Mutant ryr alleles do not produce protein products. (A) Schematic of a cross-section of the trunk of a 1-2 dpf zebrafish embryo highlighting muscle organization. SC spinal cord, noto notochord. (B-D) Transverse cryosections of 24 hpf embryo trunks illustrating the RNA expression patterns of (B) ryr1a, (C) ryr1b, and (D) ryr3 detected by WISH. (E-H″) Transverse sections through the trunks of 48 hpf wild-type and mutant embryos immunostained with the 34C (anti-RyR) antibody. Brightfield images (E-H), fluorescent images showing 34C staining (green) (E′-H′), and merged brightfield/fluorescent images (E″-H″) of embryos of indicated genotypes. Each ryr mutation is associated with loss of a distinct component of the normal expression pattern of RyR channels in embryonic muscle. Somitic muscle of triple ryr1a;ryr1b;ryr3 mutants lack all RyR protein detected by the 34C antibody.

Formation of Shh-dependent muscle in ryr mutants. Muscle cell type patterning in wild-type and mutant zebrafish 24 hpf embryos was assessed by immunohistochemical staining for expression of the Prox1 and Engrailed nuclear proteins. (A-D) Representative images of Prox1 (magenta) and Engrailed (green) staining of somitic muscle in wild-type (A), MZryr1a (B), MZryr3 (C) and MZryr1a;MZryr3 (D) embryos. Slow muscle pioneer cells (MPs) were identified as cells that expressed both Prox1 and Engrailed antigens; medial fast fibers (MFFs) were identified as cells that expressed only the Engrailed antigen; and superficial slow fibers (SSFs) were identified as cells that expressed only the Prox1 antigen. (E) Quantification of Shh-dependent muscle cell types. One-way ANOVA was used to determine statistical relationships with Sidak's multiple comparisons test used to adjust P-values. n.s., not significant; *P<0.01, **P<0.001.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Muscle fiber type-specific roles of RyR channels. (A) Schematic for generating zebrafish embryos that mosaically express GCaMP in muscle fibers. One-cell stage embryos were injected with a DNA expression plasmid driving constitutive expression from the B-actin promoter of GCaMP6-slow translationally linked, by a 2A linker peptide, with mCherry. Embryos with isolated muscle fibers that expressed the mCherry reporter were selected for analysis. (B) Experimental setup for SPIM imaging. The 2 dpf embryos, mounted in agarose in a capillary tube, were placed inside an embryo medium-filled chamber. Electrical stimulation to the head was used to evoke a single muscle contraction, and fluorescent GCaMP signal was recorded. (C) GCaMP6-slow signals were recorded from individual fast muscle fibers during a contraction. Loss of RyR1a or RyR3 channels did not alter Ca²⁺ release; however, loss of RyR1b eliminated all Ca²⁺ release in fast fibers. (D) GCaMP6-slow signals were recorded from individual slow muscle fibers during a contraction. Loss of RyR1b activity reduced Ca²⁺ release, whereas loss of RyR1a activity resulted in increased release of Ca²⁺ in slow fibers. Arrows indicate time at which electrical stimulus was delivered. Error bars indicate s.e.m.

RyR-mediated muscle contractions are required for slow fiber maturation. (A-I) Slow fibers were visualized with F59 antibody at 20 and 24 hpf and with the slow fiber-specific S58 antibody at 48 hpf. Slow fibers, which are initially wavy, mature and align with respect to each other as wild-type embryos develop. In ryr1b mutant embryos, maturation is delayed; in paralyzed triple mutants, fiber maturation is arrested. (J) Sample image indicating how slow muscle fiber (yellow) and A-P somite (magenta) lengths were determined using ImageJ. (K) Sample image of sarcomere banding indicating how sarcomere A-P lengths (white bracket) were determined. (L) Quantification of slow fiber:somite length ratios. (M) Quantification of sarcomere lengths of slow muscle fibers. One-way ANOVA was used to determine statistical relationships at each developmental time point with Tukey's multiple comparisons test used to adjust P-values. To determine fiber length or sarcomere length, five fibers were examined in each of five different embryos for each condition. ns, not significant.

Paralyzed ryr mutants have abnormal craniofacial morphology. Craniofacial features of 6 dpf larvae were visualized following staining with Alcian Blue, which marks cartilage. (A-D) Jaw structures of wild-type larvae: lateral view of intact larva (A), lateral view of stained intact larva (B), isolated upper jaw (C) and isolated lower jaw (D). (E-H) Jaw structures of paralyzed ryr1a;ryr1b;ryr3 larvae: lateral view of intact larva (E), lateral view of stained intact larva (F), isolated upper jaw (G) and isolated lower jaw (H). Jaw architecture of mutant larvae was dramatically shortened in the A-P dimension and wider than normal. Highlighted features include the shape of hypophyseal fenestre (asterisks), the angle between the midline and ceratohyal cartilages of the lower jaw (brackets) and orientation of Meckel's cartilage (arrows).

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.