In vitro indeterminate teleost myogenesis appears to be dependent on Pax3

Abstract

The zebrafish (Danio rerio) has been used extensively as a model system for developmental studies but, unlike most teleost fish, it grows in a determinate-like manner. A close relative, the giant danio (Devario cf. aequipinnatus), grows indeterminately, displaying both hyperplasia and hypertrophy of skeletal myofibers as an adult. To better understand adult muscle hyperplasia, a postlarval/postnatal process that closely resembles secondary myogenesis during development, we characterized the expression of Pax3/7, c-Met, syndecan-4, Myf5, MyoD1, myogenin, and myostatin during in vitro myogenesis, a technique that allows for the complete progression of myogenic precursor cells to myotubes. Pax7 appears to be expressed only in newly activated MPCs while Pax3 is expressed through most of the myogenic program, as are c-Met and syndecan-4. MyoD1 appears important in all stages of myogenesis, while Myf5 is likely expressed at low to background levels, and myogenin expression is enriched in myotubes. Myostatin, like MyoD1, appears to be ubiquitous at all stages. This is the first comprehensive report of key myogenic factor expression patterns in an indeterminate teleost, one that strongly suggests that Pax3 and/or Myf5 may be involved in the regulation of this paradigm. Further, it validates this species as a model organism for studying adult myogenesis in vitro, especially mechanisms underlying nascent myofiber recruitment.

Figure 1

Immunocytochemistry of Pax7 and MyoD1 expression in myogenic precursor cells cultured for 2.5 h (a, b; e, f) and 5 h (c, d; g, h) post-isolation. Anti-Pax7 detected by Texas Red fluorophore and anti-MyoD1 detected by FITC fluorophore. Total nuclei detected by DAPI. a, b; c, d; e, f; and g, h represent identical fields. White arrowheads indicate the same cells between pairs of identical fields. Images are representative of duplicated staining across triplicate experiments. Scale=100 μm. Quantification of Pax7 and MyoD1 expression (i), Pax7: One-way ANOVA, P<0.001 (different from days 1–9); MyoD1: one-way ANOVA, P=0.4643 (not different from days 1–9), n=18.

Figure 2

Immunocytochemistry of Pax3 and Pax7 expression in myogenic precursor cells, early-stage myoblasts, late-stage myoblasts, and differentiating myotubes at days 1–2 (a, b; i, j), days 3–4 (c, d; k, l), days 6–7 (e, f; m, n), and days 9–10 (g, h; o, p) of culture, respectively. Anti-Pax3 and anti-Pax7 detected by Texas Red fluorophore. Total nuclei detected by DAPI. a, b; c, d; e, f; (g, h; i, j; k, l; m; n; and o, p represent identical fields. White arrowheads indicate the same cells between pairs of identical fields. Images are representative of duplicated staining across triplicate experiments. Scale=100 μm. Quantification of Pax3 and Pax7 across culture period (q). Pax3: one-way ANOVA, P=0.0024, n=12; Pax7: one-way ANOVA, P<0.0001, n=12. Different letters indicate statistical differences at P<0.05 by Bonferroni’s post hoc test.

Figure 3

Immunocytochemistry of c-Met and syndecan-4 expression in myogenic precursor cells, early-stage myoblasts, late-stage myoblasts, and differentiating myotubes at days 1–2 (a, b; i, j), days 3–4 (c, d; k, l), days 6–7 (e, f; m, n), and days 9–10 (g, h; o, p) of culture, respectively. Anti-c-Met and anti-syndecan-4 detected by fluorescein isothiocyanate (FITC) fluorophore. Total nuclei detected by DAPI. a, b; c, d; e, f; g, h; i, j; k, l; m; n; and o, p represent identical fields. White arrowheads indicate the same cells between pairs of identical fields. Images are representative of duplicated staining across triplicate experiments. Scale=100 μm. Quantification of c-Met and syndecan-4 across culture period (q). c-Met: one-way ANOVA, P=0.2523, n=12; syndecan-4: one-way ANOVA, P<0.0001, n=12. Different letters indicate statistical differences at P<0.05 by Bonferroni’s post hoc test.

Figure 4

Immunocytochemistry of Myf5, MyoD1, and myogenin expression in myogenic precursor cells, early-stage myoblasts, late-stage myoblasts, and differentiating myotubes at days 1–2 (a, b; i, j; q, r), days 3–4 (c, d; k, l; s, t), days 6–7 (e, f; m, n; u, v), and days 9–10 (g, h; o, p; w, x) of culture, respectively. Anti-Myf5, anti-MyoD1, and anti-myogenin detected by fluorescein isothiocyanate (FITC) fluorophore. Total nuclei detected by DAPI. a, b; c, d; e, f; g, h; i, j; k, l; m; n; o, p; q, r; s, t; u, v; and w, x represent identical fields. White arrowheads indicate the same cells between pairs of identical fields. Images are representative of duplicated staining across triplicate experiments. Scale=100 μm. Quantification of Myf5, MyoD1, and myogenin expression across culture period (y). Myf5: one-way ANOVA, P=0.0205, n=12; MyoD1: one-way ANOVA, P=0.4643, n=12; myogenin: one-way ANOVA, P=0.3392. Different letters indicate statistical differences at P<0.05 by Bonferroni’s post hoc test.

Figure 5

Immunocytochemistry of myostatin expression in myogenic precursor cells (a, b) and early-stage myoblasts at days 3–4 (c, d), respectively; and late-stage myoblasts and differentiating myotubes at day 6 (e, f) and day 9 (g, h) of culture, respectively. Anti-myostatin detected by fluorescein isothiocyanate (FITC) fluorophore. Total nuclei detected by DAPI. a, b; c, d; e, f; and g, h represent identical fields. White arrowheads indicate the same cells between pairs of identical fields. Images are representative of duplicated staining across triplicate experiments. Scale=100 μm. Quantification of myostatin expression (i), myostatin: one-way ANOVA, P=0.0589, n=12.

Figure 6

a, Total giant danio MPCs and myoblasts detected in vitro by 4′,6-diamidino-2-phenylindole nuclear staining over the culture period (black) and proliferation of giant danio myogenic precursor cells and myoblasts as determined by the percentage of 5-bromodeoxyuridine (BrdU)-positive nuclei (red), *P<0.0001 by one-way ANOVA; and b, percent-change in total cells present (white) or BrdU⁺ cells (black) between X and Y days (i.e., days X–Y).

Figure 7

Schematic of relative cell proliferation (red) and expression levels of paired-box factors, myogenic precursor cell-specific markers (red), myogenic regulatory factors, and myostatin (green) in giant danio myogenic precursor cells, myoblasts, and differentiating myotubes.