Tuckermann J P, Pittois K, Partridge N C, Merregaert J, Angel P
Division of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
J Bone Miner Res. 2000 Jul;15(7):1257-65. doi: 10.1359/jbmr.2000.15.7.1257.
Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo.
软骨内成骨需要软骨细胞系和成骨细胞系细胞的作用，这些细胞在此过程中会持续分化。为了鉴定参与骨形成的静止、增殖和肥大软骨细胞以及成骨细胞亚群，我们在此鉴定了软骨内和膜内成骨中存在的两个新的标记基因。我们使用Northern印迹分析以及对小鼠胚胎和新生小鼠骨骼的平行切片进行原位杂交，比较了最近克隆的Itm2a和MMP-13（胶原酶-3）基因与已确立的骨形成标记基因（如碱性磷酸酶（ALP）、骨钙素（OC）和X型胶原）在软骨内和膜内成骨过程中的表达模式。在胚胎发育过程中，Itm2a和ALP的表达在妊娠中期（交配后11.5天[dpc]）即可检测到，并持续增加直至16.5 dpc。MMP-13和OC的表达分别始于14.5 dpc和16.5 dpc。这种时间表达反映在这些标记物在长骨生长板中的空间分布上。在经历软骨内成骨的区域，Itm2a表达见于静止区和增殖区的软骨细胞。ALP和MMP-13的表达相互排斥：仅在上层区域X型胶原阳性的肥大软骨细胞中发现ALP转录本。MMP-13的表达仅限于也表达X型胶原的肥大软骨下层区域的软骨细胞。在参与软骨内和膜内成骨的成骨细胞中不存在Itm2a。ALP、MMP-13和OC在这些细胞中相互排斥表达，提示ALP、MMP-13和OC存在依赖分化的顺序表达。Itm2a、ALP、MMP-13和OC连续顺序表达的鉴定，将使我们能够建立一系列非常适合在体内软骨细胞和成骨细胞分化过程中表征骨细胞的标记基因。
