Carin Lundmark

Expression patterns of RNAs for amelin and amelogenin in developing rat molars and incisors.

We have recently identified a novel RNA sequence in ameloblasts, coding for amelin (Cerny et al., 1996). In the present paper, its expression has been compared with that of amelogenin in developing incisors and molars of rats, by means of in situ hybridization of paraffin sections. The RNAs for both amelin and amelogenin were highly expressed in secretory ameloblasts. The expression of RNA for amelogenin gradually decreased in the post-secretory ameloblasts. In contrast, the RNA expression for amelin remained high in post-secretory ameloblasts up to the stage of fusion between dental and oral epithelia at the time of tooth eruption. We suggest that amelin might be involved in the mineralization of enamel or in the attachment of ameloblasts to the enamel surface. The whole-mount in situ hybridization procedure is described for the first time in dental research. It proved to be a useful method and confirmed the results of the conventional in situ hybridization.

RESPONSES OF BONE TO TITANIA-HYDROXYAPATITE COMPOSITE AND NACREOUS IMPLANTS : A PRELIMINARY COMPARISON BY IN SITU HYBRIDIZATION

The effect of two biomaterials on bone formation in vivo by in situ hybridization, was compared by using RNA probes complementary to collagen α1(I) RNA, osteonectin RNA and osteocalcin RNA. Holes were drilled into the midshafts of rat femurs. Titania–hydroxyapatite composite (THA) or nacre cylinders were implanted and the bone–implant regions collected 14 days after operation. Cuboidal osteoblasts, intensely labelled with the three probes, were seen to be lining the newly formed bone surrounding the THA implant. Between the implant and the new bone, a layer of un-labelled, apparently non-osteogenic cells was observed. By contrast, the nacre implant was bonded to the newly formed bone without any soft tissue interference. Osteoblasts lining the distal surface of the newly formed bone were stained with all three RNA probes, although weaker than in the THA sample. Some of the osteoblasts were flattened. We concluded from the appearance of the osteoblasts that the bone formation in the nacre samples had progressed beyond the phase of maximal synthetic activity. Around the THA implant, the labelling indicated that bone-forming activity was still high. It was concluded that the bioactivity of nacre was higher than that of THA.

RP59, a marker for osteoblast recruitment, is also detected in primitive mesenchymal cells, erythroid cells, and megakaryocytes.

We recently described a novel protein in bone marrow of rats, RP59, as a marker for cells with the capacity to differentiate into osteoblasts. In this work, its expression pattern was further investigated to learn about the origin and biological relevance of RP59 expressing marrow cells. As revealed by in situ hybridization and by immunohistochemistry of yolk sac embryos, RP59 was found in the cells of the primitive ectoderm and primitive streak as well as in blood islands and extraembryonal mesoderm. Later, RP59 occurred in fetal liver cells and in circulating blood. From the time around birth, it was found in bone marrow and spleen cells. In addition, in vitro–formed blood vessels contained RP59‐positive cells in the lumen. Endothelial cells and the vast majority of cells outside the blood vessels were not labeled. Concerning more mature hematopoietic cell types, RP59 was observed in megakaryocytes and nucleated erythroblasts, but absent from lymphoid cells. In conclusion, RP59 was induced in early mesoderm. It was maintained in the erythroid and megakaryotic lineages and, as earlier described, in young osteoblasts.

Facial development and type III collagen RNA expression: Concurrent repression in the osteopetrotic (Toothless, tl) rat and rescue after treatment with colony‐stimulating factor‐1

The toothless (osteopetrotic) mutation in the rat is characterized by retarded development of the anterior facial skeleton. Growth of the anterior face in rats occurs at the premaxillary‐maxillary suture (PMMS). To identify potential mechanisms for stunted facial growth in this mutation we compared the temporospatial expression of collagen I (Col I) and collagen III (Col III) RNA around this suture in toothless (tl) rats and normal littermates by in situ hybridization of specific riboprobes in sagittal sections of the head. In normal rats, the suture is S shaped at birth and becomes highly convoluted by 10 days with cells in the center (fibroblasts and osteoblast progenitors) expressing Col III RNA and those at the periphery (osteoblasts) expressing no Col III RNA but high amounts of Col I RNA throughout the growth phase (the first 2 postnatal weeks). In the mutant PMMS, cells were reduced in number, less differentiated, and fewer osteoblasts were encountered. Expression of Col I RNA was at normal levels, but centrosutural cells expressed Col III RNA only after day 6 and then only weakly. A highly convoluted sutural shape was never achieved in mutants during the first 2 postnatal weeks. Treatment of tl rats with the cytokine CSF‐1 improved facial growth and restored cellular diversity and Col III RNA expression in the PMMS to normal levels. Taken together, these data suggest that normal facial growth in rats is related to expression of Col III RNA by osteoblast precursors in the PMMS, that these cells are deficient in the tl mutation and are rescued following treatment with CSF‐1. Dev Dyn 1999;215:117–125.

Collagen mRNA expression during tissue development: The temporospacial order coordinates bone morphogenesis with collagen fiber formation

Bone formation of the maxilla and premaxilla of rats was studied by in situ hybridization, using probes for fibrillar collagen mRNAs. Chondroblasts, osteoblasts, fibroblasts and peripheral bone cells differed in their expression patterns. Prospective nasal chondroblasts expressed collagen alpha1(II) and alpha1(XI) RNA from day 15 post coitum. Bone formation in the adjacent maxilla and premaxilla started around day 17: groups of osteoblasts, representing ossification centers, expressed collagen alpha1(I) RNA strongly, and alpha1(V), alpha2(V) and alpha1(XI) RNA weakly, but they were deficient in collagen alpha1(III) RNA. As the centers expanded, osteoblasts in the resulting bone domains expressed collagen alpha1(I) RNA in abundance, whereas collagen alpha1(III) RNA was absent. Bone domains were surrounded by fibroblasts containing collagens alpha1(I), alpha1(III) and alpha2(V) RNA. Widely separated fibroblasts underwent condensation into densely packed periosteum and sutural soft tissues. Cells at the periphery of fast-growing bone domains also displayed, apart from collagen alpha1(I) RNA, collagens alpha2(V) and alpha1(XI) RNA. Given the continuous recruitment of cells from the periosteum, peripheral bone cells represent differentiating osteoblasts synthetizing collagens alpha2(V) and alpha1 (XI) RNA transiently. Thus, gene expression during osteoblast differentiation reflects synthesis of fiber components during bone growth, since collagen V is located in the center of fibers consisting primarily of collagen I.