Electrophoretic mobility shift assay (EMSA) of <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i>.

<p>(A) The <i>Tbx6</i>^<i>Oune</i> allele did not influence the DNA binding ability of the T-box binding consensus sequences. <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i> (Tbx6 mut) showed no difference in binding ability to the Tbind probe, two T-box gene binding sites, and to the Tbind-half probe, a single binding site, judged from intensity of shifted bands. Free binding probes were shown at the bottom. (B) DNA binding ability of mutant Tbx6 was not changed. The Tbind probe concentration was diluted to one sixteenth, but no difference was detected between Tbx6-wt and mut. mTbx6 and rTbx6 represents mouse and rat Tbx6, respectively.</p

Notochord and floor plate patterning in <i>Oune/+</i> embryos.

<p><i>In situ</i> hybridization analysis was performed on transverse sections through the trunk of E12.5 embryos using RNA probes for <i>Brachyury</i> (A, B), a notochord marker, and <i>Foxa2</i> (C, D), a floor plate marker. No expression change was observed between <i>+/+</i> and <i>Oune/+</i> embryos. Notochord (NC) is indicated by arrowheads and floor plate (FP) is enclosed by yellow lines. Scale bar, 200 μm.</p

Translational efficiency and transcription activation ability of <i>Tbx6</i> proteins.

<p>(A) Western blotting analysis of S protein-tagged mTbx6 proteins. Cell lysate from transfectant of the <i>mTbx6</i> and <i>mTbx6</i>^<i>Oune</i> expression constructs (Tbx6 and Tbx6 mut, respectively), in which the coding region of wild type and <i>Oune</i> mutant <i>mTbx6</i> are tagged with partial S protein sequences in N-terminus, was used with anti-S protein (the upper panel) and anti-USF2 (the lower panel) antibodies. Signals of S protein tagged mTbx6 are indicated by the arrow. (B) <i>In vitro</i> translation assays for mTbx6 and mTbx6 mut. Difference of translational efficiency between wild type and mutant mTbx6 was not observed. The S-tag mTbx6 constructs showed multiple translational initiations (tagged protein: asterisk). (C) Transcriptional activation properties of <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i> using a <i>Mesp2</i> promoter-luciferase reporter construct. <i>rTbx6</i>^<i>Oune</i> activates transcription less effectively than <i>rTbx6</i> when a Notch intracellular domain (NICD) expression construct was cotransfected into C2C12 cultured cells. (D) Transcriptional activation properties of a mixture of <i>Tbx6</i> and <i>Tbx6</i>^<i>Oune</i> constructs. Half-and-half of rTbx6 and rTbx6 mut constructs with NICD showed intermediate levels of luciferase activities. Assays were performed in triplicate. One-way analysis of variance was performed on data from all experiments, and significance was determined using Turkey's post hoc test. ns, not significant. mTbx6 and rTbx6 represents mouse and rat Tbx6, respectively.</p

Morphological abnormalities of vertebral column in ENU-induced <i>Oune</i> rats.

<p>(A, B) Perinatal and newborn offspring derived from (<i>Oune/+</i> x <i>+/+</i>) mating pairs. <i>Oune</i> rats were distinguished from their siblings because of kinky tails (yellow bar and arrowheads). (C-F) Ventral view of axial skeletons of newborn wild type and <i>Oune/+</i> siblings. In the cervical and thoracic region, <i>Oune/+</i> rats showed loss and malformations of vertebrae (D, boxes). In the lumbar and sacral region of <i>Oune/+</i> animals, vertebrae were malformed and laterally bent (F, asterisks). An extra lumbar vertebra was frequently observed (F, L7). (G-J) Ventral view of axial skeletons of E15.5 <i>Oune</i> siblings. Wild type embryos showed ordered thoracic vertebral blocks along the anterior-posterior axis (G, bars). In <i>Oune/Oune</i> embryos, vertebral blocks were located along two different axes (H, I bars) with loss of rib formation. Original magnification: 12.5x (C, D); 10x (E, F); 32x (G-I).</p

Altered expression of Notch pathway components in <i>Oune/+</i> embryos.

<p>Whole mount <i>in situ</i> hybridization with rat <i>Tbx6</i> (A, B), <i>Mesp2</i> (C, D), and <i>Dll1</i> (E, F) probes was performed using E12.5 +/+ and <i>Oune/+</i> embryos. Obvious changes of expression patterns and intensity were not observed. Red boxes (A, B) and arrowheads (C, D) indicate <i>Tbx6</i> expression in tail bud and <i>Mesp2</i> expression in presomitic mesoderm, respectively. The contiguous expression of <i>Dll1</i> in the presomitic mesoderm (bars, E, F) appear extended anteriorly in heterozygous mutant embryos. Arrows indicates expression borders between somites and presomitic mesoderm. Note that low levels of <i>Dll1</i> expression were observed in mutants. Original magnification: 20x (A, B); 90x (C, D); 40x (E-F).</p

Somite pattering in <i>Oune/+</i> embryos.

<p>Sagittal sections of E14.5 +/+ and <i>Oune/+</i> embryos were used for hematoxylin and eosin staining (A, B) and <i>in situ</i> hybridization with various somite markers, <i>Pax1</i> (C-H), <i>Uncx4</i>.<i>1</i> (I-N), and <i>Dll1</i> (O-T). Incomplete somite patterning in the anterior region of <i>Oune/+</i> embryos was observed (B, box a). In the same embryo, somites in the trunk and posterior regions were morphologically normal (B, boxes t and p). In <i>Oune/+</i> embryos, <i>Pax1</i> expression was decreased in the anterior region (F), and somites were dislocated in the posterior region (H). Signals of markers for the caudal half of somites, <i>Uncx4</i>.<i>1</i> and <i>Dll1</i>, were reduced in the anterior and posterior regions of <i>Oune/+</i> embryos (L and N: <i>Uncx4</i>.<i>1</i>; R and T: <i>Dll1</i>). Scale bar, 200 μm.</p