Fig. S1

RA-induced ectopic intercalvarial ossification predominantly occurs in the overlying dermis, consistent with the cutaneous col1a2 expression. (A,B) SL8-9 fish treated with DMSO (A) or RA (B) for 7 days, after consecutive in vivo AR staining (red) before and calcein staining (green) after the treatment, as shown in Fig. 1D,E; virtual transverse sections obtained from 3D projections along confocal Z-stacks (ZEN2008 software; Zeiss) at positions indicated by arrowheads in Fig. 1D,E. The weak calcein staining between the frontal calvaria of the RA-treated fish (Fig. 1E; asterisk) predominantly occurs in regions slightly dorsal of the calvarial plates (indicated in (B) with arrowhead). According to H&E and Azan novum stainings (see for instance Fig. S5A), this is the dermis. (C,D) in situ hybridizations of untreated SL10 fish for col1a1 (C) and col1a2 (D). In contrast to col1a1, whose expression is restricted to bone-lining osteoblasts (C; compare with Figs. 2F and 5C), col1a2 is expressed by basal keratinocytes (bf), dermal fibroblasts (df), consistent with former reports (Le Guellec and Sire, 2004), and in connective tissue fibroblasts (fb) between and below the calvarial plates. Upon RA-treatment, this col1a2 expression became slightly reduced (data not shown). However, the reduction was much more moderate than that of col1a1 in osteoblasts (see Figs. 2F,G and 5C,D). Abbreviations: bk, basal keratinocytes; df, dermal fibroblasts; fb, fibroblasts; fp, frontal calvarial plates