Analysis of craniosynostosis caused by mutations in fibroblast growth factor receptors and their related genes

为了了解哺乳动物颅骨穹窿发育相关基因的功能，我们首先研究了腺病毒载体介导的基因体内表达的可能性。通过子宫外手术将增强型绿色荧光蛋白（eGFP）表达载体皮下注射到小鼠胎儿颅骨。在发育中的小鼠头骨中鉴定出四个层，皮肤层、结缔组织层、成骨层和脑膜层。在成骨层中，每个颅骨骨域出现在颅骨的侧面，并向上生长扩展到顶部。感染特异性的层根据注射的阶段而变化。在胚胎（E）13.5天进行注射时，脑膜层优选地被感染，在E14胎次注射时，成骨层是主要的感染部位。E14. 5注射后，eGFP仅在结缔组织层表达，Twist转录因子在胚胎发育过程中，在成骨层未分化间充质细胞和脑膜层广泛表达，维持未分化状态。我们将Twist表达载体注射到胎儿头骨的右侧或左侧。当感染发生在成骨层时，骨结构不受影响，但注射侧的骨比未注射侧的骨稍薄。然而，当Twist在脑膜层中过表达时，注射侧骨域的生长受到抑制，并且它们停留在颅骨的外侧。骨形态发生蛋白4和7的表达水平，这是正常的表达在脑膜层，严重减少扭曲过度表达的网站。从这些结果中，我们认为，从脑膜层的信号促进骨域生长的成骨层。

In order to understand the function of the genes involved in mammalian skull vault development we first investigated the possibility of adenoviral vector-mediated gene expression in vivo. Mouse fetal skull was injected enhanced green fluorescent protein (eGFP) expression vector subcutaneously by ex-utero surgery. There are four layers identified in the developing mouse skull, skin, connective tissue, osteogenic and meningeal layers. In the osteogenic layer each skull bone domain appears at the lateral side of the skull and grow expanding up to the top. Infection specificity in the layers varied according to the stage of the injection. The meningeal layer was preferably infected when the injection was carried out on embryonic day (E)13.5, the osteogenic layer was a major infection site when E14 fetuses were injected. The injection on E14.5 resulted in eGFP expression only in the connective tissue layer.A transcription factor, Twist, is known to function in maintenance of undifferentiated condition and expressed in undifferentiated mesenchymal cells of the osteogenic layer and ubiquitously in the meningeal layer during the prenatal skull development. We injected the Twist expression vector into either right or left side of a fetal skull. When the infection was achieved in the osteogenic layer bone domain patterning was not affected but the bone on injected side became slightly thinner than that of uninjected side. When Twist was over-expressed in the meningeal layer, however, growth of the bone domains of injected side was inhibited and they stayed at the lateral side of the skull. Expression levels of bone morphogenetic protein 4 and 7, which are normally expressed in the meningeal layer, were severely decreased in Twist over-expressed site. From these results we suggest that a signal from the meningeal layer promotes bone domain growth in the osteogenic layer.