Fig. 4.

Injections of recombinant proteins affected beak development in chicken embryos. All embryos were injected at HH30 and were collected at HH38. HH, Hamburger and Hamilton stage. Bones of embryonic heads at HH38 were stained by alizarin red (a-e). a. The rule of measurements for the length of the upper and lower beak (arrow). b. Control embryos were treated with BSA. c. rFGF13-treated embryos had longer beaks. d. rITGB3 shortened the beak, especially the lower beak (arrow). e. No differences were observed in rWIF1-treated embryos. f. Quantification of treatment-dependent changes in length relative to control embryos. g. Whole mount ALP staining in the chicken head detected osteoblasts (stained purple) around the upper and lower beak skeleton (control). h. More robust ALP-positive staining in rFGF13-treated embryos (n = 3) demonstrated higher density of osteoblasts than control (n = 4). i. Levels of osteoblasts in rWIF1-treated embryos (n = 2) did not change. j. Whole mount TRAP staining detected more osteoclasts (stained purple) in the lower beak than the upper (arrowhead). k-l. Less TRAP-positive staining in rFGF13-treated (n = 2) and rITGB3-treated embryos (n = 3) demonstrated lower density of osteoclasts compared to control (n = 3)