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

Abstract

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.

Figure 1.

Characteristics of G8 ^{po s} and G8^neg epiblast cells in vivo and in vitro. The stage 4 embryo was labeled with the G8 mAb and a secondary antibody conjugated with Alexa 488, followed by in situ hybridization with Cy3 labeled dendrimers containing a recognition sequence for MyoD mRNA. (A) Nuclei were stained with bis-benzamide (blue). Cells coexpressed the G8 antigen (green) and MyoD mRNA (red). The G8^pos and G8^neg populations were isolated from stages 3 to 5 epiblasts. After 5 d in culture, cells were stained with antibodies to the skeletal muscle specific 12101 antigen, cardiac muscle specific troponin T (CTpn), neurofilament associated antigen (Nf), and type II collagen (Col). G8^pos cells differentiated into skeletal muscle when plated at high (B) or low density (C). Some G8^neg cells differentiated into cardiac muscle (D), neurons (E), and chondroblasts (F). G8^neg cells formed skeletal muscle when grown in conditioned medium from G8^pos cultures (G). Noggin (H) and soluble BMP receptor-IA (I) also stimulated skeletal myogenesis in G8^neg cultures. Bar, 10 μm.

Figure 2.

Immunofluorescence localization of cadherins and β-catenin in G8 ^{po s} and G8^neg cultures. G8^pos and G8^neg cells were isolated from stages 3 to 5 epiblasts, cultured for 5 d, and double labeled with antibodies to N- and E-cadherin (A–D), or single labeled with antibodies to cadherin 11 and β-catenin. Most G8^pos cells expressed N- but not E-cadherin (A and B). G8^neg cultures contained cells with E- but not N-cadherin (C and D). G8^neg cells stained with antibodies to β-catenin (E) and cadherin 11 (F). Bar, 10 μm.