Figure 1: Mohawk gene (Mkx) is expressed in the outer annulus fibrosus (OAF).

(a) Representative images of intervertebral discs (IVDs) of E14.5 mice. Left: Safranin O fast green stain in a wild-type mouse. Middle: IHC of a Venus knock-in mouse. Green: anti-GFP. Blue: Hoechst. Right: in situ hybridization of Mkx of a Venus knock-in mouse. Scale bars, 200 μm. (b) Representative disc images from embryonic stages to growth stages (upper and middle panels, Safranin O fast green (S-F) stain and Masson’s trichrome (M-T) stain in a wild-type mouse; lower panel, IHC of a Venus knock-in mouse; green, anti-GFP; blue; Hoechst). Scale bars, 200 μm. (c) Representative images of IVDs from 10-week-old mice (upper panel, safranin O fast green stain in a wild-type mouse; lower panel, IHC of a Venus knock-in mouse; green; anti-GFP; blue; Hoechst). Scale bars, 200 μm. (d) Immunohistochemistry of the coronal sections of a human lumbar disc IAF and OAF (#1: 20-year-old male, L1/2). Green: anti-MKX. Blue: Hoechst. Scale bars, 100 μm. (e) Overview of the observed area of the IAF and OAF. The section is a haematoxylin–eosin staining of a human lumbar disc (#1: 20-year-old male, L1/2). Dotted lines indicate borders of the NP, IAF and OAF. Scale bar, 1 cm. (f) Percentages of human IAF and OAF cells that were positive for MKX in each zone. Values are the mean of nine discs from five donors. Error bars represent s.e.m. ***P<0.001. Statistical differences were assessed with Student’s t-test. GFP, green fluorescent protein; MKX, Mohawk homeobox protein; P1, postnatal day 1.