Protein Kinase Cζ and Glycogen Synthase Kinase-3β Control Neuronal Polarity in Developing Rodent Enteric Neurons, whereas SMAD Specific E3 Ubiquitin Protein Ligase 1 Promotes Neurite Growth But Does Not Influence Polarity

Enteric nervous system (ENS) precursors migrate extensively before differentiating to form uni-axonal or multi-axonal neurons. ENS precursor survival, neurite growth, and cell migration are all directed by Ret kinase, but downstream signaling pathways are incompletely understood. We now demonstrate that proteins regulating polarity in other cells including partitioning defective 3 (PAR3), PAR6, protein kinase Cζ (PKCζ), and glycogen synthase kinase 3β (GSK3β) are expressed in developing enteric neurons with a polarized distribution. Blocking PKCζ or GSK3β reduces ENS precursor migration and induces the formation of multi-axonal neurons. Axon elongation also depends on SMURF1 (SMAD specific E3 ubiquitin protein ligase 1), which promotes RhoA degradation and associates with polarity proteins. SMURF1 inhibition, however, does not increase the number of multi-axonal neurons in ENS precursors. These data link cell surface Ret activation with molecular machinery controlling cytoskeletal dynamics and suggest that polymorphisms influencing PKCζ or GSK3β might alter Hirschsprung disease penetrance or expressivity by affecting ENS precursor migration.