Fig 1.
Vertebra defects in adult mice.
A, B) Alizarin red stained cervical vertebrae from wild type and Tgif1 null mice are shown. Note the absence of the dorsal spine on C2 in the Tgif1 null (A, arrowed in wild type), and the ectopic bone in C1 (arrowhead in A) in the Tgif1 null. In B, the partially fused C4 and C5 are indicated by a bracket, and T2 is indicated by an arrow. C) Separated alizarin red stained vertebrae, from C1 to T3 are shown from representative mice. The phenotypes of the mice shown in C and S1 Fig are listed in panel D, and summarized in panel E.
Fig 2.
Rib and vertebra defects in Tgif1 mutant E18.5 embryos.
E18.5 embryos stained with alizarin red and alcian blue are shown focused on the cervical vertebrae or upper thoracic region. A-D) Tgif1 mutant embryos with defects in cervical vertebra are shown. Affected vertebrae are indicated with numbers, where 2/3 and 3/4 represent fused vertebrae. E) A wild type embryo with the anterior most rib on T1. F, G) Tgif1 mutant embryos with ectopic ribs on C7 (arrows), which join either the lower rib (F) or the sternum (G). H) A Tgif1 mutant embryos with an ectopic C7 rib that joins both sternum and the lower rib at the junction between bone and cartilage (arrows). I, J) Examples of Tgif1 null embryos with posterior transformation (arrows) and cervical vertebra defects (stars). C7 and T1 vertebrae are indicated in panels E-J. The same embryo is shown in panels B and I.
Fig 3.
Rib defects in Tgif1 and Tgif2 mutants.
A) The number (and percentage) of embryos of each genotype with defects the cervical vertebrae are summarized. Defects observed were fusion of two adjacent vertebrae (fused, in figure), or loss of one vertebra on one side of the embryo (partial, in figure). B) Axial patterning defects are summarized for each genotype. Embryos with C6, T14, L6 are separated out depending on whether this posterior transformation was seen on both sides or on left (L) or right (R) only. The percentage with any defect is shown. C) The normal (C7, T13, L6) and defective (C6, T14, L6) patterns are shown schematically. D) Co-occurrence of the posterior transformation phenotype and defects in the cervical vertebrae is summarized for Tgif1 null embryos.
Fig 4.
Normal somites in Tgif1 null embryos.
Wild type and Tgif1 null embryos at E9.5-E10.0 (as indicated) were analyzed by whole-mount in situ hybridization, for Uncx4.1 (A) and Myogenin (B). A higher resolution image of part of the embryos is shown below.
Fig 5.
Wild type and Tgif1 null embryos at E9.5 were analyzed by whole-mount in situ hybridization, for Hoxc6. A higher resolution image of part of the embryos is shown to the right. The bracket indicates the extent of strong Hoxc6 staining in the somites, and the arrows indicate somite boundaries.
Fig 6.
Altered Hoxc6 expression in Tgif1 null embryos.
Wild type and Tgif1 null embryos at E10.5 were analyzed by whole-mount in situ hybridization, for Hoxc6 and Myogenin together (A) and for Hoxc8 and Myogenin (B). A higher resolution image of part of the embryos is shown below. The stripes of Myogenin expression in somites 8 and 12 are indicated by stars, and the approximate anterior boundaries of Hoxc6 and Hoxc8 expression are shown by arrows. C) The anterior boundaries of Hoxc6 and Hoxc8 expression in all embryos analyzed are shown schematically, together with the median boundaries (vertical lines: red—Tgif1 null, blue—wild-type).
Fig 7.
Wild type and Tgif1 null embryos at E10.5 were analyzed by whole-mount in situ hybridization, for Hoxc5. Images of representative embryos are shown, with a higher resolution image of part of the embryos below. Two of four wild type embryos had this expression pattern, with the other two having slightly higher staining in somite 9 on one side of the embryo. Of the five Tgif1 null embryos, three had the pattern shown and the remaining two had slightly weaker expression in somite 9 on one side of the embryo. Somites 8 and 12 are indicated by stars, and the arrowhead shows the expression in somite 9.
Fig 8.
Combination of Tgif1 and Tgif2 mutations.
A) The axial formulas and the presence of cervical vertebra defects is shown for E18.5 embryos from Tgif1;Tgif2 double heterozygous intercrosses. # = the embryo number. For example a total of 12 double heterozygous embryos were analyzed, with embryos 1 to 4 being normal. The phenotypes for embryos 5 to 12 are listed individually. B) The percentage of embryos of the indicated genotypes with either normal or posterior transformation is shown, based on the embryos detailed in 3B and 8A. C) Data for E18.5 embryos from Tgif1 heterozygous intercrosses, in which the embryos were exposed to 10mg/kg retinoic acid (ATRA) at E8.5. D) the percentage of embryos with different axial formulas are shown for embryos from Tgif1 intercrosses (from 3B) compared to those treated with ATRA.