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取得了巨大的进步，但哺乳动物脑发育的机制尚不完全清楚。其中一个原因是很难观察和操纵在母亲子宫内发育的哺乳动物胚胎。本研究通过体外手术对小鼠胚胎脑进行了一些实验操作，以阐明大脑皮层的发育机制。我们已经对大鼠大脑皮层的体内发育进行了研究，得到的数据表明，神经细胞粘附分子L1和TAG-1与脑特异性硫酸软骨素蛋白聚糖（CSPG）、神经蛋白聚糖和磷蛋白聚糖之间的分子相互作用参与了大脑皮层的通路形成（Fukuda et al., 1997）。在发育中的大鼠大脑皮层中，L1和TAG-1免疫反应分别特异性定位于丘脑皮质轴突和皮层传出轴突。携带l1的丘脑皮质轴突优先在神经蛋白特异性表达的皮质基质亚板中移动。相反，轴突对TAG-1的免疫反应避开了亚板，并在神经能表达不太突出的中间区域运行。此外，携带tag -1的轴突广泛侵入表达另一种CSPG的区域，即磷酸腺苷。尽管TAG-1和L1在体外均可结合神经蛋白和磷蛋白，但神经细胞粘附分子与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，我们采用体外手术将一种降解硫酸软骨素、抗L1抗体或TAG-1反义寡核苷酸的酶微量注射到E13小鼠胚胎的侧脑室，并对其脑组织进行组织学检查。该系统将为研究哺乳动物大脑发育机制提供有用的工具。

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.