exists that Hes1 is involved in the development of the pancreas [7] and pituitary gland [8] in mice. Indeed, in embryos invalidated for Hes1, the pancreas is hypoplastic. The upregulation of Ngn3 is associated with precocious and excessive differentiation [7]. Hes1 ensures proper timing of the events involved in tissue morpho- genesis, as shown for neurogenesis and gliogenesis [9]. Hes1 knock-out mice die of abnormal brain development from E12 onwards [9]. Moreover, Hes1 may contribute to maintain the pro- liferation potential of progenitors by regulating cyclin-dependent kinase inhibitors such as p21Cip1, p27Kip1, and p57Kip2 [10–12]. Notch signalling and, more specifically, Hes1 were recently found in normal and transformed human thyrocytes, in which they regulated thyroid-specific gene expression [13]. Indeed, Hes1 appears to regulate the transactivation of the sodium iodide symporter (NIS). Moreover, decreased Notch signalling has been found in follicular and medullary thyroid cancer tissue [13,14]. These data prompted us to investigate the role for Hes1 in thyroid development. We found that Hes1 mutant mouse embryos had severe thyroid hypoplasia related to low thyroid progenitor cell numbers, an abnormality that was independent from p57. Moreover, the Hes1 mutants had decreased production of both T4 and calcitonin. The decreased T4-synthesis was paralleled by a strong decrease of Nis expression. Thus, our study provides the first evidence that Hes1 is involved in thyroid development and endocrine function of both, thyrocytes and C-cells. Results To investigate the role for Hes1 in thyroid development, we measured Hes1 expression during thyroid differentiation and we studied thyroid development in mice lacking Hes1. Hes1 is expressed during thyroid murine development Hes1 mRNA expression was significantly increased in thyroid glands from E13.5 to E18.5 embryos (p,0.05 E13.5 vs. E18.5) (Figure 1A). Then, we studied expression of Hes1 on mRNA and protein level by use of in situ hybridization (ISH) and of a specific antibody on wild-type and Hes12/2 embryos (Figure S1). Hes1 protein was only expressed in a subset of Nkx2-1-positive precursors of the median anlage at E9.5, (Figure 1B, E, H) but was clearly expressed at E11.5 in both, the median anlage lining the aortic sac (Figure 1C, F, I) and in the ultimobranchial bodies in a lateral position (Figure 1D, G, J). Further, Hes1 positive cells were also present in the thyroid anlagen surrounding tissues (Figure 1E, F, G). To determine which cell types expressed Hes1, we investigated co-expression of Hes1 with antibodies against Nkx2-1, thyroglobulin (TG), calcitonin (CT), and Mash1. At E13.5, Hes1 was expressed in some Nkx2-1-positive cells (Figure 1K, O, S). From E15.5 to E18.5 and in adult glands, Hes1 showed nuclear staining in most Nkx2-1-positive (data not shown) and TG-producing thyrocytes within thyroid follicles (Figure 1M, N, Q). Hes1 was expressed at E13.5 in Mash1- positive cells (Figure 1L, P) and from E15.5 onwards in CT- producing C-cells (Figure 1R). This Hes1 expression profile in the developing as well as adult thyroid tissue suggested a role of Hes1 for differentiation and endocrine function of Nkx2-1-positive thyrocytes precursors as well as for Nkx2-1/Mash1-double- positive C-cell progenitors. Thyroid hypoplasia in Hes1 mutant embryos To determine the role for Hes1 in thyroid development, we investigated mice lacking functional Hes1 alleles. This well- characterized allele lacks the first 3 exons of the Hes1 gene, which encodes the bHLH domain of Hes1 [15]. Hes1 Hes1 in Thyroid Development embryos die during fetal development, allowing examination of the morphology of the thyroid in wild-type and Hes1 mutants at the onset of endocrine function at E16.5 but not beyond. Nkx2-1 is expressed in undifferentiated thyrocytes as well as C-cells precursors in the median anlage and the ultimobranchial bodies from E9.5 onwards and remains expressed in both cell types after onset of endocrine function. Thus, we evaluated the size of the developing thyroid by measuring the surface area of Nkx2-1- positive cells at E9.5, E11.5, E13.5, E15.5 and E16.5. We compared at each stage three Hes12/2 and three wild type embryos (Figure 2A, B and C). At E9.5, we performed sagittal sections of embryos and we located the Nkx2-1 positive thyroid median anlage within the endoderm covering the tongue in analogy with previous work [1,3] (Figure 2A). From E11.5 to E16.5, we performed transverse sections of embryos and we located the Nkx2-1 positive thyroid lobes in the typical lateral position on both sides of the trachea and larynx, and the isthmus connecting the two lobes by crossing the trachea in a pretracheal position (Figure 2A). In accordance with the general Nkx2-1 expression pattern, the tracheal epithelium showed also specific positive staining. At E9.5, the median anlage of Hes1 mutants was already significantly smaller than in wild type mice, the Nkx2-1- positive surface area being 36% smaller (p,0.05) (Figure 2B). At E11.5, the overall Nkx2-1-positive surface area of both thyroid anlages in Hes12/2 mice was 65% (p,0.01) smaller than in the wild type mice (median anlage: 236%; ultimobranchial bodies: 283%) (Figure 2B). At E13.5, E15.5, and E16.5, the thyroids of Hes12/2 mice were 65% (p,0.05), 34% (p,0.01), and 50% (p,0.01) smaller, respectively, than those of wild type mice (Figure 2C). Thyroid size of heterozygous Hes1+/2 thyroids was normal compared to wild-type littermates, but was significantly bigger than Hes12/2 thyroids (p,0.01) (data not shown). This significant reduction of thyroid size at all stages was not paralleled by a general hypotrophy or reduction of the overall body size of Hes12/2 mutants. These results suggest that Hes1 expression in the developing thyroid was required for the gland to reach its normal size. Thyroid anlagen specification, in contrast, was normal in Hes12/2 mice. Thyroid shape and fusion abnormalities in Hes12/2 mice The wild type thyroid showed a high level of organisation at E16.5: Nkx2-1 was expressed in all thyroid cells, Pax8 in TG- producing thyrocytes organized in small thyroid follicles, and CT and Mash1 in C-cells scattered throughout the gland in a parafollicular position (Figure 3A–E). Mash1 expression was maintained in the developing thyroid, a finding at variance with results by Kameda et al. [16]. The Hes12/2 thyroid at E16.5 contained Nkx2-1-, Pax8-, TG-, and T4-expressing cells scattered throughout the gland (Figure 3F–I). Unexpectedly, we found that CT- and Mash1-expressing cells were clustered in a specific region of the gland (Figure 3I–J). We sought an explanation to this difference with the wild type gland by examining thyroids at two earlier stages, E15.5 (Figure 3K–T) and E13.5 (data not shown). At E15.5, fusion of the ultimobranchial bodies with the median anlage was apparent in wild type mice but not in Hes12/2 mice (n = 3) (Figure 3K, L, P, Q). The E15.5 ultimobranchial bodies of the Hes12/2 mice expressed a moderate number of Pax8- positive cells and few CT- and Mash1-positive cells (Figure 3R, S, T). Thus, absence of Hes1 was associated with a 3-day delay in the fusion of the ultimobranchial bodies to the median anlage: fusion occurred at E16.5 in the Hes12/2 mice and at E13.5 in the wild type mice. Moreover, CT- and Mash1-expressing cells in Hes12/2 thyroids had not spread throughout the gland at E16.5 PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e16752