Tissue-specific stem cells: lessons from the skeletal muscle satellite cell

In 1961, the satellite cell was first identified when electron microscopic examination of skeletal muscle demonstrated a cell wedged between the plasma membrane of the muscle fiber and the basement membrane. In recent years it has been conclusively demonstrated that the satellite cell is the primary cellular source for muscle regeneration and is equipped with the potential to self renew, thus functioning as a bona fide skeletal muscle stem cell (MuSC). As we move past the 50(th) anniversary of the satellite cell, we take this opportunity to discuss the current state of the art and dissect the unknowns in the MuSC field.     

3H-thymidine and 3H-uridine incorporation into the heart cells of the freshwater crayfish Astacus astacus and the swimming crab Portunus holsatus

DNA and RNA syntheses in the heart cells of two decapod species were investigated with the aid of electron microscopic autoradiography. Isotopes were injected in the cavity of adult animals 4 hours before fixation. 3H-thymidine labeling was found in several satellite cell nuclei and in some particular epicardial cell nuclei. None of myonuclei was labeled. 3H-uridine incorporated in all the nuclei of muscle fibers. Satellite cells were labeled with 3H-uridine very slightly, if at all. Such a peculiarity of biosynthetic processes in the decapod heart satellite cell suggests their myoblastic nature similar to that of satellite cells of somatic muscles. The active 3H-thymidine uptake by the heart satellite cells of adult animals may be accounted for by the permanent growth of the decapods through their whole life span.     

Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.     

Fine structure and quantitative study in the otic ganglia of the rat

Observations were concentrated on the ultrastructure of perikarya and satellite cells of the otic ganglion of the adult rat. Characteristics of both cell types were morphologically analysed as well as their relationship. Quantitative data concerning the volumetric density were calculated for the following elements of the ganglion: glial cells together (28.5%), unmyelinated fibres (13.4%), myelinated fibres (2.7%), connective tissue (13.6%). This electron microscopic study is a sequence of our previous light microscopic study which determined neuronal densities (COSTA, MANDARIM-DE-LACERDA and BAUER, in press).     

Does ADP-Ribosylation of Proteins in Nuclei Contribute to Ectoderm Cell Differentiation in Sea Urchin Embryos?

In nuclei of sea urchin embryos, marked increase in ADP-ribosyltransferase activity followed by its decrease occurrs in the pre-hatching and post-hatching periods with peaks of activity at the morula and gastrula stages. Increase in its activity was blocked by cycloheximide in the pre- and post-hatching periods and by actinomycin D only in the post-hatching period. Embryo wall cells (ectoderm cells) isolated from gastrulae exhibited markedly higher activity of this enzyme than archenteron cells and mesenchyme cells. Probably, the increase in the activity of this enzyme in the post-hatching period results from expression of the gene for this enzyme mainly in ectoderm cells. In the post-hatching period, the activity increased more in animalized embryos than in normal ones, and increased little in vegetalized embryos. 3-Aminobenzamide (3-ABA), as well as luminol and nicotinamide, inhibited formation of ectoderm structures more than that of endoderm structures, such as the archenteron, in normal and animalized embryos, but had no appreciable effect on morphogenesis in vegetalized embryos. The reaction catalyzed by ADP-ribosyltransferase probably contributes to ectoderm cell differentiation. Treatment of embryos with 3-ABA in the pre-hatching period had little inhibitory effect on the morphogenesis in the post-hatching period, though it caused death of many embryos.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

Intracellular development of a large DNA bacteriophage lytic for Caulobacter crescentus

Summary The cytological alterations accompanying Cb 13 infection of Caulobacter crescentus CB13 cells were followed by electron microscopic examination of sections of cells fixed at various stages of the infection. During the first half of the latent period, the cells appear unaltered. In the second half, the nucleoplasm migrates to the cell periphery and becomes more discrete than the nucleoplasm of uninfected cells. Phage particles appear within the migrated nucleoplasm. The only further alteration apparent in the sections is the absence of the lysozyme-versenesoluble layer of the cell wall of phage-lysed cells.This work was supported by National Science Foundation grant GB-2872.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday.     

Isolation of a variant family of mouse minor satellite DNA that hybridizes preferentially to chromosome 4

Two cosmids (HRS-1 and HRS-2) containing mouse minor satellite DNA sequences have been isolated from a mouse genomic library. In situ hybridization under moderate stringency conditions to metaphase chromosomes from RCS-5, a tumor cell line derived from the SJL strain, mapped both HRS-1 and HRS-2 to the centromeric region of chromosome 4. Sequence data indicate that these cloned minor satellite DNA sequences have a basic higher order repeat of 180 bp, composed of three diverged 60-bp monomers. Digestion of mouse genomic DNA with several restriction enzymes produces a ladder of minor satellite fragments based on a 120-bp repeat. The restriction enzyme NlaIII (CATG) digests all the minor satellite DNA into three prominent bands of 120, 240, and 360 bp and a weak band of 180 bp. Thus, the majority of minor satellite sequences in the genome are arranged in repeats based on a 120-bp dimer, while the family of minor satellite sequences described here represents a rare variant of these sequences. Our results raise the possibility that there may be other variant families of minor satellites analogous to those of alphoid DNA present in humans.     

Functional morphology of the myosatellitocytes in ontogenesis of higher vertebrates and man

By means of electron microscopic radioautography of RNA and DNA synthesis, morphometry of satellite cells, transmissive electron microscopy applying photometric scanning of the image, a successive development and maturation of myosatellites has been studied in ontogenesis of hens, rats and the man. The initial muscle cells for simplasts are promyoblasts. At the stage of myotubes and young muscle fibres, structural and functional heterogeneity of the satellite cells in the myotubes takes place. It is possible to distinguish satellite promyoblasts (the cells which are at the state of proliferative rest, interact with simplasts, intensively synthesize RNA, temporarily do not fuse with simplasts but have certain metabolic connection with them). Subsequently, promyocytes form myosatellitocytes of the I type, they are constantly found in small amount in the muscle fibre composition during the whole course of the animal's ontogenesis. According to the morphological classification, promyoblasts and promyocytes belong to myosatellitocytes of the II type. When studying myosatellitogenesis in the animals, no other sources of cell production have been found.     

Entamoeba histolytica: cell cycle and nuclear division

The cell cycle of Entamoeba histolytica, the duration of its phases, and the details of the nuclear division stages are described in this paper. Trophozoites from clone L-6, strain HM1:IMSS, were synchronized by colchicine. Synchrony was observed immediately after treatment and cultures remained synchronous for at least three replicative cycles with synchrony indexes between 13 and 15 hr. The stages of nuclear division were studied by light and electron microscopy. Four stages of the nuclear division were defined: prophase, early anaphase, late anaphase, and telophase. No metaphase stage was observed by light or electron microscopy. One of the first events in the nuclear division was the presence of a bud close to the juxtanuclear body, which grew to a daughter nucleus. The karyosome and the nuclear membrane remained throughout the mitotic process. Bundles of intranuclear microtubules were observed forming a "V" from the center of the nucleus to one of the poles, and associated with them, 12 to 16 chromosomes-like structures appeared. The results of these studies strongly suggest that division of E. histolytica involved a pleuromitotic process which is carried out in about 120 min.     

Ultrastructural evidence for apoptosis of pavement cells, chloride cells, and hatching gland cells in the developing branchial area of the trout Salmo trutta

Death by apoptosis of branchial epithelial cells was studied in brown trout embryos by means of transmission electron microscopy. Superficial pavement cells are sloughed off for the renewal of the epithelium after an apoptotic degeneration with shrinkage of the cytoplasm and loss of desmosomal contacts. Chloride cells appear as immature, mature and degenerating cells. Degenerating chloride cells, which are separated from the ambient water by pavement cells, show condensation of the cytoplasm and structural alterations in the tubular system and the mitochondria. Hatching gland cells degenerate either into apoptotic bodies or into cellular debris, depending on the functional stage of the cell. There was no phagocytosis by macrophages or adjacent cells of the degenerating chloride and hatching gland cells, but an infiltration of leucocytes was always observed in the epithelium undergoing cellular degeneration. In some instances, secondary necrosis of apoptotic hatching gland cells was observed. Apoptosis occurs in the three types of cells since early stages of development. However, a massive wave of cellular death occurred in pavement and hatching gland cells during the hatching stage and in the chloride cells during post-hatching stages.     

Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD

Satellite cells are tissue-specific stem cells responsible for skeletal muscle growth and regeneration. Although satellite cells were identified almost 50 years ago, the identity of progenitor populations from which they derive remains controversial. We developed MyoD^iCre knockin mice, and used Cre/lox lineage analysis to determine whether satellite cell progenitors express MyoD, a marker of myogenic commitment. Recombination status of satellite cells was determined by confocal microscopy of isolated muscle fibers and by electron microscopic observation of muscle tissue fixed immediately following isolation, using R26R-EYFP and R26R (β-gal) reporter mice, respectively. We show that essentially all adult satellite cells associated with limb and body wall musculature, as well as the diaphragm and extraocular muscles, originate from MyoD+ progenitors. Neonatal satellite cells were Cre-recombined, but only a small minority exhibited ongoing Cre expression, indicating that most satellite cells had expressed MyoD prenatally. We also show that satellite cell development in MyoD-null mice is not due to functional compensation by MyoD non-expressing lineages. The results suggest that satellite cells are derived from committed myogenic progenitors, irrespective of the anatomical location, embryological origin, or physiological properties of associated musculature.     

Postnatal differentiation of the Golgi apparatus and the dendrites of Purkinje cells of the rat cerebellum

Summary The morphology of postnatal differentiation of the Golgi apparatus, the nucleus, the perikaryon, and the dendrites was studied in Purkinje cells of the rat cerebellum for 30 days after birth using histochemical, histological, and electron microscopic methods.The Golgi apparatus during differentiation undergoes morphological and positional changes. From the 1st to 7th postnatal day, the Golgi apparatus is found in a supranuclear position, and is connected with the axes of differentiating primary dendrites by beam-like processes. From days 8 to 11 this connection disappears, and most of the Golgi apparatus assumes a lateronuclear and infranuclear position. After the 11th or 12th day, the Golgi apparatus is found in perinuclear and peripheral cytoplasmic positions. The formation of granular endoplasmic reticulum occurs in the vicinity of the perinuclear Golgi apparatus. The differentiation of cell and nuclear forms requires approximately 20 days. The morphological changes of differentiation are discussed in relation to the participation of the Golgi apparatus in the differentiation of dendrites and in the formation of the granular endoplasmic reticulum.     

An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal

Dietary administration of a cytochrome P450 aromatase (P450arom) inhibitor (fadrozole) in genetic female juveniles of zebrafish (Danio rerio) was performed at 15-40 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, treated with 0, 10, 100 and 1000 microg fadrozole g(-1) diet(-1) were 0, 62.5, 100 and 100%, respectively. Rearing at high water temperature in genetic all-females was performed at 15-25 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, at water temperatures of 28.5, 35 and 37 degrees C were 0, 68.8 and 100%, respectively. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive oocytes of early diplotene and perinucleolar stages in fadrozole-treated genetic females (1000 microg g(-1) diet(-1)) were observed at 15-40 days post-hatching during sex-reversal. In contrast, apoptotic oocytes of early diplotene stage in high temperature-treated genetic females (at 35 and 37 degrees C) during sex-reversal and presumptive males of wild-type fish during sex differentiation were found at 15-27 days post-hatching. Our findings indicate that oocyte apoptosis, depletion of P450arom activity and differentiation of spermatogonia during gonadal sex-reversal are caused by treatments of aromatase inhibitor or high water temperature.     

Effect of moderately low and low temperatures on cellular and intracellular structures of the liver

Initial stages of the rat liver ultrastructural rearrangements after local cooling of the liver tissue down to 93 K and 243 K have been studied by means of light microscopic analysis, transmission electron microscopy and freeze-substitution. The main stages in the development of cell dystrophic changes during cryonecrosis formation in the cooled area have been outlined, the crystallization behaviour und cooling conditions studied has been described.     

Wild-type herpes simplex virus 1 blocks programmed cell death and release of cytochrome c but not the translocation of mitochondrial apoptosis-inducing factor to the nuclei of human embryonic lung fibroblasts

Programmed cell death activated by herpes simplex virus 1 mutants can be caspase dependent or independent depending on the nature of the infected cell. The recently discovered mitochondrial apoptosis-inducing factor (AIF) on activation is translocated to the nucleus and induces programmed cell death that is caspase independent. To assess the role of AIF and also to assay apoptosis-related events in primary human embryonic lung (HEL) fibroblasts, cells were mock infected or infected with wild-type virus previously shown not to induce apoptosis in continuous lines of primate cells or with the d120 mutant lacking infected cell protein no. 4 (ICP4) and were shown to induce apoptosis in all cell lines tested. Cells exposed to dexamethasone or osmotic shock induced by sorbitol were the positive controls. The results were as follows: (i) AIF was translocated to the nucleus in all infected cell cultures and in cells treated with dexamethasone or sorbitol, but cells infected with the wild type-virus showed no evidence of undergoing programmed death. (ii) Cytochrome c was released from mitochondria of cells infected with the d120 mutant or exposed to dexamethasone or sorbitol but not from mitochondria in cells treated with sorbitol and infected with the wild-type virus. (iii) Poly(ADP-ribose) polymerase was cleaved in mock-infected cells exposed to sorbitol or dexamethasone and in cells infected with the d120 mutant but not in either untreated cells infected with wild-type virus or cells exposed to sorbitol and then infected with wild-type virus. In contrast to HEp-2 cells, neither d120 infection nor treatment with dexamethasone or sorbitol caused fragmentation of DNA in HEL fibroblasts. Electron microscopic examination showed chromatin condensation and vacuolization in a fraction of cells infected with d120 but not in wild-type virus-infected cells or cells treated with dexamethasone or sorbitol. We conclude that AIF is translocated to the nucleus in infected cells but apoptosis does not ensue in wild-type-infected cells. HEL fibroblasts infected with the d120 virus exhibit symptoms of classical apoptosis, such as cytochrome c release and cleavage of poly(ADP-ribose) polymerase observed also in cells undergoing caspase 3-dependent programmed cell death in which AIF is either not involved or not a contributory factor.