Journal: Genes & Development
Article Title: Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog
Figure Lengend Snippet: Analysis of chondrocyte proliferation in the limbs by BrdU labeling. ( A – D ) Immunohistochemistry of tibiae from BrdU-labeled wild-type ( A ), Runx2 –/– ( B ), Runx2 –/– 3 +/– ( C ), and Runx2 –/– 3 –/– ( D ) embryos at E18.5 using anti-BrdU antibody. The sections were counterstained with toluidine blue. Magnified views of the boxed regions ( a,b,c ) are shown in the same columns. ( A,B ) In wild-type and Runx2 –/– tibiae, the boxed regions a, b , and c represent resting, proliferating, and hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the boxed regions a, b , and c represent chondrocytes in the epiphyses, metaphyses, and diaphyses, respectively. The growth plates were well formed in wild-type tibiae ( A ) and in Runx2 –/– tibiae ( B ) but not in Runx2 –/– 3 +/– tibiae ( C ) or Runx2 –/– 3 –/– tibiae ( D ). ( Ab – Db ) The columnar alignment of chondrocytes, which is seen in the layer of proliferating chondrocytes, is well formed in the wild-type and Runx2 –/– tibiae, deformed in the Runx2 –/– 3 +/– tibiae, and completely absent in the Runx2 –/– 3 –/– tibiae. ( C,D ) The diaphyses of Runx2 –/– 3 +/– tibiae were composed of slightly enlarged chondrocytes, whereas the entire tibiae of Runx2 –/– 3 –/– embryos were composed of homogeneously small chondrocytes. ( E – I ) Measurement of the frequency of BrdU-positive cells ( E,F ), cell number ( G ), matrix area ( H ), and cell size ( I ) using the tibiae of six wild-type embryos, two Runx3 –/– embryos, six Runx2 –/– embryos, one Runx2 –/– 3 +/– embryo, and two Runx2 –/– 3 –/– embryos. We measured these parameters in each region shown in A – D . ( A,B ) In wild-type and Runx2 –/– tibiae, the regions I, II, and III represent resting, proliferating, and hypertrophic and terminal hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the regions I, II, and III were arbitrarily determined in the proximal half of the tibiae and represent chondrocytes in the epiphysial, metaphysical, and diaphysial parts of the tibiae, respectively. ( F ) We also counted the number of BrdU-positive cells in femurs, because chondrocyte maturation in Runx2 –/– femurs is more severely inhibited than that in Runx2 –/– ). ( F ) In the measurement of BrdU-positive cells in whole tibiae and femurs, we counted the number of BrdU-positive cells and the total number of cells in regions I and II in wild-type and Runx2 –/– mice and in regions I, II, and III in Runx2 –/– 3 +/– and Runx2 –/– 3 –/– mice, and the mean of the percentage of BrdU-positive cells ± standard deviation (std. dev.) is shown. We measured all of the parameters in three sections of each bone, and the mean ± std. dev. is shown. (*) p
Article Snippet: We processed the embryos for histological analysis and detected BrdU incorporation by immunohistochemistry using anti-BrdU antibody (Dako).
Techniques: Labeling, Immunohistochemistry, Mouse Assay, Standard Deviation