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

Abstract

Abstract. Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and HI collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of eDNA 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 eDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen eDNA 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 proal(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 1I collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro~tl(ID collagen eDNA probe, and none by the type I collagen eDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen eDNA 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.T HE majority of total body collagen is found in the specialized connective tissues of the skeletal system in the form of the fibrillar collagens of types I, II, and III. Each of these collagens are synthesized as procollagens containing three proa-chains. Type I collagen is a heterotrimer of two al(I) and one a2(I) chains, while types II and HI collagens are homotrimers of al(ID and al0II) chains, respectively. All these chains share considerable homology both at the level of amino acids, mRNAs, and gene structure (5,17,19). Additionally, at least nine other collagen types with variable functions have been characterized (5).The development and growth of the skeletal tissues is a complicated process involving a number of changes in the expression of collagen genes (29,34). This is exemplified by the growth of long bones which occurs in the growth plate areas by chondrocyte division and deposition of cartilage matrix, containing type II collagen fibers. Having terminally differentiated, the hypertrophic chondrocytes degenerate, the extracellular matrix becomes calcified, and is finally invaded ...

Mouse cathepsin K: cDNA cloning and predominant expression of the gene in osteoclasts, and in some hypertrophying chondrocytes during mouse development

“…Before cell division or hypertrophy the chondrocytes must create space for the increase in cell size. In growth plates the dividing and hypertrophying cells create space by concomitantly synthesizing new matrix in the longitudinal direction resulting in long bone growth [25]. Thus the increase in cellular volume is not solely dependent on matrix removal since space is also created by longitudinal growth.…”

Urinary Bladder Transitional Cell Carcinogenesis Is Associated with Down-Regulation of NF1 Tumor Suppressor Gene in Vivo and in Vitro

“…In situ hybridizations were conducted according to previously described protocols with minor modifications. 37,38 Here, the technique is described briefly. Linearized pBluescript plasmid containing the NF1 cDNA insert was used as a template.…”

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins

“…The collagen plasmids were a gift from E. Vuorio [collagen types I (pHCALlU) (Makela et al, 1988) and III (pHFS3) (Sandberg & Vuorio, 1987)] and D. Prockop, Jefferson, Philadelphia, PA, USA (type IV, HT21; Pihlajaniemi et al, 1985). The probe for fibronectin was donated by R.O.…”