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. Author manuscript; available in PMC: 2021 Nov 22.
Published in final edited form as: Neuron. 2019 Mar 19;102(3):602–620.e9. doi: 10.1016/j.neuron.2019.02.034

Figure 6. p190 Curbs Axon Attraction to Netrin-1.

Figure 6.

(A) Schematic of 3D-motor explant attraction assay. Hb9MN::GFP+ explants are co-cultured with cell aggregates in collagen-Matrigel matrices for ~15 h, which is insufficient time for motor axon outgrowth unless cell aggregates express a chemoattractant. The proximal (P) and distal (D) sides of the explant correspond to the two halves of the ventral spinal cord, containing the left and right motor columns (MC) and the floor plate (FP) in between.

(B) Quantification of directional axon growth (GFP+ pixels on the P versus D sides of the explant). p190−/− motor axons are attracted to Netrin-1. Mean ± SEM, n explants: control AP-Fc (11); control Netrin-1 (49); p190−/−/AP-Fc (18); p190−/−/AP-Fc (54). Mann-Whitney U test, ns, p > 0.05; ***p < 0.001 Netrin-1 versus AP-Fc control.

(C–F) Motor axon growth is minimal in the presence of aggregates releasing control AP-Fc (C and E), or in control explants exposed to Netrin-1 (D). In contrast, numerous GFP+ motor axons extend from the P side in p190−/− explants (F).

(G) Schematic of 2D motor axon growth assay. Hb9MN::GFP+ motor neuron explants are cultured on surfaces coated with laminin and either recombinant Netrin-1 or control IgG-Fc.

(H) Quantification of GFP+ motor axons on Netrin-1 relative to control IgG-Fc. Control explants are insensitive to Netrin-1, while p190−/− explants show increased outgrowth. Mean ± SEM, n explants: control IgG (23); control Netrin-1 (47); p190−/−/IgG (22); p190−/−/Netrin-1 (38). Mann-Whitney U test, ns, p > 0.05; ***p < 0.001 Netrin-treated versus IgG control.

(I–L) p190−/− explants (L) display enhanced motor axon outgrowth on Netrin-1 substrate compared to control explants (J). Axon outgrowth is minimal on IgG-Fc substrate (I and K).

(M) Quantification of GFP+ motor axons. Mean ± SEM, n explants: p190−/−/IgG (7); p190−/−/Netrin-1 (13); p190R1284/−/IgG (7); p190R1284/−/Netrin-1 (9). Mann-Whitney U test, ns, p > 0.05; ***p < 0.001 Netrin-treated versus IgG control.

(N–Q) ISLMN::fGFP+ motor explants grown on either Netrin-1 (O and Q) or control IgG-Fc substrates (N and P). p190−/− motor axons display enhanced outgrowth on Netrin-1+ (O) while axon growth is minimal for p190R1284/− explants (Q).

Scale bars, 200 μm. See also Figure S6.