Funahashi J, Okafuji T, Ohuchi H, Noji S, Tanaka H, Nakamura H
Laboratory of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Dev Growth Differ. 1999 Feb;41(1):59-72. doi: 10.1046/j.1440-169x.1999.00401.x.
The mes-metencephalic boundary (isthmus) has been suggested to act as an organizer in the development of the optic tectum. Pax-5 was cloned as a candidate for regulator of the organizing center. Isthmus-specific expression of Pax-5 and analogy with the genetic cascade in Drosophila suggest that Pax-5 may be at a higher hierarchical position in the gene regulation cascade of tectum development. To examine this possibility, a gain-of-function experiment on Pax-5 was carried out. In ovo electroporation on E2 chick brain with the eucaryotic expression vector that encodes chick Pax-5 cDNA was used. Not only was a considerable amount of Pax-5 expressed ectopically in the transfected brain, but irregular bulging of the neuroepithelium was induced in the diencephalon and mesencephalon. At Pax-5 misexpressing sites, uptake of BrdU was increased. Histological examination of E7 transfected brain revealed that Pax-5 caused transdifferentiation of diencephalon into the tectum-like structure. In the bulges of the E7 mesencephalon, differentiation of laminar structure was repressed when compared to the normal side. In transfected embryos, En-2, Wnt-1 and Fgf8 were up-regulated ectopically, and Otx2 was down-regulated in the diencephalon to mesencephalon. Moreover, Ephrin-A2, which is expressed specifically in the tectum with a gradient highest at the caudal end, is suggested to be involved in pathfinding of the retinal fibers, and was induced in the bulges. When the mouse Fgf8 expression vector was electroporated, Pax-5 and chick Fgf8 were also induced ectopically. These results suggest that Pax-5, together with Fgf8, hold a higher position in the genetic hierarchy of the isthmus organizing center and regulate its activity.
中脑-后脑边界（峡部）被认为在视顶盖的发育中起组织者的作用。Pax-5被克隆为组织中心调节因子的候选基因。Pax-5在峡部的特异性表达以及与果蝇基因级联的相似性表明，Pax-5可能在顶盖发育的基因调控级联中处于更高的层次位置。为了检验这种可能性，进行了Pax-5的功能获得实验。使用编码鸡Pax-5 cDNA的真核表达载体对E2期鸡胚脑进行卵内电穿孔。不仅在转染的脑中异位表达了大量的Pax-5，而且在间脑和中脑诱导了神经上皮的不规则隆起。在Pax-5异位表达的部位，BrdU的摄取增加。对E7期转染脑的组织学检查显示，Pax-5导致间脑转分化为顶盖样结构。与正常侧相比，E7期小鼠中脑隆起处的层状结构分化受到抑制。在转染的胚胎中，En-2、Wnt-1和Fgf8在异位上调，而Otx2在间脑到中脑下调。此外，Ephrin-A2在顶盖中特异性表达，在尾端梯度最高，被认为参与视网膜纤维的路径寻找，并在隆起处被诱导。当电穿孔小鼠Fgf8表达载体时，Pax-5和鸡Fgf8也在异位被诱导。这些结果表明，Pax-5与Fgf8一起，在峡部组织中心的基因层次中占据更高的位置并调节其活性。
