Hes1 in Thyroid Development Figure 4. Decreased CT and T4 expression in Hes12/2 thyroids. CT and T4 surface areas were quantified at E16.5 in wild type and Hes12/2 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. doi:10.1371/journal.pone.0016752.g004 developing thyroid, 2) increased proportion of apoptotic cells, 3) accelerated cell differentiation, and 4) decreased pool of progenitor cells from specification. To assess proliferation, we determined the ratio of BrdU-labelled Nkx2-1-positive cells over all Nkx2-1-positive cells. At E9.5, the proportion of Nkx2-1-positive cells labelled by BrdU was 4.6% in wild type thyroids (Figure 6A–D and Figure 7A) and 13.2% (p,0.001) in Hes12/2 thyroids (Figure 6F–I and Figure 7A). At E11.5, E13.5, and E15.5, this ratio was not significantly different between Hes12/2 and wild type thyroids (Figure 6K–N, P–S and Figure 7A). Neither did we find any evidence supporting a role for increased apoptosis in the thyroid hypoplasia (data not shown). Hes12/2 and wild type thyroids were not different regarding the time of appearance of TG and T4 immunostaining in thyrocytes or of CT immunostaining in C-cells. Thus, hormone-producing cells did not differentiate earlier in Hes12/2 than in wild type mice, in keeping with data on pancreas development [7]. In contrast, the number of Nkx2-1 positive progenitor cells was significantly lower in Hes12/2 thyroids than in wild type thyroids: at E9.5 in the median anlage by 42% (p,0.05) and at E11.5 in the ultimobranchial bodies by 73% (p,0.01), respectively (Figure 7B). To investigate the cause of the progenitor cell depletion, we analysed the expression of cyclin-kinase inhibitors (CKIs). CKIs play a major role in regulating cell cycle exit and have links with Notch signalling [10]. We found that the CKI p57 was clearly expressed in the median anlage in both Hes12/2 and wild type mice at E9.5 (Figure 6E, J) but not at the other stages studied (from E11.5 to E16.5) (Figure 6O, T). These data were consistent with the low proliferation ratio at E9.5 in the wild type thyroid: 4.6% (Figure 6A–D and Figure 7A) while the rate of proliferation was on average 40% from E11.5 onwards. Thus, p57 was not involved in the thyroid progenitor cell deficiency in Hes12/2. Moreover, we also analysed expression of two further CKIs. However, we did not find differences in the expression of p21 and p27 in Hes1 wild type vs. Hes12/2 mice (data not shown). Therefore, neither accelerated differentiation, increased apop- tosis, or decreased proliferation, provided an explanation for thyroid hypoplasia. Further, the determination of the number of progenitors of the thyroid primordium by Hes1 was independent of p57, p21 and p27. Discussion Hes1 is well known in development of different tissues, particularly endoderm derived endocrine organs like pancreas, and neuroendocrine cells of lung. Hes1 controls normal develop- ment by regulating proliferation and differentiation and so cell fate. In mutant Hes1 mouse embryos, we found that the thyroid gland was hypoplastic and hypofunctional. Thus, the thyroid gland is not the first example of an endodermic tissue affected by Hes1. This study allowed us to involve a new gene in thyroid development (Figure 8). However, specification of the median anlage and the ultimobranchial bodies was independent of Hes1. Hes1 expression in the thyroid gland Nuclear Hes1 staining occurred in both thyroid anlages as early as they appeared. This expression pattern on the protein level was maintained throughout development as well as in adult tissues. Hes1 expression was heterogenous at early embryonic stages (E9.5 and E11.5) in the median anlage, as observed by others during early pancreas development (E10–11) (data not published). From E13.5 onwards we found an increasing expression of Hes1 on mRNA level with highest expression levels in the adult tissue. The maintained Hes1 expression during differentiation of thyrocytes and C-cells as well as in the mature tissue differs from decreasing Hes1 expression in endocrine pancreas development [19]. Thyroid hypoplasia and delayed fusion of median and lateral thyroid anlages in Hes12/2 mutants Hypoplasia of the thyroid anlagen in Hes12/2 mice was already present as early as E9.5 in the median anlage and E11.5 in the ultimobranchial bodies. The ratio in size reduction between mutant and wild type thyroids remained constant between E9.5 and E16.5. PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e16752