Figure 3. Netrin1 orients the initiation of axonal growth around the circumference of the spinal cord.

(A–T) Lumbar (A, B, E–H) or thoracic (C, D, I–T) level transverse sections of E10.5 netrin1^+/+(E, F, K–O), netrin1^lacZ/+ (A–D) or netrin1^lacZ/lacZ (G–J, P–T) mouse spinal cords labeled with antibodies against NF (green), β-gal (red, A–D, G–J), Robo3 (red, K–L, N, P–Q and S), netrin1 (E–F) and Tag1 (blue, K–L, O, P–Q and T).

(A–F) Ventrally-directed NF⁺ axon growth starts at a position co-incident with the dorsal-most border of both netrin1::β-gal expression in the VZ domain (chevrons, B, D) and the netrin1 substrate on the pial surface (chevrons, F). Inset in E shown in F.

(G–J) In contrast, NF⁺ axons grow more randomly in netrin1 mutant spinal cords (I), robustly extending both into zone 1, i.e. above the netrin1::β-gal dorsal boundary (brackets, H, J) and into zone 2, i.e. medially into the VZ (chevrons, H, J).

(K–O) At E10.5, control NF⁺, Robo3⁺ and Tag1⁺ commissural axons are in the process of pioneering their path to the FP at the ventral midline. The motor column and DRGs also express Tag1 at this stage.

(P–T) In netrin1 mutants, NF⁺ and Robo3⁺ axons project randomly along the dorsal-ventral axis of the spinal cord (chevrons, Q, R, S). The intermediate spinal cord in K, P is shown magnified in L, Q. Tag1⁺ (T) and Robo3⁺ (S) commissural axons additionally show an axon growth defect, either stalled growth (Serafini et al., 1996) or a delay in the initiation of axon growth.

(U) Quantification showed that NF⁺ axon growth co enced at the netrin1::β-gal dorsal border in >70% of control sections (n=35 sections, 2 mice), whereas there was growth above this border in >60% of sections taken from netrin1 mutant sections (n= 55 sections from 4 mice).

(V) Up to 4-fold more NF⁺ Robo3⁺netrin1 mutant growth cones (shown in the inset panel) are observed extending towards the lumen. n as for (W).

(W) Up to 4-fold more NF⁺ or NF⁺ Robo3⁺ axons project into the VZ in netrin1 mutant spinal cords (n=62 sections from 4 embryos) compared to control littermates (n=34 sections from 3 embryos).

(X) The NF⁺ and NF⁺ Robo3⁺ mis-projecting axons were also assigned to four zones within the spinal cord (Fig. 2O), to assess the fold change in axon extension into the netrin1^lacZ/lacZ VZ along the dorsal-ventral axis. n as for (W).

For the quantification, probability of similarity between control and mutant, *** p< 0.0005, Student’s t-test.

Scale bar: 130 µm