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Figure 1. Embryonic origin of the bony skull in five vertebrate model organisms arrayed on a simplified vertebrate phylogeny.Neural crest-derived territories (blue) have been verified experimentally in each species, although the specific contributions from individual migratory streams are reported only for chicken, axolotl and Xenopus. Derivation of remaining components from mesoderm (magenta) has been verified experimentally in mouse and chicken and is presumed for the remaining species. Arrowheads point to the neural crest–mesoderm interface in the skull roof, which is displaced caudally in Xenopus. Data for zebrafish are from refs 8, 9; diagram is based on ref. 8 (figure reproduced with permission from PLoS). Data for axolotl and Xenopus are from the present study; skulls are redrawn from refs 16, 42, respectively (figures reproduced with permission from John Wiley and Sons). Data for chicken are from ref. 43; diagram is based on ref. 44 (figure reproduced with permission from John Wiley and Sons). Data for mouse are from refs 7, 45; diagram is based on refs 4, 46 (figure reproduced with permission from John Wiley and Sons). F, frontal; Fp, frontoparietal; N, nasal; P, parietal; Px, premaxilla; Sq, squamosal.
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Figure 2. Transverse sections through an axolotl skull showing GFP labelling of bone.GFP-labelled cells are rarely seen within the bony matrix, which is largely acellular (upper row), but they are abundant in the periosteum, a connective tissue layer that invests individual bones (lower row). (a–d) A single GFP-labelled osteocyte (arrow) in the bony matrix of the premaxilla. (e–h) Four labelled cells (arrows) in the periosteum of the parasphenoid. In each row, a single section is depicted four times at the same magnification, each with a different combination of fluorescent illumination. Labelling: DAPI-stained nuclei (blue); GFP-positive cells (green); and alizarin-stained bone matrix (red). Scale bar, 100 μm.
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Figure 3. Patterns of CNC derivation of the bony skull differ between Xenopus and axolotl.Coloured regions denote contributions from individual migratory streams of CNC. Red labels denote homologous bones that have a different embryonic origin between species. Data for axolotl and Xenopus, two amphibians, are from the present study; skulls are redrawn from refs 16, 42, respectively. Data for the domestic chicken, an amniote, are from ref. 43; diagram is based on ref. 44 (figure reproduced with permission from John Wiley and Sons).
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Figure 4. CNC derivation of cartilages and bones in the skull of the adult axolotl.Most cartilages (a,c) and bones (b,d) are derived from the mandibular stream (yellow). Hyoid stream contributions (blue) are limited to (a) the stapes of the middle ear and (b) the retroarticular process of the lower jaw. There is no contribution to the skull proper from the branchial stream, which contributes extensively to the branchial or gill skeleton (not illustrated). The remainder of the skull (dark grey) is presumably derived from paraxial mesoderm, although this remains to be confirmed experimentally. Skulls are redrawn from ref. 42.
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Figure 5. Mandibular stream neural crest is the principal source of skull bones in the axolotl.Panels depict transverse sections from juvenile axolotls that received embryonic grafts of mandibular (a–j) or hyoid (k,l) stream neural crest. Schematics of skulls show bone of interest (green); dashed red lines indicate plane of section. GFP-labelled cells are green; bony matrix is stained red; and cell nuclei are counterstained blue (except c). Arrows point to labelled osteocytes within bony matrix or labelled periosteal cells. Chondrocytes (arrowheads) and mesenchymal core of teeth (*) are also labelled. Br, brain; En, external naris; Fr, frontal; Mc, Meckel’s cartilage; Na, nasal cartilage; Pa, parietal; Pt, pterygoid cartilage; Qu, quadrate; St, stapes. Scale bar, 100 μm.
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Figure 6. All three CNC streams contribute to the osteocranium in X. laevis.Transverse sections are from postmetamorphic frogs that received GFP-positive embryonic grafts of the mandibular (a–l), hyoid (m–t) or branchial (u–x) stream. Each pair of images depicts adjacent sections of the grafted (left) side. In most, the left section is stained histologically to reveal cartilage (blue) and bone (red); a,m are viewed with Nomarski (differential interference contrast) microscopy. The right section is immunostained for GFP (green); cell nuclei are counterstained blue. Note the composite origin of the premaxilla from both mandibular (a,b) and hyoid (m,n) streams. Scale bar, 50 μm.
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Figure 7. Hypothesis for the evolution of CNC derivation of the bony skull.Coloured regions depict contributions to the osteocranium from the three CNC migratory streams in four tetrapod model systems. Stream-level contributions are not known in the mouse, but they are presumed to resemble those in the chicken46. It is most parsimonious to posit that urodeles and amniotes share a common pattern of CNC derivation, which evolved no later than their common tetrapod ancestor (blue bar on the simplified phylogeny), and that the unique pattern in Xenopus evolved after the anuran clade diverged from urodeles (green bar).
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Figure 8. Grafting procedure.(a) Photograph of a living stage-16 axolotl embryo38, dorsal view, anterior at the top. Paired neural folds are about to meet in the midline and fuse postcranially, but they remain prominent and far apart in the head. (b) Drawing of stage-17 embryos depicting the seven regions within the left cranial neural fold39 that were grafted individually from GFP-positive donor embryos (green) into wild-type hosts. The approximate locations of premigratory mandibular, hyoid and branchial stream neural crest are depicted on the right side of the host embryo. (c) Stage-36 embryo in lateral view depicting migratory streams of mandibular, hyoid and branchial neural crest, which occupy the rostral region of the head and the oropharyngeal arches. (d–f) Donor-derived CNC cells (green) migrating within the first, second and posterior oropharyngeal arches are visible in living chimeric embryos following grafts of premigratory mandibular, hyoid and branchial stream neural crest, respectively. Mandibular stream neural crest also populates the rostral region of the head in d. Lateral views, anterior is to the left. Scale bar, 1 mm.
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