Knockdown of pgam2 specifically affects developing fast muscle fibers in zebrafish embryos. (A–C2) Morpholino mediated pgam2 knockdown (pgam2 MO) leads to a decrease in muscle birefringence. (A–C) Bright-field images; A2–C2 are the same embryos as in A–C under polarized light. Intensity of birefringence is color-coded. misMO, mismatch control morpholino injected embryos. (D–I) A muscle phenotype characterized by disturbance of fiber arrangement and reduced fiber diameters is only observed in fast muscle (G–I) of zebrafish pgam2 morphants. (D–F) Slow muscle fibers stained with the F59 antibody (green). (G–I) Fast muscle stained with phalloidin (red). (Scale bars: 11.9 μm.) (J and K) Fast fibers stained with Unc45-b-GFP (green) to reveal position of nuclei (arrowheads). (L) The number of nuclei per fast muscle fiber is significantly reduced in pgam2 morphants (***P = 0.0001). (M and N) The diameter (micrometers) of fast fibers (N), but not of slow fibers (M), is significantly reduced in pgam2 morphants (**P = 0.0027). A–I, M, and N: 48 hours postfertilization (hpf) embryos; J–L: 72 hpf embryos.

Control morpholino experiments in zebrafish. (A) Percentage of larvae showing a reduction in birefringence (phenotype, blue) after injection of the indicated morpholinos (Fisher’s exact test ; ***P < 0.0001). Injection of control morpholinos carrying five mismatches (misMO) does not lead to any obvious phenotype. (B–D) Coinjection of pgam2 morpholino and a construct carrying a GFP reporter of pgam2 expression revealed loss of GFP expression (D) compared with embryos only injected with the reporter construct (B) or to embryos coinjected with the mismatch morpholino (C). (E–H) Immunostaining against Pax7 revealed an increase of Pax7-positive nuclei in pgam2 morphants (G) compared with uninjected (F) or control morpholino-injected embryos (G). (Scale bar: 47.5 μm.) (H) Quantification of Pax7-positive myoblasts. All nuclei were counted in an area of 500 × 500 pixels in 10 different 48 hours postfertilization (hpf) embryos for each category and summarized in a chart. (I) Rate of heart beat in 48 hpf embryos (counted during 10 s, n = 12 embryos for each category). (J–M) Immunostaining with phalloidin in (J) uninjected, (K) control morpholino-injected embryos, and (L) pgam2 morphants; 48 hpf embryos. (Scale bar: 12 μm.) (M) Number of fibers present within a square of 500 × 500 pixels (eight squares counted for each category).