The role of basement membrane type heparan sulfate proteoglycan, perlecan, in epithelial morphogenesis -transgenic mice overexpressing perlecan in the epithelial cells-

Perlecan是一种基膜型硫酸乙酰肝素蛋白多糖，定位于釉质器官的细胞间隙。因此，它被认为在牙齿形态发生中起着重要作用。为了阐明Perlecan在牙齿发育中的作用，我们利用角蛋白5启动子建立了在上皮细胞中高表达Perlecan的转基因小鼠，并检测了Perlecan过表达对成釉器官形成的影响。用DNA构建的C57BL/C3HF1受精卵母细胞显微注射获得转基因小鼠，并在牛角蛋白5启动子的控制下表达完整的Perlecan核心蛋白。用EM、免疫组织化学和RT-PCR方法研究了它们的磨牙牙胚中Perlecan和牙胚相关分子，免疫组织化学证实Perlecan在包括釉质器官在内的上皮组织中有强表达。在形态上，TG磨牙的星状网状结构显示…细胞间隙变宽。胚胎期(E13-E18)牙板间隙较大，牙板较厚，形状不规则。出生后第1天(P1)Tg小鼠的釉器明显缺乏细胞密度，血管化程度较低。最后，TG磨牙牙冠形状变钝，下颌磨牙牙根发育不全，牙根发散。结果提示，β可能通过结合和释放生长因子来调控基质的空间排列，并可能作为营养物质在上皮细胞内扩散的载体。此外，在牙齿发育过程中，Perlecan可能有助于形成高度水合的环境，实现釉质器官中细胞的生长和分化。然而，Perlecan在上皮内表达的时间表似乎受到了严格的控制，因为持续的过度表达Perlecan干扰了正常的牙齿形态发生。较少

Perlecan, a basement membrane-type heparan sulfate proteoglycan, is localized in the intercellular space of the enamel organ. Hence, it has been suggested to play an important role in tooth morphogenesis. To elucidate the function of perlecan in odontogenesis, we generated transgenic mice overexpressing perlecan in the epithelial cells using a keratin 5 promoter, and examined the effect of perlecan overexpression on enamel organ formation. Transgenic mice were generated by pronuclear microinjection of fertilized C57BL/C3H F1 oocytes with the DNA construct, and they express the entire perlecan core protein under the control of a bovine keratin 5 promoter. Their molar tooth germs were studied by EM, immunohistochemistry, and RT-PCR for perlecan and tooth germ-related molecules.Perlecan was immunohistochemically confirmed to be expressed strongly in epithelial tissues, including the enamel organ of Tg mice. Morphologically, the stellate reticulum of the Tg molars showed widened intercellu … More lar spaces and thick, irregularly shaped dental laminae at the embryonic stage (E13-E18). In postnatal day 1 (P1) Tg mice, the enamel organ was obviously lacking cell density and was less vascularized. Finally, the crown shape of Tg molars became dull-ended, with incomplete crystallization and divergent tooth roots in mandibular molars. mRNA expression levels for FGF-2, TGF-β1, and PKC-a were elevated in Tg tooth germs at P1.From these results, it was suggested that perlecan may control the space arrangement of stromata by binding and releasing of growth factors, and it may act as a carrier for nutrient transport by diffusion within the epithelial milieu. Also in the process of odontogenesis, perlecan might contribute in forming highly hydrated circumstances to realize cell growth and differentiation in the enamel organ. However, the time schedule of the intraepithelial expression of perlecan seems to be critically controlled, because a constant overexpression perlecan has interfered normal tooth morphogenesis. Less