GDNF and Endothelin 3 Regulate Migration of Enteric Neural Crest-Derived Cells via Protein Kinase A and Rac1

Abstract

Enteric neural crest-derived cells (ENCCs) migrate from the anterior foregut in a rostrocaudal direction to colonize the entire gastrointestinal tract and to form the enteric nervous system. Genetic approaches have identified many signaling molecules regulating the migration of ENCCs; however, it remains elusive how the activities of the signaling molecules are regulated spatiotemporally during migration. In this study, transgenic mice expressing biosensors based on Förster resonance energy transfer were generated to video the activity changes of the signaling molecules in migrating ENCCs. In an organ culture of embryonic day 11.25 (E11.25) to E13 guts, ENCCs at the rostral wavefront migrated as a cellular chain faster than the following ENCCs that formed a network. The faster-migrating cells at the wavefront exhibited lower protein kinase A (PKA) activity than did the slower-migrating trailing cells. The activities of Rac1 and Cdc42 exhibited an inverse correlation with the PKA activity, and PKA activation decreased the Rac1 activity and migration velocity. PKA activity in ENCCs was correlated positively with the distribution of GDNF and inversely with the distribution of endothelin 3 (ET-3). Accordingly, PKA was activated by GDNF and inhibited by ET-3 in cultured ENCCs. Finally, although the JNK and ERK pathways were previously reported to control the migration of ENCCs, we did not find any correlation of JNK or ERK activity with the migration velocities. These results suggest that external cues regulate the migration of ENCCs by controlling PKA activity, but not ERK or JNK activity, and argue for the importance of live imaging of signaling molecule activities in developing organs.

Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice

“…We previously established a method to generate transgenic mice expressing FRET biosensors, with which the time lapse of activity changes in protein kinases can be imaged (25,26). To achieve dMSN-and iMSN-specific labeling of the PKA and ERK FRET biosensors, we created two flox lines of transgenic mice, floxed-AKAR3EV (PKA) and floxed-EKAREV (ERK) (27), both of which contained a nuclear export signal to localize these kinases in the cytoplasm (Fig.…”

Building a second brain in the bowel

“…Mechanisms must be dynamically regulated because cells at the ENCDC migration wavefront behave differently from cells behind the wavefront. Recent, elegant studies of Förster resonance energy transfer have confirmed that PKA, RAC1, and CDC42 are differentially activated in migrating ENCDCs according to proximity to the wavefront (68). These proteins regulate the mode of cell migration (69,70) and the efficiency of bowel colonization.…”

“…These proteins regulate the mode of cell migration (69,70) and the efficiency of bowel colonization. Interestingly, both PKA inhibition and exogenous cAMP analogs slow ENCDC migration in organ culture (68,71), but critical PKA targets in this context are not known. This suggests that localized PKA activation or moderate activation is needed to support migration.…”

“…in vitro and in ex vivo organ culture (68,73,76). Elevated RHOA, which often acts in opposition to RAC, also disrupts ENCDC chain migration and causes hypoganglionosis (77).…”

Maintenance of stereocilia and apical junctional complexes by Cdc42 in cochlear hair cells

“…FRET imaging under twophoton excitation microscopy was performed as described previously (Goto et al, 2013). Samples were maintained in an incubation chamber (Tokai Hit, Japan) and imaged using a BX61WI/FV1000 upright microscope equipped with 606 water-immersion objective (LUMPlanFLN; Olympus, Japan) connected to a Mai Tai DeepSee HP Ti:sapphire Laser (Spectra Physics, Mountain View, CA).…”