CLASS-6 SEMAPHORINS IN PERIPHERAL AXON GUIDANCE

Proper wiring of the peripheral nervous system (PNS) not only depends on spatial but also on temporal regulation. Axons need to know where and when to grow, but also when to pause . This timing is tightly controlled by guidance cues in the surrounding environment. Growing axons interpret these cues through receptors such as class-6 Semaphorins, which convert environmental signals into temporally regulated growth decisions. Class-6 transmembrane semaphorins (SEMA6A, SEMA6B, and SEMA6D) are expressed during PNS development, yet their roles in PNS wiring remain largely unexplored. The chicken embryo provides an excellent model to study these processes, as its PNS closely resembles the human PNS and allows precise spatiotemporal manipulation of gene expression using in ovo RNA interference. Analysis of embryos at multiple developmental stages revealed guidance defects in hindlimb-innervating nerves following SEMA6 downregulation. In particular, SEMA6A-deficient embryos showed premature muscle innervation, bypassing the normal waiting period before muscle entry. Because peripheral nerves extend over large distances and form complex three-dimensional trajectories, these phenotypes were visualized using optical clearing of whole-mount embryos combined with light-sheet microscopy. To process the resulting large-scale imaging datasets, a U-Net–based machine learning model was trained to segment peripheral nerves, followed by skeletonization to extract nerve centerlines for graph-based morphological analysis. Together, these findings identify a temporally restricted, receptor-like role for class-6 semaphorins in axon guidance and demonstrate the value of combining in ovo RNA interference, 3D imaging, and computational analysis in studies of neural development.