Planar cell polarity genes in motor axon guidance in the limb

The assembly of neuronal circuits depends on the correct wiring of axons and dendrites. Studies in our laboratory revealed a critical role of seven-pass atypical cadherin Celsr3, a member of planar cell polarity (PCP) proteins, in the development of axonal tracts in the central nervous system, such as the anterior commissure, internal capsule and corticospinal tract. Celsr3 deficiency does not alter axonal growth, but affects axon guidance in cell-autonomous or non-cell-autonomous manners, causing axon stalling at intermediate targets or rerouting. Notably, all axon guidance defects in Celsr3−/− were observed in mice bearing mutations in the PCP gene Fzd3, and some errors were reported in mice with mutations of Vangl2, another PCP gene. Despite their unequivocally role, underlying molecular mechanisms remain elusive. Furthermore, their functions in the peripheral nervous system are still largely unexplored. Here we show that Celsr3 cooperates with Fzd3 in spinal motor neurons to mediate pathfinding of motor axons innervating the dorsal limb. Celsr3 is expressed in postmitotic neurons in the developing spinal cord. Specific inactivation of Celsr3 in spinal motor neurons severely perturbs peroneal nerve development, leading to absent innervation of the tibialis anterior muscle and stiff hindlimb. Deletion of Celsr3 affects neither the specification of motor neurons nor neuronal survival or neurite outgrowth. Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall just after the branch point of the sciatic nerve. Mutant axons respond to repulsive ephrinA-EphA forward signaling and attractive glial cell–derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor a1 (GFRa1) and Fzd3. The function of Celsr3 in motor axons is Fzd3 dependent but Vangl2 independent. Our results thus revealed the crucial roles of Celsr3 and Fzd3 in motor axon guidance, and provide evidence for the first time that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb, which may help us better understand their molecular mechanisms of action.