Cited By ~ 79

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.

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

Archives of Oral Biology ◽

10.1016/0003-9969(94)90119-8 ◽

1994 ◽

Vol 39 (4) ◽

pp. 289-294 ◽

Cited By ~ 32

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

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Type VI collagen expression is upregulated in the early events of chondrocyte differentiation

Development ◽

10.1242/dev.117.1.245 ◽

1993 ◽

Vol 117 (1) ◽

pp. 245-251

Author(s):

R. Quarto ◽

B. Dozin ◽

P. Bonaldo ◽

R. Cancedda ◽

A. Colombatti

Keyword(s):

Suspension Culture ◽

Type I Collagen ◽

Type Ii Collagen ◽

Type I ◽

Type Ii ◽

Type Vi Collagen ◽

Full Spectrum ◽

Collagen Expression ◽

Hypertrophic Chondrocyte

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310–2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5–6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

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

The Journal of Cell Biology ◽

10.1083/jcb.97.4.986 ◽

1983 ◽

Vol 97 (4) ◽

pp. 986-992 ◽

Cited By ~ 28

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.

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