Fig 1.
Morphological abnormalities of vertebral column in ENU-induced Oune rats.
(A, B) Perinatal and newborn offspring derived from (Oune/+ x +/+) mating pairs. Oune 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 Oune/+ siblings. In the cervical and thoracic region, Oune/+ rats showed loss and malformations of vertebrae (D, boxes). In the lumbar and sacral region of Oune/+ 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 Oune siblings. Wild type embryos showed ordered thoracic vertebral blocks along the anterior-posterior axis (G, bars). In Oune/Oune 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).
Table 1.
Statistics of skeletal phenotypes of new born animals from crosses between Oune heterozygote and F344/NSlc wild type.
Fig 2.
Positional candidate cloning of the causative gene for Oune.
(A) Genetic mapping of the Oune locus indicates that the critical interval is located between D1Mgh36 and D1Got163 on rat chromosome 1. Oune/+ rats, originally derived from the F344 strain, were crossed to the Bs/Ss strain, and overall 202 N2 animals were genotyped using 50 genome-wide microsattelite markers. (B) T to A transversion at the 521st base pair of Tbx6 transcripts in Oune rats. The point mutation was identified from candidate sequencing of exons in the critical region. (C) The Tbx6Oune mutation causes an amino acid exchange in the T-box, the DNA binding domain, near a predictable dimerization interface (Up, arrowhead). Alignment of amino acid sequences among vertebrate and protochordate T-box genes (Down). The 174th isoleucine of rat Tbx6 (arrowhead), which is exchanged to asparagine in Oune, is conserved among mouse, human, Xenopus, and zebrafish Tbx6 and mouse Brachyury (mT). In ascidian T (As-T, As-T2) the isoleucine is changed to valine and leucine, but they are, like isoleucine, hydrophobic amino acids.
Fig 3.
Somite pattering in Oune/+ embryos.
Sagittal sections of E14.5 +/+ and Oune/+ embryos were used for hematoxylin and eosin staining (A, B) and in situ hybridization with various somite markers, Pax1 (C-H), Uncx4.1 (I-N), and Dll1 (O-T). Incomplete somite patterning in the anterior region of Oune/+ 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 Oune/+ embryos, Pax1 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, Uncx4.1 and Dll1, were reduced in the anterior and posterior regions of Oune/+ embryos (L and N: Uncx4.1; R and T: Dll1). Scale bar, 200 μm.
Fig 4.
Notochord and floor plate patterning in Oune/+ embryos.
In situ hybridization analysis was performed on transverse sections through the trunk of E12.5 embryos using RNA probes for Brachyury (A, B), a notochord marker, and Foxa2 (C, D), a floor plate marker. No expression change was observed between +/+ and Oune/+ embryos. Notochord (NC) is indicated by arrowheads and floor plate (FP) is enclosed by yellow lines. Scale bar, 200 μm.
Fig 5.
Altered expression of Notch pathway components in Oune/+ embryos.
Whole mount in situ hybridization with rat Tbx6 (A, B), Mesp2 (C, D), and Dll1 (E, F) probes was performed using E12.5 +/+ and Oune/+ embryos. Obvious changes of expression patterns and intensity were not observed. Red boxes (A, B) and arrowheads (C, D) indicate Tbx6 expression in tail bud and Mesp2 expression in presomitic mesoderm, respectively. The contiguous expression of Dll1 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 Dll1 expression were observed in mutants. Original magnification: 20x (A, B); 90x (C, D); 40x (E-F).
Fig 6.
Electrophoretic mobility shift assay (EMSA) of Tbx6 and Tbx6Oune.
(A) The Tbx6Oune allele did not influence the DNA binding ability of the T-box binding consensus sequences. Tbx6 and Tbx6Oune (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.
Fig 7.
Translational efficiency and transcription activation ability of Tbx6 proteins.
(A) Western blotting analysis of S protein-tagged mTbx6 proteins. Cell lysate from transfectant of the mTbx6 and mTbx6Oune expression constructs (Tbx6 and Tbx6 mut, respectively), in which the coding region of wild type and Oune mutant mTbx6 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) In vitro 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 Tbx6 and Tbx6Oune using a Mesp2 promoter-luciferase reporter construct. rTbx6Oune activates transcription less effectively than rTbx6 when a Notch intracellular domain (NICD) expression construct was cotransfected into C2C12 cultured cells. (D) Transcriptional activation properties of a mixture of Tbx6 and Tbx6Oune 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.