Targeted Deletion of Btg1 and Btg2 Results in Homeotic Transformation of the Axial Skeleton

Abstract

Btg1 and Btg2 encode highly homologous proteins that are broadly expressed in different cell lineages, and have been implicated in different types of cancer. Btg1 and Btg2 have been shown to modulate the function of different transcriptional regulators, including Hox and Smad transcription factors. In this study, we examined the in vivo role of the mouse Btg1 and Btg2 genes in specifying the regional identity of the axial skeleton. Therefore, we examined the phenotype of Btg1 and Btg2 single knockout mice, as well as novel generated Btg1-/-;Btg2-/- double knockout mice, which were viable, but displayed a non-mendelian inheritance and smaller litter size. We observed both unique and overlapping phenotypes reminiscent of homeotic transformation along the anterior-posterior axis in the single and combined Btg1 and Btg2 knockout animals. Both Btg1-/- and Btg2-/- mice displayed partial posterior transformation of the seventh cervical vertebra, which was more pronounced in Btg1-/-;Btg2-/- mice, demonstrating that Btg1 and Btg2 act in synergy. Loss of Btg2, but not Btg1, was sufficient for complete posterior transformation of the thirteenth thoracic vertebra to the first lumbar vertebra. Moreover, Btg2-/- animals displayed complete posterior transformation of the sixth lumbar vertebra to the first sacral vertebra, which was only partially present at a low frequency in Btg1-/- mice. The Btg1-/-;Btg2-/- animals showed an even stronger phenotype, with L5 to S1 transformation. Together, these data show that both Btg1 and Btg2 are required for normal vertebral patterning of the axial skeleton, but each gene contributes differently in specifying the identity along the anterior-posterior axis of the skeleton.

Fig 1. Characterization of Btg1^-/-, Btg2^-/- and Btg1^-/-;Btg2^-/- mice.

(A) The mouse Btg1 gene is disrupted by insertion of a neomycin resistance cassette via SacII restriction sites in the first exon. The second exon of the Btg2 gene is replaced by a neomycin cassette in the antisense direction. The arrows indicate the position of primers used for genotyping. (B) Genotyping of mice was performed by PCR on genomic DNA using primers specific for the Btg1 and Btg2 wild-type (WT) and knockout (KO) allele. (C) Number of pups obtained from wild-type, heterozygous and homozygous Btg1, Btg2 and Btg1xBtg2 breedings. Data are from at least 4 independent crossings. *, P< 0.05, **, P< 0.01, ***, P< 0.001.

Fig 2. Skeletal abnormalities within the cervical-thoracic region in mice deficient for Btg1 and Btg2.

(A-D) Lateral view of the skeletons from 18.5 dpc wild-type, Btg1^-/-, Btg2^-/- and Btg1^-/-;Btg2^-/- embryos, which were stained with alizarin red and alcian blue. (A-C) Wild-type C57Bl6/J mice show a rib anlage at C7 (indicated by asterisk), whereas Btg1^-/- and Btg2^-/- mice display a partial rib at C7 (indicated by asterisk). The extra formed T1 rib in Btg1 knockout animal is fused to T2 rib. (D) Btg1^-/-;Btg2 ^-/- double knockout mice display a full rib at C7 that is directly attached to the sternum (indicated by asterisk). (B and D) Btg1^-/- and Btg1^-/-;Btg2^-/- embryos show often a reduction in the amount of ossified sternebrae (indicated by arrow) compared to wild-type. (C) Btg2-deficient mice display occasionally an extra sternebra (indicated by arrow).

Fig 3. Targeted deletion of Btg1 and Btg2 results in malformation of the sternum.

(A-B) Ventral view of the sternum with attached ribs of 18.5 dpc wild-type and Btg1^-/-;Btg2^-/- embryos stained with alizarin red and alcian blue. Wild-type C57BL6/J mice display normal ossification of the five sternbrae, whereas an asymmetric pattern of ossification of the sternebrae is observed in mice lacking both Btg1 and Btg2 expression. The xiphoid process (Xip) of the sternum is not affected in Btg1- and Btg2-deficient mice.

Fig 4. Posterior homeotic transformation of the thirteenth thoracic vertebra in mice deficient for Btg2.

(A-D) Dorsal view of the cervicothoracic region of the skeleton in 18.5 dpc wild-type, Btg1^-/-, Btg2^-/- and Btg1^-/-;Btg2^-/- embryos stained with alizarin red and alcian blue. (A-B) Wild-type C57BL6/J mice have thirteen thoracic ribs, while Btg1-deficient mice display fourteen ribs due to the extra extensive rib at C7. (D) Although the T13 rib is absent in Btg1^-/-;Btg2^-/- mice they still have thirteen thoracic ribs due to the C7 to T1 posterior transformation.

Fig 5. Btg1;Btg2 double knockout mice display posterior homeotic transformation at the lumbo-sacral transition.

Skeletal defects in 18.5 dpc wild-type, Btg1^-/- and Btg1^-/-;Btg2^-/- embryos. (A-C) Dorsal view showing the lumbar-sacral regions. (B) Btg1^-/-;Btg2^-/- mice frequently show five lumbar vertebrae compared to six in wild-type mice. (C) Btg1^-/- mice may show asymmetric L6 in which the right side (indicated by arrow) indicates a lumbar vertebra and the left side a sacral vertebra.

Fig 6. Overview of the skeletal phenotypes in Btg1- and Btg2-deficient mice.

Axial vertebrae are indicated by different colors: green (cervical), yellow (thoracic), blue (lumbar) and red (sacral). The thoracic ribs are indicated by the grey horizontal lines, of which T1-T7 form sternocostal junctions with the sternum. Btg1- and Btg2-deficient mice display C7 to T1 posterior transformation, while Btg1 ^-/- mice show also partial or complete L6 to S1. Btg2^-/- mice display in most cases T13 to L1 and complete L6 to S1 transformations. Btg1^-/-,Btg2^-/- double knockout animals display a more pronounced phenotype with C7 to T1, T13 to L1 and L6 to S1 homeotic transformations.