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

Abstract

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.

Figure 1.

Expression of G8, MyoD, and Myf5 in the pregastrulating and gastrulating epiblast. Whole stage 1 and 4 embryos were double labeled for the G8 antigen (green) and MyoD mRNA (MyoD M), MyoD protein (MyoD P), or Myf5 mRNA (red). Nuclei were stained with Hoechst dye (blue). G8 and MyoD mRNA were expressed in the same cells in the stage 1 epiblast (arrows in A). The MyoD mAb did not label cells in the stage 1 epiblast or most cells of the stage 4 epiblast (arrowheads in B and D). One G8^pos cell in the stage 4 epiblast was stained with the MyoD mAb (arrow in C). Myf5 was not detected in the stage 1 epiblast (arrowhead in E). The stage 4 epiblast contained subpopulations of G8^pos/Myf5^neg, G8^pos/Myf5^pos (arrows), and G8^neg/Myf5^pos cells (arrowhead; F). Bar, 9 μm.

Figure 2.

Expression of skeletal muscle, cardiac muscle, and neuronal markers in G8^pos epiblast cells tracked into the heart and brain. Cells that expressed MyoD mRNA in the stage 2 epiblast were labeled with the G8 mAb (green) and tracked into the somite, heart, and brain of the 3-d embryo. Sections were stained for MyoD mRNA (MyoD M), MyoD protein (MyoD P), cardiac troponin T (CTpn), or neurofilament-associated antigen (NF; red). Nuclei were labeled with Hoechst dye (blue). Merged images with overlapping red and green fluorescence appear white. G8^pos cells that stained with the MyoD mAb were concentrated in the somites (arrows in A). G8^pos cells of the heart and brain contained MyoD mRNA (arrows in B and F), and some were labeled with the MyoD mAb (arrows in D and G). G8^pos cells that were not labeled with the MyoD mAb are shown in C and G (arrowheads). Other G8^pos cells in these organs did stain with the MyoD mAb (arrows in D and G). The brain contained G8^pos/sarcomeric myosin^pos (arrow) and G8^pos/sarcomeric myosin^neg cells (arrowhead; H). Cardiac troponin T or neurofilament-associated antigen was not detected in G8^pos cells in the heart and brain, respectively (arrowheads in E and I). MyoD mRNA was expressed in some G8^neg cells in the brain (arrowhead in J). Bar, 9 μm.

Figure 3.

Expression of skeletal and cardiac muscle markers in G8^pos and G8^neg epiblast cells microinjected into the developing heart. G8^pos (G8+) and G8^neg (G8−) epiblast cells were labeled with Hoechst dye (blue) and microinjected into the precardiac mesoderm of the stage 4 embryo. (A) Microinjection sites (arrows) above and lateral to the primitive streak (PS) are shown in the photomicrograph of the area outlined in the inset. (B) Embryos developed normally after microinjection. (C) An enlargement of the heart containing G8^pos cells. (D–O) Sections were stained for markers of skeletal and cardiac muscle (red in E, G, I, K, M, and O). Cardiac troponin T was not detected in G8^pos cells (arrowheads in D and E). G8^pos cells expressed MyoD mRNA (arrows in F and G). Subpopulations of G8^pos/MyoD mAb^pos (arrows) and G8^pos/MyoD mAb^neg (arrowheads) were present in the heart (H and I). The 12101 marker of skeletal muscle differentiation was detected in some G8^pos cells (arrows in J and K) but not in others (arrowheads in J and K). G8^neg cells microinjected into the developing heart did not express MyoD mRNA (arrowheads in L and M). Instead, they differentiated into cardiac muscle (arrows in N and O). Bars (A and B), 56 μm; (C) 27 μm; (D–O) 9 μm.

Figure 4.

Expression of skeletal muscle and neuronal markers in G8^pos and G8^neg epiblast cells microinjected into the developing nervous system. G8^pos (G8+) and G8^neg (G8−) epiblast cells were labeled with Hoechst dye (blue) and microinjected into the neural plate of the stage 7 embryo. Microinjected cells are shown in the photomicrograph of the area outlined in the inset of A. Embryos were grown for 2.5 d. Areas outlined in B are shown at higher magnification in C and D. The brain (C) and neural tube (NT; D) contained microinjected cells. Sections through the brain were stained with markers for skeletal muscle and neurons (red in F, H, J, L, N, and P). Neurofilament-associated antigen (NF) was not detected in G8^pos cells (arrowheads in E and F). MyoD mRNA (MyoD M) was present in G8^pos cells in the brain (arrows in G and H). Some G8^pos cells stained with the MyoD mAb (MyoD P; arrows in I and J). A host cell lacking the G8 marker was positive for MyoD mRNA (arrowheads in G and H). A G8^pos cell expressing sarcomeric myosin is shown in K and L. G8^neg cells expressed neurofilament-associated antigen (arrows in M and N) but not MyoD mRNA (arrowheads in O and P). Bars (A and B), 56 μm; (C and D) 27 μm; (E–P) 9 μm.

Figure 5.

Differentiation of microinjected G8^pos epiblast cells in vitro. G8^pos cells were isolated from the stage 1 epiblast, labeled with Hoechst dye (blue), and microinjected into the stage 4 precardiac mesoderm and stage 7 neural plate. 2 d later, the hearts (A), heads (B), and trunks (C) were dissociated, and the cells were cultured for 5 d and stained with the 12101 mAb. Hoechst-labeled cells differentiated into skeletal muscle, whereas surrounding host cells were not stained with the 12101 mAb. Bar, 9 μm.