Evi1^KI/+ mice show mild ossification delay. (A) Alcian blue and Alizarin red staining of Evi1^+/+ mice and Evi1^KI/+ mice at E18.5 and P0. Bars represent 5 mm. The ossified fraction was used to evaluate ossification, based on the study by Chang et al.²⁴ (B) Ossification of Evi1^+/+ and Evi1^KI/+ mice. At E18.5, although only the comparison of the humeral ossification was statistically significant, Evi1^KI/+ mice (n = 7) exhibited delayed ossification compared with Evi1^+/+ mice (n = 8). At P0, there was no significant difference between the ossification of Evi1^+/+ mice (n = 7) and Evi1^KI/+ mice (n = 6). Threshold significance level, P = .05 (2-tailed Welch t test). Underlined numbers denote the P value. Values are the mean ± SEM of each group. WT, Evi1^+/+ mice; KI, Evi1^KI/+ mice.