Application of the exo utero surgery to the study on the development of the cerebral cortical

尽管近年来基因技术有了很大的进步，但哺乳动物脑发育的机制仍不完全清楚。其中一个原因是很难观察和操作在母亲子宫中发育的哺乳动物胚胎。为阐明大脑皮层发育的机制，本研究采用体外培养的方法对胚胎小鼠脑进行了一些实验研究。我们已经对大鼠大脑皮层的体内发育进行了研究，并获得了一些数据，表明神经细胞黏附分子L1和TAG-1之间的分子相互作用，以及脑特异性硫酸软骨素蛋白多糖(CSPG)、神经胶和磷酸多糖，参与了大脑皮层通路的形成(Fukuda等人，1997)。在发育中的大鼠大脑皮质，L1和Tag-1免疫反应分别定位于丘脑皮质轴突和皮质传出轴突。含L1的丘脑皮质轴突优先运动于皮质原基底板，在该亚板中有神经钙素的特异性表达。相反，TAG-1免疫反应阳性的轴突避开亚板，运行在神经钙素表达较弱的中间区。此外，TAG-1轴突广泛侵入表达另一种类型的CSPG的区域，即磷酸聚糖。虽然TAG-1和L1在体外都被报道结合了神经胶和磷酸聚糖，但神经细胞黏附分子与CSPGs之间的各种相互作用模式可能在前脑的通路形成中发挥重要作用。在此基础上，我们利用体外手术将降解硫酸软骨素的酶、抗体L1或TAG-1的反义寡核苷酸注入E13胚胎的侧脑室，并取脑组织学观察。该系统将成为研究哺乳动物大脑发育机制的有用工具。

In spite of the great advance of the gene technology in recent years, the mechanism of mammalian brain development is not fully known. One of the reasons is the difficulty in observing and manipulating mammalian embryos which develop in the mother's uterus. In the present study, some experimental manupilations were made to the embryonic mouse brain by using exo utero sugery to clarify the developmental mechanisms of the cerebral cortex. We have already studied the in vivo development of the rat cerebral cortex and obtained the data which indicate that molecular interactions between neural cell adhesion molecules, L1 and TAG-1, and brain-specific chondroitin sulfate proteoglycan (CSPG), neurocan and phosphacan, are involved in the pathway formation of the cerebral cortex (Fukuda et al., 1997). In the developing rat cerebral cortex, L1 and TAG-1 immunoreactions were specifically localized on thalamocortical axons and cortical efferent axons, respectively. L1-bearing thalamocortical axons preferentially travel in the subplate of the cortical anlage where neurocan, was specifically expressed. In contrast, axons immunoreactive for TAG-1 evaded the subplate and run in the intermediate zone where neurocan expression was less prominent. In addition, TAG-1-bearing axons extensively invaded regions expressing another type of CSPG,phosphacan. Although both TAG-1 and L1 have been reported to bind both neurocan and phosphacan in vitro, various pattern of interactions between neural cell adhesion molecules and CSPGs may play important roles in the pathway formation of the forebrain. Based on these observation, we made microinjection of an enzyme which degrades chondrotin sulfate, antibody ageinst L1 or antisense oligo DNA of TAG-1 into the lateral ventricle of E13 mouse embryos using exo utero surgery, and exmined the brain histologically. This system will be a useful tool to study the developmental mechanism of the mammalian brain.