MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle.

The epiblast of the chick embryo gives rise to the ectoderm, mesoderm, and endoderm during gastrulation. Previous studies revealed that MyoD-positive cells were present throughout the epiblast, suggesting that skeletal muscle precursors would become incorporated into all three germ layers. The focus of the present study was to examine a variety of organs from the chicken fetus for the presence of myogenic cells. RT-PCR and in situ hybridizations demonstrated that MyoD-positive cells were present in the brain, lung, intestine, kidney, spleen, heart, and liver. When these organs were dissociated and placed in culture, a subpopulation of cells differentiated into skeletal muscle. The G8 antibody was used to label those cells that expressed MyoD in vivo and to follow their fate in vitro. Most, if not all, of the muscle that formed in culture arose from cells that expressed MyoD and G8 in vivo. Practically all of the G8-positive cells from the intestine differentiated after purification by FACS. This population of ectopically located cells appears to be distinct from multipotential stem cells and myofibroblasts. They closely resemble quiescent, stably programmed skeletal myoblasts with the capacity to differentiate when placed in a permissive environment.

DNA dendrimers localize MyoD mRNA in presomitic tissues of the chick embryo.

MyoD expression is thought to be induced in somites in response to factors released by surrounding tissues; however, reverse transcription-PCR and cell culture analyses indicate that myogenic cells are present in the embryo before somite formation. Fluorescently labeled DNA dendrimers were used to identify MyoD expressing cells in presomitic tissues in vivo. Subpopulations of MyoD positive cells were found in the segmental plate, epiblast, mesoderm, and hypoblast. Directly after laying, the epiblast of the two layered embryo contained approximately 20 MyoD positive cells. These results demonstrate that dendrimers are precise and sensitive reagents for localizing low levels of mRNA in tissue sections and whole embryos, and that cells with myogenic potential are present in the embryo before the initiation of gastrulation.

The role of stably committed and uncommitted cells in establishing tissues of the somite.

Somites are blocks of embryonic mesoderm tissue that give rise to skeletal muscle, cartilage, and other connective tissues. The development of different tissues within the somite is influenced by adjacent structures, in particular, the neural tube and notochord. Results of experiments performed in vivo and in vitro suggest that somites contain populations of cells stably programmed to undergo either skeletal myogenesis or chondrogenesis and a population uncommitted to either pathway. The fate of the uncommitted cells would depend on a transfer of information from the committed cells. Communication between committed and uncommitted cells is regulated by cell and tissue interactions that either activate or inhibit this process.

In vitro development of precursor cells in the myogenic lineage.

Expression of the muscle-specific integral membrane protein H36 and the intermediate filament protein desmin, detected by immunofluorescence, was used to identify cells at distinct stages in the skeletal myogenic lineage. These proteins were coordinately expressed in cultures of rat hindlimb myoblasts from 17- and 19-day fetuses and newborn pups, and in satellite cells from juveniles. Both H36+ and desmin+ cells were present in cultures from 13.5- and 15-day embryonic hindlimbs, but desmin expression was more prevalent: H36-/desmin+ myoblasts predominate during this early stage of development. H36 was not detected in Day 12 embryo hindlimb bud cells in vivo nor in cultures soon after plating. Initially, only 1% of the Day 12 limb bud cells expressed desmin. When these cells were serially passaged every 3-4 days, cells with all three possible myogenic phenotypes developed: that is, H36+/desmin-, H36+/desmin+, and H36-/desmin+ cells. There was a progressive increase in the frequency of H36+ cells, with 75% of cells positive by passage 6 (Day 27 in vitro). The maximum frequency of cells that expressed desmin occurred in passage 5 (Day 23 in vitro). These results demonstrate that precursors to the cells that express H36 and desmin are present in the 12-day embryo hindlimb bud and that the transition from H36-/desmin- precursors to cells with a myogenic phenotype can occur in vitro. MyoD1 and myogenin were not detected in these cells, suggesting that the initial expression of H36 and desmin in the myogenic lineage may precede and/or is independent of these regulatory proteins. The conversion of precursor cells in the 12-day limb bud to a more advanced stage of development serves to define additional cells in the myogenic lineage. The ability to monitor in vitro these stages of development affords the opportunity to study how they are regulated.

Phospholipase C activity in palate mesenchyme cells: calcium and pH requirements, substrate specificity, and subcellular localization.

Primary cultures of mouse embryo palate mesenchyme cells were incubated with [3H]arachidonic acid and [14C]stearic acid in order to radiolabel their lipids. The cells were then washed, collected by centrifugation, and homogenized. Incubation of the homogenates under various conditions revealed that deoxycholate inhibited phospholipase A activity and stimulated a phospholipase C activity in these cells which preferentially degraded phosphatidylinositol (PI) compared to phosphatidylcholine (PC), -ethanolamine (PE), and -serine (PS). Expression of this phospholipase C (E.C. 3.1.4.10) activity was dependent on Ca2+ and had a pH optimum of no more than 7.0-7.5. Centrifugation of the homogenates at 105,000g for 30 min produced a membranous fraction that contained phospholipase C activity with characteristics similar to those of the enzyme found in the supernatant. Such a dual distribution of this enzyme may reflect that mouse embryo palate mesenchyme cells are neural crest in origin.

Combinations of monoclonal antibodies distinguish mesenchymal, myogenic, and chondrogenic precursors of the developing chick embryo.

Monoclonal antibodies (MAbs) were used as probes for molecular differences in the surfaces of nonterminally differentiated cells of the developing chick limb. The specificity of the MAbs was determined by immunofluorescent localization performed on cultured breast muscle and limb bud cells and cryosections of a variety of embryonic (stages 15-37) and neonatal tissues. Subpopulations of MAb-positive and -negative cells were isolated by fluorescence-activated cell sorting and their developmental potential was assessed in vitro. Cells of the compacted somite, lateral plate mesoderm, and early limb bud were labeled with the CSAT MAb. Myogenic precursors of the dermatome and limb bud were labeled with the CSAT and L4 MAbs. Chondrogenic precursors of the sclerotome and limb bud were labeled with the CSAT, L4, and C5 MAbs. These precursors were distinguished from fibroblasts which were labeled with the CSAT and C1 MAbs. The differentiation and maturation of muscle and cartilage were accompanied by alterations in the labeling patterns of the MAbs. These results indicate that combinations of these MAbs can be used to distinguish mesenchymal, myogenic, and chondrogenic precursors, identify their site of origin during development, and isolate subpopulations of embryonic cells.

In vitro and in vivo expression of alpha 7 integrin and desmin define the primary and secondary myogenic lineages.

Skeletal muscle fibers form during two periods of development and differ biochemically, functionally and in their morphology. Primary fibers develop in the rat hindlimb during Days 14 to 16 of embryogenesis. These fibers are subsequently surrounded by secondary fibers that eventually constitute the bulk of muscle mass in the limbs. We have used the expression of the alpha 7 muscle laminin binding integrin (Song et al., J. Cell Biol. 117, 643-657, 1992) and the intermediate filament protein desmin to identify myogenic cells at distinct stages of development both in vitro and in vivo. The phenotypes of these cells, determined by immunofluorescence microscopy, discriminate two lineages and indicate that the development of primary and secondary muscle fibers is regulated by multiple mechanisms. The cells which compose the primary myogenic lineage are derived from a population of precursor cells that is in part present in the Day 12 embryo limb bud and which do not express either alpha 7 integrin or desmin. These precursor cells develop into cells that express desmin, but not alpha 7, and which subsequently mature into replicating myoblasts that are competent to undergo terminal differentiation. This maturation process requires the in vivo environment of the Day 13 embryo limb. The alpha 7 integrin and slow myosin heavy chain are first expressed in primary muscle cells well after the onset of terminal differentiation. Some cells that give rise to secondary muscle fibers also are present in the Day 12 embryo hindlimb. The precursors of secondary fibers will develop into cells which express either alpha 7 integrin or desmin and subsequently into replicating myoblasts that express both proteins. Upon terminal differentiation of secondary myoblasts there is an increase in the expression of both alpha 7 integrin and desmin. The temporal regulation of expression of these proteins indicates that the environment of the limb plays a role in the maturation of precursors of both lineages. At least two roles of alpha 7 integrin during myogenesis are related to its association with beta 1 integrin and its function as a laminin receptor. Laminin selectively maintains the proliferation of secondary myoblasts and modulates their shape and mobility in vitro. This responsiveness of secondary myoblasts to laminin corresponds to the time when laminin is a major component of the extracellular matrix, when there is an expansion of the population of secondary myoblasts, and when the alpha 7 integrin is expressed on secondary myoblasts in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Maturation of myogenic and chondrogenic cells in the presomitic mesoderm of the chick embryo.

The establishment of cells with myogenic or chondrogenic potential is temporally and spatially separated from terminal differentiation in the developing chick embryo. Both cell types arise from tissue adjacent to the neural tube and notochord, the paraxial mesoderm. A cell culture system was developed in order to study the maturation and differentiation of myogenic and chondrogenic cells along the length of the paraxial mesoderm at different stages of development. Somite and segmental plate cells obtained from 36- to 52-h (stages 10-15) embryos were plated as a monolayer on substrata of gelatin, fibronectin, or laminin. A substratum of gelatin plus fibronectin was most effective in supporting adhesion and differentiation. Maximal increase in number of cells in somite cultures occurred 24 h earlier than that in segmental plate cultures. Fewer skeletal muscle cells and chondroblasts were present in cultures prepared from progressively more caudal regions of the paraxial mesoderm and from younger embryos. Some cells present within the somites and the rostral two-thirds of the stage 13 segmental plate differentiated without replication after placement in culture. Only the progeny of cells from its caudal third, and from stage 10 somites and segmental plates, differentiated under these conditions. The results suggest that some myogenic and chondrogenic cells obtain the ability to differentiate under these in vitro conditions after stage 10 of development, as they occupy more rostral positions within the segmental plate relative to the addition of cells at its caudal end. Although some stage 13 segmental plate cells form skeletal muscle and cartilage directly after removal from the embryo, differentiation is not observed in ovo until these cells are incorporated into somites, a minimum of 10 h later. Three-dimensional tissue interactions, and/or cell-cell interactions, while not required for segmental plate cells to undergo myogenesis and chondrogenesis, may play a role in regulating the timing of terminal differentiation within the embryo.

N-cadherin promotes the commitment and differentiation of skeletal muscle precursor cells.

Cells with the potential to form skeletal muscle are present in the chick embryo prior to gastrulation. Muscle differentiation begins after gastrulation within the somites. The role of cadherin-mediated adhesion in the commitment and differentiation of skeletal muscle precursor cells was examined by analyzing the expression of cell-cell adhesion molecules in cultures of epiblast, segmental plate, and somite cells and by determining the effects of adhesion-perturbing antibodies on the accumulation of MyoD and sarcomeric myosin. Cultured primitive streak stage epiblast cells downregulate E-cadherin and upregulate N-cadherin. This switch in cadherin expression also occurs in vivo as epiblast cells enter the primitive streak. Although MyoD protein is present in cells with N- or E-cadherin, only cells with N-cadherin differentiate into skeletal muscle. In contrast to the primitive streak stage epiblast cells, prestreak epiblast cells maintain the expression of E-cadherin in vitro. While the majority of prestreak cells contain MyoD, only a few synthesize myosin. Treatment of primitive streak stage epiblast cells with function-perturbing antibodies to N-cadherin resulted in an inhibition of myosin accumulation and a decrease in the percentage of cells with MyoD. Segmental plate and somite cells are similar to primitive streak stage epiblast cells in that most differentiated into skeletal muscle when cultured in serum-free medium. While function-perturbing antibodies to N-cadherin inhibited the accumulation of myosin in these mesoderm cells, the number of MyoD positive cells was unaffected in somite cultures and only partially reduced in segmental plate cultures. These results suggest that N-cadherin-mediated cell-cell adhesion is involved in both the commitment of muscle precursors and their terminal differentiation.

Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro.

The epiblast layer of the chick embryo gives rise to all embryonic tissues. In vitro analyses were carried out to determine whether epiblast cells could form skeletal muscle prior to entry into the primitive streak. Epiblasts were separated from the mesoderm, hypoblast, and primitive streak, dissociated to produce a single cell suspension, and plated at high density. Myogenesis began on the first day in culture, and by the fifth day most cells had differentiated into skeletal muscle. Some cells differentiated without replicating. MyoD messenger RNA was present in epiblast tissue and translated in practically all cells in culture. Cells from regions of the epiblast which do not form muscle later in the embryo did so in vitro. Epiblasts cultured for 2 days as an intact epithelium, or in the presence of the mesoderm and hypoblast, did not undergo myogenesis. These findings demonstrate that myogenic potential is wide-spread within the primitive streak stage epiblast, and that muscle differentiation, which occurs relatively autonomously in culture, can be prevented by cell and tissue interactions.

Hepatocyte growth factor/scatter factor promotes a switch from E- to N-cadherin in chick embryo epiblast cells.

Epiblast cells downregulate E-cadherin and upregulate N-cadherin as they ingress through the primitive streak and when placed in culture. The factors that promote the alteration in cadherin expression during gastrulation are unknown. The effects of hepatocyte growth factor/scatter factor (HGF/SF) on cadherin expression were tested in cultures of prestreak epiblast cells. HGF/SF decreased the expression of E-cadherin and increased the percentage of cells with N-cadherin and sarcomeric myosin. Cells with N-cadherin but not E-cadherin differentiated into skeletal muscle. HGF/SF also stimulated proliferation and the formation of cellular aggregates. Sensitivity to HGF/SF in vitro depended on the original position of cells within the epiblast. More cells from the lateral epiblast switched cadherins and proliferated in response to HGF/SF than medial epiblast cells. HGF/SF may affect gastrulation by altering cadherin expression, modulating cell adhesion, and stimulating proliferation within the epiblast.