F. Wachtler

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice.

 It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle.

The nucleolus: A structural and functional interpretation

Abstract This review addresses the problem of understanding nucleolar morphology in terms of nucleolar function. Nucleolar morphology and function are outlined to serve as a basis for reviewing in situ-cytochemical results that have, so far, not led to a generally accepted view on the structure-function correlation for nucleoli. Nucleoli in meiotic cells are dealth with in detail, because they illustrate the relationship between chromosomes and nucleoli particularly well. Spontaneous and induced changes in nucleolar morphology are presented and an attempt is made to explain particular morphologies in terms of functional states. The use of nucleolar morphology as a tool in diagnostics is critically evaluated.

On the position of nucleolus organizer regions (NORs) in interphase nuclei. Studies with a new, non-autoradiographic in situ hybridization method.

The distribution of 18S and 28S ribosomal RNA (rRNA), i.e. the chromosomal nucleolus organizer regions (NORs) was visualized in interphases and metaphases of non-stimulated and phytohemagglutinin (PHA)-stimulated human lymphocytes with a recently developed non-autoradiographic in situ hybridization method. This procedure involves mercurated RNA as a probe and a sulfhydryl-trinitrophenyl-mercury binding ligand and FITC-labelled antibodies as detection system. Silver staining was used to visualize nucleoli in interphase. In the secondary constriction of all ten acrocentric chromosomes, varying amounts of rDNA were detected. In the interphase nuclei of most of the non-stimulated human lymphocytes, only one small nucleolus could be seen. The in situ hybridization, however, revealed several agglomerations of rDNA scattered over the whole nuclear area, clearly outnumbering the number of nucleoli in these cells. This means that not all of the NORs are transcriptionally active in non-stimulated lymphocytes and that these inactive NORs lie at a distinct distance from the active ones. With PHA stimulation (transforming the small lymphocytes from peripheral blood into large, lymphoblast-like cells) the number of nucleoli increased slightly, whereas the number of separable rDNA spots decreased. This means that in the course of PHA-induced cellular activation, formerly inactive NORs become transcriptionally active and tend to associate with one another. This indicates the occurrence of movements of the NORs within the nucleus, depending on their transcriptional activity.

Nucleolus organizer regions and nucleoli

In the last few years numerous papers have been published on the nucleolus and its relation to the nucleolus organizer region (NOR). Several, even very recent, publications still reveal differences in the interpretation of these results. It is, however, our opinion that at the present state most of the observations on NORs and nucleoli can be interpreted so as to achieve a general understanding of the nucleolus. For this purpose we shall summarize some morphological results and problems with particular reference to the example of resting and stimulated lymphocytes. This may supplement recent reviews covering molecular aspects (Perry 1981; Moss and Birnstiel 1982), the cytological visualization of nucleolar genetic activity (Miller 1981; Scheer et al. 1982), the amplification of ribosomal genes (Macgregor 1982), problems of nucleolus formation in plants (Flavell and Martini 1982; de la Torre and Gimenez-Martin 1982), as well as reviews dealing mostly with the functional morphology of nucleoli in vertebrate and also human cells (Goessens and Lepoint 1979; Jordan and McGovern 1981; Bouteille et al. 1982; Stahl 1982). A few notes on the history of the early work on nucleoli are given in the beginning, supplementing the introduction to the review by Miller (1981), since we feel that the very careful observations of the early cytologists should be remembered and reread, if not for their face value, then at least as an example of exact descriptive morphology. Needless to say, this short review article can give only a small selection of the many papers on our topic and will be far from complete.

Connexin 26 mutations in cases of sensorineural deafness in eastern Austria

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G→T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.

Grafting experiments on determination and migratory behaviour of presomitic, somitic and somatopleural cells in avian embryos

SummaryThe state of determination of somites, parts of somites, unsegmented paraxial mesoderm and of somatopleure was investigated by grafting these tissues from quail embryos to the wing buds of chicken embryos. It was found that muscular and chondrogenic determination occur before the formation of somites. Muscular determination takes place earlier than previously assumed and ahead of chondrogenic determination. Somatopleure yields cartilage, but no skeletal muscle. Prospective sclerotomes are primarily capable of differentiating into muscle and loose this potency in the course of development. Myogenic cells extensively migrate within the wing bud in a proximo-distal direction, whereas chondrogenic cells both of somitic and somatopleural origin show no overt migratory tendency.

Ribosomal DNA is located and transcribed in the dense fibrillar component of human Sertoli cell nucleoli

The distribution of rDNA and the uptake of tritiated uridine was investigated in nucleoli of human Sertoli cells. The nucleolar components in these cells are spatially arranged in a highly ordered and invariable way and can be recognized in both light and electron microscopy. The pattern of distribution of rDNA and the pattern of uridine uptake in these nucleoli correspond to the distribution of the dense fibrillar component but cannot be correlated to the shape and size of the fibrillar centers in these cells. It therefore can be concluded that the dense fibrillar component, and not the fibrillar centers, is the site of rDNA location and transcription in nucleoli of human Sertoli cells.

Electron microscopic in situ hybridization and autoradiography: localization and transcription of rDNA in human lymphocyte nucleoli

The distribution of ribosomal DNA (rDNA) in the nucleoli of human lymphocytes was revealed by in situ hybridization with a nonautoradiographic procedure at the electron microscopic level. rDNA is located in the dense fibrillar component of the nucleolus but not in the fibrillar centers. In the same cells the incorporation of tritiated uridine takes place in the dense fibrillar component of the nucleolus as seen by autoradiography followed by gold latensification. From these findings it can be concluded that the transcription of ribosomal DNA takes place in the dense fibrillar component of the nucleolus.

Nucleolar changes in human phytohaemagglutinin-stimulated lymphocytes

SummaryThe nucleoli of lymphocytes from circulating peripheral blood and from phytohaemagglutinin (PHA)-stimulated cultures (from 2 h–96 h) were studied using a silver method, RNA-specific fluorescent staining, and electron microscopy of ultrathin sections. In peripheral blood about 75% of the lymphocytes have one “ring-shaped” nucleolus composed of a distinct fibrillar centre surrounded by a dense pars fibrillaris and little granular material; the remaining lymphocytes showing two or more small “ring-shaped” nucleoli. With PHA stimulation, the number of cells with several nucleoli increases first (from 2 h–12 h). Next, cells containing one or, at most, two large nucleoli with nucleolonema devoid of fibrillar centers are seen (from 4 h on). 34 h after PHA, nucleoli of the “compact” type containing one or more fibrillar centres appear and comprise about 60% of the cells after 72 h. The appearance of more than one nucleolus per cell shortly after PHA administration suggests an activation of additional nucleolar organizer regions (NOR), which fuse to form one or two large nucleoli with nucleolonema. These are then transformed into “compact” nucleoli. The fibrillar centers stain preferentially with silver. They contain nonchromosomal proteins and may serve as stores for nucleolar proteins. The fusion of activated NORs during the first cell cycle explains the relatively high frequency of satellite associations in first mitoses compared to later mitoses after stimulation.

Ontogeny of avian extrinsic ocular muscles

SummaryLight- and electron-microscopic studies were performed on those tissues that are supposed to deliver the anlagen of the extrinsic ocular muscles. Since the blastemata of the ocular muscles can be traced back into the prechordal mesoderm, it can be concluded that this tissue is the source of these muscles. In embryos from stage 8–10 according to Hamburger and Hamilton (HH) cells are found to detach from the lateral border of the prechordal mesoderm. These cells are assumed to give rise to the trochlearis and abducens musculature. In stage-14 embryos the paired premandibular cavity arises within the lateral wings of the prechordal mesenchyme. In 4-day embryos the lateral wall of each premandibular cavity becomes denser forming a premuscular mass, which is subdivided into the anlagen of the oculomotorius muscles in 5-day embryos. The head cavities are not homologous to somites because their structures, origins and sites are very different.