Skip to main content
. 2009 Dec 15;136(24):4143–4153. doi: 10.1242/dev.043281

Fig. 7.

Fig. 7.

Reactivation of Hh signaling by purmorphamine rescues limb defects in Atf4−/− embryos. (A) Purmorphamine, an agonist of the Hh signaling pathway, restores the length of the Atf4−/− forelimb in organ cultures. (a-f) Limb explants at the beginning (t0, a-c) and the end (4 days, t4, d-f) of culture. n=3. Purmorphamine partially rescued the longitudinal growth of the Atf4−/− radius (double-headed arrow, f) as compared with the control (e). Arrowheads indicate BrdU-positive cells. (g-i) In situ hybridization of Gli1 expression in WT (g), vehicle-treated Atf4−/− (h), and purmorphamine-treated Atf4−/− (i) radii. n=3. Purmorphamine restored Gli1 expression in Atf4−/− radii (i) to a level similar to that in WT controls (h), indicating activation of Hh signaling in Atf4−/− explants. (B) Quantification of the increase in radius length upon purmorphamine treatment of WT and Atf4−/− limbs. The percentage increase in radius growth after 4 days in culture is shown. P=0.01 by paired Student's t-test. (C) Purmorphamine partially corrects the delayed chondrocyte hypertrophy in Atf4−/− growth plates in organ culture. (a-c) Alcian Blue, Alizarin Red and Hematoxylin staining of radius sections of 4-day cultures. (d-f) In situ hybridization for Col10a1 in purmorphamine-treated and vehicle-treated limb explants. n=3. (D) Quantification of the relative length of the non-hypertrophic and hypertrophic zones upon purmorphamine treatment of WT and Atf4−/− radii. Error bars indicate s.e.m. of the length of the non-hypertrophic (left) and hypertrophic (right) zones normalized to the respective WT controls at 4 days in culture. (E) Purmorphamine increases chondrocyte proliferation in Atf4−/− growth plates. Immunohistochemistry of BrdU-labeled chondrocytes in limbs cultured for 4 days in the absence and presence of purmorphamine. The sections were counterstained with Hematoxylin. Boxed regions are magnified beneath to show BrdU-positive cells (arrowheads). n=3. (F) Quantification of proliferation rate represented by the ratio of BrdU-positive cells (brown) to total cells upon purmorphamine treatment of WT and Atf4−/− radii. Error bars indicate s.e.m. Scale bars: 0.2 mm in A,C; 0.5 mm in E.