Fig. 3.

Short- and long-range phenotypes observed in the absence of NPC- or FP-derived netrin1. (A) Summary of the distribution of netrin1 transcript (red) and protein (green). NPCs and FP cells express netrin1, while netrin1 protein is present on the pial surface and on commissural axons. (B) In netrin1 loss of function mutants, spinal axons are highly defasciculated, extending into the VZ, and wandering across the motor column. (C, D) Short range phenotypes for NPC-derived netrin1: When netrin1 expression is removed from either a large (C) or small (D) number of NPCs using conditional genetic approaches (C=Pax3::cre driver; D=Dbx1::cre driver) (Varadarajan et al., 2017), axons defasciculate specifically and locally in the region where netrin1 activity is absent. In particular, the introduction of ectopic netrin1 on::off boundaries (D) locally reshapes axon trajectories in a manner consistent with the hederal boundary model. Long-range phenotypes for NPC-derived netrin1: none observed. (E–I) Short-range phenotypes for FP-derived netrin1: In netrin1ΔFP mice (Shh::cre driver), commissural axons project ventrally toward the FP (E) in a manner largely indistinguishable from controls. However, errors occur ~10μm from the FP (yellow box, G), when axons reach the “off” edge of the ventral domain pial-netrin1, where they locally defasciculate (arrowheads, I). Axons start to cross the FP from this “off” edge, resulting in a laterally displaced “U” shaped trajectory (G), distinct from the “V” shape observed in controls (F). Control commissural axons then turn rostrally to project longitudinally beside the FP in the ventral funiculus (H). Most axons correctly make the rostral turn in netrin1ΔFP mice, however, we (S.A. and S.J.B., personal communication) and others (Moreno-Bravo et al., 2019) have observed that a subset of commissural axons fail to cross the FP, and turn ipsilaterally (I), resulting in a thinning of the FP commissure (Wu et al., 2019). It remains unresolved whether this ipsilateral turn is the result of axons following an ectopic hederal boundary created by the specific loss of netrin1 in the FP (I), or a direct requirement for FP-netrin1 in axon crossing. Long-range phenotypes for FP-derived netrin1: While we have not observed this phenotype, other reports (Wu et al., 2019) have shown that commissural axons are modestly defasciculated in the ventral spinal cord in the absence of FP-derived netrin1 (yellow arrowheads, G) i.e. ~100μm from the FP. This phenotype is significantly enhanced when netrin1ΔFP is combined with a Boc mutation (G), the non-canonical receptor that mediates the axon guidance activities of Shh (Wu et al., 2019).