Fig 1.
(WAM 11.9.1). A, lateral and B, medial views of synarcual comprising three fused vertebral elements (neural arches, spines). Fusion is dorsal and ventral with space between these elements remaining, for the neural spines. The dorsalmost part of the three spines has become enlarged, large white arrows indicate lines of perichondral bone deposition on the developing heterotopic cartilage. Ventrally, the neural arches are also flared (asterisk), particularly the most anterior arch remnant; the third element retains more of the typical circular base shape. Arrowheads indicate preservation of the sharp edges along the incorporated neural spines, as are seen in more posterior separate vertebra of the posterior axial skeleton. Large white arrows indicate anterior direction.
Fig 2.
Callorhinchus milii, stage 22.
A-D, lateral view showing general morphology as well as Sox9 (neural crest cells, prechondrogenic cartilage, red) and Mf20 (muscle fibres, developing skeletal musculature, green) staining. Developing vertebrae are separate and distinct at this early ontogenetic stage. Asterisk indicates rear of developing braincase. Abbreviations: h, heart; op, optic capsule; ov, otic vesicle; pa, pharyngeal arches, v1, 3, 5, differentiating vertebrae 1, 3, 5.
Fig 3.
Callorhinchus milii, stage 23.
A-D, lateral view showing A, staining as in Fig 2, also DAPI (cell nuclei, white) staining. Blue colour indicates collagen type II staining in the notochord. B, As in A, but DAPI staining not visualised; C, Sox9 and Mf20 staining, showing dorsal and ventral vertebral elements (neural and haemal arches) and skeletal musculature; D, Sox9 staining alone. All vertebral elements separate and distinct at this stage. Abbreviations as in Fig 1, also: nc, notochord; oc.v, occipital vertebrae; skel.musc, skeletal musculature; rb, rostral bulb [28].
Fig 4.
A-E, Callorhinchus milii, stages 25 (A, B), 27 (C, D).
A, Cell nuclei (white). Sox9 (red) marks dorsal and ventral vertebral elements and neural crest cells; the vagus and accessory nerves (X and XI) are clearly visible. Three anteriormost occipital vertebrae (oc.v) lack the spinal nerves associated with more posterior vertebrae; they appear closer together differ in shape relative to the more posterior vertebrae, which are better developed and still distinct from each other. B, notochord (blue) and developing skeletal musculature are visualized. The pectoral fin bud is beginning to develop (p.fin), while the pectoral fin musculature is differentiating from the ventral myotome (m.pre). C, Stage 27, Sox9 no longer stains the vertebral elements (differentiated beyond the prechondrogenic cartilage stage); they are best seen via DAPI staining (cell nuclei). All vertebral elements are still separate from one another. Developing pectoral fin musculature is more distinctly bifid in shape. Abbreviations as in previous figures, also: drg, dorsal root ganglia; m.pre, pectoral fin muscle precursor; p.fin, pectoral fin; sg, sympathetic ganglia; vr, ventral root.
Fig 5.
Callorhinchus milii, stage 28, A-C.
Cleared and stained specimen (Alcian blue). A, lateral view, showing embryo with external gills and developing vertebrae. White arrow indicates anterior vertebrae that have become misshapen, indicating fusion and coalescence. The position of these relative to the pharyngeal arches and pectoral fin indicates that these are fusing to the occipital region of the braincase, rather than as part of a more posterior synarcual. More posterior vertebrae appear normal at this stage. B, C, dorsal view, arrowhead indicates occipital anteriorly and vertebrae posteriorly. White arrow indicates foramina Abbreviations, as in previous figures, also: ext.gills, external gills.
Fig 6.
A, B, Stage 29 (maximum projection, Sox9 red, DAPI white, Mf20 green) shows that the occipital vertebrae are still distinct from each other but that the braincase is now well developed. The posterior elements are still separate but show distinct dorsal and ventral elements for each vertebra. In stage 30 (C, F; one plane in Z, Sox9 red, DAPI white), the synarcual has started to form and can be recognised medially. The individual vertebral elements are still visible laterally (v). The vagus and accessory nerves are visible lateral to the mineralised braincase (C). D, E show distribution of the cranial nerves in a C. milii hatchling (Khonsari et al. 2013). Abbreviations as in previous figures, also Br, Braincase; E, Eye; R, Rostrum; Syn, synarcual. Images in Fig 6D and 6E taken as screenshots from a 3-D reconstructed CT scan model; Wikimedia Commons datafile, Khonsari et al. 2013. BMC Biology. doi:10.1186/1741-7007-11-27.
Fig 7.
A-E, Callorhinchus milii, adult, A-E.
A, 3D volume rendering (Aviso), anterior view. Asterisk indicates comparable region in 7E. B, 3D volume rendering, lateral view (Drishti), arrow indicates anterior. False color differences indicate surficial mineralization (yellow) and unmineralized cartilage (brown). Levels of Fig 7C and 7E indicated. Note that mineralization proceeds internally via the spinal nerve foramina (C, virtual section, Drishti). D, virtual section (Drishti) showing pairs of spinal foramina anteriorly, continuous foramina posterior. Arrowhead indicates region in Fig 7E, arrow indicates ventral area where vertebral elements have not fully fused or coalesced. E, virtual section (Drishti) through ventral part of synarcual showing unfused elements (arrowheads) that have become mineralized, preventing further fusion and preserving a portion of the original shape.