Extended Data Figure 9: Progressive reduction of collagen secreting cells from the AER of different species in the gnathostome phylogeny.

a–h, Embryos of the epaulette shark H. ocellatum stained for Col1a1a (red), skeletal muscle (green) and nuclei (blue). a, b, At 36 mm (approximately S. canicula stage 26/27; ref. 32), Col1a1-expressing cells are present at low numbers within the AER initially, with cells evident traversing the AER basement membrane from the fin mesenchyme (arrows in b, b’). The density of Col1a1a-expressing cells within the AER increases notably with the extension of the fin, and the cells are present in large numbers in 48 mm embryos (c, d), (approximately S. canicula stage 30/31; ref. 28). Col1a1a-expressing cells are maintained at the edge of the fin fold at advanced stages of fin development (85 mm, e, f) and cluster into discrete foci at the edge of the fin fold by 90 mm (g, h). a, c, e, g, Dorsal views proximal to the top, inserts are high-magnification views of the boxed regions showing only Col1a1a expression in red in each fin. b, d, f, h, Maximum projection confocal renders of the distal edge of the AER/AF region of each fin in a, c, e and g, respectively. b’, d’, f’, h’, Sagittal views of the rendered images in b, d, f and h, respectively, revealing the persistent localization of Col1a1a-expressing cells at the distal most edge of each fin (arrows). i–p, Embryos of the paddlefish P. spathula, sectioned in the transverse plane and stained for Col1a1a expression (brown, DAB) at 1 dpf, (i, j), 2 dpf (k, l) and 4 dpf (m, n). Colonization of the AER by Col1a1a-expressing cells follows a very similar pattern temporally and spatially to that evident in the zebrafish fin, but appear at a lower density than in the epaulette shark. o, p, Control immunohistochemistry on 1-dpf sections with no primary antibody. j, l, n, p, High-magnification views of the boxed regions in i, k, m and o, respectively. Arrows in j, l and n mark Col1a1a-expressing cells in the AER. q–x, N. forsteri embryos in transverse section and stained for Col1a1a and counterstained with H&E. Very few Col1a1a-expressing cells colonize the AER initially (q, r, stage 42–44), and they continue to be present in low numbers up to stage 48 (s, t) and are no longer evident at fin extension and AF formation stages (u, v, stage 50). w, x, Control immunohistochemistry on stage-48 sections with no primary antibody. r, t, v, x, High-magnification views of the boxed regions in q, s, u and w, respectively. Arrows in inset images in r and t mark the limited number of Col1a1a-expressing cells in the AER of lungfish embryos. Scale bars, 200 μm (a, c), 50 μm (b, f), 100 μm (d, h), 400 μm (e) and 500 μm (g, i, k, m, o, q, s, u, w).