Alborzi A, Mac K, Glackin C A, Murray S S, Zernik J H
University of Connecticut Health Center, Farmington, USA.
J Craniofac Genet Dev Biol. 1996 Apr-Jun;16(2):94-106.
We have previously studied the expression of alkaline phosphatase (ALP) and alpha2(I) collagen (two phenotypic markers of osteoblastic cell differentiation) during development of the rat mandible, and the spatial and temporal distribution of the respective transcripts. Our current studies utilize the rat mandible and hind foot as in vivo model systems to investigate the relationship between osteoblastic differentiation and proliferation during intramembranous and endochondral bone formation. Pregnant rats, at 15.17, and 19 days of gestation were intraperitoneally injected with various doses of [3H]-thymidine, and sacrificed at various time intervals in order to label dividing embryonic osteoblastic and preosteoblastic cells. Cross sections through the mid-body of 15-day embryos showed [3H-thymidine dose-dependent labeling of a relatively high percentage of cells in the liver (49 +/- 8% at 440 muCi) and a lower percentage of cells of the developing vertebral cartilage (29 +/- 6% at 440 muCi). ALP-positive condensed mesenchyme--consisting of mandibular preosteoblast (15 days of gestation) showed a relatively high (32 +/- 5%) level of [3H]-thymidine labeling, compared to surrounding ALP-negative loose mesenchymal cells (22 +/- 1%). Similar results were observed in the developing hind foot of 19-day embryos for ALP-positive cells (15 +/- 6%) and surrounding ALP-negative cells (13 +/- 5%). In both the hind foot and the mandible an overall decrease in labeling was observed during bone development. RNA samples from these tissues show increasing amounts of ALP mRNA, and decreasing amounts of histone H4 mRNA between days 15 and 19 of gestation. These data indicate that a general inverse correlation between osteoblastic differentiation and proliferation, similar to the correlation previously described in cultured osteogenic cells, is also present in developing bones in vivo. However, these results indicate that ALP-positive preosteoblasts, committed to the osteoblastic lineage, maintain their proliferative capacity. In an attempt to elucidate underlying molecular mechanisms, we further investigated the levels of expression of m-twist in these tissues. This member of the basic helix-loop-helix family of transcription regulators has been previously implied as playing a role in osteoblast differentiation in culture. Our results demonstrate a decrease in m-twist levels during bone development in both the mandible and the hind foot.
我们之前研究了大鼠下颌骨发育过程中碱性磷酸酶（ALP）和α2（I）型胶原蛋白（成骨细胞分化的两个表型标志物）的表达，以及各自转录本的时空分布。我们目前的研究利用大鼠下颌骨和后足作为体内模型系统，以研究膜内成骨和软骨内成骨过程中成骨细胞分化与增殖之间的关系。在妊娠15、17和19天的孕鼠腹腔注射不同剂量的[3H] - 胸腺嘧啶核苷，并在不同时间间隔处死，以便标记正在分裂的胚胎成骨细胞和前成骨细胞。对15天胚胎中部的横断面显示，[3H] - 胸腺嘧啶核苷对肝脏中相对较高比例的细胞（440μCi时为49±8%）呈剂量依赖性标记，而对发育中的椎体软骨细胞的标记比例较低（440μCi时为29±6%）。由下颌前成骨细胞组成的ALP阳性浓缩间充质（妊娠15天）显示出相对较高（32±5%）的[3H] - 胸腺嘧啶核苷标记水平，相比之下，周围ALP阴性的疏松间充质细胞的标记水平为（22±1%）。在19天胚胎发育中的后足，对于ALP阳性细胞（15±6%）和周围ALP阴性细胞（13±5%）也观察到了类似结果。在后足和下颌骨中，在骨骼发育过程中均观察到标记总体减少。来自这些组织的RNA样本显示，在妊娠第15天至19天期间，ALP mRNA的量增加，组蛋白H4 mRNA的量减少。这些数据表明，成骨细胞分化与增殖之间存在普遍的负相关，类似于先前在培养的成骨细胞中描述的相关性，在体内发育中的骨骼中也存在。然而，这些结果表明，致力于成骨细胞谱系的ALP阳性前成骨细胞保持其增殖能力。为了阐明潜在的分子机制，我们进一步研究了这些组织中m - twist的表达水平。转录调节因子基本螺旋 - 环 - 螺旋家族的这个成员先前被认为在培养的成骨细胞分化中起作用。我们的结果表明，在颌骨和后足的骨骼发育过程中，m - twist水平降低。
