Figure 1.
Sema6A Is Highly Expressed in the Developing Embryonic Thalamus
(A and B) Coronal section of an E14.5 mouse brain showing in situ hybridization for Sema6A in the forebrain (A). Sema6A is expressed in neurons and axons (arrowheads) of the dorsal geniculate nucleus (dLGN) and ventrobasal complex (VB) of the developing dorsal thalamus (dTh; [B]).
(C and D) PLAP staining on comparable coronal sections of Sema6A+/− mouse brains. PLAP-positive axons, presumably TCAs, can be seen projecting from the dTh through the internal capsule towards the neocortex (NCx; arrowheads).
Hyp, hypothalamus; vLGN, ventrolateral geniculate nucleus. Scale bars in (A and C) indicate 500 μm; in (B and D) 200 μm.
Figure 2.
Early Thalamocortical Guidance Defects in Sema6A−/− Mouse
Coronal sections showing half the brain or close-ups of ventral telencephalon.
(A and B) TCAs follow a normal route along the ventral telencephalon and striatum (Str) in wild-type embryos as revealed by DiI tracing from the dorsal thalamus (dTh, [A]) as well as by neurofilament staining (B).
(D–H) In Sema6A−/− embryos, a large proportion of TCAs are derailed at the ventral telencephalon (filled arrowheads in [D and H]), as shown by DiI tracing from the internal capsule (ic) at E15.5 (D) and neurofilament staining at E16.5 (E and F), and an overlaid series of images at E17.5 (G and H). Note that some TCAs follow their normal route through the striatum (Str) in the Sema6A−/− brains (open arrowhead in [D]). Also, note that misrouted TCAs in the Sema6A−/− brains appear to bifurcate in the ventral telencephalon (open and filled arrowheads in [H]).
(C and I) Schematic diagrams of the trajectory of TCAs in wild-type (C) and Sema6A−/− brains (I).
Scale bars in (A and D) indicate 1 mm; in (B and E) 500 μm, in (F) 300 μm; and in (G and H) 250 μm.
Hyp, hypothalamus; NCx, neocortex; Str, striatum.
Figure 3.
Visual Thalamocortical Neurons Misproject to the Ventral Telencephalon in the Absence of Sema6A Function
(A) DiI crystals were placed at the surface of the ventral telencephalon in wild-type and Sema6A−/− brains at P0 (ventral view). All other panels are coronal sections.
(B and C). No back-labeled cells were seen in the dorsal thalamus (dTh) after ventral telencephalic DiI placements in wild-type brains.
(D–I) Rostrocaudal coronal sections of a Sema6 −/− brain showing abnormal axonal projections (filled arrowheads in [E and H]) from the ventral telencephalon to the dTh. Many back-labeled cells were observed located at the dorsal lateral geniculate nucleus (dLGN; open arrowheads in [G and H]). At caudal levels, some back-labeled cells were located at the primary visual cortex (V1; open arrowhead in [I]).
Asterisks in (A, B, D, and E) indicate the DiI crystal placement sites. Scale bars in (A) indicate 2.5 mm; in (B and D–F) 1 mm; and in (C and G–I) 200 μm.
Str, striatum; VB, ventrobasal nucleus; vLGN, ventral lateral geniculate nucleus.
Figure 4.
DiI Crystal Placements to the Dorsal Lateral Geniculate Nucleus (dLGN) Confirmed That Visual Thalamic Axons Are Severely Affected by the Absence of Sema6A Function
Coronal sections showing half the brain or close-ups of specific areas.
(A–H) Small crystals of DiI were implanted into the dLGN of wild-type (A–D) and Sema6A−/− (E–H) brains at P0. Asterisks in (A, B, and E) indicate the DiI crystal placement sites. (A–D) DiI injections in the dLGN of wild-type brains, labeled thalamic axons extending through the internal capsule and striatum (Str), and entering to the neocortex (NCx; filled arrowheads in [B]). Many back-labeled cells were seen at the primary visual cortex (V1, open arrowheads in [C]).
(E–H) DiI placements in the dLGN of Sema6A−/− brains labeled bundles of axons misrouted at the ventral telencephalon (filled arrowheads in [E and F]). Fewer back-labeled cells were observed at V1 in Sema6A−/− brains (open arrowhead in [G]) compared to control.
Scale bars in (A) indicate 3 mm; in (B) 1 mm; in (C and G) 100 μm; and (F) 200 μm.
H, hippocampus; Hyp, hypothalamus.
Figure 5.
Early Topographical Defects in Thalamocortical Connectivity in Sema6A−/− Mice
(A–J) Coronal sections showing back-labeling in dorsal thalamus.
(A and B) In Sema6A+/− brains, DiI crystal placement in the occipital cortex back-labels cells in the dorsal lateral geniculate nucleus (dLGN; filled arrowheads), whereas a DiA crystal placement in parietal cortex back-labels cells in the ventrobasal complex (VB; open arrowheads).
(C–D) In Sema6A−/− brains, identical dye crystal placements resulted in back-labeled red (DiI) and green (DiA) cells in the VB of the dorsal thalamus, indicating a medial shift of visual thalamocortical connectivity in these mice. Note that at E16.5 (C), the visual shift was more pronounced than at P0 stages (D).
(E–H) In Sema6A+/− brains at E16.5, retinothalamic projections, labeled by a DiI crystal placed in the retina, entered the ventral and dorsal lateral geniculate nucleus (vLGN and dLGN, respectively; open arrowheads in [E and F]). A DiA crystal placement in the occipital cortex back-labeled dLGN cells (filled arrowheads in [E and F]). In contrast, in Sema6A−/− brains, whereas the retinal projection enters the vLGN and dLGN (open arrowheads [G and H]) normally, thalamic cells in the VB are back-labeled from a DiA crystal in the occipital cortex (filled arrowheads, [G and H]).
(I–L) At early stages (E16.5), the vast majority of cells back-labeled from the occipital cortex are found in the dLGN in Sema6A+/− mice, with few if any, being found in the VB. In Sema6A−/− mice at the same age, the majority of cells back-labeled from the occipital cortex are found in the VB, with very few being found in the dLGN.
These differences are highly statistically significant (triple asterisks [***] indicates p <0.0001).
At P0, the proportion of cells back-labeled from the occipital cortex and found in the dLGN is significantly greater than that at E16.5 but still significantly less than in Sema6A+/− mice (p < 0.0001). By P4, there was no significant difference between wild-type and Sema6A−/− mice in the proportion of cells back-labeled from the occipital cortex and found in the dLGN. Error bars indicate standard error mean (s.e.m.).
Scale bars in (A–D) indicate 200 μm; in (E and G) 200 μm; in (F and H) 200 μm; and in (I and J) 100 μm.
MeA, medial amygdala.
Figure 6.
Postnatal Recovery of Visual Thalamocortical Connectivity in Sema6A−/− Mice
All sections are coronal.
(A and B) In wild-type brains, DiI and DiA crystal placement in occipital and parietal cortex, respectively, back-labeled cells in the dorsal lateral geniculate nucleus (dLGN; filled arrowhead) and the ventrobasal complex (VB) (open arrowhead) at P4. Dashed box indicates the area in (A) that is magnified in (B).
(C and D) In contrast to what happened at earlier stages, in Sema6A−/− brains at P4, DiI crystal placement in the visual cortex back-labeled cells predominantly in the dLGN (filled arrowhead). DiA crystal placement in the somatosensory cortex back-labeled cells in the VB (open arrowhead). Dashed box indicates the area in (C) that is magnified in (D).
(E, F, H, and I) Red and green cholera toxin (CT) was injected into the visual (V1, open arrowhead in [H]) and somatosensory (S1, open arrowhead in [E]) cortex, respectively, of wild-type and Sema6A−/− adult mice. In wild-type and Sema6A−/− mice, red CT back-labeled cells and axon terminals in the dLGN (filled arrowheads in [F and I]), and green CT back-labeled cells and axon terminal in the VB (open arrowheads in [F and I]).
(G and J) Schematic diagrams showing the location of cell somata containing either red or green retrobeads, from injections in the visual (red) and somatosensory (green) cortices of adult wild-type and Sema6A−/− brains. In wild-type and Sema6A−/− brains, red retrobeads were found in the dLGN and never the VB in the dorsal thalamus. Green retrobeads were only ever observed in the VB.
Scale bars in (A and C) indicate 1 mm; in (B and D) 500 μm; in (E and H) 1 mm; and in (F and I) 300 μm.
MeA, medial amygdala; NCx, neocortex.
Figure 7.
PLAP Staining Reveals Potential Recovery Pathways of Visual TCAs in Sema6A−/− Adult Brains
All sections are coronal.
(A and F) Low-power images of coronal sections of adult Sema6A+/− (A) and Sema6A−/− (F) brains, stained for PLAP. Misrouted TCAs are clearly visible in the ventral telencephalon of Sema6A−/− brains (arrowheads in [F]).
(B, C, G–I) High-power view of these aberrant axon tracts (G), some of which project towards and into the external capsule, whereas others take a more ventral position and can be seen extending dorsally, close to the pial surface, up to the neocortex ([F, H, and I] red arrowheads). These axon tracts are never seen in wild-type or Sema6A+/− brains (B and C).
(D, E, J, and K) Caudal coronal sections, showing the primary visual cortex (V1) of adult Sema6A+/− (D) and Sema6A−/− (J) brains. Note the presence of misrouted axons close to the pial surface of V1 in Sema6A−/− brains (red arrowheads in [K]). These axon tracts are never seen in wild-type or Sema6A+/− brains (E).
(L and M). Overlaid serial sections through the same Sema6A−/− brain shown in (F) illustrates more clearly the persistent misrouted axons entering the external capsule (black arrowhead in [L and M]) or projecting more ventrally and close to the pial surface (red arrowheads in [L and M]) and up to the neocortex.
(N) Schematic representation of the aberrant routes taken by misrouted TCAs to the neocortex in Sema6A−/− brains.
Scale bars in (A, F, D, J, and L) indicate 1 mm; in (B, C, E, G, H, I and K) 200 μm; in (M) 500μm.
I, layer I; II–III, layers II and III; dTh, dorsal thalamus; H, hippocampus; MeA, medial amygdala; NCx, neocortex; Str, striatum.
Figure 8.
Recovery Pathways in Sema6A−/− Mouse Brains Revealed by Retrograde Labeling with DiI from the Primary Visual Cortex
All sections are coronal.
An injection of dissolved DiI in the V1 of Sema6A−/− mouse brains at P7 retrogradely labels cells bodies in the dLGN (A). The axons of these cells could be followed through consecutive sections extending ventrally into the ventral telencephalon and turning laterally and rostrally towards the external capsule (white arrowheads in [B]; black arrowheads in [D1–D6]). Many axons were also observed to initially project ventrally before turning to loop back to enter the internal capsule (black arrowheads in [D7]; schematic [C]). The entire pathway could be observed by overlaying consecutive coronal sections (B) as illustrated in the schematic diagram (C). Fibers back-labeled from V1 are labeled in blue. VB axons are not back-labeled from V1 at this stage, but their trajectories are shown for comparison in red.
Scale bars in (A) indicate 2 mm, in (B) 500 μm.
dLGN, dorsal lateral geniculate nucleus; ic, internal capsule; MeA, medial amygdala; NCx, neocortex; VB, ventrobasal complex.
Figure 9.
Effects of Loss of Sema6A on the Structure and Extension of Both dLGN and the Visual Cortex
(A–E) Coronal sections of wild-type (A and B) and Sema6A−/− (D and E) adult mouse brains stained with Nissl (A and D) and cytochrome oxidase (B and E). Note the large reduction in size and volume of the dorsal lateral geniculate nucleus (dLGN) in Sema6A−/− brains (C).
(F–J) Increased apoptosis in the dLGN in Sema6A−/− brains at P4 detected by caspase 3 antibody (filled arrowheads in [I and J]).
(H) Graphical representation of the increased levels of apoptosis in the dLGN of Sema6A−/− compared to wild-type mice.
(K and L) Tangential sections of wild-type (K) and Sema6A−/− (L) mouse brains stained for serotonin immunohistochemistry to reveal the cortical sensory domains at P7. Rostrocaudal and mediolateral directions are indicated by the cross-bars. Note that the size of the primary visual cortex (V1) in Sema6A−/− brains is reduced compared to that of wild-type brains.
(M) Graphical representation of the area of the V1 in wild-type and Sema6A−/− brains at P7.
Quantifications shown in (C, H, and M) are average + s.e.m. Double asterisks (**) indicate p < 0.001; triple asterisks (***) indicate p < 0.0005, t-test. Scale bars in (A, B, D, and E) indicate 300 μm; in (F, G, I, and J) 500 μm; and in (K and L) 2.5 mm.
A1, primary auditory cortex; PMBSF, posteromedial barrel subfield; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; vLGN, ventral lateral geniculate nucleus; VPM, ventroposterior medial nucleus.