Hes1 in Thyroid Development 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 Hes12/2 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. doi:10.1371/journal.pone.0016752.g007 expression in Hes12/2 thyroids. However, in variance to published data, Mash1 expression was maintained until E16.5 in wild type thyroids [16]. In contrast, Hes12/2 thyroids showed very few Mash1-expressing cells, and a strongly reduced ratio of CT/Nkx2-1 positive surface area, suggesting a reduced endocrine function of C-cells. Then, we can not conclude that the expression of Hes1 and Mash1 show an inverse correlation in mouse thyroid development. It was shown in Hoxa-3 and Eya1 mutant mice that the fusion of the thyroid anlagen plays a crucial role for normal differentiation [24,25]. Disordered differentiation is only observed in Eya1 knock- out mice after fusion of the ultimobranchial bodies expressing Eya1 and the median anlage which does not express Eya1 [25]. Thus, the delayed fusion in Hes12/2 mice provides a possible explanation for impaired endocrine function. T4 production and, thyroid size depend on TSH/TSH-receptor signalling in differentiated thyrocytes. A marked reduction in TSH production has been reported in the pituitary of Hes12/2 mice [8]. As TSH is secreted from E14.5 by the pituitary gland and TSH- receptor protein is expressed in the thyroid from E14.5 onwards, thyroid hypoplasia noted before E14.5 in Hes12/2 mice cannot be ascribed to TSH/TSH-receptor activity [26]. However, we cannot exclude an additional role of TSH deficiency for decreased T4 production seen at E16.5 in the Hes12/2 mice. These data allowed us to postulate that Hes1 involved in the thyroid differentiation of the thyrocytes and the C cells. PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e16752