The Role of Rho/ROCK Pathway on the Sacral Neural Crest Cell Migration in the Mouse Gut

Abstract

The enteric nervous system (ENS) originates from neural crest cells (NCCs) at two different levels along the neuraxis: vagal (somites 1–7) and sacral (caudal to somite 24 in the mouse) levels. While the development of vagal NCCs has been widely studied, little has been known about sacral NCCs, particularly in mammals. Our objective was to test whether inhibition of small GTPases such as ROCK (an effector of Rho) affects sacral NCC migration in the mouse gut.First, cultured sacral NCCs from pelvic ganglia were treated with different concentrations of Y‐27632 to suppress active ROCK in vitro, and results showed that inhibition of ROCK significantly decreased neurite outgrowths and migration distances of sacral NCCs. Immunofluorescent localization demonstrated that the expression of cofilin which is a downstream protein of Rho/ROCK pathway remained unchanged while its phosphorylated form p‐cofilin (inactive state) was increased significantly. Expression of phalloidin which is a marker of stress fibers decreased after Y‐27632 treatment. Time‐lapse live cell imaging also showed that the speed and persistence of sacral NCC migration decreased, and the directional migration was disrupted after Y‐27632 treatment. Interestingly, expression of both phalloidin and p‐cofilin in sacral NCCs was restored to normal levels by Rho activator treatment.Next, we established an embryonic gut explant culture, where a pelvic ganglion (serving as a source of sacral NCCs) was recombined with a recipient hindgut segment ex vivo. Whole‐mount preparation and cryosection staining showed that the migration and differentiation of endogenous vagal NCCs in the hindgut explant cultured ex vivo was similar to those in the hindgut developed from E12.5 to E16.5 in vivo. Hence the gut culture explant system could support the normal development of the hindgut explant ex vivo. The recipient hindgut segment into which sacral NCCs from the pelvic ganglion had migrated 2 day after cultured ex vivo was then treated with different concentrations of Y‐27632. It was found that Y‐27632 reduced the migration distance, cell number and axonal extension of sacral NCCs within the hindgut segment. Moreover, time‐lapse live cell imaging also suggested that the speed and persistence of axonal extension of sacral NCCs decreased, and directional migration was disrupted after Y‐27632 treatment.In summary, these observations provide evidence that Rho/ROCK pathway regulates stress fiber formation and directional migration during sacral NCC migration.Support or Funding InformationThe work was supported by grants from RGC General Research Fund (Project Reference: 14102214) and RGC Theme‐based Research Scheme (Project Reference: T12C‐714/14‐R).