Figure 1.
Runx2 gene construct and splicing patterns.
Runx2 transcription is induced by distal and proximal promoters (P1 and P2, respectively). Although the first exon of Runx2-II contains a 197-bp mini-intron, the two parts of the exon are often shown as a single exon (exon 1), as we have done here (gray box). Exon 2, the first exon for Runx2-I consists of a Runx2-I-specific sequence (black box; includes a coding sequence that is translated into five amino acids unique to Runx2-I, MRIPV) and a common sequence (white box; corresponds to the second exon of Runx2-II).
Figure 2.
Generation of Runx2-I-targeted mice.
(A) Schematic representation of the targeting strategy. A targeting construct was designed to replace the start codon (ATG) of Runx2-I with a stop codon (TGA). Relevant EcoR V (EV) recognition sites are indicated. (B) PCR analysis of the indicated Runx2-I-targeted mice. The primers depicted in panel (A) were used to detect the wild-type allele (WT-Fw and Rv) or the mutated allele (Neo-Fw and Rv). The PCR products were 1.1 kb in size for both the wild-type and the mutated allele. +/+, wild-type; neo/+, heterozygous; neo/neo, homozygous. (C) Confirmation of recombination by Southern blot analysis using the genome of Runx2-I-targeted littermates after digestion by EcoR V. The membrane was blotted with the probe shown in panel (A). The size of the band was 7.2 kb in the wild-type allele and 2.2 kb in the mutated allele. (D) Sequence analysis of the first ATG of Runx2-I in the mice of each genotype. The genome sequence was amplified by using the set of primers flanking the first ATG region, and the amplified fragment was read by the DNA sequencer.
Figure 3.
The neo transgene perturbed Runx2 expression.
(A) Expression of total Runx2, Runx2-I, and Runx2-II in Runx2-I-mutated mice. Expression was analyzed by quantitative real-time PCR using RNA prepared from E18.5 calvarial cells of wild-type (+/+), Runx2-Ineo/+ (neo/+) and Runx2-Ineo/neo (neo/neo) mice. The results were normalized against the expression of Gapdh. Data are representative of three independent experiments and are means ± SEM (n = 3/group); *p<0.05, **p<0.01. (B) PCR primers (arrows) were placed on Runx2 exons and the neo transgene. (C) Semiquantitative RT-PCR analysis of RNA prepared from E18.5 calvarial cells of wild-type (+/+) and Runx2-Ineo/neo (neo/neo) mice by using the primer set depicted in panel (B). Gapdh was simultaneously amplified as an internal control (lanes 9 and 10). (D) Schematic diagram of alternative splicing pattern for Runx2-neo fusion transcripts. (E) Sequences of the fusion transcripts and proteins. The nucleotide sequence shown in italics is from neo, and the asterisk indicates the stop codon in the reading frame. The amino acids corresponding to each codon, and their numbers in the sequence of each Runx2 isoform are also indicated.
Figure 4.
The lethality in Runx2-Ineo/neo mice was rescued by neo cassette deletion.
Runx2-Ineo/+ mice crossed with a Cre deleter mice to remove the neo cassette. (A) Schematic illustration of depletion of the neo cassette. Relevant EcoR V (EV) and Sal I (SI) recognition sites are indicated. (B) Deletion of the neo cassette was confirmed by PCR. The primers depicted in panel (A) were used to detect the wild-type allele (WT-Fw and Rv) and the neo-deleted allele (loxP-Fw and Rv). The PCR products were 1.1 kb in size for both the wild-type and the neo-deleted allele. +/+, wild-type; TGA/+, heterozygous; TGA/TGA, homozygous. (C) Southern blot analysis using the genome of the indicated Runx2-I-targeted littermates after digestion by EcoR V and Sal I. The membrane was blotted with the probe shown in panel (A). The size of the band was 7.2 kb in the wild-type allele and 1.9 kb in the neo-deleted allele. (D) Fetal calvarial cells were prepared from generated mice. RNA was purified from mice of each genotype and then analyzed by quantitative real-time PCR for expression levels of total Runx2, Runx2-I, and Runx2-II. Data are representative of three independent experiments and are means ± SEM (n = 3/group).
Figure 5.
Runx2-I was expressed in Runx2-ITGA/TGA mice as N-terminal truncated Runx2.
(A) Anti-Runx2 mAb (clone: 8G5) could recognize Runx2-II, Runx2-I, and their truncated versions. HEK293 cells were transiently transfected with the indicated expression vectors (see Materials and Methods). Forty-eight hours after transfection, nuclear proteins were obtained from the cells and then immunoblotted with anti-Runx2 mAb. (B) Nuclear proteins of calvarial cells freshly prepared from embryos (18.5 dpc) with the genotypes indicated in the figure were immunoblotted with anti-Runx2. The membranes were also blotted with anti-Lamin A/C mAb as an internal control. The data are representative of three independent experiments.
Figure 6.
Osteoblastic gene expression profile during fetal development.
Pregnant C57BL/6 mice were euthanized for examination of fetuses on days 11, 13, 15, 16, 17, and 18 postcoitum. The calvaria of fetuses were enzymatically digested, and collected cells were subjected to the quantitative real-time PCR analyses. Because the fetuses at days 11 and 13 did not yet have firm calvarial structures, we instead used the upper part of head tissue, which might have contained cells other than calvarial cells (e.g., brain and skin cells). Data are means ± SEM (n = 7–9/group).
Figure 7.
Osteoblastic gene expression was diminished in Runx2-Ineo/neo calvarial cells.
Expression of the indicated osteoblastic genes in calvarial cells (A and C) and osteoblast differentiated cells (B and D) from Runx2-I-mutated (A and B; +/+, neo/+, neo/neo, C and D; +/+, TGA/+, TGA/TGA). Quantitative real-time PCR analysis was performed by using the same RNA used for the analyses shown in Figure 3A (neo-possessing mice) and Figure 4D (neo-deleted mice). Data are representative of three independent experiments and are means ± SEM (n = 3/group); *p<0.05, **p<0.01.
Figure 8.
Runx2-Ineo/neo mice showed severely delayed bone ossification.
Skeletal tissues of E18.5 fetuses of wild-type (+/+), Runx2-Ineo/+ (neo/+) and Runx2-Ineo/neo (neo/neo) mice were stained with Alcian blue to detect cartilage, and Alizarin red to detect calcified bone. After whole-body images were captured (A), specimens were separated into parts: (B) clavicle and forelimb; (C) skull; (D) hyoid bone; (E) sternum and rib.
Figure 9.
Runx2-Ineo/neo mice showed decreased bone density.
(A) Mineralization of E18.5 fetuses of wild-type (+/+), Runx2-Ineo/+ (neo/+) and Runx2-Ineo/neo (neo/neo) mice was assessed by microcomputed tomography of the whole body (upper panels) and the skull (lower panels). Scale bars: 5 mm. (B) Quantitative bone mineral parameters were calculated from femur measurements. All the parameters of Runx2-Ineo/neo mice showed significantly severe deficiency of bone formation. Interestingly, Runx2-Ineo/+ mice did not exhibit any abnormalities compared with wild-type littermates. Data are means ± SEM (n = 4–9/group); *p<0.05, **p<0.01.
Figure 10.
Calvarial cells of Runx2-Ineo/neo mice showed defective osteoblast development.
Calvarial cells recovered from wild-type (+/+) and Runx2-Ineo/neo (neo/neo) mice were cultured in osteogenic medium for 14 days and then fixed and stained with Alizarin red to visualize bone nodule formation. Stained culture wells (A) and numbers of bone nodules normalized by culture area (B). Data are means ± SEM (n = 2–6/group); *p<0.05. (C) At the same time that the Alizarin red assay was conducted, cells were lysed, and alkaline phosphatase activity was tested. Data are representative of four independent experiments and are means ± SEM (n = 3–7/group); *p<0.05.