Fig 2: FP long-range chemoattractive activity is not correlated with CA guidance phenotypes. (a–e) Cocultures of dorsal hindbrain explants with FP explants prepared from Ntn1 +/+ (a), Ntn1 Ko (b), Ntn1 FP-Ko (c), Ntn1 VZ-Ko (d) and Ntn1 dVZ-Ko (e) mice. After culture for 28–32 h, the explants were immunostained for Robo3. FP explants from Ntn1 +/+ (a) and Ntn1 VZ-Ko (d) and Ntn1 dVZ-Ko mice (e) elicit outgrowth of Robo3+ neurites from the dorsal hindbrain explants, whereas few axons emanate from the dorsal hindbrain explants in coculture with FP explants from Ntn1 Ko (b) and Ntn1 FP-Ko (d) mice. (f) Histograms representing the Robo3+ neurite outgrowth from the proximal side facing the FP explant (Ntn1 +/+, n = 13; Ntn1 Ko, n = 10; Ntn1 FP-Ko, n = 7; Ntn1 VZ-Ko, n = 11; Ntn1 dVZ-Ko, n = 11; n.s. P > 0.05, *P < 0.05, ***P < 0.001, Kruskal-Wallis test followed by Steel-Dwass post hoc test; P = 5.32 × 10−4 for Ntn1 +/+ versus Ntn1 Ko, P = 9.83 × 10−5 for Ntn1 +/+ versus Ntn1 FP-Ko, P = 0.974 for Ntn1 +/+ versus Ntn1 dVZ-Ko). Error bars indicate SEM. FP explants from Ntn1 VZ-Ko mice do elicit Robo3+ neurite outgrowth from dorsal hindbrain explants (P = 7.26 × 10−4 for Ntn1 VZ-Ko versus Ntn1 Ko, P = 1.52 × 10−4 for Ntn1 VZ-Ko versus Ntn1 FP-Ko), albeit to a lesser extent than those from Ntn1 +/+ or Ntn1 dVZ-Ko mice (P = 0.0377 for Ntn1 VZ-Ko versus Ntn1 +/+, P = 0.0146 for Ntn1 VZ-Ko versus Ntn1 dVZ-Ko).

Fig 3: Normal CA growth in Ntn1 pMN-Ko mice. (a,b) Ntn1 ISH in whole-mount preparations of E12.5 control (Olig2 +/Cre ;Ntn1 +/flox) (a) and Ntn1 pMN-Ko (b) mouse hindbrains (control, n = 6; Ntn1 pMN-Ko, n = 6). In Ntn1 pMN-Ko mouse hindbrains, Ntn1 hybridization signals are not detected in pMN adjacent to the FP (arrowheads). (c,d) Robo3 immunostaining in whole-mount preparations of E12.5 control (c) and Ntn1 pMN-Ko (d) mouse hindbrains (control, n = 8; Ntn1 pMN-Ko, n = 5). In both genotypes, Robo3+ axons grow ventrally and reach the midline. (e,f) DiI labeling of CAs in whole-mount preparations of E12.5 control (e) and Ntn1 pMN-Ko (f) mouse hindbrains (control, n = 8; Ntn1 pMN-Ko, n = 6). DiI-labeled CAs grow towards the midline (arrowheads) and cross it in both genotypes. (g,h) Histograms representing the fluorescence intensity of Robo3+ axons within the ventral one-fourth of the hindbrain normalized to that in the preparation (control, n = 8; Ntn1 pMN-Ko, n = 5; n.s P > 0.05, Mann–Whitney U-test; P = 0.724) (g) and the fluorescence intensity of DiI-labeled axons within the FP normalized to that at the implantation site (control, n = 8; Ntn1 pMN-Ko, n = 6; n.s P > 0.05, Mann–Whitney U-test; P = 0.573) (h), respectively. Error bars indicate SEM. There are no significant differences in both Robo3+ and DiI-labeled axon growth between genotypes. Dorsal is upwards and rostral is towards the left. Hindbrains at the rhombomere 6 to 8 level are represented. The bar in (a), (c) and (e) apply to (a,b), (c,d) and (e,f), respectively.

Fig 4: Commissural Axon Guidance Defects in Ndfip Double Mutants(A and B) Representative confocal images of E11.5 transverse spinal cord sections that were taken from Ndfip2+/− or Ndfip2−/− or Ndfip1−/−; Ndfip2−/− mouse embryos. All sections were processed for immunohistochemistry for Robo3 (A) and TAG1 (B). Bottom rows show the ventral commissure bundle at higher magnification. Ndfip1−/−; Ndfip2−/− mutant embryos exhibit significant reduction in ventral commissure thickness compared with Ndfip2−/− and Ndfip2+/− embryos.(C and D) Quantification of Robo3-positive (C) and TAG1-positive (D) commissure thickness normalized with the length of the spinal cord at E11.5 in Ndfip2+/− or Ndfip2−/− or Ndfip1−/−; Ndfip2−/− mouse embryos. Robo3+ commissure thickness in Ndfip2−/− (0.8 ± 0.013, p = 0.0015) and Ndfip1−/−; Ndfip2−/− (0.6 ± 0.014, p < 0.0001) and TAG1 + commissure thickness in Ndfip2−/− (0.73 ± 0.012, p = 0.0004) and Ndfip1−/−; Ndfip2−/− (0.55 ± 0.008, p < 0.0001). The quantifications show the mean and SEM of five to eight sections per embryo, with n = 3 embryos were analyzed for each indicated genotype.(E–L) Confocal images of Dil injections in E12.5 spinal cord open-book preparations labeling commissural axons. The majority of axons in open-book preparations of wild-type embryos cross the floor plate and turn anteriorly on the contralateral side (E and F). In contrast, labeled axons in Ndfip2−/− spinal cords frequently stop short and fail to make the correct anterior turn (G). In a few embryos, we also observed that some axons take an abnormal posterior turn in Ndfip2 mutant spinal cords (denoted with asterisk in H).(I–L) In Ndfip1−/−; Ndfip2−/−, these phenotypes are significantly stronger than those observed in the Ndfip2 single-mutant cords (G and H). In addition to stalling phenotypes, we sometimes observe ipsilateral mis projections in Ndfip1−/−; Ndfip2−/− spinal cords (denoted with asterisk in L).(M) The graph represents the percentage of the axons with the indicated phenotype. The percentage of axons that turned anteriorly is significantly decreased in Ndfip2−/− and Ndfip1−/−; Ndfip2−/− mouse embryos compared with wild-type control. Percentage of axons that turned anteriorly in Ndfip2−/− (50% ± 0.40, p = 0.0016) and in Ndfip1−/−; Ndfip2−/− (18% ± 0.34, p < 0.0001). Wild-type; n = 4 with number of injection sites 17, Ndfip2−/−; n = 5 with number of injection sites 22, Ndfip1−/−; Ndfip2−/−; n = 3 with number of injection sites 11 (n, number of embryos analyzed for each genotype). Significance was assessed using Student’s t test (**p < 0.0001 and *p < 0.01). FP, floor plate. Scale bars represent 50 μm in (A) and (B); higher magnification images in (A) and (B) are 20 μm and 20 μm in (E)–(L).

Fig 5: Aberrant CA growth caused by deletion of Ntn1 from the VZ. (a–d) Robo3 immunostaining in whole-mount preparations of E12.5 Ntn1 +/+ (a), Ntn1 Ko (b), Ntn1 FP-Ko (c) and Ntn1 VZ-Ko (d) mouse hindbrains (Ntn1 +/+, n = 7; Ntn1 Ko, n = 5; Ntn1 FP-Ko, n = 8; Ntn1 VZ-Ko; n = 7). Robo3+ axons grow ventrally and reach the FP in Ntn1 +/+ and Ntn1 FP-Ko mice (a,c). Ventrally growing Robo3+ axons were highly disorganized and their number is markedly reduced in Ntn1 Ko and Ntn1 VZ-Ko mice (b,d). (e) Histograms representing the fluorescence intensity of Robo3+ axons within the ventral one-fourth of the hindbrain normalized to that in the preparation (Ntn1 +/+, n = 7; Ntn1 Ko, n = 5; Ntn1 FP-Ko, n = 8; Ntn1 VZ-Ko, n = 7; n.s P > 0.05, *P < 0.05, **P < 0.01; Kruskal-Wallis test followed by Steel post hoc test; P Ko = 0.0126, P FP-Ko = 0.159, P VZ-Ko = 0.00493). Error bars indicate SEM. (f–i) DiI labeling of CAs in whole-mount preparations of E12.5 Ntn1 +/+ (f), Ntn1 ko (g), Ntn1 FP-ko (h) and Ntn1 VZ-ko (i) mouse hindbrains (Ntn1 +/+, n = 6; Ntn1 Ko, n = 9; Ntn1 FP-Ko, n = 9; Ntn1 VZ-Ko, n = 8). DiI crystals are implanted into the dorsal hindbrain at the rhombomere 7/8 level. In Ntn1 +/+ mice, DiI-labeled CAs grow straight towards the FP (arrowheads) and crossed it (f). Longitudinally extending axons in the dorsal margin and ipsilaterally turning axons at the middle along the dorsoventral axis are also normally labeled (f). In Ntn1 Ko mice, ventrally directed axons are foreshortened, spread rostrocaudally and fail to invade ventral hindbrain (g). Ntn1 FP-Ko mice exhibit axon trajectories almost identical to those of Ntn1 +/+ mice (h, arrowheads), whereas Ntn1 VZ-Ko mice exhibit CA guidance defects similar to those of Ntn1 Ko mice (i). (j) Histograms representing the fluorescence intensity of DiI-labeled axons within the FP normalized to that at the implantation site (Ntn1 +/+, n = 6; Ntn1 Ko, n = 9; Ntn1 FP-Ko, n = 9; Ntn1 VZ-Ko, n = 8; n.s P > 0.05, **P < 0.01, ***P < 0.001; Kruskal-Wallis test followed by Steel post hoc test; P Ko = 8.65 × 10−4, P FP-Ko = 0.334, P VZ-Ko = 0.00533). Error bars indicate SEM. Dorsal is upwards and rostral is towards the left. Hindbrains at the rhombomere 6–8 level are represented. The bar in (d) and (i) apply to (a–d) and (f–i), respectively.