Sullivan S A, Barthel L K, Largent B L, Raymond P A
Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor 48109-0616, USA.
Dev Genet. 1997;20(3):208-23. doi: 10.1002/(SICI)1520-6408(1997)20:3<208::AID-DVG4>3.0.CO;2-B.
Members of the Notch gene family are thought to be involved in the regulation of cell fate decisions in a variety of embryonic tissues, particularly in the developing central nervous system (CNS) in Drosophila and vertebrates. In goldfish the CNS continues to develop and add neurons well into adulthood and has the capacity to regenerate new neurons. Using probes derived from Xenopus Notch to screen an adult goldfish retinal cDNA library, followed by 5' RACE, we isolated a partial cDNA for a goldfish Notch homologue, G-Notch. Sequence alignment supported assignment of G-Notch to the Notch-3 class. Northern blot analysis revealed a single transcript of > 8 kb, and RNase protection assays indicated that G-Notch is expressed in eye and brain but not muscle of adult goldfish. The spatiotemporal pattern of expression of G-Notch was defined from early embryonic stages to adulthood by in situ hybridization. Expression in the embryonic CNS was localized to neurogenic regions and was downregulated in differentiated cell populations. In adult goldfish, expression persisted in and adjacent to the germinal zones in the retina and the brain. Weak expression was seen in scattered cells in the inner nuclear layer of the retina, which might include neurogenic stem cells. Following retinal lesions (puncture wounds or laser lesions restricted to photoreceptors in the outer nuclear layer), G-Notch was upregulated in proliferating cell populations throughout the retina, in association with a generalized mitogenic response. In the region of the laser lesion, where earlier studies have demonstrated that photoreceptors are regenerating at 1-3 weeks following the lesion, G-Notch expressing cells were abundant in the outer nuclear layer. These observations suggest that retinal regeneration involves the re-expression of an important developmental signaling molecule in neuroepithelial cells resident in the differentiated retina.
Notch基因家族的成员被认为参与了多种胚胎组织中细胞命运决定的调控，特别是在果蝇和脊椎动物发育中的中枢神经系统（CNS）。在金鱼中，中枢神经系统在成年后仍持续发育并添加神经元，并且具有再生新神经元的能力。我们使用源自非洲爪蟾Notch的探针筛选成年金鱼视网膜cDNA文库，随后进行5' RACE，分离出了金鱼Notch同源物G-Notch的部分cDNA。序列比对支持将G-Notch归为Notch-3类。Northern印迹分析显示有一个大于8 kb的单一转录本，核糖核酸酶保护分析表明G-Notch在成年金鱼的眼睛和大脑中表达，但在肌肉中不表达。通过原位杂交确定了G-Notch从胚胎早期到成年期的时空表达模式。在胚胎中枢神经系统中的表达定位于神经源性区域，并在分化的细胞群体中下调。在成年金鱼中，表达在视网膜和大脑的生发区及其附近持续存在。在视网膜内核层的散在细胞中可见弱表达，这些细胞可能包括神经源性干细胞。视网膜损伤（穿刺伤口或仅限于外核层光感受器的激光损伤）后，G-Notch在整个视网膜的增殖细胞群体中上调，与普遍的促有丝分裂反应相关。在激光损伤区域，早期研究表明光感受器在损伤后1至3周再生，外核层中有丰富的G-Notch表达细胞。这些观察结果表明，视网膜再生涉及分化视网膜中驻留的神经上皮细胞中一种重要的发育信号分子的重新表达。
