Figure 4

Functional screening of miRNAs in molar tooth germ explants and validation of miRNA target genes.

(a–e) Representative molar tooth germ phenotypes generated by miRNA antagomir transfections into mandibular explants. Coronal sections (10 μ) of molar tooth germs show nuclear labeling with DAPI (blue), epithelial E-cadherin immunostaining (red) and EdU incorporation (green). Note increased proliferation (EdU) in the lingual (L) side to the tooth germ (in b,c,e) and the progression of epithelial morphogenesis (from cap to bell stage) after targeting miR-429-3p (b), miR-325-3p (c) and miR-200c-3p (e), whereas epithelial proliferation and morphogenesis were inhibited with miR-590-5p antagomir transfection, compared to off-target antagomir (a). (f) Coronal and sagittal sections of E13.5 mandible explants showing molar tooth germ epithelium (EpiCam, green) and nuclei (DAPI, red). Defects in molar tooth germ morphogenesis after 48 h of antagomir transfection targeting miR-590-5p (Anta-590) include lack of the normal cap stage morphology observed in the Anta-off-target control and arrest of tooth morphogenesis at the bud stage after transfection of the miRNA-590-5p mimic (arrow in the lower panel). B and L correspond to buccal and lingual orientation and P and A to posterior and anterior orientation, respectively. Scale bar: 100 μ. (g–h) qRT-PCR analysis shows significantly increased expression for the miR-590-5p targets Chd7, Msx1 and Bcl11b, 24 h after antagomir transfection (g) and corresponding decreases in expression after mimic transfections (h). Graphs represent fold-change in gene expression normalized to the control housekeeping gene Rps29 and to an off-target antagomir transfection group (red dotted lines). (i) qRT-PCR analysis shows decreased fold change in miR-590-5p expression after transfection of the corresponding antagomir (A-590) and increased detection of miR-590-5p after mimic transfection (M-590). A-Off: Antagomir off-target; M-Off: Mimic off-target. Graph shows relative fold-change in qRT-PCR expression, normalized to control non-coding RNA (snoRD61) and to an off-target antagomir control (red dotted line). (j) Enrichment of miR-590-5p targets in the pulldown fraction after transfecting a biotinylated miR-590-5p mimic in E13.5 mandible explants (24 h) and performing the pulldown (miR-PD) assay. Graph shows relative percent enrichment for miR-590-5p predicted targets Chd7, Msx1 and Bcl11b, compared to non-target genes (Shh, Sox2, Bmp4). Details on qRT-PCR data normalization are in Suppl. Fig. 2.