Creatine kinase, myokinase, and acetylcholinesterase activities in muscle-forming primary cultures of mouse teratocarcinoma cells.

Multipotential mouse teratocarcinoma cells in embryoid bodies were explanted on plastic or collagen substrates. Various modes of cell determination, including myogenesis, occurred. The predominant avenue of differentiation soon became myogenesis: many multinucleated myotubes formed and yielded an extensive network of skeletal muscle fibers. The process does not proceed to normal completion, as the fibers have a paucity of striations and are not contractile. Activities of several enzymes ordinarily associated with muscle differentiation were examined. Acetylcholinesterase activity increases, especially during myotube formation, as in normal myogenesis. However, creatine kinase activity rises during myotube formation and then drops abnormally, and myokinase activity fails to increase appreciably. The fetal isozymic form of creatine kinase is expressed in the cultures, although well differentiated solid tumors taken from mice show attainment of the adult muscle isozyme type if skeletal muscle is demonstrably present. The results are consistent with the interpretation that coordinately regulated changes in gene expressions controlling these functions may be required for later stages of myogenesis.     

Reptilian myotomal myogenesis-lessons from the sand lizard Lacerta agilis L. (Reptilia, Lacertidae)Update

Reptilian myotomal myogenesis is poorly understood. This paper reports on structural, ultrastructural and immunocytochemical studies of muscle differentiation in sand lizard (Lacerta agilis) embryos. During somitogenesis, the somites are composed of epithelial vesicles with a centrally located somitocoel. At later developmental stages the ventral portion of the somite cortex disaggregates into the sclerotome mesenchyme, while the dorsal wall of the somite differentiates into dermomyotome. At these developmental stages, mononucleated cells of the dermomyotome are Pax3-positive. The dermomyotome layer forms the dorsomedial and ventromedial lips. The myotome is first composed of mono- and then of multinucleated myotubes and small mononucleated cells that occur in the vicinity of the myotubes. These mononucleated cells exhibit low proliferative potential as revealed by the use of PCNA antibody. At subsequent stages of myogenesis the mononucleated cells express Pax7 protein, a marker of satellite cells, and assume ultrastructural features characteristic of satellite cells. Some of the mononucleated cells contribute to muscle growth, being involved in fusion with differentiating muscle fibers. This study revealed similarities of myotomal myogenesis in reptiles to that of other vertebrates.     

Transdifferentiation of Adult Frog Iris in Retina or Lens by Exogeneous Influences

The mechanisms of transdifferentiation of iris epithelial cells of Rana temporaria (Anura) in culture depending on influences from different sources were studied. In terminally differentiated iris cells, the process of transdifferentiation is initiated by dedifferentiation. Melanosomes are shed from iris cells due to cell surface activity. After depigmentation, iris epithelial cells become capable of proliferating and competent to react to the influences of various exogenous factors. Under the influence of retinal factors secreted by lentectomized tadpole eyes, both dorsal and ventral irises are converted to neural retina. Under the influence of factors from eye vesicles, the irises are converted to neural retina as well. Similar results were obtained in transfilter experiments, in which a 3-day period of transfilter interaction between the irises and eye vesicles ensured depigmentation of the iris followed by transdifferentiation into complete NR with visual receptor. Lentoid formation occurred under the influence of adult frog lens epithelium. Immunofluorescent analysis confirmed the lens nature of the lentoids. In control experiments under the conditions of the tadpole eye orbit, in which programming influences were absent, iris epithelial cells remained unaffected.
The problem of true cell-reprogramming to new differentiation in contrast to expression of inherent properties of the iris epithelial cells is discussed.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

Cytologische Veränderungen während der Metamorphose des CubopolypenTripedalia cystophora (Cubozoa,Carybdeidae) in die Meduse

The life cycle ofTripedalia cystophora includes a sessile saclike polyp — the asexual reproducing form — and a pelagic tetraradial medusa — the sexually reproducing generation. Medusan development can be induced by temperature increase. It reveals neither budding nor strobilation, but a real metamorphosis of a polyp to only one medusa. According to morphological and anatomical criteria the metamorphosis can be subdivided into four different stages: (1) four longitudinal furrows segment the polyp, the tentacles of which are apportionated on the four quadrants of the body. (2) The subumbrellar cavity develops by invagination of the peristom; the relicts of the fused tentacles change to four rhopalia buds. (3) Medusan architecture including four new interradial tentacles, four rhopalia and the subumbrellar swimming musculature is completed. (4) A young tetraradial medusa starts swimming. Ultrastructural analysis of those metamorphic stages show the different processes of morphogenesis: (a) Gastrodermal cells — absorptive and spumous cells — undergo transdifferentiation and proliferation to medusan cells of the same structure and function. (b) Epidermal cells, excluding the epithel muscle cells, dissociate and are autolytically withdrawn. Dedifferentiated epithel muscle cells — interstitial cells — regain the ability to develop a complete new set of somatic cells, not originally present in the polyp. They include amongst others cross-striated muscle cells, medusan typic nematocyts and particularly sensory and nervous cells. Those cells establish a nervous system with lens-eyes, simple ocelli, statocysts, diffuse nerve net and an additional nerve ring.     

Experimentelle Untersuchungen zum Stockwachstum und zur Medusenbildung bei dem marinen HydrozoonEirene viridula

The development of both slide-grown and non-substrate bound colonies ofE. viridula (Thecata-Leptomedusae) ranging in size from 1 to 50 hydranths was investigated under various temperature conditions. The majority of slide-grown colonies reached a larger final size than non-substrate bound ones, in 20°, 25° and 29° C. Raising the temperature did not stimulate propagation of hydranths as expected. Most of the colonies transferred to 25° or 29° C finally were even smaller than those reared at 20° C. This was partially due to resorption of several hydranths about 9 days after the temperature rise; the influence of “physiological competition” between development of new hydranths and budding of medusae on colony growth is discussed. Transfer from higher to lower temperatures affected colony growth negatively. Raising the temperature from 20° to 25° or 29° C initiated formation of gonozooids from the distal part of hydranth stalks and development of medusa buds in both types of colonies. With the exception of slide-grown colonies transferred to 25° C, also young medusae were budded off. There was a remarkable coincidence in predominance of colony growth in slide-grown colonies and of medusa budding in non-substrate bound cultures. In the latter, medusa buds developed 1 to 2 days earlier. Most buds did not differentiate into liberated medusae, but were resorbed. Transformation of medusa buds into hydranths was not observed. In the clone ofE. viridula, onset of medusa budding did not depend on a “minimal colony size”. Even single hydranths were able to produce medusa buds after transfer to higher temperatures; budded off medusae were recorded from non-substrate bound colonies with an initial size of 3 hydranths. In slide-grown cultures, medusa buds developed into colonies with an initial size of only 3 hydranths. No hydranth propagation prior to medusa budding occurred in these cases. After raising temperature from 25° to 29° C medusa buds were observed in nonsubstrate bound colonies only; a small number of medusae were budded off from some of these colonies. Lowering the temperature from 29° or 25° to 20° C caused resorption of existing medusa buds. In several non-substrate bound colonies, transfer from 29° to 25° C induced development of gonozooids with medusa buds and, in some cases, of young medusae. Incubation with the alkylating cytostaticTrenimon and transfer from 20° to 25° C caused irreversible resorption of all hydranths when 4 × 10^?2 mg/ml were administered for 10 mins. Thereafter, only development of stolonial structures was observed. With one exception, the colonies treated with 4 × 10^?3 mg/ml, and all others submitted to 4 × 10^?4 mgTrenimon/ml were able to produce new hydranths and also medusa buds; some of the colonies first had to overcome a degressive phase. Treatment with 4 × 10^?2 mg destroyed all interstitial cells (I-cells). Incubation with 1 × 10^?3 or 1 × 10^?4 mg/ml left the I-cells at least partially intact. It is concluded that I-cells are indispensable for hydranth and medusan morphogenesis inE. viridula.     

Evolution of striated muscle: jellyfish and the origin of triploblasty

The larval and polyp stages of extant Cnidaria are bi-layered with an absence of mesoderm and its differentiation products. This anatomy originally prompted the diploblast classification of the cnidarian phylum. The medusa stage, or jellyfish, however, has a more complex anatomy characterized by a swimming bell with a well-developed striated muscle layer. Based on developmental histology of the hydrozoan medusa this muscle derives from the entocodon, a mesoderm-like third cell layer established at the onset of medusa formation. According to recent molecular studies cnidarian homologs to bilaterian mesoderm and myogenic regulators are expressed in the larval and polyp stages as well as in the entocodon and derived striated muscle. Moreover striated and smooth muscle cells may have evolved directly and independently from non-muscle cells as indicated by phylogenetic analysis of myosin heavy chain genes (MHC class II). To accommodate all evidences we propose that striated muscle-based locomotion coevolved with the nervous and digestive systems in a basic metazoan Bauplan from which the ancestors of the Ctenophora (comb jellyfish), Cnidaria (jellyfish and polyps), as well as the Bilateria are derived. We argue for a motile tri-layered cnidarian ancestor and a monophyletic descent of striated muscle in Cnidaria and Bilateria. As a consequence, diploblasty evolved secondarily in cnidarian larvae and polyps.     

Transdifferentiation,Metaplasia and Tissue Regeneration

Transdifferentiation is defined as the conversion of one cell type to another. It belongs to a wider class of cell type transformations called metaplasias which also includes cases in which stem cells of one tissue type switch to a completely different stem cell. Numerous examples of transdifferentiation exist within the literature. For example, isolated striated muscle of the invertebrate jellyfish (Anthomedusae) has enormous transdifferentiation potential and even functional organs (e.g. tentacles and the feeding organ (manubrium) can be generated in-vitro. In contrast, the potential for transdifferentiation in vertebrates is much reduced, at least under normal (non-pathological) conditions. But despite these limitations, there are some well-documented cases of transdifferentiation occurring in vertebrates. For example, in the newt, the lens of the eye can be formed from the epithelial cells of the iris. Other examples of transdifferentiation include the appearance of hepatic foci in the pancreas, the development of intestinal tissue at the lower end of the oesophagus and the formation of muscle, chondrocytes and neurons from neural precursor cells. Although controversial, recent results also suggest the ability of adult stem cells from different embryological germlayers to produce differentiated cells e.g. mesodermal stem cells forming ecto- or endodermally-derived cell types. This phenomenon may constitute an example of metaplasia. The current review examines in detail some well-documented examples of transdifferentiation, speculates on the potential molecular and cellular mechanisms that underlie the switches in phenotype, together with their significance to organogenesis and regenerative medicine.     

Gene expression profiles during differentiation and transdifferentiation of bovine myogenic satellite cells

The molecular mechanisms underlying myogenic satellite cells (MSCs) differentiation into myotube-formed cells (MFCs) and transdifferentiation into adipocyte-like cells (ALCs) are unclear. As a step towards understanding the molecular mechanisms underlying MSC differentiation and transdifferentiation, we attempted to identify the genes differentially expressed during differentiation and transdifferentiation using gene microarray analysis (GMA). Thirty oligonucleotide arrays were used with two technical replicates and nine and six biological replicates for MFCs vs. MSCs and ALCs vs. MSCs, respectively, to contrast expression profile differences. GMA identified 1,224 differentially expressed genes by at least 2-fold during differentiation and transdifferentiation of MSCs. To select the highly expressed genes for future functional study, genes with a 4-fold expression difference were selected for validation by real time RT-PCR and approximately 96.9% of the genes were validated. The up-regulation of marker genes for myogenesis (MYL2, MYH3) and adipogenesis (PPAR??, and FABP4) was observed during the differentiation and transdifferentiation of MSCs into MFCs and ALCs, respectively. KOG analysis revealed that the most of the genes up-regulated during differentiation and transdifferentiation of MSCs were related to signal transduction. Again the exact location of 109 differentially expressed genes by 4-fold were analyzed by chromosome mapping. Among those, co-localization of 29 genes up-regulated during transdifferentiation with QTL for marbling score and intramuscular fat percentage supports the involvement of these genes in cellular transdifferentiation. Interestingly, some genes with unknown function were also identified during the process. Functional studies on these genes may unfold the molecular mechanisms controlling MSC differentiation and transdifferentiation.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.     

Cnidarians as a model system for understanding evolution and regeneration

Hydra and Podocolyne are two cnidarian animals which provide complementary advantages for analysing developmental mechanisms possibly reflecting the basic developmental processes shared by most bilaterians. Interestingly, these mechanisms remain accessible all along the life of these animals, which bud and regenerate, whatever their age. The Hydra polyp permits a direct study of the molecular cascades linking amputation to regeneration. Podocoryne displays a complete life cycle, polyp and medusa stages with a fast and inducible sexual cycle and an unparalleled In vitro transdifferentiation potential. In both cases, a large number of evolutionarily conserved molecular markers are available, and analysis of their regulation highlights the molecular mechanisms which underly pattern formation in these two species.     

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells

The transforming growth factor (TGF)-β inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-β, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.     

Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.     

TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors

The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E- cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta- mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition.