Clonal analysis of vertebrate myogenesis. VIII. Fibroblasts growth factor (FGF)-dependent and FGF-independent muscle colony types during chick wing development

The effect of bovine fibroblast growth factor (FGF) on the in vitro differentiation of various stage-specific populations of skeletal muscle colony-forming (MCF) cells from the developing chick wing bud was examined. The results show that bovine FGF (3 ng/ml daily) delays the onset of differentiation of MCF cells obtained from Day 4-12 wing buds by about 1 day; but, in addition, the results demonstrate that a subset of colony-forming cells derived from stage 23-27 (Day 4-5) embryos require FGF for myogenic differentiation. The FGF-dependent MCF cells attach and grow in the absence of FGF, but do not differentiate unless given FGF within 1-3 days after inoculation. Thus, between stages 23 and 27 the myogenic population contains discrete subclasses that are FGF dependent and others that are FGF independent. Both subclasses are found within two of the previously classified MCF cell populations, the early and late MCF cells. FGF-dependent and independent early MCF cells are present within the wing bud until stage 25, after which only the FGF-independent early MCF subclass persists. Similarly, both FGF-dependent and -independent late MCF cells are present between stages 25 and 27, but only the FGF-independent late MCF subclass remains after stage 31. The mechanisms responsible for relative changes in the proportions of MCF cell subclasses and for the FGF requirements are not understood. In addition, while FGF is required, there is no evidence suggesting that FGF triggers skeletal muscle terminal differentiation within the FGF-dependent MCF cell subclasses.     

Clonal analysis of vertebrate myogenesis. I. Early developmental events in the chick limb

Early developmental events occurring in the prospective muscle tissue region of chick embryo leg buds have been subjected to an in vitro clonal analysis. Colony-forming cells are present at stage 20 (72 hr incubation), but none of the colonies exhibit morphological signs of muscle differentiation. After an additional 8 hr of incubation (stage 21), approximately 10% of the colony-forming cells have acquired the capacity to form multinucleated cells in vitro, and the percentage of clonable myoblasts increases to a level of approximately 60% during the next 3 days of incubation. Clonal analysis of myoblast populations within regions of the developing limb have indicated that, between stages 21 and 27, the dorsal and ventral segments of the myogenic region contain appreciably more clonable muscle cells than the anterior and posterior segments. In addition, during stages 21 and 22 there is a 3-fold difference in muscle-colony-forming cells between the proximal and distal halves of the dorsal-ventral segments, as well as between the proximal and distal halves of the anterior-posterior segments. Thus at least two temporal and regional gradients—proximal to distal and medial to lateral—of clonable myoblast content can be delineated within the developing chick limb. In addition to changes in the proportions of muscle-colony-forming cells, the extent of multinuclearity within individual muscle colonies increases with the developmental age of the embryo from which the clonable myoblasts are derived. The progressive changes in the relative proportions of muscle-colony-forming cells and in clonal morphology are discussed in terms of their possible cell lineage implications.     

Bcl-2 Expression Identifies an Early Stage of Myogenesis and Promotes Clonal Expansion of Muscle Cells

We show that Bcl-2 expression in skeletal muscle cells identifies an early stage of the myogenic pathway, inhibits apoptosis, and promotes clonal expansion. Bcl-2 expression was limited to a small proportion of the mononucleate cells in muscle cell cultures, ranging from ∼1–4% of neonatal and adult mouse muscle cells to ∼5–15% of the cells from the C2C12 muscle cell line. In rapidly growing cultures, some of the Bcl-2–positive cells coexpressed markers of early stages of myogenesis, including desmin, MyoD, and Myf-5. In contrast, Bcl-2 was not expressed in multinucleate myotubes or in those mononucleate myoblasts that expressed markers of middle or late stages of myogenesis, such as myogenin, muscle regulatory factor 4 (MRF4), and myosin. The small subset of Bcl-2–positive C2C12 cells appeared to resist staurosporine-induced apoptosis. Furthermore, though myogenic cells from genetically Bcl-2–null mice formed myotubes normally, the muscle colonies produced by cloned Bcl-2–null cells contained only about half as many cells as the colonies produced by cells from wild-type mice. This result suggests that, during clonal expansion from a muscle progenitor cell, the number of progeny obtained is greater when Bcl-2 is expressed.     

Onset of troponin synthesis in the chick wing bud.

Indirect antibody labeling techniques were used to determine when cells in the chick embryo wing bud begin to synthesize troponin. Frozen sections of stage 22 through stage 27 wing buds were treated with antibodies to the troponin complex and fluorescein-labeled antiimmunoglobulin. Cells producing detectable quantities of troponin were found first in late stage 24 or early stage 25 wing buds; all wing buds stage 25 and older contained labeled cells. Cells synthesizing troponin were initially localized in the muscle-forming areas of the wing bud nearest to the body wall. As the wing bud developed, cells located in more distal areas of the wing bud became labeled with fluorescent antibody, and the number of cells engaged in troponin synthesis increased in all areas. At all stages in which labeling occurred, some cells contained fluorescent cross-striations. When placed in the context of recent studies on the appearance of myofibrillar proteins, these results indicate that myogenic cells in the chick limb bud begin to synthesize large quantities of troponin at approximately the same time as the other muscle contractile proteins.     

Spatial analysis of limb bud myogenesis: Elaboration of the proximodistal gradient of myoblasts requires the continuing presence of apical ectodermal ridge

Myogenic tissue from embryonic chick wing and leg buds is composed of several subpopulations of myoblasts. These clonally distinct subpopulations first appear at different developmental stages, and are distributed differently along the proximo-distal axis of the buds, giving the appearance of a gradient of myoblast cell types. This myoblast distribution pattern has been utilized to investigate the dependence of muscle tissue outgrowth and development on the presence of the apical ectodermal ridge (AER). Wing buds which have had the AER removed at stages 17–18 (2 days) subsequently develop normal proximal regions, but fail to elaborate skeletal structures distal to the humerus. The myoblast pattern of operated buds is also normal proximally, but distal portions of the pattern are not observed. Removal of the AER at stage 20 (3 days) results in buds which develop slightly more distal skeletal structures and the coinciding portions of the myoblast pattern, but in which the more distal portions of the normal myoblast gradient are truncated. These data suggest that elaboration of the myogenic pattern in early limb buds is dependent on the continuing presence of the AER, and that early removal of the AER leads to the subsequent cessation of myoblast pattern specification.     

Clonal analysis of vertebrate myogenesis: VII. Heritability of muscle colony type through sequential subclonal passages in vitro

Muscle colony-forming (MCF) cells from vertebrate limb buds show stage-specific differences in media requirements and clonal morphology (N. K. White, P. H. Bonner, D. R. Nelson, and S. D. Hauschka, 1975, Develop. Biol.44, 346–361). The cell lineage relationship, if any, between the different myogenic cell types is unknown. To investigate whether early MCF cells convert or mature into the late type, MCF cells from stage 23–33 chick embryo leg buds were grown in vitro and sequentially subcloned. Results indicate that colony type, no matter what the stage of origin, is heritable in vitro through at least five passages (45 in vitro doublings) or until the subclones reach proliferative senescence. These results are discussed with respect to various alternatives for the generation of MCF cell diversity during limb development.     

The effects of local application of retinoids to different positions along the proximo-distal axis of embryonic chick wings

Summary Two retinoids, all-trans-retinoic acid and a synthetic analog, TTNPB, were locally applied to different positions along the proximo-distal axis of embryonic chick wing buds using controlled release carriers. Truncations or limbs with duplicated structures across the antero-posterior axis develop after retinoid application to distal positions in buds from stage 20–24 embryos. Phocomelic limbs develop when the retinoids are applied more proximally to buds of stage 23–24 embryos. Duplications of the pattern of structures along the proximo-distal axis never occur.Using TTNPB that is relatively stable, the amount of retinoid in the wing tissue when phocomelia is induced was measured. There is twice as much retinoid per cell in the proximal half of the bud as in the distal half of the bud. The concentration of TTNPB in proximal tissue is estimated to be three times higher than in distal tissue in which pattern formation and cartilage morphogenesis are relatively normal.At early stages in the development of phocomelia, the shape of the bud changes and the indentation that marks the elbow does not arise. Neither retinoid-induced cell killing nor effects on the pattern of programmed cell death were detected.The induction of phocomelia by retinoids appears to be based on effects on proximal cells, whereas retinoids produce pattern changes by acting on distal cells. Furthermore, compared with pattern changes, higher concentrations of retinoid in the bud tissue are required to produce phocomelia.     

Separation of the myogenic and chondrogenic progenitor cells of undifferentiated limb mesenchyme

Undifferentiated limb bud mesenchyme consists of at least two separate, possibly predetermined, populations of progenitor cells, one derived from somitic mesoderm that gives rise exclusively to skeletal muscle and one derived from somatopleural mesoderm that gives rise to the cartilage and connective tissue of the limb. In the present study, we demonstrate that the inherent migratory capacity of myogenic precursor cells can be used to physically separate the myogenic and chondrogenic progenitor cells of the undifferentiated limb mesenchyme at the earliest stages of limb development. When the undifferentiated mesenchyme of stage 18/19 chick embryo wing buds or from the distal subridge region of stage 22 wing buds is placed intact upon the surface of fibronectin (FN)-coated petri dishes, a large population of cells emigrates out of the explants onto the FN substrates and differentiates into an extensive interlacing network of bipolar spindle-shaped myoblasts and multinucleated myotubes that stain with monoclonal antibody against muscle-specific fast myosin light chain. In contrast, the cells of the explants that remain in place and do not migrate away undergo extensive cartilage differentiation. Significantly, there is no emigration of myogenic cells out of explants of stage 25 distal subridge mesenchyme, which lacks myogenic progenitor cells. Myogenic precursor cells stream out of mesenchyme explants in one or occasionally two discrete locations, suggesting they are spatially segregated in discrete regions of tissue at the time of its explantation. There are subtle overall differences in the morphologies of the myogenic cells that form in stage 18/19 and stage 22 distal subridge mesenchyme explants. Finally, groups of nonmyogenic nonfibroblastic cells which are fusiform-shaped and oriented in distinct parallel arrays characteristically are found along the periphery of stage 18/19 wing mesenchyme explants. Our observations provide support for the concept that undifferentiated limb mesenchyme consists of independent subpopulations of committed precursor cells and provides a system for studying the early determinative and regulatory events involved in myogenesis or chondrogenesis.     

Local extrinsic signals determine muscle and endothelial cell fate and patterning in the vertebrate limb

Both the muscle and endothelium of the vertebrate limb derive from somites. We have used replication-defective retroviral vectors to analyze the lineage relationships of these somite-derived cells in the chick. We find that myogenic precursors in the somites or proximal limb are not committed to forming slow or fast muscle fibers, particular anatomical muscles, or muscles within specific proximal/distal or dorsal/ventral limb regions. Somitic endothelial precursors are uncommitted to forming endothelium in particular proximal/distal or dorsal/ventral limb regions. Surprisingly, we also find that myogenic and endothelial cells are derived from a common somitic precursor. Thus, local extrinsic signals are critical for determining muscle and endothelial patterning as well as cell fate in the limb.     

The independence of myogenesis and chondrogenesis in micromass cultures of chick wing buds

Micromass cultures prepared from stage 23, 24, or 25 chick wing buds and cultured under identical conditions produce similar numbers of myoblasts. After treatment with the DNA synthesis inhibitor cytosine-1-beta-D-arabinofuranoside, [3H]thymidine labeling and autoradiography of the cultures show that the increase in myoblast number during the first 48 hr of culture is due primarily to cell division. Micromass cultures prepared from proximal and distal portions of stage 23 or 24 wing buds have very different chondrogenic potentials in vitro (B.J. Swalla, E.M. Owens, T.F. Linsenmayer, and M. Solursh (1983). Dev. Biol. 97, 59-69) but a similar myogenic potential under these culture conditions. Medium supplements that significantly enhance chondrogenesis by proximal cell cultures, such as low serum or 1 mM db cyclic AMP, do not affect the number of myoblasts per unit area of culture during the first 3 days. Muscle cells are eventually reduced in number in whole limb micromass cultures, yet persist as long as 6 days in proximal and distal cultures. These results suggest that myogenic cells are already committed in the early limbs but are inhibited from differentiation in situ until a later time. Myogenesis and chondrogenesis occur independently in culture, consistent with the idea that these two differentiated cells are derived from two separate cell populations. Furthermore, treatments which enhance chondrogenesis do not act indirectly by killing the myoblast population in these cultures.     

A comparative study of myogenic cell invasion of the avian wing and leg bud.

The borders of myogenic cell invasion of avian wing and leg buds were determined using the interspecific grafting technique between quail and chick embryos. Distal parts of quail limb buds were grafted ectopically into the coelomic cavity of chick embryos. The presence or absence of skeletal muscle was investigated in histological sections of the reincubated grafts. A comparison between the borders of myogenic cell invasion of the wing and leg buds showed that the differences in the position of the distal most muscles in the adult avian limbs could be a consequence of the cranio-caudal sequence of development.     

Two distinct classes of prechondrogenic cell types in the embryonic limb bud

Differences are demonstrated in the chondrogenic potential of cells derived from the distal and proximal halves of chick wing buds from as early as stage 23, prior to the appearance of overt cartilage differentiation. In high cell density cultures, cells obtained from the distal portions of stage 23 or 24 limb buds are spontaneously chondrogenic in micromass cultures. Cells obtained from the proximal portions, however, become blocked in their differentiation as protodifferentiated cartilage cels, since these cells in micromass cultures make detectable type II collagen, but fail to synthesize significant levels of cartilage proteoglycan or to accumulate an extracellular matrix that will stain for sulfated glycosaminoglycans. Such cultures of proximal limb bud cells can be stimulated to form alcian blue staining nodules by the addition of 1 mM dbcAMP or 50 micrograms/ml ascorbate, or by mixing proximal cells with small numbers of distal cells (1 distal cell to 10 proximal cells). These results demonstrate the existence of two distinct stages among prechondrogenic mesenchyme cells. The earlier stage appears to be able to provide a chondrogenic stimulus to proximal cells.     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.     

Neurogenic and myogenic motor patterns of rabbit proximal, mid, and distal colon

The rabbit colon consists of four distinct regions. The motility of each region is controlled by myogenic and neurogenic mechanisms. Associating these mechanisms with specific motor patterns throughout all regions of the colon has not previously been achieved. Three sections of the colon (the proximal, mid, and distal colon) were removed from euthanized rabbits. The proximal colon consists of a triply teniated region and a single tenia region. Spatio-temporal maps were constructed from video recordings of colonic wall diameter, with associated intraluminal pressure recorded from the aboral end. Hexamethonium (100 μM) and tetrodotoxin (TTX; 0.6 μM) were used to inhibit neural activity. Four distinct patterns of motility were detected: 1 myogenic and 3 neurogenic. The myogenic activity consisted of circular muscle (CM) contractions (ripples) that occurred throughout the colon and propagated in both antegrade (anal) and retrograde (oral) directions. The neural activity of the proximal colon consisted of slowly (0.1 mm/s) propagating colonic migrating motor complexes, which were abolished by hexamethonium. These complexes were observed in the region of the proximal colon with a single band of tenia. In the distal colon, tetrodotoxin-sensitive, thus neurally mediated, but hexamethonium-resistant, peristaltic (anal) and antiperistaltic (oral) contractions were identified. The distinct patterns of neurogenic and myogenic motor activity recorded from isolated rabbit colon are specific to each anatomically distinct region. The regional specificity motor pattern is likely to facilitate orderly transit of colonic content from semi-liquid to solid composition of feces.     

The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells

Using chimeras consisting of chick embryos that had received substitution grafts of quail somites, we have determined the distalmost extension of the myogenic primordia in the outgrowing wing bud at 5 days of incubation. At Hamburger-Hamilton stage 25 the most distal premuscle cell is consistently 300 mum or more from the apex of the wing mesoblast. The stage 25 wing tip resembles very early whole limb buds in not having proceeded beyond the mesenchymal state or having expressed markers of terminal differentiation. However, unlike early whole limb buds it is free of a myogenic subpopulation. We therefore propose that the stage 25 wing tip is the appropriate system for in vitro and molecular studies of cartilage differentiation.     

Distribution and changes of glycoconjugates in rat colonic mucosa during development

Summary A study was made of the modifications of glycoconjugates in rat colonic mucosa during development. Sections of the caecum, and proximal and distal portions of the colon from Sprague Dawley rats at different stages of development (embryos, fetuses, suckling, weaning and adult rats) were examined. The sections were incubated with a battery of eight fluoresceinated lectins: DBA, SBA, WGA, LFA, PNA, GS-I, UEA-I and Con A. Some sections were treated with neuraminidase, and others were submitted to sequential saponification-neuraminidase treatment prior to incubation with the lectin (WGA, PNA or LFA). The intensity of the fluorescence was evaluated and graded from absent (–) to very positive (4+). Gradual and progressive changes were seen in colonic glycoconjugates during development. These changes revealed a unique developmental pattern for each lectin, which was independent for each cellular compartment (goblet cells, luminal surface and supranuclear region). Local and regional differences, observed between the different colonic sections, were already present from early stages of development. Moreover, our study showed that for several glycoconjugates, the differentiation process in colonic mucosa began in the distal region and continued through to the proximal region, the former being the first to reach the adult pattern. In the caecum, some lectins maintained a fetal pattern throughout all the periods of development up to the adult stage.     

The determination of myogenic and cartilage cells in the early chick embryo and the modifying effect of retinoic acid

Summary The time of determination of cartilage and skeletal muscle was studied by making chimeric grafts or explants of small tissue pieces from several stages of early chick or quail embryos. Chondrogenesis was assessed by histology or with antibodies directed against type II collagen or cartilage proteoglycan, while myogenesis was detected immunohistochemically with antibodies directed against 3 different muscle markers, including muscle myosin. Grafts from Hensen's node, primitive streak and segmental plate of donor embryos of Stage 3–5 (Hamburger and Hamilton) were transplanted under the ectoderm in the extraembryonic area of Stage 12 host embryos. In addition, explants and mesodermal cells were cultured on glass in DMEM+F12 medium supplemented with 10% FCS. The results showed that determined myogenic cells could first be detected in Hensen's node and the primitive streak at Stage 3⁺–4 and that they developed from mesodermal cells located between the epiblast and hypoblast. Myogenic cells also appeared in grafted and explanted segmental plate with or without notochord from Stage 5 embryos. On the other hand, cartilage cells only formed in grafted and explanted segmental plate that also contained notochord. RA (1 ng/ml) could induce the formation of cartilage cells in the explanted primitive streak without Hensen's node or notochord taken from Stage 3–5 embryos and could also promote the differentiation of myogenic cells in primitive streak from Stage 3 embryo. Thus RA can substitute for Hensen's node or the notochord in the induction of cartilage cells and has some stimulatory effects on the differentiation of myogenic cells. Additional evidence indicates that the hypoblast might play an inductive role in the formation of the notochord which may subsequently promote the differentiation of cartilage cells. Offprint requests to: M. Solursh     

Localization of nucleotide pyrophosphatase in the rat kidney

Summary Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freezedried sections. Nucleotide pyrophosphatase activity, expressed in mol·min^–1·mg protein^–1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichement of the activity during the purification of brush border vesicles was measured. A tenfold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.Supported by the Swiss National Foundation, grant nr. 3.813.084