Skeletal staining was attempted with the PBS- and 5E1-injected mice. (A) 6-week old mice exposed to 5E1 at E12.5 exhibit limb, skull, tail and trunk bones that are reduced in size and length, but mice exposed to PBS show the same bone length as wild-type mice. (B) The forelimbs and hindlimbs of E14.5, one-day-old, one-week-old, three-week-old and six-week-old mice were stained with alcian blue and alizarin red. Mice injected with 5E1 have both shorter forelimbs and hindlimbs, and their bones grow similarly to those of PBS-injected mice. (C) The length of the humerus, ulna, and radius in 5E1-injected mice are shorter than in the PBS-injected mice at one-week old, three-week old, and 6-week old; the skeletal staining results agree with those of the microCT analysis. Student’s t-test was employed for statistical analysis, with the level of statistical significance set at (*) p<0.01 or (**) p<0.05 (Sc; Scapula, S; Stylopod, Z; Zeugopod, A; Autopod, scale bar = 3 mm).