Hes1 in Thyroid Development Figure 5. Nis protein expression and quantification in Hes12/2 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 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.g005 Further analysis revealed that the number of progenitors was clearly decreased in the median anlage and more so in the ultimobranchial bodies. This correlates with the more abundant expression of Hes1 in the ultimobranchial bodies at E11.5 rather than in median anlage at E9.5. This observation is in accordance with data showing that the number of embryonic progenitor cells is a limiting factor for pancreatic size [20]. Thus, Hes1 influences the number of thyroid progenitor cells of thyrocytes as well as C- cells available in both thyroid anlages, most likely by a cell- autonomous way. CKI play a major role in regulating cell cycle exit and have links with Notch signalling [10]. In the pancreas, lack of effect of Hes1 on p57, a member of the CKI family, caused decrease of pancreatic progenitors and resulted in accelerated differentiation [7,10]. To characterize the mechanism leading to reduced progenitor pool in the Hes12/2 thyroid, we compared the expression of p57 in mutant and wild-type tissues. At E9.5 cell proliferation was limited and the cells were in cycle arrest due to strong p57 expression in the median anlage in Hes12/2 and wild-type mice. In contrast, at E11.5, in the median anlage as well as in the ultimobranchial bodies, the cells proliferated actively, and very few cells expressed p57. The fact, that p57 expression was not different in mutant and wild type thyroids from E9.5 onwards suggests that Hes1 regulates the progenitor pool in a p57 independent manner. PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e16752