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.

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 ∼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 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.

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.

MyoD mRNA is expressed in a subpopulation of cells within the embryonic epiblast. Most of these cells are incorporated into somites and synthesize Noggin. Ablation of MyoD-positive cells in the epiblast subsequently results in the herniation of organs through the ventral body wall, a decrease in the expression of Noggin, MyoD, Myf5, and myosin in the somites and limbs, and an increase in Pax-3–positive myogenic precursors. The addition of Noggin lateral to the somites compensates for the loss of MyoD-positive epiblast cells. Skeletal muscle stem cells that arise in the epiblast are utilized in the somites to promote muscle differentiation by serving as a source of Noggin.