Cells that express MyoD mRNA in the epiblast are stably committed to the skeletal muscle lineage.

The epiblast of the chick embryo contains cells that express MyoD mRNA but not MyoD protein. We investigated whether MyoD-positive (MyoDpos) epiblast cells are stably committed to the skeletal muscle lineage or whether their fate can be altered in different environments. A small number of MyoDpos epiblast cells were tracked into the heart and nervous system. In these locations, they expressed MyoD mRNA and some synthesized MyoD protein. No MyoDpos epiblast cells differentiated into cardiac muscle or neurons. Similar results were obtained when MyoDpos cells were isolated from the epiblast and microinjected into the precardiac mesoderm or neural plate. In contrast, epiblast cells lacking MyoD differentiated according to their environment. These results demonstrate that the epiblast contains both multipotent cells and a subpopulation of cells that are stably committed to the skeletal muscle lineage before the onset of gastrulation. Stable programming in the epiblast may ensure that MyoDpos cells express similar signaling molecules in a variety of environments.

Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage.

Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.

NeuroM and MyoD are expressed in separate subpopulations of cells in the pregastrulating epiblast.

Epiblast cells form skeletal muscle and neurons in culture and some express mRNA for the skeletal muscle specific transcription factor MyoD in vivo. The following experiments were designed to determine whether the neurogenic transcription factor NeuroM is expressed in the epiblast and if NeuroM and MyoD are present in separate subpopulations of epiblast cells that can differentiate into neurons and muscle, respectively. In situ hybridization revealed that NeuroM was present in the anterior region of the pregastrulating epiblast. Some cells with NeuroM were proliferating and expressed two molecules present in neurogenic cells, NCAM and the Zn-12/HNK-1 carbohydrate. The G8 antibody labeled cells with MyoD but not NeuroM. When G8 positive cells were isolated by magnetic cell sorting and placed in culture, nearly all differentiated into skeletal muscle in serum free medium. A subpopulation of cells isolated with antibodies that bound to cells expressing NeuroM formed neurons when cultured in medium supplemented with sera and embryo extract. These experiments demonstrate that NeuroM and MyoD are present in separate subpopulations of cells in the pregastrulating epiblast. Epiblast cells with NeuroM are more dependent on exogenous factors to differentiate than those with MyoD.

Visualizing the Needle in the Haystack: In Situ Hybridization With Fluorescent Dendrimers.

In situ hybridization with 3DNA trade mark dendrimers is a novel tool for detecting low levels of mRNA in tissue sections and whole embryos. Fluorescently labeled dendrimers were used to identify cells that express mRNA for the skeletal muscle transcription factor MyoD in the early chick embryo. A small population of MyoD mRNA positive cells was found in the epiblast prior to the initiation of gastrulation, two days earlier than previously detected using enzymatic or radiolabeled probes for mRNA. When isolated from the epiblast and placed in culture, the MyoD mRNA positive cells were able to differentiate into skeletal muscle cells. These results demonstrate that DNA dendrimers are sensitive and precise tools for identifying low levels of mRNA in single cells and tissues.