Extracellular matrix is required for muscle differentiation in primary cell cultures of larval Mytilus trossulus (Mollusca: Bivalvia)

Abstract

Components of the extracellular matrix may modulate the growth factor effects that play important roles in the proliferation and differentiation of precursor cells. We developed an in vitro cultivation protocol for cells of the larval marine bivalve Mytilus trossulus to study the role that extracellular matrix components may play in myodifferentiation and replication-mediated DNA synthesis using immunofluorescence and confocal laser scanning microscopy. Here, we demonstrate that the extracellular matrix regulates the expression of muscle proteins, leading to their assembly and the terminal muscle differentiation of larval cells during cultivation. We further show that the myogenesis process progresses in cells cultivated on fibronectin, carbon or poly-L-lysine but is inhibited in cells grown on a collagen carpet. Consistent with a decrease in muscle protein expression in cells cultivated on collagen, we demonstrate an increase in the number of BrdU-positive cells in comparison with cells cultured on other substrates during the entire cultivation period. Moreover, we demonstrate that the matrix-dependent myogenic differentiation of larval mussel cells is reversible. Round-shaped cells cultivated on collagen were able to differentiate into muscle cells after reseeding on fibronectin, carbon or poly-L-lysine. In addition, cells cultured on collagen and then transplanted to fibronectin exhibited distinct cross-striation and contractile activity. Taken together, our data suggest that the extracellular matrix participates in the regulation of the proliferation and myodifferentiation of mussel trochophore progenitor cells and validate novel approaches for successfully culturing cells from bivalves over extended periods.

Fig. 1

Specificity of primary antibodies to mussel muscle proteins. Antibodies were raised against samples from highly purified fractions of individual muscle proteins [twitchin (530 kDa), myosin (200 kDa) and paramyosin (100 kDa)] isolated from the mussel Crenomytilus graynus and were subjected to SDS-PAGE (a). The antibodies were tested by western blot analysis (b) against the total muscle (anterior adductor) protein extracts from the mussel M. trossulus. Immunofluorescence staining using the different muscle antibodies labeled the striated myofibrils from the pecten Chlamys nipponensisi (c, e, g) and the smooth muscle fibers from the mussel M. trossulus (d, f, h) but not other tissues from adult molluscs (data not shown)

Fig. 2

Effect of substrates on larval mussel cell morphology after 2, 24 h, or 10 days of cultivation. Cultivation of trochophore-derived cells plated on fibronectin (a–c), poly-l-lysine (d–f), carbon (g–i), or collagen I (1 mg/ml) (j–l)

Fig. 3

Effect of substrates on larval mussel cell myodifferentiation after 2, 12, 24 h, or 10 days of cultivation. Immunocytochemistry of muscle proteins in larval cells cultivated on fibronectin (a–d), poly-l-lysine (e–h), carbon (i–l), or collagen I (1 mg/ml) (m–p). The cells were fixed in 4 % formaldehyde in 1× PBS and incubated with anti-myosin primary antibodies, AlexaFluor 488 (green) secondary antibodies, phalloidin tagged with AlexaFluor 546 (red) and DAPI (blue). (Color figure online)

Fig. 4

Restoration of the myogenic differentiation of larval mussel cells after cultivation on collagen (a). Larval cells cultivated on collagen were seeded on type I collagen (b, c), poly-l-lysine (d, e), carbon (f, g), or fibronectin (h, i). Analysis of the cellular morphology of the larval cells (b, d, f, h) and the detection of myofibrils by immunofluorescence was performed after 2 days in culture (c, e, g, i)

Fig. 5

Effect of substrates on DNA synthesis in larval mussel cells during cultivation (1, 10, or 20 days). Analysis of BrdU incorporation revealed a decreased proliferation of cells cultivated on fibronectin (a–c), poly-l-lysine (d–f), carbon (g–i), or collagen I (1 mg/ml) (j–l). By 20 days of cultivation, the larval cells plated on collagen were positive for BrdU (arrows)