The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

Abstract

The mechanisms involved in the development of skeletal muscle fibers have been studied in the last 70 years and yet many aspects of this process are still not completely understood. A myriad of in vivo and in vitro invertebrate and vertebrate animal models has been used for dissecting the molecular and cellular events involved in muscle formation. Among the most used animal models for the study of myogenesis are the rodents rat and mouse, the fruit fly Drosophila, and the birds chicken and quail. Here, we describe the robustness and advantages of the chick primary muscle culture model for the study of skeletal myogenesis. In the myoblast culture obtained from embryonic chick pectoralis muscle it is possible to analyze all the steps involved in skeletal myogenesis, such as myoblast proliferation, withdrawal from cell cycle, cell elongation and migration, myoblast alignment and fusion, the assembly of striated myofibrils, and the formation of multinucleated myotubes. The fact that in vitro chick myotubes can harbor hundreds of nuclei, whereas myotubes from cell lines have only a dozen nuclei demonstrates the high level of differentiation of the autonomous chick myogenic program. This striking differentiation is independent of serum withdrawal, which points to the power of the model. We also review the major pro-myogenic and anti-myogenic molecules and signaling pathways involved in chick myogenesis, in addition to providing a detailed protocol for the preparation of embryonic chick myogenic cultures. Moreover, we performed a bibliometric analysis of the articles that used this model to evaluate which were the main explored topics of interest and their contributors. We expect that by describing the major findings, and their advantages, of the studies using the embryonic chick myogenic model we will foster new studies on the molecular and cellular process involved in muscle proliferation and differentiation that are more similar to the actual in vivo condition than the muscle cell lines.

Keywords: chick embryo; muscle differentiation; myoblast; myogenesis; myotube; skeletal muscle.

FIGURE 1

Schematic representation of in vitro chick embryonic skeletal myogenesis. (A) Specific cell phenotypes can be characterized during skeletal muscle development (for more details, see Table 1). Presumptive myoblasts (or pre-myoblasts, shown in beige) are mononucleated (nuclei in blue), round shaped and proliferative cells that do not express myofibrillar proteins. When these cells withdraw cell cycle, they begin to express desmin (green in the cytoplasm) and they change to a bipolar shape. These bipolar myoblasts begin to express myofibrillar proteins (red) and they will align with other myoblasts. Cell adhesion and fusion happens between steps 3 and 5. Muscle differentiation culminates with the formation of long and multinucleated myotubes filled (steps 4 and 5) with contractile myofibrils (sarcomeres in red). Pre-myoblasts (beige cells), post-mitotic myoblasts (round green cells), bipolar myoblasts (elongated green cells), and fibroblasts (triangular-shaped and carmine-colored cells) are present in all steps of chick myogenic cultures (steps 1–5). (B) The intracellular distribution of the structural and signaling proteins flotillin-2 (light blue), beta-catenin (yellow), Lmo7 (brown), and myofibrillar proteins (red) are shown for each specific cell phenotype (myoblasts, fibroblasts and myotubes) found in chick myogenic cell cultures.

FIGURE 2

Cell shape changes during in vitro chick myogenesis. Primary cultures of 11-day old chick myoblasts were grown for 24, 48, and 72 h and images were acquired under phase contrast microscopy. Bipolar myoblasts aligned in a pre-fusion chain-like structure (white arrow) are seen in a 24-h culture (A), while thin myotubes are already present in a 48-h culture (B) and multinucleated myotubes are present in a 72-h culture (C). Note the presence of fibroblasts in all the images (A–C, yellow arrows). Scale bar = 10 μm.

FIGURE 3

Myoblast adhesion and myotube formation are hallmarks of skeletal myogenesis. (A) Cadherin and beta-catenin accumulate at cell-cell adhesion contacts in pre-fusion chick myoblasts. In chick myogenic cultures 24 h after cell plating, primary fusion (myoblast-myoblast fusion) can be observed as an intense immunolabeling of beta-catenin (green, white arrow in A) in continuous lines at cell-cell contacts. Note that beta-catenin is also present in a dot-like pattern in the whole sarcolemma (green, yellow arrow in A). Scale bar in (A) = 10 μm. (B) Myotubes from chick primary skeletal muscle cultures are multinucleated cells that contain highly organized myofibrils. Chick skeletal muscle cells were grown in culture for 72 h and stained with antibodies anti-sarcomeric alpha-actinin (green), anti-desmin (red, yellow arrow in B) and with the nuclear dye DAPI (blue). Note in the merged image the periodical labeling of alpha-actinin in the Z-bands of myofibrils (white arrow in B), the presence of desmin filaments at the periphery of the myotube (yellow arrow in B) and well-aligned nuclei. Scale bar in B = 10 μm. (C,D) Myoblasts adhere to myotubes in a secondary fusion process. Chick skeletal muscle cells were grown in culture for 72 h and stained with antibodies anti-alpha-tubulin (green, C) and with the nuclear dye DAPI (blue, D). Small mononucleated myoblasts (white arrow in C) can be seen at the top of a multinucleated myotube, which is evidenced by the microtubule staining. DAPI shows the presence of a high number of nuclei in each chick myotube cell (blue, D). Image (A) shows a 24-h primary myogenic culture, where most of the myoblasts are undergoing primary myoblast fusion (myoblast-myoblast fusion), while image (C) shows a 72-h primary myogenic culture, where secondary myoblast fusion (myoblast-myotube fusion) is frequently observed. Scale bar in (D) = 20 μm.

FIGURE 4

Protocol for primary culture of embryonic chick myogenic cells. Schematic representation of the main steps involved in the culture of embryonic chick pectoral breast muscle cells.

FIGURE 5

Number of papers on primary culture of embryonic chick myogenic cells per year. Distribution of publications retrieved from PubMed. The retrieved dataset only included papers until the year of 2020.

FIGURE 6

Journals that published most papers on primary culture of embryonic chick myogenic cells. Number of papers by journal, as indicated. The retrieved PubMed dataset only included papers until the year of 2020.

FIGURE 7

Number of apers by last author. (A) Number of papers by journal, as indicated. (B) Pie chart depicting the relative proportion of journals grouped by the number of published papers on the subject. The retrieved PubMed dataset only included papers until the year of 2020.

FIGURE 8

Countries and institutions that harbored most papers on primary culture of embryonic chick myogenic cells. (A) Number of papers by country of the leading institution, as indicated. (B) Number of papers by leading institution. We defined the leading institution as the first affiliation depicted in the retrieved Dimensions dataset, which included publications until the year of 2020.

FIGURE 9

Vocabulary analysis of the articles using chick muscle cultures. (A) A word cloud was generated using the titles of articles on primary culture of embryonic chick myogenic cells. This method gives greater prominence to frequent words. (B) Word frequency list. The retrieved PubMed dataset only included papers until the year of 2020.