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1.
Fig. 7.

Fig. 7. From: miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

miR-200c counterbalances proliferation by regulating Vldlr-dependent expression of FGFR genes during epithelial branching morphogenesis. We propose that miR-200c targets Vldlr function by decreasing Vldlr and Reln expression, which counterbalances FGF-dependent proliferation and differentiation in the epithelial end buds. Dab1 is the intracellular adapter protein that transduces Vldlr signaling and Dab1–/– mice have smaller SMGs than control mice (supplementary material Fig. S6). Our working model also includes the previously described miR-200c target genes Zeb1 and Hs3st1, which regulate Cdh1 and heparan sulfate (HS) modification, respectively (gray). Genes associated with epithelial cell identity (Cdh1), progenitor maintenance (Krt5) and end bud differentiation (Aqp5) are downstream to miR-200c.

Ivan T. Rebustini, et al. Development. 2012 January 1;139(1):191-202.
2.
Fig. 1.

Fig. 1. From: miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

Identification of miR-200c in the submandibular gland end bud epithelium, a miRNA that influences branching morphogenesis. (A) Mouse embryonic submandibular glands (SMGs) were separated into epithelium and mesenchyme, and end buds and primary duct, prior to miRNA expression analysis using TaqMan low density arrays. (B-E) Relative expression of miRNAs that show higher expression in the epithelium than in the mesenchyme (B), in the epithelial end buds than in the main duct (C), and in the main duct than in the end buds (D). The miR-200 family components are also ubiquitously expressed throughout the epithelium (E). miRNAs were selected for loss-of-function studies based on their relative expression in the end buds (miR-34a and miR-200c, blue), and in the main duct (miR-204 and miR-135a, purple). (F) Epithelial branching increases with miR-200c and miR-34a and decreases with miR-204 and miR-135a loss-of-function. E12 SMGs were cultured with an off-target miR-control, miR-200c, miR-34a, miR-204 and miR-135a antagomirs for 60 hours. Branching morphogenesis was quantitated by counting the number of end buds.*P<0.05, **P<0.01 (ANOVA). Error bars indicate s.e.m.

Ivan T. Rebustini, et al. Development. 2012 January 1;139(1):191-202.
3.
Fig. 6.

Fig. 6. From: miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

Increased epithelial proliferation with miR-200c antagomir is Vldlr- and MAPK-dependent, and reelin treatment recapitulates miR-200c loss-of-function. (A-E) Epithelial proliferation was analyzed by EdU incorporation after (A) miR-control treatment, (B) 100 ng/ml reelin, (C) 500 ng/ml reelin, (D) miR-200c antagomir, and (E) a combination of miR-200c antagomir and anti-Vldlr function-blocking antibody. (F) Quantitation of proliferation normalized to nuclei staining and miR-control treatment. (G) qPCR analysis shows a decrease in miR-200c expression after reelin and miR-200c antagomir treatments and an increase with Vldlr function-blocking antibody treatment. (H) Reelin treatment (as in D) reduces gene expression (fold change) of Cdh1 (0.5) and Krt5 (0.4), and increases Fgfr1b (2.1), Fgfr2b (2.0), Etv4 (2.0) and Aqp5 (3.8). qPCR analysis was normalized to 29S and miR-control. (I) The Vldlr function-blocking antibody reduces, and recombinant reelin stimulates, MAPK activation after 24 hours. The bar charts show the ratio (normalized to control treatment) of phospho-MAPK to total MAPK (right) and phospho-AKT to total AKT (left). *P<0.05, **P<0.01, ***P<0.001 (ANOVA). Scale bar: 50 μm.

Ivan T. Rebustini, et al. Development. 2012 January 1;139(1):191-202.
4.
Fig. 5.

Fig. 5. From: miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

Vldlr function influences epithelial proliferation and Fgfr2b-dependent gene expression in the intact SMG. (A) Vldlr was detected in SMG epithelial end buds. The single laser-scanning microscopy sections show that Vldlr localizes in the epithelial end bud (left panels). E-cadherin (green) and VLDR (red) are shown together in the right panels. (B) SMGs were cultured with reelin and a Vldlr function-blocking antibody, and proliferation was detected by EdU incorporation after 24 hours. Reelin increases, whereas a Vldlr-blocking antibody decreases, branching morphogenesis and proliferation. The bar charts beneath show quantitation of branching morphogenesis and proliferation. (C) Gene expression analysis after reelin and Vldlr function-blocking antibody treatments was performed by qPCR. Recombinant reelin and Vldlr function-blocking antibody showed opposite effects on the expression of Cdh1, Fgfr1b, Fgfr2b, Fgf1, Etv4, Aqp5 and Krt5. The data were normalized to 29S and control treatment (red line). *P<0.05, **P<0.01, ***P<0.001 (ANOVA). Scale bars: 100 μm in A top and B; 10 μm in A bottom. Error bars indicate s.e.m.

Ivan T. Rebustini, et al. Development. 2012 January 1;139(1):191-202.
5.
Fig. 4.

Fig. 4. From: miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

Expression of the predicted miR-200c target genes Vldlr and Hs3st1 is localized in the epithelial end buds and increases after miR-200c loss-of-function. Target genes for miR-200c were predicted using MicroCosm, TargetScan and PicTar databases (supplementary material Fig. S2 and Table S5), and their relative expression in E13 SMG was verified in the SGMAP database. (A) Microarray analysis comparing the fold change in gene expression of predicted miR-200c target genes in the epithelial end buds compared with the main duct detects Vldlr and Hs3st1 expression in the end buds. The red line represents genes that are expressed equally in the epithelial end buds and in the main duct. (B) Gene expression (fold change) of predicted targets was measured 18 hours after antagomir treatment in isolated epithelia and intact SMGs (inset). miR-200c loss-of-function increases expression of Hs3st1 (4.0), Vldlr (4.2), Reln (18.0), Zeb1 (3.7), Asap1 (4.6), Dusp1 (7.0) and Rlf (4.8). The inset shows gene expression of miR-200c target genes in the intact SMG: Reln (5.4), Vldlr (4.6), Hs3st1 (4.5) and Zeb1 (1.9). Zeb1 is a positive control for miR-200c loss-of-function and is shown in gray. **P<0.01, ***P<0.001 (Student’s t-test). Error bars indicate s.e.m.

Ivan T. Rebustini, et al. Development. 2012 January 1;139(1):191-202.
6.
Fig. 3.

Fig. 3. From: miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

Loss-of-function of miR-200c in the epithelium increases Fgf10-dependent proliferation and the expression of genes associated with Fgf10/Fgfr2b signaling. SMG epithelia were cultured with antagomirs for 42 hours. (A) miR-200c loss-of-function increases epithelial branching morphogenesis and proliferation as determined by EdU incorporation (top panels) and reduces E-cadherin staining (bottom panels). Scale bar: 50 μm. (B) Quantification of fluorescence after miR-200c loss-of-function in the epithelia, normalized to miR-control. *P<0.05 (Student’s t-test). (C) The miR-200c antagomir specifically reduces expression of miR-200c and does not reduce expression of other miR-200 family members. SMGs were analyzed by TaqMan PCR, normalized to snRNA-U6 expression, 42 hours after antagomir treatment. ***P<0.001 (ANOVA). (D) miR-200c loss-of-function increases the expression (fold change) of Fgfr1b (3.0), Fgfr2b (2.7), Fgf1 (2.0), Etv4 (4.1), Zeb1 (3.1), Aqp5 (4.9), and decreases expression of Cdh1 (0.6) and the epithelial progenitor cell marker Krt5 (0.3). Expression was normalized to 29S and compared with miR-control (red line). *P<0.05, **P<0.01, ***P<0.001 (ANOVA). (E) The increase in epithelial morphogenesis after miR-200c antagomir treatment is dependent on FGFR signaling. Morphogenic index = number of buds × length of ducts in arbitrary units, normalized to untreated control epithelia. SU5402 inhibits FGFR phosphorylation. Error bars indicate s.e.m.

Ivan T. Rebustini, et al. Development. 2012 January 1;139(1):191-202.
7.
Fig. 2.

Fig. 2. From: miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

The expression of miR-200c increases during SMG development, is higher in the epithelial end buds, and its loss-of-function increases proliferation during branching morphogenesis. (A) miR-200c expression was analyzed by qPCR throughout SMG development and normalized to expression at E12 (red line). miR-200c increases 3-fold at E14, ∼6-fold at E15 and E17, and 84-fold in the adult SMG. (B) miR-200c was detected by fluorescent in situ hybridization (red), combined with E-cadherin immunohistochemistry (green) in E13 SMG end buds. miR-200c is localized in the cytoplasm of the epithelial tissue (right-hand image). White lines outline the epithelium. (C) Epithelial proliferation significantly increases with miR-200c loss-of-function. The bottom panels show higher magnification views of end buds. Proliferation was analyzed by EdU incorporation (green) and normalized to the epithelial area (bar chart). (D) The miR-200c antagomir specifically reduces expression of miR-200c and does not reduce expression of other miR-200 family members. SMGs were analyzed by TaqMan PCR, normalized to snRNA-U6 expression, 18 hours after antagomir treatment. (E) Gene expression was analyzed after antagomir treatment and normalized to 29S. There is decreased expression (fold change) of the epithelial marker Cdh1 (0.6) and the epithelial progenitor marker Krt5 (0.5), and increased expression of Fgfr1b (2.0), Fgfr2b (2.0), Fgf1 (2.4), Etv4 (1.8) and Aqp5 (5.0) as compared with the miR-control (red line). *P<0.05, **P<0.01, ***P<0.001 (ANOVA). Error bars indicate s.e.m. Scale bars: 20 μm in B; 50 μm in C.

Ivan T. Rebustini, et al. Development. 2012 January 1;139(1):191-202.

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