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

Suppression of Dicer leads to a glomerulocystic disease.

<p>Representative pictures of hematoxylin and eosin (A-F) and Masson’s Trichrome (G,I). Staining of Ctr (A, D, G), Dicer cKO kidneys at P30 (B, E, H) and Dicer cKO kidneys at P50 (C, F, I). Dicer induced cyst formation is limited to the cortex (C). Dicer cKO mice present a glomerulocystic phenotype and tubular dilatation at P50 (F). These alterations are not so prominent at P30, where only sites of dilatation in Bowman capsule can be sporadically seen (E). Interstitial fibrosis is evident in Dicer cKO mice at P50 (I). (A, B, C) Magnification 5X. (D- I) Magnification 40X.</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

Alterations of GSK3β / β-catenin are associated with the development of the glomerulocystic phenotype.

<p>Representative pictures of renal cortex from 30 (A, B) and 50 days old (C, D) mice. Ctr (A, C) and Dicer cKO (B, D) sections were stained with anti β-catenin antibody. In Dicer cKO, β-catenin expression is limited to the basolateral domain in both tubules and parietal cells of the Bowman’s capsule, at both P30 and P50 (B-D). (A-D) Magnification 63X, zoom1.5. Panel E and F show immunoblotting of Cortex/OSOM samples of 30 (E) and 50 (F) days old mice. GSK3β expression is already upregulated at the pre-cystic stage (P30) in Dicer cKO mice (E) and this is persistent at P50 (F). Glomerular cyst formation is associated with loss of cytosolic β-catenin expression and general downregulation of cortical protein level (F). No significant changes were observed in the fraction of their main regulating phosphorylated forms. Panel G represent qPCR analysis of mRNA of β-catenin in 30 (right) and 50 days old mice (left). mRNA level of β-catenin is downregulated in Dicer-cKO mice at P50. Data are expressed as mean ± sem; n power is 5 vs 5. * is for p value < 0.05; ** is for p-value <0.01.</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

Selective Dicer Suppression in the Kidney Alters GSK3β/β-Catenin Pathways Promoting a Glomerulocystic Disease - Table 1

<p>Data are expressed as mean ± sem; n is expressed in brackets.</p><p>** is for p <0.01;</p><p>*** is for <0.001</p><p>Selective Dicer Suppression in the Kidney Alters GSK3β/β-Catenin Pathways Promoting a Glomerulocystic Disease - Table 1 </p

Tubular dilatation mainly involves the distal segments of the nephron.

<p>Representative pictures of renal cortex from Ctr mice (A, D) and Dicer cKO at P30 (B, E) and at P50 (C, F) mice stained with LAH (Lectin from Arachis Hypogaea) (A- C) and AQP2 (D- F). Dilated tubules in P50 Dicer cKO mice are positive for LAH (C) and AQP2 (F), reflecting their origin from distal convolute tubules, connecting and collecting ducts. (A-F) Magnification 63X.</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

Dicer suppression is associated with downregulation of AQP2, NKCC2 and lower density of collecting ducts.

<p>Representative pictures of the inner stripe of the outer medulla of control (A, C) and Dicer cKO (B, D) mice double labeled with anti-AQP2 (green) and anti-H⁺ATPase (red) antibodies. At P50, Dicer cKO mice (D) showed no alteration in the distribution pattern of principal (AQP2 +) and intercalated cells (H⁺ATPase +), but a lower density of collecting ducts compared to the controls (C). (A-D) Magnification 40X. (E) Western blot analysis for AQP2 and NKCC2 expression at P30 (upper) and P50 (lower) of samples of IM and ISOM, respectively. AQP2 expression is significantly downregulated in Dicer cKO mice compared to Ctr at P50. NKCC2 expression is significantly downregulated in Dicer cKO mice compared to Ctr at P30 and P50. Data are expressed as mean ± sem; n power is 5 vs 5. *** is for p value < 0.001.</p