1 min). Peptidylpropyl isomerase A was used as an endogenous control. Adult thyroid cDNA was used as a calibrator sample. The data were analysed using the comparative cycle threshold method and presented as the fold change in gene expression, normalized for a calibrator whose value equalled 1. Histological analyses After sacrifice of pregnant mice, whole embryos or dissected thyroids were fixed in 3.7% formalin, and embedded in paraffin. Sections (4 mm thick) were collected and processed for immuno- histochemistry (sagittal sections at E9.5 and E11.5; and transverse sections at E13.5, E15.5, and E16.5). The antibodies were used at the following dilutions: rabbit anti-Hes1 (1/500, gift from Dr T. Sudo), rabbit anti-Nkx2-1 (1/2500, Biopat, Milan, Italy), rabbit anti-Pax8 (1/2000, Biopat, Milan, Italy), rabbit anti-Mash1 (1/20, Becton-Dickinson, Franklin Lakes, NJ), mouse anti-TG (1/100, Dako, Glostrup, Denmark), rabbit anti-calcitonin (1/400, Dako, Glostrup, Denmark), mouse anti-T4 (1/10000, AbD Serotec, Raleigh, NC), rabbit anti-Nis (1/200, gift from Thierry Pourcher), mouse anti–E-cadherin (1/100, Becton-Dickinson, Franklin Lakes, NJ), rabbit anti-p57 (1/10, Abcam, Cambridge, UK) and mouse anti-BrdU (1/4, Amersham, Fairfield, CT). The fluorescent secondary antibodies used were Alexa Fluor 488 goat anti-rabbit and anti-mouse and Alexa Fluor 633 goat anti-rabbit and anti- mouse antibodies (1/400, Alexa, Invitrogen, Carlsbad, CA). The paraffin-embedded sections were deparaffinised by serial passages in toluene and in alcohol. The sections were then heated for 15 minutes in Antigen Retrieval solution (Biogenex, San Ramon, CA) in a microwave, cooled, blocked with TBS-BSA 3%- Triton 0.3%, and incubated with the primary antibody overnight at 4uC. After three 5-minutes washes in TBS-Tween 0.1%, the sections were incubated with the appropriate fluorescent second- ary antibody for 2 hours at room temperature then mounted with mounting liquid. Photographs were taken using a fluorescence microscope (Leitz DMRB; Leica, Nussloch, Germany) and digitised using a Hamamatsu C5810 chilled 3CCD camera (Middlesex, NJ). For Mash1, the detection protocol was adapted from the Dako kit protocol for peroxidase-labelling amplification (Dako, Glostrup, Denmark). For Hes1 antibody, a different protocol was used: tissues were fixed at 4uC in 4% paraformaldehyde in PBS, cryoprotected in 15% sucrose-PBS at 4%uC overnight, embedded in 15% sucrose27.5% gelatine in PBS, and frozen at 250uC in isopentane. Cryosections 4 mm in thickness were prepared. Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide in methanol for 30 minutes. The sections were microwaved in EDTA solution, cooled, and incubated with blocking solution (TBS-BSA 3%-Tween 0.1%) then with the primary antibody for 1 h at 37uC. Then, the sections were incubated with biotinylated secondary antibody for 1 hour. The next step was immunostaining using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manu- facturer’s instructions. The sections were then incubated in 3,39- diaminobenzidine tetrahydrochloride and counterstained with hemalun-eosin. Specificty of Hes1 antibody was tested on Hes12/2 embryos. No Hes1 staining was detected in Hes12/2, References Hes1 in Thyroid Development while positive Hes1 staining was reproducibly detected on wild- type tissues (Figure S1). Quantification Photographs were taken using a fluorescence microscope (Leitz DMRB; Leica) and digitalised using a chilled 3CCD camera (C5810; Hamamatsu). To measure the surface area labelled with Nkx2-1, CT, Nis, or T4, immunohistochemistry was performed on every second section from each embryo throughout the whole tissue. The sections were then analysed using ImageJ 1.32 s (public domain software, www.rsbweb.nih.gov/ij). The Nkx2-1-positive surface areas per section were summed to obtain the total surface area per 2 thyroid, in mm . The surface areas positive for CT, Nis and T4, markers of late thyroid differentiation, were normalized for the Nkx2-1 surface area representing the total thyroid. At least three thyroids were analysed per condition. The results are reported as mean6SEM. In each tissue analysed with ImageJ 1.32 s, represen- tative sections were also analyzed manually by two different persons to validate fluorescent surface area measurements. TUNEL experiments were done using an in situ cell death detection kit (Roche, Neuilly-sur-Seine, France) according to the manufacturer’s instructions. Nkx2-1 immunostaining was then performed. To determine the percentage of apoptotic thyroid cells, we counted the frequency of TUNEL-positive cells among 500 Nkx2-1-positive cells. Proliferation of Nkx2-1-positive cells was estimated by counting BrdU-positive nuclei among Nkx2-1-positive cells on every second section throughout the complete tissue. The chi-square test was used to determine whether differences were statistically significant. For all tests and for all stages, we systematically analyzed three Hes12/2 and three wild type embryos. Supporting Information Figure S1 Expression of Hes1 by ISH and by IHC (immunohistochemistry) on wild-type and Hes12/2 em- bryos. A: Hes1 ISH and Nkx2-1 IHC on sagittal sections of wild-type and Hes12/2 embryos at E10.5 stage. B: Hes1 IHC and Nkx2-1 IHC on sagittal sections of wild-type and Hes12/2 embryos at E10.5 stage. No staining for Hes1 by ISH and IHC in Hes12/2 embryos in the Nkx2-1 positive thyroid anlage, while clear and reproducible staining by HIS and IHC was found in wild-type embryos. (TIF) Acknowledgments We thank Dr Palle Serup, Dr Mette Jørgensen, and Dr Jan Jensen (Gentofte, Denmark) for their help including providing Hes12/2 mice, Dr Tetsuo Sudo (Tokyo, Japan) for providing anti-Hes1 antibody, Dr Thierry Pourcher (Nice, France) for providing anti-Nis antibody, Dr Rapha¨el Scharfmann for helpful discussion and Marie-Therese Simon for technical assistance. Author Contributions Conceived and designed the experiments: AC GS MP. Performed the experiments: AC ET BR. Analyzed the data: AC LR GS MP. Wrote the paper: AC GS. Reviewed the paper: LR MC MS J-MB MP. 1. De Felice M, Di Lauro R (2004) Thyroid development and its disorders: genetics and molecular mechanisms. Endocr Rev 25: 722–746. 2. Kameda Y, Nishimaki T, Chisaka O, Iseki S, Sucov HM (2007) Expression of the epithelial marker E-cadherin by thyroid C cells and their precursors during murine development. J Histochem Cytochem 55: 1075–1088. 3. Fagman H, Andersson L, Nilsson M (2006) The developing mouse thyroid: embryonic vessel contacts and parenchymal growth pattern during specification, budding, migration, and lobulation. Dev Dyn 235: 444–455. 4. Szinnai G, Lacroix L, Carre A, Guimiot F, Talbot M, et al. (2007) Sodium iodide symporter (NIS) gene expression is the limiting step for the onset of thyroid function in the human fetus. J Clin Endocrinol Metab 92: 70–76. 5. Kageyama R, Ohtsuka T, Tomita K (2000) The bHLH gene Hes1 regulates differentiation of multiple cell types. Mol Cells 10: 1–7. 6. Kageyama R, Ohtsuka T, Kobayashi T (2007) The Hes gene family: repressors and oscillators that orchestrate embryogenesis. Development 134: 1243–1251. PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e16752