Figure 1.
Hes1 expression during thyroid development.
A: RT-QPCR analysis of Hes1 mRNA expression in embryonic and adult thyroids.Hes1 expression increased significantly from E13.5 until E18.5 (p<0.05). B-T: Immunohistochemistry for Hes1 and Nkx2-1 in whole embryos at E9.5 and E11.5 (sagittal sections) (B–J). At E9.5, Nkx2-1 stained the median thyroid anlage evaginating of the endoderm. At E11.5, Nkx2-1 stained the median anlage along the aortic sac. At E11.5, Nkx2-1 marked the symmetrical ultimobranchial bodies from the endoderm of the fourth pharyngeal pouch. Hes1 co-localised with Nkx2-1 cells in the median anlage and in the ultimobranchial bodies. At E13.5, immunohistochemistry for Hes1, Nkx2-1 and Mash1 is shown in dissected thyroids at E13.5 (K, L, O, P, S). In dissected thyroids at E17.5 (M, N, Q, R) Hes1/TG, and CT/E-cadherin co-labelling was analysed in adjacent sections. H, I, J, Q, R, S represent enlarged areas. Hes1 is expressed in progenitors of and in mature thyrocytes and C-cells. ma: median anlage; UB: ultimobranchial body, as: aortic sac.
Figure 2.
Thyroid hypoplasia in Hes1−/− embryos.
A: Nkx2-1 staining at E9.5 (sagittal sections) and at E11.5, E13.5, E15.5, and E16.5 (transverse sections) in littermates. Total thyroid surface area (µm2) was quantified using Nkx2-1 staining at each embryonic stage, B: The results represent the surface area of three embryos per stage and per genotype. UB: ultimobranchial body; ma: median anlage; tr: trachea, oe: oesophagus. Student's test, * p<0.05 and ** p<0.01.
Figure 3.
Thyroid morphology and expression of thyrocyte and C-cell markers at late embryonic stages.
Staining for Nkx2-1 (A, B, F, G, K, L, P, Q), Pax8 and TG (C, H, M, R), CT and T4 (D, I, N, S), and Mash1 (E, J, O, T) at E16.5 (A–J) and E15.5 (K–T) in transverse sections from wild type and Hes1−/− mice. Boxed areas were enlarged (B–E, G–J, L–O, Q–T). Thyroid fusion was delayed by 3 days in Hes1−/− thyroids compared to wild type thyroids. tr: trachea; thy: thyroid; ma: median anlage; pt: parathyroid; UB: ultimobranchial body.
Table 1.
Morphological abnormalities in Hes1−/− mutant thyroid.
Figure 4.
Decreased CT and T4 expression in Hes1−/− thyroids.
CT and T4 surface areas were quantified at E16.5 in wild type and Hes1−/− mice and compared to the Nkx2-1-positive total thyroid surface area. Three embryos per genotype were used for quantification.Chi-square test, * p<0.05.
Figure 5.
Nis protein expression and quantification in Hes1−/− thyroids.
The Nkx2-1 staining in A and D localizes the thyroid gland. In B, C, E, F, Nis protein labelled thyrocytes. In C and F, co-staining of Nis and Hoechst visualizes Nis in the basolateral membrane of the thyrocytes and Hoechst in the nucleus of the thyrocytes. In G, Nis surface areas were quantified at E16.5 in wild type and Hes1−/− mice and compared to the Nkx2-1-positive total thyroid surface area. Three embryos per genotype were used for quantification.Chi-square test, * p<0.05.
Figure 6.
Cell proliferation and p57 expression in thyroids at E9.5 and E15.5.
Immunohistochemistry of Nkx2-1 (A, B, F, G, K, L, P, Q), BrdU (C, H, M, R), and p57 (E, J, O, T) in transverse sections from wild type mice (A–E and K–O) and Hes1−/− mice (F–J and P–T) at E9.5 (A–J) and E15.5 (K–T). Boxed areas were enlarged (B–E, G–J, L–O, Q–T). tr: trachea; thy: thyroid; ma: median anlage; to: tongue.
Figure 7.
Proliferation ratio and progenitor cell number.
A: The proliferation ratio was calculated as the proportion of Nkx2-1-positive cells labelled with BrdU at E9.5, E11.5, E13.5, and E15.5 in wild type and Hes1−/− thyroids. At E11.5, before fusion the median thyroid anlage (ma) and the ultimobranchial bodies (UB) were quantified separately. Three embryos per stage and per genotype were used for quantification. Chi-square test, ** p<0.01. B: Number of progenitor cells at E9.5 in the median thyroid anlage (ma) and at E11.5 in the ultimobranchial bodies (UB). Three embryos per stage and per genotype were used for quantification.Student's test, * p<0.05 and ** p<0.01.
Figure 8.
Proposed roles of Hes1 during murine thyroid development and function.
Hes1 controls the number of thyrocyte progenitors in the median anlage (in grey) at E9.5 and C-cell progenitors in the ultimobranchial bodies (in white) at E11.5. Hes1 has a role for the correct fusion of the two anlages at E13.5. At E16.5, Hes1 is involved in normal endocrine function in differentiated thyrocytes and C-cells.