Figure 3. Live imaging for osteoblasts.

Evaluation of osterix-DsRed (a–d) and osteocalcin-DsRed (e–h) fluorescent signals. (a,b,e,f) Stacked ventral images in the pharyngeal region at day 1 for the ground (a,e) and flight (b,f) groups. The images were captured under the same conditions between flight and ground samples shown in Supplementary Table S2 and constructed by Z-projection done with ImageJ software. Yellow arrows show bones with osteoblasts localized on them. up: upper pharyngeal bone, lp: lower pharyngeal bone, c: cleithrum. Scale bars = 50 μm. (c,g) The fluorescence intensity of osterix (c) and osteocalcin (g) constantly increased during the flight. The numbers of osterix line fish: ground, n = 9 (day 1), 10 (day 2), 10 (day 3), 10 (day 4), 11 (day 5), 10 (day 6), 11 (day 7), and 11 (day 8); flight, n = 6 (day 1), 6 (day 2), 6 (day 3), 7 (day 4), 7 (day 5), 6 (day 6), 5 (day 7), and 4 (day 8). Numbers of osteocalcin line fish: ground, n = 6 (day 1), 7 (day 2), 7 (day 3), 6 (day 4), and 3 (day 5); flight, n = 3 (day 1), 3 (day 2), 5 (day 3), 4 (day 4), and 4 (day 5). These numbers are shown in Supplementary Table S1. *P < 0.05, Student’s t-test. Error bars, s.e.m. (d) The osterix-positive area was significantly increased in the flight group at days 6 and 7. *P < 0.05, Student’s t-test. Error bars, s.e.m. (h) Schematic diagram of the ventral view of a medaka larva head. The dotted rectangle shows the observation area in Fig. 3a,b,e,f and Fig. 4a,b,d,e. up: upper pharyngeal bone, lp: lower pharyngeal bone, c: cleithrum.