Hes1 in Thyroid Development 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. doi:10.1371/journal.pone.0016752.g001 due to delayed fusion of the ultimobranchial bodies with the median anlage. At this stage, CT- and Mash1-expressing cells were localized at the cranial part of the thyroid lobes. Additional morphological abnormalities such as abnormalities of the isthmus are further detailed in Table 1 for each stage. We found no abnormalities in the expression of E-cadherin, a protein known to be involved in cell-cell adhesion (data not shown) [17]. These findings indicated that Hes1 was required to ensure normal shape, and size. However, formation of thyroid follicles was normal in Hes12/2 thyroids. Decreased CT, Nis and T4 surface area in the Hes12/2 thyroid To investigate onset of thyroid function in Hes12/2 mice, we determined the endocrine signature of the thyroid cells by measuring CT and T4 immunoreactivity. The CT-positive surface area relative to the Nkx2-1-positive surface area was smaller than in wild type mice, by 64% at E16.5 (p,0.05) (Figure 4, Figure 3D, I). Moreover, the T4-positive surface area normalized to the Nkx2-1 positive surface area at E16.5 was 78% lower in Hes12/2 mice than in wild type mice (p,0.05). Total thyroid surface area (Nkx2-1-positive surface area) in Hes12/2 thyroids was lower by 50% at E16.5, compared to wild type thyroids. The decreases in CT- and T4-positive surface areas were more pronounced than the decrease in total thyroid surface area. These data indicate that the hypoplastic thyroids in Hes12/2 animals show also intrinsically decreased endocrine function at E16.5. Ferretti et al. have shown that Nis was an apparent Hes1 target gene. Indeed, HES1 transactivated NIS directly in human adult thyrocytes [13]. Nis promotes the active iodide uptake by the thyrocytes, which is a crucial step for the onset of thyroid hormone synthesis and is expressed from E15.5 onwards in mice [18]. Thus, we quantified the Nis-positive surface area in relation to the total Figure 2. Thyroid hypoplasia in Hes12/2 embryos. A: Nkx2-1 staining at E9.5 (sagittal sections) and at E11.5, E13.5, E15.5, and E16.5 (transverse 2 sections) in littermates. Total thyroid surface area (mm ) 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. doi:10.1371/journal.pone.0016752.g002 PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e16752