Dicer inactivation affects expression of thyrocyte cell adhesion proteins.

<p>(A) Immunofluorescence analysis of Cdh16 (red signal) in Ctr (A–C), Het (D–F) and cKO (G–I) thyroids at 1 month (scale bar: 20 µm). Nuclei are stained with DRAQ5 (blue signal). (B) Immunofluoresence analysis of Cdh1 (red signal) in Ctr (A–C), Het (D–F) and cKO (G–I) thyroids at 1 month (scale bar: 20 µm). Nuclei are stained with DRAQ5 (blue signal). (C) Western blot analysis of Cdh16 and Cdh1 proteins from Ctr, Het and cKO thyroids at 1 month. Ctr denotes Dcr1^Flox/Flox or Pax8^Cre/+ mice, used as controls.</p

Oxyphilic cells in postnatal Dicer cKO thyroid gland do not express thyroid markers.

<p>Hematoxylin and eosin (H&E) staining (A, B) and immunohistochemical analysis of Nkx2.1 (C, D), Pax8 (E, F), Calcitonin (Calc) (G, H) in Ctr and cKO thyroid sections at 1 month. Immunohistochemical analysis of Chromogranin A/B expression is shown in thyroid (I) and in chief cells of the parathyroid glands (J, indicated with *) of cKO mice (scale bar: 50 µm). Dashed lines delimit representative oxyphilic cells patches.</p

Late differentiation markers expression in Dicer cKO thyroids at one month after birth.

<p>(A) Western blot analysis of Tg and Nis proteins in Ctr, Het and cKO thyroids. (B) Q-PCR analysis of TSHr and Tpo expression in Ctr, Het and cKO thyroids at 1 month. Each RNA sample is obtained by pooling four thyroids/genotype. Ctr denotes Pax8^Cre/+ mice, used as controls.</p

Thyroid morphology and differentiation in Dicer cKO E15.5 embroys and newborn mice.

<p>(A) Hematoxylin and eosin (H&E) staining and immunohistochemistry for Pax8, Nkx2.1 and Tg of Ctr, Het and cKO mice embryos at E15.5 (100× magnification). (B) The same analysis as in A was performed on thyroids of newborn mice (200× and 400× magnification for H&E staining and immunohistochemistry, respectively). In both panels Ctr denotes Pax8^Cre/+ mice, used as controls.</p

Hypothyroidism in Dicer cKO mice.

<p>(A) One month old cKO mice are smaller than Ctr littermates. (B) Body weight of male and female mice at one month after birth (n = 12 for each sex and genotype). P-value (***:p<10e-11) was calculated with Student's t-test comparing both Ctr and Het to cKO mice of the same sex. (C) ELISA assay of TSH serum level of Ctr (n = 10), Het (n = 10) and (n = 20) cKO mice at one month after birth (the same number of female and male mice have been analyzed). P-value (***:p<10e-13) was calculated with Student's t-test comparing both Ctr and Het to cKO mice. (D) Survival rates for Ctr, Het and cKO mice (n = 15 for each) during 12 weeks after birth. In all panels Ctr denotes Dcr1^Flox/Flox mice, used as controls.</p

Generation of thyroid conditional Dicer knockout mice.

<p>(A) Gene targeting strategy for Dicer conditional inactivation achieved by Cre mediated removal of RNase III domains. (B) PCR analysis of thyroid genomic DNA extracted from newborn and one month old mice of the indicated genotypes. Pax8 alleles were amplified with a primer set enabling to distinguish the wild-type allele (lower band) from the Cre-encoding one (upper band). For the amplification of Dicer alleles two different primer sets were used, one (F2-R2) amplifying the floxed region (Dcr1^Flox excised, middle panels), and the other (F1-R1), amplifying part of the exon 21, used as a control (bottom panels). Dicer-amplifying primers positions are shown in A. (C) Q-PCR analysis of mature miR-24, miR-23a, miR-29b relative expression in mice thyroids. P-value (*:p<0,05; **:p<0,005) was calculated with Student's t-test comparing Ctr to cKO mice. Ctr denotes Pax8^Cre/+ mice, used as controls.</p

Immunofluorescence analysis of differentiation in Dicer cKO thyroids at one month after birth.

<p>(A) Immunofluorescence analysis of Pax8 (green signal) in Ctr (A–C), Het (D–F) and cKO (G–I) thyroids at 1 month (scale bar: 20 µm). Nuclei are stained with DRAQ5 (blue signal). (B) Double immunofluorescence analysis of Nis and Nkx2.1 (green and red signal, respectively) in Ctr (A–D), Het (E–H) and cKO (I–L) thyroids at 1 month (scale bar: 20 µm). Nuclei are stained with DRAQ5 (blue signal). Ctr denotes Dcr1^Flox/Flox mice, used as controls.</p

Mutant AKT1^E17K accelerates tumor formation induced by chemical carcinogens.

<p><b>A.</b> Lung tumor multiplicity in <i>R26-AKT1</i>^<i>E17K</i> infected with Ad-Cre (6 and 9 months after the treatment, respectively). Each point represents one mouse; bars represent means ± SD. **p<0.01, *p<0.05. <b>B.</b> Diameter of lung tumors generated by urethane in <i>R26-AKT1</i>^<i>E17K</i> mice treated with increasing doses of Ad-Cre. Bars represent mean diameter of tumor ±SD. *p<0.05. <b>C, D.</b> Representative H&E staining of lung lesions developed in <i>R26-AKT1</i>^<i>E17K</i> mice treated with solvent or Ad-Cre, as indicated, 9 months after urethane administration. <b>E, F.</b> Phosporylated AKT staining of lung lesions developed in <i>R26-AKT1</i>^<i>E17K</i> mice treated with solvent or Ad-Cre, as indicated, 9 months after urethane administration. Magnification as indicated.</p

Generation of transgenic <i>R26-AKT1</i>^<i>E17K</i> mouse.

<p><b>A.</b> Schematic representation of the targeting construct used for the conditional knock-in in the <i>R26</i> locus. The human <i>AKT1</i>^<i>E17K</i> cDNA preceded by a loxP-flanked transcriptional stop cassette, was recombined into the R26 locus. Cre-mediated removal of the stop cassette links Rosa26 exon 1 to the exogenous cDNA allowing expression of the transgene. <b>B.</b> Southern blot of EcoRV digested genomic DNA derived from mESCs transfected with the targeting construct carrying mutant AKT1. Lane 1: DNA from non-targeted mESCs, lane 2 and 3: DNA from DNA from two different <i>R26-AKT1</i>^<i>E17K</i> mESCs strains. Endogenous allele corresponds to the 11.5 kb band (<i>WT</i>); mutant allele corresponds to the 4.3 kb band (<i>REC</i>). <b>C.</b> Relative mRNA expression of human AKT1^E17K by Q-RT-PCR in targeted mESCs and in the corresponding cells transfected with the Flp (pFlpE-IRES-Puro) or Cre recombinase (pCre-IRES-Puro). Data are from replicate experiments as the mean±SD. ***p<0.001. <b>D</b>. Genotype analysis by PCR on tail-tip DNA of genetically modified mice as indicated.</p

Immunostaining analysis of <i>R26-AKT1</i>^<i>E17K</i> mice.

<p>pAKT immunostaining of lung tissues from <i>R26-AKT1</i>^<i>E17K</i> mice treated with solvent alone (A) or infected with Ad-Cre after 9 and 18 months from infection, respectively (B, C).</p