Protocadherin-αC2 is required for diffuse projections of serotonergic axons.

Serotonergic axons extend diffuse projections throughout various brain areas, and serotonergic system disruption causes neuropsychiatric diseases. Loss of the cytoplasmic region of protocadherin-α (Pcdh-α) family proteins, products of the diverse clustered Pcdh genes, causes unbalanced distributions (densification and sparsification) of serotonergic axons in various target regions. However, which Pcdh-α member(s) are responsible for the phenotype is unknown. Here we demonstrated that Pcdh-αC2 (αC2), a Pcdh-α isoform, was highly expressed in serotonergic neurons, and was required for normal diffusion in single-axon-level analyses of serotonergic axons. The loss of αC2 from serotonergic neurons, but not from their target brain regions, led to unbalanced distributions of serotonergic axons. Our results suggest that αC2 expressed in serotonergic neurons is required for serotonergic axon diffusion in various brain areas. The αC2 extracellular domain displays homophilic binding activity, suggesting that its homophilic interaction between serotonergic axons regulates axonal density via αC2's cytoplasmic domain.

Spinal RacGAP α-Chimaerin Is Required to Establish the Midline Barrier for Proper Corticospinal Axon Guidance.

In the developing CNS, the midline barrier, which comprises guidance molecule-expressing midline glial somata and processes, plays a pivotal role in midline axon guidance. Accumulating evidence has revealed the molecular mechanisms by which the midline barrier ensures proper midline guidance for axons. In contrast, the mechanisms for establishing the midline barrier remain obscure. Here, we report that Rac-specific GTPase-activating protein (RacGAP) α-chimaerin is required for both axonal repulsion at and establishment of the midline barrier in the spinal cord. We generated cortex-specific and spinal-cord-specific α-chimaerin gene (Chn1) knock-out mice (Cx-Chn1KO and Sp-Chn1KO mice, respectively) and found that both showed aberrant corticospinal tract (CST) axon midline crossing in the spinal cord. Strikingly, Sp-Chn1KO mice had breaks (holes) in the ephrinB3(+) spinal midline barrier and EphA4(+) CST axons aberrantly crossed the midline through these holes. During normal embryonic development, EphA4(+) spinal cells are located in juxta-midline areas but are excluded from the midline. In contrast, in Chn1KO embryos, several EphA4(+) cells were aberrantly relocated into the midline and the midline barrier was broken around these cells. Similarly, the spinal cord midline of Epha4KO mice was invaded by juxta-midline EphA4 cells (i.e., Epha4 promoter-active cells) during the embryonic stage and holes were formed in the midline barrier. Juxta-midline EphA4 cells in the spinal cord expressed α-chimaerin. We propose that spinal α-chimaerin aids in establishing an intact spinal midline barrier by mediating juxta-midline EphA4(+) cell repulsion, thus preventing these cells from breaking into the ephrinB3(+) midline barrier.SIGNIFICANCE STATEMENT The midline barrier plays a critical role in midline axon guidance, which is fundamental to the formation of neural circuits that are responsible for proper left-right coordination of the body. Studies have revealed some of the mechanisms underlying how the midline barrier navigates axons. In contrast, the establishment of the midline barrier during embryonic development remains unclear. In this study, we determined that α-chimaerin is required for the formation of an intact midline barrier. Spinal-cord-specific α-chimaerin knock-out mice had spinal midline barriers with numerous breaks (holes), through which corticospinal axons aberrantly crossed the midline. We propose that α-chimaerin protects the midline barrier by mediating cell-repulsive signaling in juxta-midline cells, which prevents these cells from invading the midline.