Scube gene function in tooth development.

<p>(A–I) <i>Scube</i> gene expression in the developing first molar at E14.5–16.5 assayed by <i>in situ</i> hybridization on frontal sections. (A–C) <i>Scube2</i>; (D–F) <i>Scube1</i>; (G–I) <i>Scube3</i>. (J–O) Ultra-structural view of incisor tooth development in wild type and <i>Scube3^−/−</i> mice at P18. In the incisor, amelogenesis commences apically and proceeds in an coronal direction, demonstrating (J, M) Secretion; (K, N) Maturation; (L, O), Post-maturation. These different stages of amelogenesis all show a characteristically normal morphology in wild-type and <i>Scube3^−/−</i> mice. a, ameloblasts; em, enamel matrix.</p

Skeletal morphogenesis in wild type and <i>Scube3^−/−</i> mice.

<p>Comparison of E17.5 wild type and <i>Scube3^−/−</i> skeletal structures differentially stained for bone (alizarin red) and cartilage (alcian blue). (A, B) Gross appearance of the skeleton, including sternum and forelimb; (C, D) Skull, including lateral view (norma lateralis, left panels), cranial base (norma basalis, middle panels; note, the mandible has been removed), cranial vault (norma verticalis, right panels; note, the cranial base and nasal bones have been removed); (E) Mandible (lateral view); (F) Middle ear skeletal elements (right ear in wild type; left ear in mutant). ab, alveolar bone of mandible; agp, angular process of mandible; au, autopod; bb, basal bone of mandible; bm, body of malleus; bo, basioccipital; bs, basisphenoid; cdp, condylar process of mandible; cli, crus longus of incus; cna, cupola nasi anterior; crp, coronoid process of mandible; cs, coronal suture; eo, exoccipital; fpmx, frontal process of maxilla; fr, frontal; go, gonial; hu, humerus; in, incus; ip, interparietal; jg, jugal; li, lower incisor; lo, lamina obturans; ma, mandible; MC, Meckel's cartilage; mm, manubrium of malleus; mx, maxilla; na, nasal; ns, nasal septum; pbm, processus brevis of malleus; pca, pars canalicularis; pbm, processus brevis of malleus; pco, pars cochlearis; pl, palatine; pmx, premaxilla; pp, parietal plate; ppmx, palatine process of maxilla; pppl, palatine process of palatine; pppx, palatal process of premaxilla; pr, parietal; ptg, pterygoid; ra, radius; sc, scapula; so, supraoccipital; sq, squamosal; st, stapes; tr, tympanic ring; ui, upper incisor; ul, ulna; vm, vomer; zpsq, zygomatic process squamosal.</p

Targeted disruption of the <i>Scube3</i> gene in mice.

<p>(A) Domain organization of Scube3 encoded by the <i>Scube3-001</i> splice variant. This protein consists of a signal peptide sequence (S), nine EGF-like repeat domains, a spacer region containing three cysteine-rich repeats, a CUB domain and C-terminal sequence; (B) The murine <i>Scube3</i>-<i>001</i> splice variant incorporates 6699 base pairs and contains 22 exons. Exon 1 encodes the signal peptide sequence, exons 2–10 encode the nine EGF-like domains, exons 11–18 encode the spacer region and cysteine residues in proximity to the CUB domain, exons 19 and 20 encode the CUB domain itself and exons 21–22 encode the amino acid sequence present at the C-terminal region of the protein, downstream of the CUB domain; (C) The <i>Scube3-004</i> splice variant incorporates 4103 base pairs and contains 18 exons. The splice variants are aligned to each other, meaning that exon 1 of <i>Scube-004</i> corresponds to exon 3 of <i>Scube-001</i> and so forth. This is interrupted by exon skipping at exon 16 in <i>Scube-001</i>, such that intron 13–14 of <i>Scube-004</i> corresponds to intron 15–16, exon 16 and intron 16–17 of <i>Scube3-001</i> (a total region of 714 base pairs). Exon 18 of <i>Scube3-004</i>, which corresponds in part, to exon 21 of <i>Scube-001</i> is also modified by an alternative 5′ splice site, incorporating 1684 ‘extra’ base pairs from the adjacent intronic sequence (intron 21–22 of <i>Scube-001</i>). Red boxes represent exons, white boxes represent untranslated regions of exon 1 and 22; (D) The targeting vector contains a ZEN-Ub1 cassette consisting of a <i>lacZ-p(A)</i> reporter and <i>hUbCpro-neo-p(A)</i> selectable marker flanked by <i>loxP</i> sites, which replaces the whole <i>Scube3</i> coding region following homologous recombination in ES cells (A–D are not to scale); (E) PCR analysis of DNA extracted from tail samples using wild type and mutant pairs of primers for wild type and mutant alleles, respectively. The wild type set of primers recognize a 25 base pair intronic sequence situated between exon 1 and 2 (blue arrows) and amplify a DNA fragment of 190 base pairs; whilst the mutant pair of primers amplify a 605 base pair DNA fragment within the Neo cassette (green arrows). For each sample, two separate PCR reactions were run (1) 100 base pair DNA ladder; (2) Negative control; (F) Western blot analysis reveals an absence of Scube3 in protein lysate derived from <i>Scube3^−/−</i> embryos. Protein lysates derived from human osteoblasts and <i>Scube3^WT</i> mouse embryos were used as positive controls. α-tubulin was used as a loading control. As predicted, the Scube3 protein detected in human osteoblasts and wild type embryos was approximately 110 kDa in size.</p

<i>Scube3^{+/tm1(KOMP)Vlcg}</i> reporter gene activity at E10.5 visualized by β-galactosidase staining.

<p>(A) Lateral view; (B) Frontal view (of the craniofacial region); (C) Posterior view. Note reporter expression in the central nervous system and ectoderm of the facial processes, otic vesicle and limb buds. Strong posterior-restricted expression is also seen in the limb bud mesenchyme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055274#pone.0055274-Xavier1" target="_blank">[13]</a> (only visible in the hindlimb of this sample). dd, dorsal dermomyotome; flb, forelimb bud; hb, hindbrain; hlb, hindlimb budlb, limb bud; mb, midbrain; md, mandibular process; mx, maxillary process; oe, oral ectoderm; ov, otic vesicle; pa2, pharyngeal arch 2; rh, rhombencephalon; vd, ventral dermomyotome.</p

Hedgehog signaling in wild type and <i>Scube3^−/−</i> mice.

<p>Comparison of <i>Ptch1</i> expression in the developing craniofacial region of wild type and <i>Scube3^−/−</i> mice at E14.5 assayed by <i>in situ</i> hybridization on frontal sections. (A–D) Wild type; (G–H) <i>Scube3^−/−</i>. Orientation is through the primary palate (A, E), central region (B, F) and posterior region (C, G) of the secondary palate. Red square in B, F highlights <i>Ptch1</i> expression in the cap stage maxillary molar teeth shown in D, H (red hatched line outlines the enamel organ).</p

Expression of <i>Scube3</i> and <i>Shh</i> in the early craniofacial region.

<p>Radioactive <i>in situ</i> hybridization on frontal sections. (A–D) <i>Scube3</i> expression. (A, B) At E10.5, expression is seen primarily in early ectoderm of the facial processes with transcripts also dispersed within the underlying mesenchyme; (C, D) At E11.5, expression is seen throughout the oral ectoderm, including early thickenings of the developing tooth germs, and in the craniofacial mesenchyme. (E–H) <i>Shh</i> expression. Regions of overlap between <i>Shh</i> and <i>Scube3</i> transcription are indicated by red arrows and include: (E, F) At E10.5, ectoderm of the medial nasal and maxillary processes in the midline; (G, H) At E11.5, ectoderm at the base of the nasal pits and early ectoderm of the developing teeth. fnp, fronto-nasal process; lnp, lateral nasal process; md, mandibular process; mdi, mandibular incisor tooth germs; mnp, medial nasal process; mx, maxillary process; mxm, maxillary molar tooth germs; np, nasal pit; oe, oral ectoderm.</p

Craniofacial development in wild type and <i>Scube3^−/−</i> mice.

<p>Histological comparison of the developing craniofacial region in wild type and <i>Scube3^−/−</i> mice at E13.5 and E15.5. (A–C) E13.5 wild type; (D–F) E13.5 mutant; (G–I) E15.5 wild type; (J–L) E15.5 mutant. All sections are orientated in the frontal plane and stained with Haematoxylin and Eosin. Orientation is through the primary palate (left panels), central region (middle panels) and posterior region (right panels) of the secondary palate. mdi, mandibular incisor tooth germs; mdm, mandibular molar tooth germs; mxi, maxillary incisor tooth germs; mxm, maxillary molar tooth germs; nc, nasal cavity; ns, nasal septum; ps, palatal shelf; t, tongue; vi, vibrissae; vno, vomeronasal organ.</p

Expression of <i>Scube3</i> and <i>Shh</i> at later stages of craniofacial development.

<p>Radioactive <i>in situ</i> hybridization on frontal sections. (A–E) <i>Scube3</i> expression. (A, B) At E13.5, expression is seen in ectoderm of the bud stage tooth buds, early palatal shelves and tongue; (C) At E14.5, expression is intense in the cap stage tooth germ; (D, E) At E15.5, strong expression continues in ectodermal tissues of the developing teeth, the vibrissae and cartilaginous condensations of the nasal cavity. (F–J) <i>Shh</i> expression. Regions of overlap are between <i>Shh</i> and <i>Scube3</i> are indicated by red arrows and include: (F, G) At E13.5, ectoderm of the early tooth buds and palatal shelves; (H) At E14.5, cells of the enamel knot within the cap stage tooth germs; (I–J) At E15.5, within the internal enamel epithelium of the bell stage tooth germs and the vibrissae. mdi, mandibular incisor tooth germs; mdm, mandibular molar tooth germs; mxi, maxillary incisor tooth germ; mxm, maxillary molar tooth germs; nc, nasal capsule; ps, palatal shelf; t, tongue; vi, vibrissae.</p

Shh and Fgf signaling in <i>Lrp4</i> and <i>Wise</i> mutants during palatal rugae development.

(A, B, Q, R) Sagittal sections of developing palate between midline and molar region showing in situ hybridization of Gli1 (A, B) and Erm (Q, R) in wild-type (A, Q), Shh-K14 (B) mice and Lrp4 mutant mice (R) at E13.5. Arrowheads indicating Erm expression in developing palatal rugae. (A) Box showing large magnification images of developing rugae region. (C, D) SEM image of palatal rugae of wild-type (C) and Shh-K14 (D) mice at birth. Arrowheads indicating palatal rugae presumptive region. (E-P) Whole mount explants showing Shh (E-N) and Erm (O, P) transcription in palatal rugae of wild-type (E, G, I, K, M, O), Lrp4 (N) and Wise (F, H, J, L, P) mutant mice at E12.5 (G, H), 13.5 (I, J, O, P), E14.0 (K, L) and E14.5 (E, F, M, N). Scale bar; 200μm.</p

<i>Wise</i> expression during palatal rugae development.

Diagrammatic representation of Wise expression during the developmen of palatal rugae. Light blue representing Wise expression. A, B; E11.5-E12.0, C; E12.0-E12.5, D; E12.5-E13.0, E; 13.0-E13.5, F; E13.5-E14.0, G; E14.5–15.0, H;16.0-E16.5. P2; Wise expression domain posterior to ruga 2, A8; Wise expression domain anterior to ruga 8, P4-1; Wise expression domain posterior to ruga 4, P4-2; Wise expression domain between P4-1 and A8 Wise expression domain, P6; Wise expression domain posterior to ruga 6.</p