Figure 5

Addition of CG-1A increases condensation number and density in vitro and induces ectopic cartilage in vivo. (a) PNA staining of a 2-day control leg culture. (b) Addition of 10 μg/ml CG-1A increases the condensation number, with a concomitant decrease in size. (c) Treatment with 20 μg/ml CG-1A causes the condensations to fuse. (d) Untreated control 2-day micromass culture fixed and stained with DAPI. (e) Culture treated with 10 μg/ml anti-CG-1A antibody, fixed at day 2, and stained with DAPI. Photomicrographs (a-e) are at same level of magnification; bar in (a) represents 0.25 mm. (f) Graph showing change in condensation number upon addition of 10 μg/ml CG-1A and 10 μg/ml anti-CG-1A antibody, respectively. (g) Graph showing change in inter-condensation distance upon addition of 10 μg/ml CG-1A and 10 μg/ml anti-CG-1A antibody, respectively. (h) Graph showing change in condensation size upon addition of 10 μg/ml CG-1A and 10 μg/ml anti-CG-1A antibody, respectively. All three variables (condensation number, size and inter-condensation distance) were statistically analyzed using one-way analysis of variance comparing means by the Tukey-Kramer post-hoc analysis. Results are shown as means ± S.E.M. (* p < 0.05). (i) 5½ day wing bud injected on with CG-1A, fixed and stained with Alcian blue on day 8, shows the presence of an ectopic digit in the site of injection (black arrowhead). Inset of i shows the ectopic digit in detail. (j) Untreated 8½ day control wing stained with Alcian blue showing three digits; with discernible patency of joint space between the second digit phalange and 1^st metacarpal in the inset of (j). (k) Contralateral wing bud of (j), injected with CG-1A on the 5^th day, fixed and stained with Alcian blue showing fusion of the phalange and 1^st metacarpal of the second digit and obliteration of their joint space (inset of k).