Fuhrmann S, Kirsch M, Hofmann H D
Institute of Anatomy I, University of Freiburg, Germany.
Development. 1995 Aug;121(8):2695-706. doi: 10.1242/dev.121.8.2695.
Previous in vitro studies have convincingly demonstrated the involvement of diffusible factors in the regulation of photoreceptor development. We now provide evidence that ciliary neurotrophic factor (CNTF) represents one of these regulatory molecules. In low density monolayer cultures prepared from embryonic day 8 chick retina, photoreceptor development was studied using the monoclonal antiopsin antibody rho-4D2 as a differentiation marker. The number of cells acquiring opsin immunoreactivity, determined after 3 days in vitro, was increased up to 4-fold in the presence of CNTF to maximally 10.5% of all cells. Basic fibroblast growth factor or taurine both of which have been reported to stimulate opsin expression in rat retinal cultures and other neurotrophic factors tested (nerve growth factor, brain derived neurotrophic factor) had no effect. The EC50 of the CNTF effect (2.6 pM) was virtually identical to that measured for other CNTF receptor mediated cellular responses. Conditioned medium produced by cultured retinal cells (most likely glial cells) exhibited opsin stimulating activity identical to that of CNTF. Stimulation of opsin expression was specific for morphologically less mature photoreceptors and obviously restricted to rods, since changes in the number of identifiable cone photoreceptors expressing opsin immunoreactivity (10% of all cones) were not detectable. Measurement of the kinetics of the CNTF response revealed that the factor acted on immature opsin-negative progenitors and that CNTF effects were unlikely to reflect enhanced cell survival. Proliferation of photoreceptors was also unaffected, as demonstrated by [3H]thymidine autoradiography. With prolonged culture periods a gradual decrease in the number of opsin-positive cells was observed both in controls and in the continuous presence of CNTF. This decrease could be partly prevented by the addition of 1 mM taurine. Our results suggest that CNTF acted as an inductive signal for uncommitted progenitor cells or during early stages of rod photoreceptor differentiation, whereas other extrinsic stimulatory activities seemed to be required for further maturation.
以往的体外研究令人信服地证明了可扩散因子参与光感受器发育的调控。我们现在提供证据表明睫状神经营养因子（CNTF）是这些调控分子之一。在从胚胎第8天鸡视网膜制备的低密度单层培养物中，使用单克隆抗视蛋白抗体rho-4D2作为分化标志物研究光感受器发育。体外培养3天后测定的获得视蛋白免疫反应性的细胞数量，在CNTF存在下增加了4倍，最高可达所有细胞的10.5%。碱性成纤维细胞生长因子或牛磺酸，这两种物质在大鼠视网膜培养物中均被报道可刺激视蛋白表达，而测试的其他神经营养因子（神经生长因子、脑源性神经营养因子）则没有作用。CNTF作用的半数有效浓度（EC50）（2.6皮摩尔）与其他CNTF受体介导的细胞反应所测得的数值几乎相同。培养的视网膜细胞（很可能是神经胶质细胞）产生的条件培养基表现出与CNTF相同的视蛋白刺激活性。视蛋白表达的刺激对形态上不太成熟的光感受器具有特异性，并且明显仅限于视杆细胞，因为未检测到表达视蛋白免疫反应性的可识别视锥光感受器数量的变化（占所有视锥细胞的10%）。CNTF反应动力学的测定表明，该因子作用于未成熟的视蛋白阴性祖细胞，并且CNTF的作用不太可能反映细胞存活率的提高。[3H]胸腺嘧啶核苷放射自显影显示，光感受器的增殖也未受影响。随着培养时间的延长，在对照组和持续存在CNTF的情况下，均观察到视蛋白阳性细胞数量逐渐减少。添加1毫摩尔牛磺酸可部分防止这种减少。我们的结果表明，CNTF作为未分化祖细胞的诱导信号或在视杆光感受器分化的早期阶段起作用，而进一步成熟似乎需要其他外在刺激活性。
