Nakazawa-Tanaka Nana, Miyahara Katsumi, Fujiwara Naho, Urao Masahiko, Akazawa Chihiro, Yamataka Atsuyuki
Department of Pediatric Surgery, Juntendo University Nerima Hospital, 3-1-10 Takanodai, Nerima-ku, Tokyo, 177-8521, Japan.
Department of Pediatric Surgery, Juntendo University School of Medicine, Tokyo, Japan.
Pediatr Surg Int. 2016 Jan;32(1):3-7. doi: 10.1007/s00383-015-3806-9. Epub 2015 Nov 6.
BACKGROUND/AIM: The behavior of enteric neural crest-derived cells (ENCC) during enteric nervous system (ENS) development is being gradually understood with the introduction of live-cell imaging. However, many of the analyses to date are two-dimensional and the precise multidirectional migration of ENCC has been challenging to interpret. Mice lacking the endothelin-B receptor gene, Ednrb (-/-) mice, are widely used as a model for Hirschsprung's disease (HD). We have recently developed a Sox10 transgenic (Tg) mouse to visualize ENCC with enhanced green fluorescent protein (Venus). By breeding these two models, we have created a Venus-positive, Sox10 Tg mouse with a deletion of the Ednrb gene, Sox10-Venus(+)/Ednrb (-/-) mouse, to investigate the ENS in HD. The aim of this study was to investigate the behavior of migrating ENCC in the hindgut of the Sox10-Venus(+)/Ednrb (-/-) mouse using three-dimensional and four-dimensional image analysis software.
To compare the ENCC behavior when the wavefront of ENCC reaches the mid-hindgut between HD mouse and control, we harvested the fetal hindguts of Sox10-Venus(+)/Ednrb (-/-) mice on embryonic day 15.5 (E15.5) and Sox10-Venus(+)/Ednrb (+/+) mice on E12.5, which was used as control. Dissected hindguts were cultured for 360 min and the time-lapse images were obtained using a confocal laser-scanning microscope. Each ENCC at the wavefront was tracked after adjusting the longitudinal axis of the gut to the Y axis and analyzed using Imaris software.
Track displacement (TD)-Y indicates ENCC advancement in a rostral-caudal direction. TD-X and TD-Z indicate ENCC advancement perpendicular to the rostral-caudal axis. Mean TD-Y was 34.56 µm in HD, but 63.48 µm in controls. TD-Y/TD-XZ was not significantly different in both groups. However, the mean track speeds were significantly decreased in HD (72.87 µm/h) compared to controls (248.29 µm/h).
Our results showed that the track speed of ENCC advancement was markedly decreased in the HD mice compared to controls. This technique provides added information by tracking ENCC with depth perception, which has potential for further elucidating the altered behavior of ENCC in HD.
背景/目的：随着活细胞成像技术的引入，人们对肠神经嵴衍生细胞（ENCC）在肠神经系统（ENS）发育过程中的行为有了逐渐深入的了解。然而，迄今为止的许多分析都是二维的，ENCC精确的多向迁移难以解释。缺乏内皮素B受体基因的小鼠，即Ednrb（-/-）小鼠，被广泛用作先天性巨结肠（HD）的模型。我们最近培育出一种Sox10转基因（Tg）小鼠，可通过增强型绿色荧光蛋白（Venus）使ENCC可视化。通过将这两种模型杂交，我们创建了一种Venus阳性、Ednrb基因缺失的Sox10 Tg小鼠，即Sox10-Venus（+）/Ednrb（-/-）小鼠，以研究HD中的ENS。本研究的目的是使用三维和四维图像分析软件研究Sox10-Venus（+）/Ednrb（-/-）小鼠后肠中迁移的ENCC的行为。
为了比较HD小鼠和对照小鼠中ENCC波前到达中后肠时ENCC的行为，我们在胚胎第15.5天（E15.5）收集了Sox10-Venus（+）/Ednrb（-/-）小鼠的胎儿后肠，并在E12.5收集了Sox10-Venus（+）/Ednrb（+/+）小鼠的胎儿后肠作为对照。解剖后的后肠培养360分钟，使用共聚焦激光扫描显微镜获取延时图像。在将肠的纵轴调整到Y轴后，追踪波前的每个ENCC，并使用Imaris软件进行分析。
轨迹位移（TD）-Y表示ENCC在头-尾方向上的推进。TD-X和TD-Z表示ENCC在垂直于头-尾轴方向上的推进。HD组的平均TD-Y为34.56μm，而对照组为63.48μm。两组的TD-Y/TD-XZ无显著差异。然而，与对照组（248.29μm/h）相比，HD组的平均轨迹速度显著降低（72.87μm/h）。
我们的结果表明，与对照组相比，HD小鼠中ENCC推进的轨迹速度明显降低。该技术通过对ENCC进行具有深度感知的追踪提供了额外信息，这对于进一步阐明HD中ENCC行为的改变具有潜力。
