Figure 4

Increase in the TRAP-positive area and change in the mitochondrial structure in the flight group.

(A) Histochemical demonstration of mineralized matrices (black area) and osteoclasts (red area) in the lower pharyngeal bones and teeth of ground medaka (left) and flight medaka (right) at day 56, as demonstrated by von Kossa/TRAP double staining. Arrows indicate the rostral direction; and scale bars, 200 μm. (B) Percent ratio of TRAP-positive osteoclast area relative to von Kossa-positive mineralized area of pharyngeal bones and teeth in randomly selected sections (9 or 10 sections per medaka) prepared from ground (n = 3) and flight fish (n = 3) as shown in “A” and “C.” Proportion of osteoclast area relative to mineralized area was significantly increased in the flight-group medaka at day 56. ***P < 0.0005, by Student’s t-test. Error bars, s.e.m. (C) Partial 3D models of pharyngeal bones/teeth and osteoclasts from the ground (left) and flight (right) groups at day 56 reconstructed from 4-μm-thick Technovit sections that had been doubly stained for von Kossa and TRAP reactions. The black arrows show large osteoclasts in the flight group. The white arrow indicates a gap in the von Kossa staining due to possible dissolution of minerals from bones and teeth in the flight-group medaka. (D) Low degree of roundness of mitochondria in the flight group. (a–c) Day-56 jaw from the ground group. This multinucleate osteoclast (OC) appears to be devoid of its ruffled border (a). High magnification of an osteoclast shows numerous mitochondria in its cytoplasm (b) and an even more enlarged view reveals their round shape (c). (d–f) Day-56 jaw from the flight group. Low-power view of a gigantic multinucleate osteoclast (OC), with the cell contour outlined in red, is shown (d). A clear zone-like structure (CZ) of an osteoclast attached to bone is evident (e). An enlarged view of mitochondria with heterogeneous shapes and sizes (f).