Figure 1.
miR-17-92 cluster is required for midface and palate morphogenesis.
(A–D) Scanning electron microscopy (SEM) of embryos with the designated genotypes and designated stages (frontal views). miR-17-92 mutants and miR-17-92; miR-106b-25 compound mutants exhibit broad orofacial morphogenesis defects including cleft lip (black arrows), mandibular hypoplasia (green arrows), and cleft palate (red arrowheads). (E–H) Histologic analysis with Hematoxylin-eosin (HE) staining of embryos with labeled genotypes and stages. Transverse sections of E13.5 mouse embryos (E, F) and coronal sections of E14.5 mouse embryos (G, H). Black arrows designate cleft lip and red arrowheads designate cleft palate. (I–L) Immunofluorescence (coronal sections) shows proliferation defects in E12.5 mutant embryos: proliferating cells (green)-stained with Phospho-Histon3 (PHH3); Nuclei (red)-stained with DAPI.
Figure 2.
Expression pattern of miR-17-92 at different embryonic stages.
β-Gal stained miR-17-92-lacZ BAC transgenic whole embryo (A), embryonic head (K) and craniofacial sections (I, J, L) with the designated stages. Arrowheads indicate positive structures. In situ analysis of pri-miR-17-92 (E, F) and mature miR-17 and miR-92a (B–D, G–H) at facial area. Boxed areas in C and G are correspondingly shown at higher magnification in D and H. e, eye; fl, fore-limb; fnp, frontonasal process; md, mandibular process; mnp, medial nasal process; mxp, maxillary process; lnp, lateral nasal process; ns, nose; t, tongue.
Figure 3.
miR-17-92 represses genes important for craniofacial development.
(A–D) Whole mount in situ hybridization with indicated probes in mouse embryos with designated genotypes. Black arrows and arrowhead designate expression pattern. (E) qRT-PCR data indicate that expression of Fgf10, Shox2, Tbx1, Tbx3 and Osr1 is elevated in miR-17-92; miR-106b-25 compound knock out mutants. (F) Overexpression of miR-17-92 in cranial neural crest results in repression of Fgf10, Shox2, Tbx1, Tbx3 and Osr1. (G) Luciferase reporter assays with Tbx3 reporters and miRs as labeled (See Fig. S5 for miR seed sites and mutations). mean ±s.e.m., * indicates statistically significant difference, Student's t-test (P<0.05).
Figure 4.
Bmp signals and AP-2α regulate the miR-17-92 complex in craniofacial structures.
(A–C) miR-17-92 overexpression rescue the orofacial cleft in Bmp mutant mice. Scanning electron microscopy (SEM) of embryos with the designated genotypes and designated stages (frontal views). Broad orofacial morphogenesis defects including severe bi-lateral cleft (black arrows) and mandibular hypoplasia (green arrows) are observed in a NestinCre, Bmp4 flox/flox, Bmp7 flox/+ (Bmp CKO) mutant embryo (B) while not in a control (A) and a NestinCre, Bmp4 flox/flox, Bmp7 flox/+, miR-17-92OE embryos (Bmp CKO, miR-17-92 OE) (C). (D–G) Bmp signals directly regulate miR17-92. (D) qRT-PCR data indicate that miR-17-92 was reduced in Bmp mutants. (E) In vivo Smad-ChIP-qPCR analysis of the regulatory region in the miR17-92 promoter. (F) miR-17-92 upstream Luc reporter activity co-transfected with constitutively active Alk3 (caAlk3). (G–L) AP-2α directly regulates miR-17-92. (G) qRT-PCR data indicate that mir-17-92 is reduced in AP-2α mutants. (H) ChIP-seq peaks indicate AP-2α binding regions in miR-17-92 chromatin. (I) AP-2α binding sites information based on overrepresented motifs enriched by ChIP-Seq of neural crest cells [16]. (J) Sequence alignment of two predicted AP-2α binding sites in region-2 of miR-17-92. (K) In vivo AP-2α ChIP-qPCR analysis. (L) Co-transfection data of miR-17-92 Luc reporter and AP-2α. mean±s.e.m., * indicates statistically significant difference, Student's t-test (P<0.05).