A Unique “Reversed” Migration of Neurons in the Developing Claustrum

Migration profile of the CLA neurons at each stage of development. A–E, FlashTag was injected at E11.5, and the brains were fixed at E12.5 (A), E13.5 (B), E14.5 (C), E15.5 (D), and E16.5 (E). B, White arrow indicates cortical migratory stream. A′–E′, A′′, Higher magnifications of the boxed areas in A–E. B–E′′, C′′′, Higher magnifications of the labeled-boxed areas in B′–E′. FlashTag-labeled Nurr1-positive cells were rarely observed at E13.5, but they became visible clearly at E15.5 (cyan arrowheads in D′′). F, The percentages of Nurr1-positive cells among FlashTag-positive cells in the presumptive CLA and insular cortex were measured. Values shown are the mean ± SE (from four different slices at E13.5-E15.5 or three different slices at E16.5). Total number of cells counted: 1169. **p < 0.01 (Tukey–Kramer test). Scale bars: A–D, 90 µm; A′-D′, 30 µm; A′′-D′′, C′′′, 20 µm. Left, Superficial (lateral) side. Right, Deep (medial) side.

Time-lapse imaging of migrating CLA neurons. A, A′, Time-lapse images were captured from the CLA slices prepared 3 d after electroporation of the GFP-plasmid at E11.5. The time displayed above each panel indicates the elapsed time since the beginning of the observation. The two-way arrow on the right of the panel indicates that the top is the lateral (superficial) side, and the bottom is the medial (deep) side. The orientation is consistent with B–E. The trajectories of representative cells were shown in cyan (A′), and the cell bodies were indicated by white arrowheads (A, A′). Some cells migrated from the superficial region toward the deep region. B, C, Higher magnifications of the cell shown in A′. The representative cell turned its leading process toward the superficial region at first (B, 4:00, arrowhead) and then turned it into the deep direction (C, 24:00, arrowhead). D, Immunohistochemistry for Nurr1 indicated that some GFP-labeled cells were Nurr1-positive. D′, High magnification of the labeled-boxed area in D. E, The angle of migration was defined as θ. The orientation is consistent with A–D′. F, The distribution of migration direction was bimodal: ∼100° (toward the superficial region) and –100° (toward the deep region). G, The maximum migration speeds of cells migrating inward (n = 28) and outward (n = 26) were measured. Each data point represents a different cell in a representative brain. H, I, The trajectories of migrating cells moving inward and outward were traced, and the speeds of the cells in each period were measured. The speed of representative cells migrating inward (H) and outward (I). J, K, Mean of the average migration speeds (J) and mean of the maximum migration speeds (K) of the cells migrating inward and outward. **p < 0.01 (Student's t test). F, J, K, Values are the mean ± SE of four different brains (n = 54, 61, 70, and 77). Scale bars: A-A′, 25 µm; B, C, 15 µm; D, 50 µm; D′, 25 µm.

Detailed analysis of time-lapse images of migrating CLA neurons. A, The radial speed of representative actively inward-migrating cells relative to the LCS was measured. Error bar indicates mean ± SE (n = 6, 6, 8, and 11 from four different slices). B, The ratios of the number of cells which extended their leading process toward superficial, deep, or both directions to the total number of analyzed GFP-positive cells were measured. Values shown are the mean ± SE (n = 16, 18, 19, and 31, from four different slices). C, D, Time-lapse images were captured from the CLA slices prepared 3 d after electroporation of the GFP-plasmid at E11.5. The time displayed above each panel indicates the elapsed time since the beginning of the observation. Scale bars, 15 µm. Top, Superficial (lateral) side. Bottom, Deep (medial) side.

Sparse-cell labeling of migrating neurons. tdTomato plasmid was transfected into the embryos at E11.5, and the brains were fixed 3.5 d later. A–C, Immunostaining for RFP (magenta) and Nurr1 (green). Representative cells with the major process directed toward the superficial region (A), the deep region (B), or both directions (C) are shown (white arrowheads). The nuclei were labeled with DAPI (blue). Top, Superficial side. Right, Dorsal side. D–F, Higher magnifications of the labeled-boxed areas in A–C are shown, respectively. Some tdTomato-labeled cells directed their leading process toward the deep region (a white arrowhead), and they also oriented a thin process toward the superficial region (a cyan arrowhead). G, The mean ± SE of cells extending their process toward the deep region as a proportion of the tdTomato-labeled cells in the Nurr1-negative superficial area and the Nurr1-positive deep area in four different brains (n = 19, 24, 44, and 45) were calculated. H, Immunostaining for RFP (magenta) and Nestin (green). Top, Superficial side. Right, Ventral side. I–I′′, Higher magnifications of the boxed area in H are shown. J, Immunoelectron microscopic analysis of an inward-migrating neuron. The embryos at E11.5 were transfected with GFP-expressing plasmid, fixed 3.5 d later, and observed through an immunoelectron microscope. Migrating neurons were immunolabeled with an anti-GFP antibody (visualized with nanogold, black spots). A representative immunostained cell that turned its major process toward the deep region (cyan arrow) was shown. Its cell body was indicated by a white arrow. K–M, Higher magnifications of the labeled-boxed areas in J. The cell had contact with multiple fibers (cyan arrowheads). Top, Superficial side. Bottom, Deep side. Scale bars: A–C, 50 µm; D–F, 25 µm; H, 100 µm; I–I′′, 50 µm; J, 2 µm; K–M, 500 nm.

The CLA expressed ApoER2 during development. A–C, Brains were immunostained with anti-ApoER2 (green) and anti-Reelin (magenta) antibodies at E13.5 (A), E14.5 (B), and E15.5 (C). White arrows indicate cortical migratory stream. The nuclei were labeled with DAPI (blue). A′–C′, Higher magnifications of the labeled-boxed area in A–C, respectively. Scale bars: A–C, 200 µm; A′–C′, 100 µm. Left, Deep (medial) side. Right, Superficial (lateral) side. Top, Dorsal side. Bottom, Ventral side.

Reelin mutation or KO of Apoer2 resulted in malposition of CLA neurons. A, Immunostaining for Nurr1 (magenta) and Ctip2 (green) was performed in reeler (Reelin-deficient) mice and heterozygous reeler mice at E18.5. C, Immunostaining for Nurr1 (magenta) and Reelin (green) was performed in Apoer2 homozygous and heterozygous KO mice. B, D, A bin analysis was performed to evaluate cell distribution by dividing the space between the pial surface and the medial border of LCS into 10 equal areas (10 bins), and the number of Nurr1-positive cells in each bin was calculated as a percentage of the total number in all 10 bins (B and D correspond to A and C, respectively). Mean ± SE of data obtained from four different brains in rl/+, rl/rl, and Apoer2^+/−, and three different brains in Apoer2^−/− are shown. The ratio of each corresponding bin was statistically compared between homozygous and heterozygous mutant/KO mice. *p < 0.05; **p < 0.01; Tukey–Kramer test. Scale bars: A, C, 100 µm. Top, Dorsal side. Bottom, Ventral side. Left, Superficial (lateral) side. Right, Deep (medial) side.

Vldlr KO resulted in no remarkable difference in the alignment of CLA neurons. Immunostaining for Nurr1 (magenta) and Reelin (green) was performed in Vlder homozygous and heterozygous KO mice at E18.5. The nuclei were labeled with DAPI (cyan). Scale bars, 100 µm. Top, Dorsal side. Bottom, Ventral side. Left, Superficial (lateral) side. Right, Deep (medial) side.