Figure 1.
Two-dimensional information on muscle insertions.
(A) Dissected forelimb of a Xenopus tropicalis specimen. (B–C) Thin section of the humerus after embedment in resin; picture taken under differential interference contrast (DIC). (B) Overview and (C) Details of extrinsic fibres showing that 2D thin sections can only be partially informative about the histology of a muscle attachment: the exact 3D architecture of the extrinsic fibres (ef) remains elusive, in particular in relationship to the fibres of the attaching muscles (m). (D,E) Muscle attachment on the unossified proximal part of an immature humerus. (D) Muscle fibres (m) initially only associate (white arrows) with cells on the surface of this cartilaginous humerus (c) and do not penetrate into the interior of the element. Polarized light. (E) Confocal transmission image in higher resolution showing the fibrous connection between muscle fibres and the outermost cartilage layer (white arrow).
Figure 2.
Samples and muscle insertions.
(A) Illustrated phylogeny of gnathostomes showing the position and morphology of the studied taxa: Compagopiscis, Eusthenopteron and Desmognathus. (B) Schematic representations of the different types of muscle/tendon attachments. The fibrocartilaginous (FCE) and unmediated fibrous entheses (UMFE) present extrinsic fibres embedded in the bone cortex, while some periosteally mediated fibrous entheses (PMFE) may have no fibre embedded in the bone matrix.
Figure 3.
Bone histology of the humerus of the salamander Desmognathus.
(A) Longitudinal virtual thin section made from scan data (approximately 70 µm thick, voxel size = 0.678 µm) through muscle insertions (located in the white frame). The humerus is oriented with the left side close to the proximal epiphysis (EP) and the right side to the mid-shaft. Abbreviations: cb = cortical bone; eb = endosteal bone; pb = periosteal bone; mc = medullar cavity. (B) Detail of the framed region in Figure 3A, 3D reconstruction of the virtual thin section with osteocytes modelled in blue, extrinsic fibres and canaliculi in white, bone surfaces in gold. The left white arrow shows the unmediated fibrous enthesis (UMFE) and the right white arrow shows the periosteally mediated fibrous enthesis (PMFE). (C) Locations of the four cubes of bone-cell lacunae, on which measurements and statistical tests were performed. (D) Details of bone surface at the location of the four cubes, showing a more rugose surface above cube 1 than above the others. (E) Details of the four cubes after treatment to remove noise and edge-cut lacunae.
Figure 4.
Coloured maps of bone cells in the humerus of the salamander Desmognathus.
(A) Map of the orientation of the maximum length of each osteocyte in the humerus of Desmognathus. Colour coding is represented by a RGB tripod. The orientation of the long bones is given relatively to the proximal epiphysis (PE). The surface of the bone has been added to the right model to visualize the bone-cell orientation in the cortical context. (B) Map of the orientation of the maximum length of each osteocyte lacuna in the humerus of Desmognathus, in the same virtual thin section as Figure 3B,C. (C) Map of the density of bone-cell lacunae in the humerus of Desmognathus in the same virtual thin section as Figure 3B,C. Colour coding shows the gradation between the densest (+) and least dense (−) regions. (D) Map of the volumes of bone-cell lacunae in the whole humerus of Desmognathus (left model) and in the same virtual thin section as Figure 3B,C. (right model). Colour coding is given with the distribution of lacunae volumes within the whole humerus.
Table 1.
Comparison of bone-cell lacuna characteristics in different samples.
Figure 5.
Bone histology of the humerus of Eusthenopteron.
(A) 3D model of humerus, proximal epiphysis at the top, anatomical articulation with the ulna preserved at the bottom (voxel size = 20.24 µm). The muscle attachment area scanned at 0.678 µm voxel size (Figure 5B–F) is indicated by a blue square with an arrow rising from it that shows the approximate orientation of the muscle. Successive views from left to right: dorsal, mesial and ventral face. (B) Transverse modelled virtual thin section (left) and virtual thin section created from scan images (right) of the high-resolution scan through the muscle attachment area. The proximal end of the humerus is towards the left. The vascular mesh (in pink, v) is surface-parallel and gives off numerous vertical vascular canals that are slightly inclined towards the proximal end of the bone; the fibres (in white) slope obliquely down from proximal to distal. Internally the fibres end at the border of the endosteal bone (eb); externally the great majority do not reach the surface, stopping at the first line of arrested growth (LAG), like most of the vertical vascular canals. (C) Top views showing successively the bone surface of the region of muscle attachment (left), the spatial distribution of the bundles of extrinsic fibres (middle), and the four regions of interest where measurements and statistical tests on bone-cell lacunae were performed (right). The proximal end of the humerus is towards the top in all views (and also in D-F). (D) Top-view map of the density of bone-cell lacunae. In this and the two following images the distribution of the fibres in represented in transparent overlay. (E) Top-view map of the volumes of bone-cell lacunae. (F) Top-view map of the orientation of the maximum lengths of bone-cell lacunae. Same colour codings as for the maps in Figure 3.
Figure 6.
Osteocyte lacunae in the humerus of Eusthenopteron.
Four cubes of osteocyte lacunae from the humerus of Eusthenopteron, in plan view, after treatment to remove noise and edge-cut lacunae.
Figure 7.
Bone histology of the interolateral plate of the placoderm, Compagopiscis.
(A) 3D model of interolateral plate (IL) with part of anterior ventrolateral plate (AVL), in anteroventrolateral view (5.05 µm voxel size). Reproduced from [12] with permission. The external (ventral) surface is oriented downwards, anterior to the right. High-resolution scan was done at location of white arrow. (B) Transverse virtual thin section modelled from high-resolution scan (0.678 µm voxel size), showing vascular mesh (pink), bone-cell lacunae (blue), extrinsic fibres (white), lines of arrested growth (brown) and surfaces (gold). Orientation approximately same as (A). (C) Transverse classical thin section through an isolated interolateral of Compagopiscis (WAM12.6.03). The white arrows point to a bundle of extrinsic fibres (ef) in the dorsal periosteal bone (pb; approximately corresponding to areas 1 and 2). The periosteal bone surrounds an internal core bone (icb). Picture taken under polarized light. (D) Close-up of growth arrest surface in anteriormost part of interolateral, from a second scan at 0.678 µm voxel size, showing embedded attachment fibres, each associated with a dimple in the surface possibly left by the cell producing the fibre. Clockwise from top left, edge-on view, oblique external view, oblique internal view, oblique internal view without fibres. Holes in the surface are openings for blood vessels. (E) Anteriormost region of interolateral plate showing rows of fibres alternating with vascular layers, same scan as (D). Anterior at top. (F) Virtual thin section (same as B) showing regions where measurements on bone-cell lacunae were performed. (G) Density of bone-cell lacunae in the thin section. (H) Orientation of maximum lengths of bone-cell lacunae. (I) volumes of bone-cell lacunae. Same colour codings as in Figure 4. Note distinctive region of internal core bone on the left-hand side of the section (icb); this tissue is deposited around internal vascular spaces and is never associated with muscle attachments. By contrast, the bone below, to the right of, and above this region (pb) has all been deposited by an external periosteum. (J) 3D model of fibres showing three distinct fibre orientations indicated by the white arrows in side and plan views (orientation of anterior and dorsal indicated with arrows). (K) Interpretative representation of muscle attachments, blue arrows showing approximate orientations of muscles.
Figure 8.
Bone surface of the IL of Compagopiscis.
(A) On the left, external surface of the anteriormost muscle attachment on the interolateral of Compagopiscis, modelled from scan with 0.678 µm voxel size. The bone is oriented obliquely. Top, dorsolateral view; middle, anterior view; bottom, anteroventral view. The dorsal surface shows a dimpled texture identical to that on the arrested growth surfaces in the muscle attachment, whereas the ventral surface is smoother. Scale bar: 1 mm. On the right, close-ups of surfaces showing transition from dimpled (top) to non-dimpled (bottom) surface. The dimples are the size of single cells, and each appears to form the entry point for an attachment fibre that is cemented into the bone. The dimples themselves may have housed cell bodies or reflect delayed mineralisation around the fibres [18]. Scale bar: 100 µm. (B) Smooth external surface of the posteriormost region of muscle attachment. Scale bar: 250 µm.
Figure 9.
Osteocyte lacunae in the interolateral plate of Compagopiscis.
Details of the four cubes of osteocyte lacunae in the interolateral plate of Compagopiscis after treatment to remove noise and edge-cut lacunae. Top views taken at 0 degrees ( = plan view); bottom views taken at 90 degrees.