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doi: 10.1242/10.1242/dev.00166

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The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice

¹ Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California San Francisco, San Francisco, CA 94143-0984, USA
² Gene Center and Institute of Biochemistry, University of Munich, Feodor Lynenstrasse 25, 81377 Munich, Germany

View larger version (87K): [in a new window]   Fig. 1. dLGN axons are misrouted in the amygdalar region of Ebf1-/- embryos. DiI axonal tracing in controls (left) and Ebf1-/- mutant embryos (right). (A-D) Coronal hemisections of E16 brains in which a DiI crystal was placed in the lateral part of the dorsal thalamus (stars in A,B). In controls, DiI-labeled thalamic axons exit the dorsal thalamus and enter the internal capsule (open arrowhead in A). More rostrally, thalamic axons travel through the striatum and reach the neocortical intermediate zone (open arrowheads in C). In homozygous Ebf1 mutants, at caudal levels, some thalamic axons grow ectopically into the amygdalar region (white arrowhead in B) and very few axons leave this aberrant tract to navigate through the striatum and towards the neocortex (open arrowhead in B). However, rostrally, Ebf1 mutant thalamic axons are normally positioned in the striatum and neocortex (open arrowheads in D). (E,F) Coronal hemisections of E16.5 brains where a DiI crystal was placed in the amygdalar region (stars in E,F). The stria terminalis is stained in both controls and Ebf1-/- mutant embryos (open arrowhead in E,F). However, in Ebf1-/- embryos DiI labeling of the abnormal amygdalar tract (white arrowhead) retrogradely labels cells in a thalamic nucleus that has the shape and position of the dLGN (arrow). Am, amygdala; dTH, dorsal thalamus; Ncx, neocortex; Str, striatum.

View larger version (71K): [in a new window]   Fig. 2. Ebf1 inactivation induces a shift in thalamocortical and corticothalamic connections. Coronal hemisections of E16.5 brains where crystals of DiI (A-M) or DiI and DiA (N-P) were placed in several regions of the neocortex of controls (left) and Ebf1-/- mutants (right). Schematic representations of a dorsal view of the brain (A,D,G,K,N) indicate the position of DiI and DiA crystals in the occipital (A,D), parietal (G), frontal (K), or parietal and occipital (N) neocortex. The insertion sites of DiI crystals are also visible in B and C (stars). The retroflexus tract, which can be used as a morphological landmark, is indicated by a white arrowhead (E,F,H,I,L,M,O,P). Open arrowheads indicate the medial boundary of the thalamic domain where cell bodies and axons are labeled in wild-type embryos (E,H,L,O). This wild-type boundary is indicated in Ebf1-/- embryos (open arrowheads) and shows that position of the labeled thalamic domains are shifted medially (F,I,M,P). dTH, dorsal thalamus; Fr, frontal neocortex; IC, internal capsule; Ncx, neocortex; OB, olfactory bulb; Occ, occipital neocortex; Par, Parietal neocortex; Str, striatum.

View larger version (30K): [in a new window]   Fig. 10. Schematic representation of the phenotypes observed in Ebf1-/- and Dlx1/2-/- mutant embryos. Schematic coronal serial sections of the forebrain at the level of the occipital, parietal and frontal neocortex. In the wild-type brain, neocortical domains and thalamic nuclei that are normally interconnected have the same color and are connected by an axon tract of the same color. The axons grow through the internal capsule (open circle) and they pass through the basal ganglia (broken black lines). In Ebf1-/- embryos, the basal ganglia domain has molecular defects (indicated by the light gray), dLGN axons (dark blue) grow ectopically into the amygdalar region; the remainder of thalamic projection show a shift in their positions in the internal capsule and in the neocortex. In Dlx1/2-/- embryos, the basal ganglia develop abnormally (indicated by dark gray). The internal capsule is perturbed and numerous thalamic axons, the identity of which could not be clearly determined, grow into the amygdalar region and then travel rostrally (gray bundle). These probably contain dLGN axons, as these were not detected in the neocortex. Other axons grow towards the neocortex in the internal capsule; as in the Ebf1 mutants, these axons show a shift in their position within the internal capsule and in the neocortical domain that they enter.

View larger version (69K): [in a new window]   Fig. 3. Ebf1 is expressed in the dorsal thalamus and in the basal ganglia, along the path of thalamic axons. Coronal hemisections of E13.5 (A,A',B,B') or E14.5 (C,C') wild-type brains processed for Ebf1 in situ hybridization and Hoechst fluorescent cell stain. On the left, brightfield pictures showing Ebf1 expression are presented. On the right are fluorescent pictures of the same sections, where Ebf1 expression (open arrowheads in A',B',C') as well as low cell-density fiber tracts such as the thalamic axons (white arrowheads in A',B',C') appear in black. At E13.5, Ebf1 expression is observed in the mantle of the dorsal thalamus and of the ganglionic eminences (open arrowheads in A,B). Note that by E13.5, Ebf1 expression is undetected in the layer I of the neocortex. At E14.5, Ebf1 expression is observed in the amygdala and striatum (arrows in C) as well as in a group of cells located near the telencephalon-diencephalon boundary (open arrowhead in C). Ebf1-expressing cells are located on the path of thalamic axons at both ages (A',B',C'). dTH, dorsal thalamus; GE, ganglionic eminence; Ncx, neocortex.

View larger version (91K): [in a new window]   Fig. 4. Neocortical regionalization is not affected by Ebf1 inactivation. Sagittal sections of E17.5 heterozygous (left) and homozygous (right) Ebf1 mutants processed for in situ hybridization with the following probes: Id2 (A,B), COUP-TFI (C,D), Cdh6 (E,F), Epha7 (G,H) and Cdh8 (I,J). Arrowheads indicate rostrocaudal boundaries or changes in gene expression. These boundaries or gradients of expression are not changed in Ebf1-/- mutant embryos. H, hippocampus; Ncx, neocortex; OB, olfactory bulb; Str, striatum.

View larger version (52K): [in a new window]   Fig. 5. Ebf1 inactivation does not affect the molecular regionalization of the dorsal thalamus. Coronal hemisections of E16.5 heterozygous (left) and homozygous (right) Ebf1 mutant brains processed for in situ hybridization with the following probes: COUP-TFI (A,B), Cdh6 (C,D), Epha7 (E,F), Epha4 (G,H), Lhx9 (I,J), Gbx2 (K,L) and Sema6a (M,N). The expression domains of COUP-TF1, Lhx9 and Gbx2 cover large domains of the dorsal thalamus (A,I,K), Epha7 and Sema6a are excluded from dLGN (E,M), Cdh6 and Sema6a are low in medial nuclei (C,M), and Epha4 shows a lateromedial gradient within VB (G). Expression of these genes is not apparently perturbed by Ebf1 inactivation. Black arrowheads indicate the position of the retroflexus tract. A broken line indicates the pial surface of the thalamus in E,F,M,N. dLGN, dorsal lateral geniculate nucleus; dTH, dorsal thalamus; IC, internal capsule; Ncx, neocortex; VB, ventrobasal complex.

View larger version (100K): [in a new window]   Fig. 6. Ebf1 inactivation affects the pathfinding of early thalamic axons and Sema6a expression in the primordium of the basal ganglia. (A-H) Coronal hemisections of E13.5 (left) and E15.5 (right) wild-type and Ebf1-/- brains where a DiI crystal was introduced in the lateral part of the neocortex (A-D) or of the dorsal thalamus (E-H). In both wild-type and Ebf1-/- embryos, axons labeled by cortical DiI crystals make a sharp turn and enter the ganglionic eminences (open arrowhead in A-D). Crystals placed in the dorsal thalamus of wild-type embryos show that DiI-labeled thalamic axons (white arrowhead in E,G) make a sharp turn into the ganglionic eminences in the direction of the neocortex. On the contrary, in Ebf1-/- embryos thalamic axons run closer to the pial surface of the brain (white arrowhead in F,H). In E13.5 brains, a group of retrogradely labeled cells is detected (arrow in F) at the position where thalamic axons are located in wild-type embryos (compare arrow in F with arrowhead in E). These cells of the perireticular nucleus are intermingled with the thalamic axons in wild-type embryos. (I-P) Coronal hemisections of E13.5 (left) and E15.5 (right) wild-type and Ebf1-/- brains processed for Sema6a (I-L) and netrin 1 (M-P) in situ hybridization. In E13.5 controls (I), Sema6a is expressed in the mantle of the dorsal thalamus (black arrowhead), in a group of cells in the mantle of the ganglionic eminences (open arrow) and in the ventral pallium (black arrow). In Ebf1-/- mutant E13.5 embryos (J), although the thalamic expression is unaffected (black arrowhead), Sema6a expression in the mantle of the ganglionic eminences is greatly reduced. In E15.5 wild-type embryos (K), thalamic fibers (open arrowhead) grow dorsally to a group of Sema6a-expressing cells (open arrow). However in Ebf1-/- mutant embryos (L), Sema6a expression is strongly reduced and thalamic axons (open arrowhead) invade a zone that would be Sema6a positive in wild-type embryos. Netrin 1 expression, in E13.5 controls (M) is detected in the dorsal thalamus mantle (black arrowhead), in a group of cells in the mantle of the ganglionic eminences (open arrow) and in the ventral pallium (black arrow). At E15.5 (O), two groups of netrin 1-positive cells (black arrows) are located on both sides of the incoming thalamic axons (open arrowhead) and another group of labeled cells is located more laterally (open arrowhead). In Ebf1-/- mutant embryos (N,P) this expression pattern was not clearly changed, except for a slight reduction at E13.5, in the expression domain in the ganglionic eminences (compare M with N). dTH, dorsal thalamus; GE, ganglionic eminence; Ncx, neocortex.

View larger version (71K): [in a new window]   Fig. 7. A caudal shift of thalamic axons is detected inside the internal capsule of Ebf1-/- embryos. Horizontal hemisections of E16.5 wild-type (left) and Ebf1-/- mutant (right) brains in which one crystal of DiI and one of DiA were introduced in: (1) the frontal and parietal neocortex (A-C); (2) the occipital and parietal neocortex (D-F); and (3) in two putative thalamic nuclei — the dorsal lateral geniculate nucleus (dLGN) and ventrobasal complex (VB) (G-K). Schematic diagrams show the locations of DiI and DiA injection sites (A,D,G). (A-F) Horizontal sections of either frontal and parietal neocortex double injections (A-C) or occipital and parietal neocortex double injections (D-F). In wild-type embryos, bundles of labeled axons are restricted to regions of the internal capsule. The position of neocortical axons was not detectably altered in Ebf1 mutants (compare B with C and E with F). (G-K) Horizontal sections at ventral levels (H,I) and more dorsal levels (J,K) of brains following a thalamic double injection. Even though the tracer crystals were relatively small, a large number of axons were stained in our experiments because of the small size of the thalamus. In wild-type animals, putative dLGN axons (red) and VB axons (green) turn into the striatum (H) and remain as two separate bundles in the caudal and intermediate regions of the internal capsule, respectively (J). In Ebf1-/- mutant embryos, dLGN axons are detected as an abnormal tract going towards the amygdala (white arrowhead in I). In dorsal sections, only VB axons are detected (green) and they are located in a more caudal region of the internal capsule than in controls (compare J with K). dTH, dorsal thalamus; Fr, frontal neocortex; Ncx, neocortex; Occ, occipital neocortex; Par, parietal neocortex; Str, striatum.

View larger version (72K): [in a new window]   Fig. 8. Both cortical and thalamic axons pathfinding defects contribute to the formation of an abnormal internal capsule in Dlx1/2-/- embryos. Coronal hemisections of E14 (A-F) and E16.5 (G-J) Dlx1/2 heterozygous (upper panel) and homozygous (lower panel) mutant brains where DiI crystals were introduced in the lateral part of the neocortex (A,B) or of the dorsal thalamus (C-J). Stars in E,F,I,J indicate DiI crystal positions. (A,B) DiI crystals in the lateral neocortex of control embryos label axons that make a turn and enter the ganglionic eminences (open arrowhead in A). In Dlx1/2-/- embryos, these axons, do not make a sharp turn and grow into the ganglionic eminences towards the pial surface (open arrowhead in B). (C-F) DiI crystals in the dorsal thalamus of control embryos label thalamic axons that make a sharp turn into the ganglionic eminences (white arrowhead in E). In more rostral sections (C), axons grow in direction of the neocortex (white arrowhead in C) reaching the region where cortical axons enter the ganglionic eminences (compare C with A). In Dlx1/2-/- embryos, thalamic axons fail to make a sharp turn (white arrowhead in F) and grow towards the pial surface of the ganglionic eminences (white arrowhead in D). Note that misrouted thalamic and cortical axons both travel in a more superficial domain of the ganglionic eminences (compare A with C and B with D). (G-J) A DiI crystal (star in I,J) in the lateral part of the dorsal thalamus labels the internal capsule, which is displaced closer to the pial surface in Dlx1/2-/- mutant embryos. Furthermore, in addition to axons that navigate to the neocortex (open arrowheads), axons diving into the amygdalar region (white arrowhead in J) were detected. These axons were detected in more rostral sections (H), running in the ventral pallium (white arrowhead), whereas axons of the superficially displaced internal capsule run dorsally into the necortex (compare open arrowheads in G,H). dTH, dorsal thalamus; GE, ganglionic eminences; IC, internal capsule; Ncx, neocortex; Str, striatum.

View larger version (68K): [in a new window]   Fig. 9. The topography of thalamocortical projections is shifted in Dlx1/2-/- embryos. Coronal (A-L) or horizontal (M-Q) hemisections of E16.5 Dlx1/2 heterozygous (left) and homozygous (right) mutant brains where DiI crystals (A-I) or DiI and DiA crystals (J-Q) were introduced in the occipital neocortex (A-C), the parietal neocortex (D-I), the occipital and parietal neocortex (J-L), or the putative dorsolateral geniculate nucleus (dLGN) and ventrobasal (VB) complex (M-Q). Schematic diagrams indicate the position of DiI and DiA crystals (A,D,G,J,M) and stars indicate their actual position in B,C. (A-L) Morphological landmarks, including the pial surface of the thalamus (broken line) and the retroflexus tract (white arrowhead), are used to position presumptive thalamic nuclei. In controls, injections in the occipital neocortex label cells in the putative dLGN (B,K) and injections in the parietal neocortex label cells in the VB complex (E,H,K). Open arrowheads indicate the medial boundary of the thalamic domain stained in wild-type embryos (B,E,H,K). This boundary is indicated in Dlx1/2-/- embryos and shows that thalamic domains labeled by occipital and parietal injections are shifted medially (C,F). Note that the number of cells labeled is reduced in homozygous mutant embryos (compare B with C and E with F). In some cases, the region containing labeled cells was broader in mutant embryos, partially including the domain labeled in controls as well as a more medial domain (H,I). Similarly, double occipital and parietal injections show that the labeled domains in mutant embryos are medially displaced (K,L). In this case there is some overlap in the regions labeled by each dye (arrow in L). (M-Q) Horizontal sections at ventral levels (N,O) and more dorsal levels (P,Q) of brains after a thalamic double injection in the presumptive dLGN and VB. Even though the tracer crystals were relatively small, a large number of axons were stained in our experiments because of the small size of the thalamus. In wild-type animals, putative dLGN axons (red) and VB axons (green) turn into the striatum and remain as two separate bundles in the caudal (open arrowheads in N and P) and intermediate regions of the internal capsule, respectively (N,P). In Dlx1/2-/- mutant embryos, dLGN axons are primarily detected ventrally (open arrowhead in O), and are mixed with VB axons. In more dorsal sections (Q), very few dLGN axons are visible (open arrowhead), indicating that they remain in ventral regions. On the contrary, a large number of VB axons are detected in a caudal region where normally dLGN axons travel (compare P with Q). dTH, dorsal thalamus; Fr, frontal neocortex; IC, internal capsule; Ncx, neocortex; Occ, occipital neocortex; Par, parietal neocortex; Str, striatum.