(A) From left to right: The sagittal diagram shows the projection stacks for the post-commissural fornix (pf; red) and mammillo-thalamic tract (mt, purple); the green line indicates the level of the coronal section. Representative coronal section corresponding to the onset of the anterior part of the anterior hypothalamic area (AHA) in postnatal day 0 (P0) wild-type (WT) and CRMP4-KO brains immunolabeled with anti-neuropilin-1 (Nrp1) antibody. Fornix and mammillo-thalamic tract were squared. Scale bar, 1 mm. Higher-magnification images show representative post-commissural and mammillo-thalamic tracts in CRMP4-KO and WT brains after Nrp1 immunolabeling. Scale bars, 100 µm. Dot plots show quantifications of both fornix and mammillo-thalamic tract diameters. Mean ± s.e.m. WT n = 6, KO n = 5, Mann-Whitney test, *p < 0.001. (B) From left to right: The coronal diagram shows the projection stack of post-commissural fornix (red) on a coronal plane. The green line indicates the plane of the sagittal sections. Representative sagittal sections with a cutting angle of 30° for P0 WT and CRMP4-KO brains were immunolabeled with anti-Nrp1 antibody. The lengths of post-commissural fornices were measured using ImageJ, taking the anterior commissure as the starting point. All traces measured from WT (n = 5; red) and CRMP4-KO (n = 6; green) were superimposed and are shown in the right panel. The dot plot shows quantifications of post-commissural fornices. Scale bar, 250 µm. Mean ± s.e.m. Mann-Whitney test, *p < 0.05. (C) The sagittal diagram shows the projection stack of the post-commissural fornix (pf, in red) and black lanes indicate the segmented portion of the fornix. The anterior commissure (ac, in orange) was used as the starting point to segment the fornix (upper left diagram). A representative P2 segmented fornix is shown in 3D reconstruction (Nrp1 in red) in the whole brain (autofluorescence in grey), illustrated in lateral view and ventral view (upper right panel). Reconstructions were produced using the ImageJ 3D Viewer plugin. Scale bar, 1 mm. Representative ventral z projections of WT and CRMP4-KO fornices at embryonic day 18.5 (E18.5), P0, and P2 are presented (bottom left panels). The fluorescence intensity of the segmented fornix is represented by a red-to-white color scale. White arrowheads point to axon bundles splitting off from the fornix. Scale bar, 500 µm. Histogram shows quantifications of the segmented post-commissural fornix volume at E18.5 (WT = 3; KO = 2), P0 (WT = 4, KO = 5) and P2 (WT = 3; KO = 2) for WT and CRMP4-KO mice. Bars indicate the mean volume ± s.e.m. Kruskal-Wallis non-parametric test followed by Dunn’s multiple comparisons and one-way ANOVA to compare WT vs. KO, *p < 0.05, **p < 0.01.