Fig. S5

The role of osteoblasts and osteoclasts in axon patterning, ECM formation and scale regeneration, related to Figures 5 and 6

(A) Transverse TEM of scale radius. Note that the position of the nerve is variable with respect to other radial cell types (compare to Figure 3E). en, endothelium; ob, osteoblast; bc, basal cell; mc, mucus secreting cell; n, nerve; r, radius; ms, mineralized scale. (B) Lateral DIC and fluorescent images of an isolated scale. f, focus. r, radius. Dashed boxes indicate regions of magnification. Inset, a single radius. (C) Scale nerve and radial patterns in csf1ra mutants. (D) Lateral view of intact skin. (E) Posterior edge of an isolated scale immunostained as indicated. (F) Single z-plane near the posterior edge of an isolated scale immunostained as indicated. Due to the curvature of the scale, the z-plane is an oblique section through the epidermis. In panel F’’ the laminin channel has been overexposed (o.e.) to highlight the intracellular puncta within basal cells. Basal cells are distinguishable by their characteristic nuclear size and cell borders. ep, episquamal osteoblasts; hyp, hyposquamal osteoblasts; bc, basal cell. Dashed boxes indicate region of magnification. Yellow line, plane of orthogonal section. (G) Brightfield and fluorescent images of control and osteoblast-ablated adult animals 7 days after removal of two dorsolateral rows of anterior scales (brackets). Note the lack of scale regeneration in osteoblast-ablated animals compared with robust scale regeneration in control animals (asterisks). Transgenes: (B) endothelium [Tg(fli1a:EGFP)] and axons [Tg(p2rx3a>mCherry)]; (D) Tg(sp7:mCherry-nfsB); (G) all cells [Tg(actb2:BswitchR)] and osteoblasts [Tg(sp7:mCherry-nfsB)]. Staining: (C) axons (acTubulin) and nuclei (DAPI); (E) laminin (anti-laminin), F-actin (phalloidin) and nuclei (DAPI); (F) laminin (anti-laminin), osteoblasts (zns-5), F-actin (phalloidin) and nuclei (DAPI). Scale bars, 1 μm (A), 250 μm (B), 50 μm (C-F), 10 μm (F’) and 500 μm (G).