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First published online September 16, 2005
Journal of Experimental Biology 208, 3675-3687 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01826

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Effects of decreased muscle activity on developing axial musculature in nic^b107 mutant zebrafish (Danio rerio)

T. van der Meulen^*, H. Schipper, J. L. van Leeuwen and S. Kranenbarg

Experimental Zoology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, NL-6709 PG Wageningen, The Netherlands

View larger version (26K): [in a new window]   Fig. 1. (A) Schematic presentation of the XzYzZz and XfYfZf coordinate systems. The XzYzZz coordinate system in blue is associated with the optical sections taken by the CLSM. The XfYfZf coordinate system in red is defined relative to the fish body. (B) Dorsal view of the postanal part of a fish embryo, anterior to the top. The body axis is indicated by a black line. Muscle fibres are indicated by black arrows and run in a particular direction in the left half of the body. The direction of muscle fibres in the right half of the body is the mirror image of that in the left half. When the angle of sectioning is perpendicular to the body axis, the observer (red eye) looks at right angles to the sections that are perpendicular to the body axis (red lines). When the sections are not made at an angle of 90° to the body axis, the observer (blue eye) still looks at right angles to the sections (blue lines), but these present the fibres as if they run asymmetrically (C). (D) Rotating the section mathematically in such a way that the view of the red observer is obtained will yield the correct symmetrical muscle fibre field shown in E. Dotted lines indicate parts of muscle fibres and body axis that are not covered by the sections.

View larger version (127K): [in a new window]   Fig. 2. (A) Wild-type and (B) nicb107 embryos aged 120 hpf. The mutants display different degrees of body curvature. About half the mutant embryos in a clutch are curved. Scale bar, 200 µm.

View larger version (17K): [in a new window]   Fig. 3. Morphometric data on developing wild-type and nicb107 zebrafish during the first five days of development. (A) Illustration of parameters that were measured. Abbreviations: b, total length (the broken box indicates location of measurements at anus level); c, muscle height; d, notochord height; e, somite size; f, somite angle (). (B) Total length of the embryos. (C) Muscle height at anal somite. (D) Notochord height at anal somite level. (E) Somite size at anal somite level. (F) Somite angle at anal somite level. Black lines indicate wild-type data; grey lines indicate nicb107 data. *Wild-type and nicb107 are significantly different (P<0.01).

View larger version (93K): [in a new window]   Fig. 4. Sections at anus level of wild-type larvae aged (A) 48 hpf, (B) 72 hpf and (C) 96 hpf, and nicb107 larvae aged (D) 48 hpf, (E) 72 hpf and (F) 96 hpf stained for propidium iodide with muscle fibre trajectories superimposed. Trajectories are visualised by a white circle for the anterior starting position and a line segment that represents the direction and projected distance travelled over the next 5 µm. Azimuth and elevation can be inferred from the components in the X and Y direction of the line segment (see main text). All sections are viewed from the anterior side, and the projections are of fibres running from anterior to posterior (below the surface of the figure) over a distance of 5 µm. This implies that the left side of the embryos is to the right and vice versa in this view. Neural tube (nt) and notochord (nc) are indicated. Scale bars, 20 µm.

View larger version (60K): [in a new window]   Fig. 5. F59 antibody staining showing slow muscle fibres in green on cross sections of 100 hpf wild-type (A) and nicb107 embryos (B). Abbreviations: fm, fast muscle fibres; nc, notochord; nt, neural tube. Arrows indicate slow muscle fibres. Scale bars, 20 µm.

View larger version (197K): [in a new window]   Fig. 6. Electron microscope images of wt and nicb107 white muscle mass. (A) 72 hpf wild-type; (B) 96 hpf wild-type; (C) 72 hpf nicb107; (D) 96 hpf nicb107. Abbreviations: n, nucleus; s, sarcoplasmic reticulum; sm, sarcomere. Arrowheads indicate Z-line in adjacent myofibrils; note irregular Z-line stacking in nicb107 embryos when compared with wild-type embryos. Scale bars, 1 µm.

View larger version (32K): [in a new window]   Fig. 7. Expression of genes in total nicb107 embryos as fold increase versus gene expression in total wild-type embryos during the first four days of development. Expression of (A) col12 (collagen type 1 2) and (B) col21 (collagen type 2 1) is indicated by black bars. Expression of sarcomere components (C) mylz2 (myosin light chain 2); (D) myhz2 (fast muscle specific myosin heavy chain 2); (E) myhz5 (slow muscle specific myosin heavy chain 5); (F) mylz3 (fast muscle specific myosin light chain 3); (G) titin; (H) tropC fast (fast muscle specific troponin C) and (I) tropC slow (slow muscle specific troponin C) is indicated by grey bars. Expression of growth factors (J) IGF-1 Ea2 (insulin-like growth factor splice form Ea2); (K) IGF-Ra (insulin-like growth factor receptor a) and (L) IGF-Rb (insulin-like growth factor receptor b); (M) gdf8 (growth and differentiation factor 8/myostatin) and (N) myogenin is indicated by vertically shaded bars. Expression of (O) oxygen carrier myoglobin, (P) muscle-specific glycolysis enzyme pfk-m (phosphofructokinase in muscle); and mitochondria specific oxidative phosphorylation genes (Q) SDHa (succinate dehydrogenase a) and (R) NADHd (diaphorase) are indicated by open bars. All scales are linear, except in M, which is logarithmic. Values are means of five embryos + S.D. A dotted line indicates where the fold increase is one and no difference exists between expression in nicb107 and wild-type embryos. Asterisks indicate significant difference between nicb107 and wild-type expression (P<0.05).

View larger version (28K): [in a new window]   Fig. 8. Effects of increased (IMA) and decreased muscle activity (DMA) on muscle growth and differentiation. Two main fibre types exist: fast and slow fibres. Each type expresses a set of specific genes (e.g. fast or slow myosins and troponins), with some overlap (e.g. titin). IMA promotes muscle growth and promotes a fast-to-slow transition of fibre types, whereas DMA inhibits muscle growth and promotes a slow-to-fast transition. Muscle growth is promoted by IMA, partly by increased insulin-like growth factor (IGF) and myogenin signalling, and is inhibited by DMA, partly by growth and differentiation factor 8 (gdf8) signalling. Gdf8 signalling acts a.o. by downregulation of myogenin and IGF expression. Overcrowding stress inhibits gdf8 expression in zebrafish, even though it also represses muscle growth. Open arrows indicate the fast-to-slow and slow-to-fast fibre type transitions. Filled arrows indicate a positive influence, and T-shaped arrows indicate a negative influence on the process or tissue it points at. The asterisk denotes that high relative myogenin expression promotes a shift towards a slow muscle phenotype. SDHa, succinate dehydrogenase a; NADHd, diaphorase; pfk-m, phosphofructokinase in muscle.