Two-photon microscopic observation of cell-production dynamics in the developing mammalian neocortex in utero
Two-photon microscopic observation of cell-production dynamics in the developing mammalian neocortex in utero
复制标题
双光子显微镜观察子宫内发育中的哺乳动物新皮质的细胞产生动态
DOI：
10.1111/dgd.12648
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Kawasoe;R;Shinoda;T;Hattori;Y;Nakagawam M;Quang Pham;T;Tanaka;Y;Sagou;K;Saito;K;Katsuki;S;Kotani;T;Sano;A;Fujimori;T;Miyata;T.
形态发生和器官发育应该基于对细胞动力学的全面描述来理解。最近的研究利用切片培养物探索了哺乳动物神经祖细胞（NPC）的动态行为，其中三维系统在相当程度上保存了体内的类似环境。然而，像长期以来对斑马鱼 NPC 进行的那样，对真正存在于体内的 NPC 进行实时观察，尚未在哺乳动物中建立。在这里，我们对子宫内 H2B-EGFP 或 Fucci 转基因小鼠发育中的大脑皮层中的 NPC 进行了活体双光子显微镜观察。使用多种装置固定子宫囊中的胎儿，并通过子宫壁和羊膜上的窗口进行观察，同时监测血液循环。从胎儿头皮表面获得了 300 μm 的清晰可见度，这使我们能够定量评估胚胎第 (E) 13 和 E14 天 (E) 脑室区的神经上皮结构内的 NPC 行为，例如分裂和相互运动的核迁移。在子宫内接受 60 分钟健康监测的胎儿中，在心尖表面观察到的有丝分裂频率与在切片培养物和新鲜固定的体内标本中观察到的频率相似。尽管子宫内观察的成功率和持续时间仍然有限（≥10 分钟为 33%，60 分钟为 14%），但基于本研究的进一步改进将有助于未来了解器官发生细胞行为如何发生或如何受到全身母体状况和/或母胎关系的病理影响。
Morphogenesis and organ development should be understood based on a thorough description of cellular dynamics. Recent studies have explored the dynamic behaviors of mammalian neural progenitor cells (NPCs) using slice cultures in which three‐dimensional systems conserve in vivo‐like environments to a considerable degree. However, live observation of NPCs existing truly in vivo, as has long been performed for zebrafish NPCs, has yet to be established in mammals. Here, we performed intravital two‐photon microscopic observation of NPCs in the developing cerebral cortex of H2B‐EGFP or Fucci transgenic mice in utero. Fetuses in the uterine sac were immobilized using several devices and were observed through a window made in the uterine wall and the amniotic membrane while monitoring blood circulation. Clear visibility was obtained to the level of 300 μm from the scalp surface of the fetus, which enabled us to quantitatively assess NPC behaviors, such as division and interkinetic nuclear migration, within a neuroepithelial structure called the ventricular zone at embryonic day (E) 13 and E14. In fetuses undergoing healthy monitoring in utero for 60 min, the frequency of mitoses observed at the apical surface was similar to those observed in slice cultures and in freshly fixed in vivo specimens. Although the rate and duration of successful in utero observations are still limited (33% for ≥10 min and 14% for 60 min), further improvements based on this study will facilitate future understanding of how organogenetic cellular behaviors occur or are pathologically influenced by the systemic maternal condition and/or maternal‐fetal relationships.