Tilmann Wurtz

A Pre-mRNA-Binding Protein Accompanies the RNA from the Gene through the Nuclear Pores and into Polysomes

In the larval salivary glands of C. tentans, it is possible to visualize by electron microscopy how Balbiani ring (BR) pre-mRNA associates with proteins to form pre-mRNP particles, how these particles move to and through the nuclear pore, and how the BR RNA is engaged in the formation of giant polysomes in the cytoplasm. Here, we study C. tentans hrp36, an abundant protein in the BR particles, and establish that it is similar to the mammalian hnRNP A1. By immuno-electron microscopy it is demonstrated that hrp36 is added to BR RNA concomitant with transcription, remains in nucleoplasmic BR particles, and is translocated through the nuclear pore still associated with BR RNA. It appears in the giant BR RNA-containing polysomes, where it remains as an abundant protein in spite of ongoing translation.

Assembly and disassembly of spliceosomes along a specific pre-messenger RNP fiber.

Transcriptionally active Balbiani ring (BR) genes in the salivary glands of the dipteran Chironomus tentans were studied by immunoelectron microscopy to establish the distribution of spliceosome components along a specific pre‐messenger ribonucleoprotein (pre‐mRNP) fiber. The BR genes are 35‐40 kb in size with three introns close to the 5′ end and one close to the 3′ end; a very large middle portion lacks introns. As a rule the 5′ introns are spliced concomitant with transcription in the promoter proximal third of the gene, while the 3′ intron is spliced post‐transcriptionally. The BR genes with growing pre‐mRNPs were visualized in situ, while completed and released pre‐mRNPs were isolated from the nucleoplasm and studied unfolded on a grid surface. An anti‐snRNP antibody (Y12) bound mainly to the promoter proximal third of the BR gene (86%) and only to a minor extent to the middle and distal thirds (7 and 7% respectively). An antibody to an hnRNP protein reacted with the proximal, middle and distal regions to an increasing extent (17, 38 and 45% respectively), reflecting the increase in size of the growing transcription product. In the nucleoplasmic pre‐mRNP particle only one end of the RNP fiber was labeled by Y 12, presumably the 3′ end; the anti‐hnRNP antibody decorated the entire RNP fiber. Thus, the snRNPs do not associate along the whole pre‐mRNP fiber but rather bind to the 5′ and 3′ ends, i.e. the regions containing the introns. The results also imply that the spliceosomes both assemble and disassemble rapidly on the pre‐mRNP fiber.

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.

Tissue responses to nacreous implants in rat femur: an in situ hybridization and histochemical study

The interface of bone and aragonite nacre (Margaritifera, fresh water pearl mussel) was studied by in situ hybridization and a tartrate-resistant acid phosphatase (TRAP) histochemical assay. Columnar implants were inserted into rat femora for 4, 7, 14, 28 and 56 days. In medullary region, a burst of transient bone formation was observed, which propagated from the periphery towards the nacre implant. A fused interface of bone and nacre was observed at 14 days. Later, the new medullary bone was resorbed and bone marrow was re-established while a thin layer of bone tissue remained covering the implant surface. Expressions of collagen alpha1(I), osteocalcin, osteopontin mRNAs and TRAP in the surrounding tissue were monitored. Correlated with the histology events, a strong transient induction of collagen alpha1(I) and osteocalcin mRNAs as well as TRAP expression, exhibiting a peak signal intensity on day 7 and subsequent down-regulation after day 14 was observed. Osteopontin mRNA, in contrast, was expressed continuously. The degrading nacre surface appeared in direct contact with macrophages and multinucleated giant cells at both days 14 and 28. These cells expressed osteopontin mRNA intensively and some TRAP enzyme activity occasionally.

Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.

Influence of titanium ion on mineral formation and properties of osteoid nodules in rat calvaria cultures

The effect of the addition of titanium ion (Ti) on osteoblast function and the mineralization of osteoid nodules in rat calvaria cultures was characterized. Concentrations of 10 ppm of Ti or more inhibited cell proliferation; 5 ppm or less either had no effect or stimulated proliferation. The number of nodules formed was not influenced by 5 ppm of Ti, but mineral deposition in nodules was suppressed, as revealed by von Kossa staining. Likewise, 5 ppm of Ti inhibited the incorporation of [(45)Ca] in cultures during nodule formation even if the Ti was withdrawn from the medium when mineralization was initiated. In order to test whether the synthesis of osteoid components was affected, the expression of osteonectin (OSN), osteopontin (OPN), osteocalcin (OSC), and alkaline phosphatase (ALP) mRNAs as well as ALP enzyme activity was analyzed. The expression of OSN and OPN mRNAs was reduced dramatically, but OSC mRNA was little affected by 5 ppm of Ti. Ti delayed the development of ALP mRNA expression and enzyme activity relative to the controls. Thus Ti treatment changed the proportional composition of cellular mRNA contributing the osteoblast phenotype.

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.

Distribution of Protein Kinase Ca and Accumulation of Extracellular Ca2+ During Early Dentin and Enamel Formation

Activation of the protein kinase C (PKC)-related signal transduction system has been associated with phenotypic expression in a wide variety of cell types. In in vitro studies, it has often been activated by relatively small increases in the Ca2+ concentration ([Ca2+]) in the medium. The studies reported here explored the hypothesis that localized increases in the extracellular [Ca2+] and activation of the PKC-related pathway may be involved in early dentin and enamel formation. Whole-head, freeze-dried sections through the developing molars of 5-day-old rats were evaluated by methods that localized non-crystalline Ca2+. Immunohistochemical methods were adapted for use with the freeze-dried sections, and two monoclonal antibodies were used to localize PKCa in the formative cells of the developing teeth. Low concentrations of extracellular Ca2+ were observed in the early, unmineralized dentin in the area of ameloblast differentiation. Increased concentrations occurred at the point of initial dentin mineralization, immediately before the beginning of enamel matrix deposition. PKCa was localized in the differentiating odontoblasts, at the beginning of dentin matrix deposition. It was intensely localized in the distal borders of the pre-ameloblasts, and appeared to redistribute in the cells during ameloblast differentiation. These observations suggest that local increases in the extracellular [Ca2+] and the PKC signal transduction pathway may be involved in key inductions in the early stages of dentin and enamel formation.

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.