Callaghan D A, Dong L, Callaghan S M, Hou Y X, Dagnino L, Slack R S
Neuroscience Research Institute, Ottawa General Hospital Research Institute, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada.
Dev Biol. 1999 Mar 15;207(2):257-70. doi: 10.1006/dbio.1998.9162.
The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.
携带视网膜母细胞瘤（Rb）基因无效突变的胚胎出现严重神经功能缺损，这表明Rb在神经发生过程中起关键作用。虽然发育中的神经元在体内会发生凋亡，但从Rb基因缺陷胚胎中培养出的神经前体细胞似乎能够分化并存活。为了确定Rb是否是调节终末有丝分裂的内在途径的关键调节因子，我们研究了来自Rb基因缺陷小鼠的原代皮质祖细胞和碱性成纤维细胞生长因子（bFGF）依赖性神经干细胞的终末分化。尽管Rb基因敲除的神经前体细胞能够在体外分化，但我们发现这些细胞相对于野生型细胞在终末有丝分裂方面存在显著延迟。此外，在体外存活的Rb基因敲除细胞中，与E2F3形成复合物的p107上调。这表明p107可能部分补偿神经前体细胞中Rb的缺失。通过腺病毒介导递送E1A N端突变体对Rb家族蛋白进行功能缺失，会导致Rb基因缺陷细胞发生凋亡，这与其他Rb家族蛋白可能促进分化和存活的解释一致。虽然p107在神经前体细胞中上调并与假定的Rb靶标E2F3相互作用，但我们的结果表明它显然无法恢复正常的E2F调节。在整个分化过程中，Rb基因缺陷细胞中E2F 1和3的活性显著增强，同时伴有E2F诱导基因的异常表达。在这些研究中，我们表明Rb对于调节神经前体细胞中E2F 1和3的活性以及终末有丝分裂的开始至关重要。
