Hes1 in Thyroid Development Figure 8. Proposed roles of Hes1 during murine thyroid development and function. Hes1 controls the number of thyrocyte progenitors in the median anlage (in grey) at E9.5 and C-cell progenitors in the ultimobranchial bodies (in white) at E11.5. Hes1 has a role for the correct fusion of the two anlages at E13.5. At E16.5, Hes1 is involved in normal endocrine function in differentiated thyrocytes and C-cells. doi:10.1371/journal.pone.0016752.g008 Hes1 function in the developing and mature thyroid We found that the expression of both Hes1 mRNA and Hes1 protein increased during development and persisted in the adult mouse thyroid. The Notch pathway has been shown to be involved in human thyroid function and in thyroid carcinomas arising from thyrocytes as well as C-cells [13,14]. Ferretti et al. found that Hes1 directly activated the expression of sodium iodide symporter [13], crucial for the onset of thyroid function [4]. In our study, T4- producing cells were found in Hes12/2 thyroids. Therefore, Hes1 is one potential transcriptional co-factor for NIS-expression but seems not to be indispensable for T4 synthesis. The maintained expression of Hes1 in the adult thyroid suggests nevertheless an important role for thyroid function. These results are in contrast to pancreas development: during early pancreas development, Hes1 downregulates a key transcription factor of endocrine cells named Ngn3 [19]. Additional studies are needed to elucidate the role for Hes1 in maintaining thyroid function. In conclusion, our data provide for the first time evidence that Hes1 is an additional regulator of thyroid development and differentiation (Figure 8), beside the known thyroid enriched transcription factors such as Nkx2-1, Foxe1, and Pax8: Hes1 maintains an adequate pool of thyroid progenitor cells via a p57- independent mechanism, determining normal thyroid size. Moreover, Hes1 is necessary for normal endocrine function of both, thyrocytes and C-cells in the differentiated thyroid gland. Materials and Methods Animals Hes12/2 mice were previously generated by replacing the first three Hes1 exons, including the bHLH domain, with a neomycin- resistance cassette [15]. CD1-Hes1 mice were maintained under specific pathogen-free conditions at the Bomholtgaard breeding centre (Ry, Denmark). Genotyping was performed as previously described [7]. Embryos at specific ages were obtained from Hes1+/2 females mated with Hes1+/2 males. Hes12/2 embryos die just before or at birth. Thyroid morphology was examined in wild type and Hes12/2 embryos. Given the variability of phenotype penetrance in the nervous system and pancreas [7,15], we chose Hes12/2 embryos whose visual nervous system was abnormal. All experiments were conducted in accordance with the principles and procedures outlined in the NIH Guide to the Care and Use of Experimental Animals and with approval from the Danish regulatory authorities use of experimental animals (document 2006/561-1125) and approval from Hagedorn Research Institute. For bromodeoxyuridine (BrdU) experiments, BrdU (20 mg/kg) was injected intraperitoneally to each pregnant mouse 1 h before sacrifice. Dissection Embryos were removed from pregnant mice at different days of gestation. Briefly, in a first step, the proximal part of the trachea, the larynx and the thyroid were visualized under a dissecting loupe (Leica, Leitz DMRB, Reuil-Malmaison, France) and in a second step, the thyroid glands were micro-dissected in analogy to the procedure used in adult mice. Only bilobed tissues with intact isthmus were assigned as thyroids and used for further experiments. Real-Time PCR To determine the Hes1 transcript level in thyroid rudiments from E13.5 to E18.5 and in adult thyroid, real-time PCR was performed using the 7300 Fast real-time PCR system (Applied Biosystem, Paris, France). Total RNA was isolated using the Qiagen RNeasy Microkit (Qiagen, Courtaboeuf, France) and treated with DNase I to eliminate genomic contamination. We used 2 to 5 thyroid rudiments for RNA extraction depending on embryonic stages. The Superscript Invitrogen protocol (Invitro- gen, Carlsbad, CA) was used for reverse transcription of 100 ng of each RNA sample. The synthesized cDNA was diluted to 1/20, and 5 ml was used per PCR reaction. Each reaction consisted of TaqmanH universal PCR master mix (Applied Biosystems, Carlsbad, CA), primers, and a Taqman-labelled probe specific for each gene (Applied Biosystems, Carlsbad, CA). Each reaction was run using the universal thermal cycling protocol (95uC for 10 min followed by 40 cycles at 95uC for 15 s and 65uC for PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e16752