The Chinese University of Hong Kong (CUHK), Hong Kong
Abstract: Enteric neural crest-derived cells (ENCCs) migrate long distances to the developing gastrointestinal tract to form enteric ganglia. Abnormal migration of ENCCs in the gut may result in congenital gut motility/peristalsis disorders such as Hirschsprung’s disease. However, the molecular mechanisms governing ENCC migration remain elusive. In this study, the role of RhoA, a key molecule of the RhoA/ROCK signalling pathway which is an important signal transduction system for cell migration, in ENCC migration was investigated. Specifically, small interfering RNAs (siRNAs) were employed to knock down the RhoA expression, which resulted in significant alterations of the migration behaviour of ENCCs in vitro. To visualize the dynamic change of intracellular RhoA activities during migration, time-lapse live cell imaging combined with a Förster resonance energy transfer (FRET)-based biosensor was employed. It was found that active RhoA molecules were dynamically redistributed inside ENCCs, differentially accumulating at the rear end and the leading edges of migrating ENCCs which suggested the involvement of the RhoA in ENCC migration. Next, bulk RNA sequencing was performed on ENCCs following RhoA knockdown. The expression of other molecules related to cell migration and tight junctions was altered. These suggested that the RhoA/ROCK signalling might cross talk with other pathways to regulate ENCC migration. Particularly, Igfbp5 demonstrated a decrease in expression after supressing RhoA expression. siRNAs were employed to lower the Igfbp5 expression. The wound healing assay showed a delay in filling up the wound gap with Igfbp5-suppressed ENCCs. Besides, the live cell tracking demonstrated the loss of directionality of ENCCs 24 hours after suppression under a GDNF gradient. qPCR results also showed the decrease expression in migration-related genes. In summary, this study indicated the intricate interplay of different signalling pathways during ENCC migration. Results found that RhoA was recruited during ENCC migration, and RhoA may be involved in regulating ENCC migration through Igfbp5, which may be one of the downstream targets of the RhoA/ROCK pathway mediating the directional migration. These results provided information for potential therapeutic targets for congenital gut motility disorders.
Funding Source: The work was supported by the General Research Fund from the Research Grants Council of Hong Kong Special Administrative Region, China (Ref. no.: CUHK14120522).
