Cartilage and bone formation in the zebrafish vertebral column. (A,C,E) Alcian green staining for cartilage. (B,D,F) Alizarin red staining for bone. (A) At 9 dpf cartilage is only found in the Weberian apparatus (arrows), a specialisation of the four most anterior vertebrae. The Weberian apparatus is described as arising from V1-5 by Morin-Kensicki et al. (Morin-Kensicki et al., 2002), although V1 and V2 give the appearance of a single vertebra in our experiments (see also Coburn and Futey, 1996). Staining is not seen in the centra (asterisks). The weak staining in the bulk of the centra is non-specific, acellular background staining, indicating an absence of chondrocytes and therefore cartilage in these regions. (B) Despite the absence of cartilage, bone is seen in cervical centra at 9 dpf. (C) Cartilage is only seen in the rib heads (arrows) and not in the centra, neural (na) or haemal (ha) arches, or distal ribs (arrowheads) at 17 dpf. (D) Ossified centra are present along the trunk by 17 dpf. (E,F) By 23 dpf, neural (arrows) and haemal (arrowheads) arches have also formed in the tail. Centra and arches ossify without forming cartilage. Scale bars: A,B,~ 50 µm; C-F, ~100 µm.

Ossification in the zebrafish vertebral column. (A,B) Simultaneous labelling at 20 dpf with alizarin red (red) for bone, zns5 (green) for osteoblasts, and DAPI (blue) for nuclei. (A) Confocal microscopic image of a sagittal section through the intramembranous parasphenoid bone (arrowhead) separating the jaw cavity (jc) from the brain (br). Osteoblasts in bone matrix are indicated by co-localization of alizarin red and zns5 (yellow). (B) Representative confocal microscopic image of a sagittal section through a thoracic centrum showing bone (red) but not zns5 labelling, indicating the absence of osteoblasts (all centra were examined in 10 serially sectioned embryos). Arrowheads indicate centrum boundaries. da, dorsal aorta; sc, spinal cord. (C,D) Osteoblasts assessed by alkaline phosphatase activity (green) using wide-field fluorescent microscopy. DAPI (blue) labels cell nuclei. (C) Representative transverse section through the epiphyseal bar of the skull (arrowheads) at 20 dpf showing large numbers of osteoblasts. (D) Representative transverse section (20 µm) through a mid-trunk centrum at 20 dpf, with no osteoblast labelling in the notochord (n) and surrounding centrum (arrowheads); inset shows a view at ~0.1× (all centra were examined in 10 serially sectioned embryos). (E-H) Analysis of bone by transmission electron microscopy (20 dpf). (E) Intramembranous ossification in the parasphenoid bone; osteoblast nuclei are clearly seen within bone matrix (arrowheads). (F) A centrum in transverse section showing acellular ossification. Bone matrix (arrowheads) lies adjacent to muscle (asterisk), encircling the notochord (n). In all samples (15 sections, 5 embryos) no osteoblasts were found. A notochord cell nucleus (arrow) lies adjacent to the matrix on the inner surface of the centrum. (G,H) High magnification images of E and F, respectively. Cell nuclei (arrows) are conspicuous within the intramembranous bone (G) but not centrum matrix (H). The latter is electron dense and lamellated (arrowheads). (I) Centrum in sagittal section showing bone matrix (arrowheads) lacking osteoblasts; the gap between bone and muscle (asterisk) is due to shearing during processing; arrow marks a nucleus from the notochord (n). Scale bars: A,B, ~10 µm; C,D, ~5 µm; E,F,~ 2 µm; G,H, ~1 µm; I, ~4 µm.

The notochord deposits bone matrix. (A,B) Notochords were dissected before centrum formation (4 dpf), cultured, and then labelled with quercetin for bone matrix (n=10). (A) After 12 days′ culture, matrix is seen as bright transverse bands. (B) Bright field image of A using DIC optics; bone labelling is seen between cell boundaries. (C) Phase-contrast image of a notochord dissected and fixed at 4 dpf. (D) Composite phase-contrast and DAPI image of C showing nuclei (blue) only within the boundaries of the notochord sheath (adjacent to phase-bright lines); no cells are seen outside the sheath (n=8). (E) Notochord in C labelled with quercetin, showing no positive staining for bone matrix; the notochord is delineated by background fluorescence, and faint nuclear (DAPI) fluorescence is observed in the FITC channel. Scale bars: A,B, ~100 µm; C-E, ~80 µm.

Ablation of notochord cells prevents formation of centra. (A) Unablated embryo (12 dpf) stained with quercetin. Centra appear as stripes along the anterior-posterior axis. (B) DIC image of embryo in A; the anterior boundary of each centrum is level with each myoseptal border (arrows mark the myoseptal border cleft, anterior to left; extrapolation of the arrow vectors in A and B shows the alignment of the V-shaped myoseptal borders with the anterior edges of the developing centra). (C,D) Bright field images of a 4-dpf embryo, showing appearance of notochord cells prior to targeting with the laser. Cross hairs were aligned on the myoseptal borders at the focal plane of muscle segments (C), and the focus was then adjusted to the plane of notochord cells (D). Asterisks denote single notochord cells spanning the diameter of the notochord; these have a typically cylindrical appearance, and would be selected for ablation. The position of two myoseptal borders at the dorsoventral level of the notochord is indicated by dark lines. (E-H) Ablation of notochord cells before centrum formation (at 4 dpf) preventing subsequent development of centra. Cells were targeted at several positions along the anterior-posterior axis, each level with the myoseptal border (16 embryos). (E) DIC image of an embryo after notochord ablation showing the ablation site at 10 dpf (arrows); adjacent notochord cells have altered shape to fill the site (arrowheads). (F) Embryo in E at a focal plane above the notochord showing no damage in adjacent muscle (m); arrow indicates ablation site. (G) Targeting of three notochord cells from consecutive segments prevents development of three centra (arrows); quercetin labelling at 12 dpf. (H) Targeting of notochord cells from alternate segments prevents development of alternate centra (arrows); quercetin labelling at 12 dpf. (I,J) Ablation of notochord cells at 4 dpf in the centre of prospective centra does not affect their development (8 embryos). (I) Quercetin labelling at 20 dpf after ablation at 4 dpf. (J) DIC image of embryo in I, arrows indicate ablation sites. Scale bars: A,B, ~100 µm; C,D, ~50 µm; E,F, ~100µ m; G,H, ~100 µm; I,J, ~100 µm.

Vertebral segmentation in fused somite (fss) mutant fish. (A-D) Alizarin red staining of 30 dpf larvae. (A) In wild-type larvae, neural (arrows) and haemal (arrowheads) arches extend from each centrum in a regular manner. (B) As reported previously (van Eeden et al., 1996), defects in the position and projection of the neural and haemal arches are conspicuous in fss embryos. (C,D) Higher magnification of A and B, respectively. In wild-type (C) and fss (D) embryos, centra (asterisk) are of uniform size and no fusions or abnormalities are seen in fss centra. (E,F) Pax9 expression in sclerotome of wild-type and mutant embryos at 24 hours post-fertilization (hpf). (E) Wild-type expression in discrete cell clusters, one cluster per somite (arrowheads). (F) In fss mutants, expression is not segmented. (G,H) Smad1 expression in sclerotome of wild-type and fss embryos at 36 hpf. (G) Similar to Pax9, Smad1 expression is seen in one cell cluster per somite (arrowheads). (H) In fss mutants the segmented pattern of Smad1 expression is disrupted. (I,J) Ablation of notochord cells (arrow) in 4-dpf fss embryos prevents development of centra (8 embryos). (I) DIC image of 12-dpf embryo indicating wound site and local cell reorganisation resulting from ablation (arrow). (J) Bone staining of embryo in I showing absence of centrum (arrow indicates the probable anterior centrum boundary) after ablation. Scale bars: A,B, ~500 µm; C,D, ~100µ m; E,F, ~20 µm; G,H, ~20 µm; I,J, ~100 µm.