Figure 3: Nell-1 signalling activates Wnt/β-catenin signalling activity in vivo.

(a–d) Evaluation of Wnt/β-catenin signalling in Nell-1+/6R animals. (a) Axin2 immunohistochemistry in aged (18-month old) lumbar spine specimens of Nell-1+/+ and Nell-1+/6R animals, appearing brown. Haemotoxylin counterstain appears purple. (b) Semiquantification of Axin2 immunohistochemical staining among trabecular bone-lining cells (N=32 and 22 images). (c) Axin2 mRNA expression in bone marrow flush of aged (18-month old) Nell-1+/+ and Nell-1+/6R mice, evaluated using qRT–PCR (N=4 samples per genotype). (d,e) Evaluation of Wnt/β-catenin signalling in TOPGAL reporter mice in the context of Nell-1 overexpression. Wnt/β-catenin signalling activity was assessed after intrafemoral injection of either Nell-1-expressing adenovirus (Ad-Nell-1 or Ad-Null control, 5 × 1012pt ml−1). (d) Xgal staining in TOPgal femurs at 1 week post injection. Blue staining indicates Wnt-responsive cells, indicated by black arrows. cb: cortical bone (N=3 mice per treatment group). (e) FACS analysis of femoral TOPgal bone marrow flush for β-gal positivity, indicating Wnt-responsive cells. Percentage of β-gal+ cells expressed as a portion of CD45− marrow cells (non-haematopoietic cells); analysis performed 2 weeks post Ad-Nell-1 injection (N=4 mice per treatment group). Black scale bar, 25 μm. Data points indicate the means, while error bars represent one s.e.m. In vivo experiments were performed without replicate, unless otherwise described. Parametric data were analysed using an appropriate Student’s t-test or a one-way ANOVA, followed by a post hoc Tukey’s test. Nonparametric data were analysed with a Mann–Whitney U-test or a Kruskal–Wallis one-way analysis. **P<0.01.