McDonald S A, Tuan R S
Department of Biology, University of Pennsylvania, Philadelphia 19104.
Dev Biol. 1989 May;133(1):221-34. doi: 10.1016/0012-1606(89)90313-8.
The development of the chick embryonic calvarium, an intramembranous bone, is characterized by direct differentiation of cranial ectomesenchymal cells into osteoblasts without the formation of a cartilage anlage. Collagen biosynthesis remains predominantly as type I in the calvaria. However, in severely calcium-deficient chick embryos maintained in shell-less (SL) culture, cartilage-specific type II collagen is synthesized by the calvaria. Immunohistochemistry localized the cells expressing type II collagen to undermineralized regions of the SL bone. In this study, collagen gene expression in bones of normal (N) and calcium-deficient SL chick embryos was examined at Incubation Day 14 by in situ cDNA-mRNA hybridization. A critical step in the procedure, which used biotinylated cDNA probes, was the selection of fixation conditions which maximized RNA retention and maintenance of tissue morphology. Tissues fixed in modified Carnoy's fixative (58% ethanol, 30% choloroform, 10% acetic acid, 2% formaldehyde) for 2-4 hr at -20 degrees C sectioned well and retained their cell morphology and cytoplasmic RNA. Other treatments important for the procedure included demineralization in 0.25 M HCl and removal of matrix by hyaluronidase digestion. In situ hybridization with type-specific collagen cDNA probes revealed that type II collagen mRNA was present in cells throughout the SL calvaria. More importantly, cells with type II collagen mRNA were also present in N calvaria which do not synthesize the protein. The overall abundance of type II-positive cells in N calvaria was not significantly different from that in SL calvaria, but their distribution throughout the bones differed. In general, the regional distribution of type II cells was inversely correlated with the extent of matrix mineralization. In the N calvaria, cells containing collagen type II mRNA were absent in the extensively mineralized superior zone, but were found in the temporal zone which showed limited mineralization. On the other hand, in the SL calvaria, which were substantially undermineralized overall, cells with type II mRNA were found throughout the tissue. Interestingly, the overall ratio of type I cells to type II cells was approximately 50% higher in N calvaria. These findings suggest that collagen type mRNA expression in the chick embryonic calvarium is correlated with, and perhaps dependent on, the extent of tissue matrix mineralization.
鸡胚颅盖骨是一种膜内成骨，其发育特征是颅外间充质细胞直接分化为成骨细胞，而不形成软骨雏形。颅盖骨中的胶原蛋白生物合成主要仍为I型。然而，在无壳（SL）培养中维持的严重缺钙鸡胚中，颅盖骨会合成软骨特异性的II型胶原蛋白。免疫组织化学将表达II型胶原蛋白的细胞定位到SL骨的矿化不足区域。在本研究中，通过原位cDNA - mRNA杂交，在孵化第14天检测了正常（N）和缺钙SL鸡胚骨骼中的胶原蛋白基因表达。该程序使用生物素化cDNA探针的一个关键步骤是选择能使RNA保留最大化并维持组织形态的固定条件。用改良的卡诺固定液（58%乙醇、30%氯仿、10%乙酸、2%甲醛）在 - 20℃固定组织2 - 4小时，切片良好，保留了细胞形态和细胞质RNA。该程序的其他重要处理包括用0.25 M HCl脱矿以及用透明质酸酶消化去除基质。用类型特异性胶原蛋白cDNA探针进行原位杂交显示，II型胶原蛋白mRNA存在于整个SL颅盖骨的细胞中。更重要的是，在不合成该蛋白的N颅盖骨中也存在含有II型胶原蛋白mRNA的细胞。N颅盖骨中II型阳性细胞的总体丰度与SL颅盖骨中的没有显著差异，但它们在整个骨骼中的分布不同。一般来说，II型细胞的区域分布与基质矿化程度呈负相关。在N颅盖骨中，广泛矿化的上区没有含有II型胶原蛋白mRNA的细胞，但在矿化有限的颞区发现了这类细胞。另一方面，在总体矿化严重不足的SL颅盖骨中，整个组织都发现了含有II型mRNA的细胞。有趣的是，N颅盖骨中I型细胞与II型细胞的总体比例大约高出50%。这些发现表明，鸡胚颅盖骨中胶原蛋白mRNA的表达与组织基质矿化程度相关，甚至可能依赖于该程度。
