Abstract
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 360min 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 34.56µm in HD, but 63.48µm in controls. TD-Y/TD-XZ was not significantly different in both groups. However, the mean track speeds were significantly decreased in HD (72.87µm/h) compared to controls (248.29µm/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.
Published Version
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