Heart myofibrillogenesis occurs in isolated chick posterior blastoderm: a culture model

Abstract

Early cardiogenesis including myofibrillogenesis is a critical event during development. -Recently we showed that prospective cardiomyocytes reside in the posterior lateral blastoderm in the chick embryo. Here we cultured the posterior region of the chick blastoderm in serum-free medium and observed the process of myofibrillogenesis by immunohistochemistry. After 48 hours, explants expressed sarcomeric proteins (sarcomeric alpha-actinin, 61%; smooth muscle alpha-actin, 95%; Z-line titin, 56%; sarcomeric myosin, 48%); however, they did not yet show a mature striation. After 72 hours, more than 92% of explants expressed I-Z-I proteins, which were incorporated into the striation in 75% of explants or more (sarcomeric alpha-actinin, 75%; smooth muscle alpha-actin, 81%; Z-line titin, 83%). Sarcomeric myosin was -expressed in 63% of explants and incorporated into A-bands in 37%. The percentage incidence of expression or striation of I-Z-I proteins was significantly higher than that of sarcomeric myosin. Results suggested that the nascent I-Z-I components appeared to be generated independently of A-bands in the cultured posterior blastoderm, and that the process of myofibrillogenesis observed in our culture model faithfully reflected that in vivo. Our blastoderm culture model appeared to be useful to investigate the mechanisms regulating the early cardiogenesis.

Fig. 1

Explant from prestreak chick blastoderm. EK-stage X–XI blastoderms were collected. Posterior regions (hatched square) containing epiblast and hypoblast were isolated and cultured in serum-free medium. Note that the EK-stage indicates the embryonic stages before gastrulation by Eyal-Giladi and Kochav (1976) [10]. A, anterior; P, posterior.

Fig. 2

Cultured posterior blastoderm expresses the heart-specific transcription factor Nkx-2.5. After 48 hr in culture, explants were fixed and stained with antibodies against heart specific transcription factor (Nkx-2.5) and early heart sarcomeric protein (sarcomeric α-actinin). Cells that were expressing sarcomeric α-actinin (A) expressed Nkx-2.5 (B) in their nuclei (C, D). Bar=20 µm.

Fig. 3

EK-stage X–XI posterior blastoderm generates premature myofibrils after 48 hr in culture. Tiny bead-like deposits of sarcomeric α-actinin (arrowheads in A1) appeared along filamentous smooth muscle α-actin (arrowheads in A2–3). Z-line titin was also seen as tiny bead-like deposits and co-localized with the sarcomeric α-actinin (arrowheads in B1–3). Sarcomeric myosin exhibited a diffuse cytoplasmic staining (C1) and was distributed independently of the bead-like deposits of sarcomeric α-actinin (arrowheads in C2 and C3). Bar=20 µm.

Fig. 4

EK-stage X–XI posterior blastoderm generates mature myofibrils after 72 hr in culture. Sarcomeric α-actinin (A1) and smooth muscle α-actin (A2) were incorporated into mature I-Z-I structures (arrowheads in A1–3). Z-line titin was co-localized with striated sarcomeric α-actinin (arrowheads in B1–3). Sarcomeric myosin was also incorporated into nascent sarcomeres (arrowheads in C1) and associated with sarcomeric α-actinin (arrowheads in C2–3). Bar=20 µm.

Fig. 5

Percentage incidence of the expression of sarcomeric proteins and striation in cultured posterior blastoderm shown in Figures 3 and 4. After 48 hr (h) in culture, explants expressed sarcomeric proteins (sarcomeric α-actinin, 61%; smooth muscle α-actin (SMA), 95%; Z-line titin, 56%; sarcomeric myosin, 48%); however, they did not yet show an apparent striation (hatched column). After 72 hr, explants expressed sarcomeric proteins (sarcomeric α-actinin, 92%; SMA, 100%; Z-line titin, 94%; sarcomeric myosin, 63%). I-Z-I proteins were incorporated into the striation in 75% of explants or more (sarcomeric α-actinin, 75%; SMA, 81%; Z-line titin, 83%), and sarcomeric myosin in 37% of explants. There was a significant difference in the percentage incidence of expression or striation among four sarcomeric proteins. The results of each individual pairwise comparison were indicated. Note that there was no significant difference in the incidence of expression or striation of I-Z-I proteins and that the incidence of expression or striation of I-Z-I proteins was significantly higher than that of sarcomeric myosin. Asterisk (*) indicates that there is 5% significant difference (P<0.05/6, using Bonferroni’s correction). NS, no significant difference; n, number of explants tested; SMA, smooth muscle α-actin.