Hyperactive alpha2-chimaerin reveals the complexity of axon guidance signaling pathways in motor neuron development

Abstract

α2-chimaerin has striking importance for proper neural circuit formation. Individuals with gain-of-function mutations in α2-chimaerin have Duane retraction syndrome, a common neurogenic eye movement disorder, and α2-chimaerin knockout mice have a rabbit-like hopping gait, resulting from aberrant motor neuron wiring. While molecular mechanisms underlying altered gait circuitry in the loss-of-function mouse have been characterized, mechanisms underlying the etiology of DRS caused by mutant CHN1 remain unclear.

Here, we report the first Chn1 gain-of-function DRS mouse model (Chn1-KI), which harbors a knock-in point mutation identified in human patients. Chn1-WT/KI and Chn1-KI/KI embryonic mice exhibit abducens nerve stalling, aberrant trochlear nerve branching, and first cervical spinal segment (C1) misrouting, which result from axon guidance defects. The Chn1-KI mouse recapitulates the human DRS phenotype, thus providing a novel mouse model of DRS. Chn1-KO/KO embryos display abducens nerve wandering and defasciculation, establishing that human DRS-causing mutations are indeed gain-of-function.

We combine detailed 3D whole embryo imaging with novel in vitro approaches to demonstrate that hyperactivated α2-chimaerin acts downstream of ephrin forward and reverse signaling selectively in abducens neurons to modulate nerve development, as C1 neurons use only ephrin forward signaling, and trochlear neurons do not significantly use ephrin signaling during nerve guidance. In vivo, we find that selectively removing ephrin forward or reverse signaling via EphA4 dramatically impacts the development of the abducens nerve, distinct from bidirectionally removing EphA4 signaling.

Further experimentation reveals that alpha2-chimaerin and EphA4 can signal through other pathways. We find that hyperactivated alpha2-chimaerin modulates BDNF, GDNF, NGF, and HGF signaling in vitro, thus suggesting its role as a broad regulator of axon guidance pathways. Our studies lend insight into the complexity of axon guidance during development and highlight mechanisms that cause the abducens nerve to be selectively vulnerable to alpha2-chimaerin misregulation in DRS.