Diverse and location-specific roles of PlexinA2, PlexinA4, and NCAM in developing hippocampal mossy fibers

Mossy fibers (MFs) originate from dentate granule cells and innervate area CA3 of the hippocampus. Upon entry of CA3, MFs partition into two prominent axon bundles, the suprapyramidal tract (SPT) and infrapyramidal tract (IPT) and form lamina specific synaptic contacts in the stratum lucidum (SL) and stratum oriens (SO), respectively. Here we mapped cell type specific gene expression and subcellular distribution of Sema6A and its receptors PlexinA2 (PlxnA2) and PlxnA4. Using a mouse genetic approach, including newly generated mouse lines, we dissected the function of these genes in developing MFs. In Sema6a^-/- mice, MF partitioning into SPT and IPT bundles is incomplete and IPT axons in the SO are overextended, while the SPT correctly innervates the SL. Loss of Sema6a in granule cells results in defective MF patterning and we show that this involves Sema6A reverse signaling. Plxna4 controls MF partitioning, SPT axon bundling, and laminar targeting to the SL, as well as IPT length. Many of these defects are recapitulated in mice lacking PlxnA4 GAP catalytic activity, underscoring the importance of this GAP domain. In Plxna2^-/- mice, MFs are tightly fasciculated and fail to separate into SPT and IPT bundles, and defects are significantly reduced in PlxnA2 GAP mutants, highlighting the involvement of GAP-independent signaling events. To further explore the molecular basis of aberrant axon fasciculation, we employed anti-PlxnA2 dependent proximity biotinylation and identified several PlxnA2-associated Ig-CAM family members. Follow-up studies revealed a genetic interaction between Plxna2 and Ncam1, but not Plxna4 and Ncam1, for SPT and IPT formation and axon targeting. Together, our studies provide insights into the multifaceted and overlapping, yet distinct, functions of PlxnA family members in orchestrating specific guidance decisions in developing MFs.