Confocal microscopy images of thyroid budding, migration, and expansion from 48 to 72 hpf with 3D simulated images. All embryos shown are oriented anterior to the top in the ventral views. (A, B) at 48 h postfertilization (hpf). The thyroid primordium is located near the OFT, is surrounded by aortic arch artery 1 (aa1), and possesses a globular appearance (A). 3D simulated images reveal that the thyroid is sphere‐shaped with a small number of small protrusions (B). (C‐D) From ~ 55 hpf, mature thyroid follicles begin to appear, and the morphology of the thyroid gland undergoes severe transformation with numerous protrusions extending in all directions, thus suggesting that the thyroid has begun to migrate. The morphology was easily recognized using the simulated 3D image. In the early development of the thyroid, an increased number of long protrusions can be observed more clearly in 3D images than they can in images produced using confocal microscopy stacking imaging techniques (B, D). (E‐F) Thyroid expansion and proliferation begin at 72 hpf along with the extension of the HAs. The thyroid extends along the HAs, and the shape of the thyroid becomes an inverted ‘Y’ when viewed in the ‘head‐up’ position (E). Three‐dimensional microscopy and image fusion reconstructions were conducted to visualize the thyroid volume (F). Magnified views of the yellow box are presented below the corresponding images. * aa1: aortic arch artery 1; ** as: aortic sac. Arrowhead: protrusions of the thyroid in 3D view. Scale bar: 50 μm.

Confocal microscopy images of thyroid expansion from 96 to 120 hpf with 3D simulated images. All embryos shown are oriented anterior to the top in the ventral views. (A‐B) the thyroid is slimmer and shaped more like a long column at 96 hpf. (C‐D) Expansion of thyroid tissue moving away from the heart along the pharyngeal midline occurs at 120 hpf. (E) Thyroid volume expands gradually from 48 to 72 hpf and more drastically from 96 to 120 hpf. (F) Length increases gradually from 48 to 120 hpf, while the width remains almost constant, and this is consistent with the volume changes at the corresponding time point. Magnified views of the yellow box are presented below the corresponding images. * aa1: aortic arch artery 1; *** aa3: aortic arch artery 3. Scale bar: 50 μm. The error bars represent SEM. Data are representative of three independent experiments with similar results.

Treatment of embryos with the FGFR1‐selective inhibitor PD166866 from 36 to 96 hpf caused severe defects in thyroid morphology and volume and resulted in abnormal or absent in HAs. All embryos shown are oriented anterior to the top in the ventral views. (A) An analysis of DMSO‐treated embryos showed normal vascular development with normal thyroid morphology at 96 hpf. (B) Treatment with PD 166866 from 36 to 96 hpf caused a reduction in tg signal intensity and resulted in abnormal HAs (stubby and malformed) in 8 of 20 embryos. (C) The absence of HAs was observed in 12 of 20 embryos during 36–96 hpf drug treatment, and this occurred along with abnormal thyroid expansion. The percentage of abnormal or absent HAs in PD 166866‐treated embryos is presented in the lower right corner of B and C. Arrow: HA. *: VA. **: the absence of HA. Scale bar: 50 μm.

Treatment of embryos with the FGFR1‐selective inhibitor PD166866 from 36 to 96 hpf caused severe defects in thyroid morphology and volume and in HAs. All embryos shown are oriented anterior to the top in the ventral views. (A, C, E) Analysis of DMSO‐treated embryos showed normal vascular development with normal thyroid morphology. (B) Treatment with PD 166866 from 36 to 48 hpf caused a reduction in the signal intensity of tg. (D) During 48 to 72 hpf drug treatments, the typical inverted ‘Y’ shapes of the thyroid were barely observed in embryos after drug treatment. (F) In response to drug treatment from 72 to 96 hpf, the thyroids possessed loose and short morphology compared with those in the nondrug treatment group according to 3D simulated images, and no obvious abnormal pharyngeal vasculature surrounding the thyroid was observed. (C’’‐F’’) To better visualize HA development in embryos, the slices of C to F were presented separately. (D’’) Abnormal HAs (stubby and malformed) were observed in all embryos during 48–72 hpf drug treatment. (G) Treatment with PD166866 from 36 to 48 hpf resulted in a reduction in thyroid volume compared with that observed in vehicle‐treated embryos (P < 0.05). Drug treatment from 36 to 96 hpf resulted in a significant reduction in thyroid volume (P < 0.05). Drug treatment from 48 to 72 hpf and 72 to 96 hpf resulted in a mild effect on thyroid volume. Arrow: HA. Scale bar: 50 μm. The error bars represent SEM. Data are representative of three independent experiments with similar results. We conducted statistical comparisons using Student's t‐test for quantitative measures. *P < 0.05.