Hes1 in Thyroid Development Figure 6. Cell proliferation and p57 expression in thyroids at E9.5 and E15.5. Immunohistochemistry of Nkx2-1 (A, B, F, G, K, L, P, Q), BrdU (C, H, M, R), and p57 (E, J, O, T) in transverse sections from wild type mice (A–E and K–O) and Hes12/2 mice (F–J and P–T) at E9.5 (A–J) and E15.5 (K–T). Boxed areas were enlarged (B–E, G–J, L–O, Q–T). tr: trachea; thy: thyroid; ma: median anlage; to: tongue. doi:10.1371/journal.pone.0016752.g006 Previous work has already described a weak progenitor cell proliferation in the wild-type median anlage as it evolves through the budding stage [3]. These authors proposed recruitment of new progenitors from neighbouring Nkx2-1 negative endoderm as possible mechanism for normal bud growth. On the other hand, Lania et al. showed recently, that Tbx1 regulates, via Fgf8, the size of the early thyroid primordium by regulating the proliferation of endoderm derived thyrocytes precursors [21]. Other causes of hypoplasia such as modification of the onset of differentiation or increased apoptosis within the Hes12/2 thyroids were excluded. Further, proliferation ratio did not differ in mutant and wild type mice from E11.5 onwards, in accordance with constant size reduction ratio between thyroids of mutants vs. wild type until E16.5. In contrast to what could have been expected, proliferation ratio at E9.5 was 2.8 times higher in the median anlage of Hes12/2 mutants than in wild type thyroids. This might reflect a compensatory mechanism to produce a normal number of thyroid progenitor cells. However, this compensation has not reached the normal number of progenitor cells leading to hypoplasia of the thyroid. In summary, the mechanism of thyroid hypoplasia remains open and is different to what is decribed during pancreas development. In Hes12/2 mice, Jensen et al described disturbed fusion of the hypoplastic dorsal and ventral pancreatic evaginations at E16 [7]. In wild type mice, the median anlage reaches the definitive paratracheal position at E12.5 and the ultimobranchial bodies originating from the fourth pharyngeal pouch fuse with the upper pole of the symetrical thyroid lobes at E13. Interestingly, and in accordance with defective fusion of the ventral and dorsal pancreas, we found a three day delay of the fusion of the hypoplastic thyroid anlages in Hes1 mutant mice. The complete fusion occurred only at E16.5. Hes1 plays an important role for correct organogenesis of the thyroid gland. Reduced endocrine thyroid function in Hes12/2 mutants Onset of T4 synthesis occurs at E16.5 in mice, as soon as structural and functional differentiation is completed. Structural differentiation comprises thyroid follicle formation, the functional unit of the thyroid gland. In contrast to general hypoplasia and fusion abnormalities, follicle formation was normal in both, wild type and mutant mice. However, the ratio of T4/Nkx2-1 positive surface area was strongly reduced in Hes1 mutant mice, suggesting reduced endocrine function of thyrocytes lacking Hes1. Hes1 has recently been shown to directly transactivate the promoter of Nis. In Hes12/2 mice, Nis/Nkx2-1 ratio was also clearly reduced. Thus, reduced Nis-expression with consecutive decreased iodide accumulation could provide a possible explanation for decreased endocrine function of thyrocytes. On the other hand, C-cell differentiation depends on Mash1 expression. According to Kameda et al, Mash1 expression in the thyroid is present at a high level at E13.5 and absent from E14.5 onwards, which allows C-cell differentiation [16]. In neuronal and lung tissues, Mash1 and Hes1 expressions were inversely correlated [22,23]. Therefore, we expected to see an increase in Mash1 PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e16752