Tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation

Abstract

Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain.

Figure 1. TCAs Enter the Telencephalon through a Restricted Corridor in the MGE

(A) E13.5 coronal mouse telencephalic section showing axonal tracing of dorsal thalamic (dTh) axons (arrowhead) by insertion of a DiI crystal. Coronal sections through the telencephalon of E13.5 embryos showing the expression pattern of Nkx2-1, Islet1, Ebf1, and Calretinin. (B) Nkx2-1 expression is not detected in a corridor of cells (bracket) between the ventricular and subventricular zones (VZ/SVZ) of the medial ganglionic eminence (MGE) and the globus pallidus (GP), where TCAs navigate (arrowhead). (C) Complementary expression of Islet1 and Nkx2-1 proteins. (D) Higher magnification of the area boxed in (C). (E) Coexpression of Ebf1 mRNA and Islet1 protein in the striatum (Str) and in the MGE corridor but not in preoptic area (POa, solid arrow). (F) Higher magnification of the area boxed in (E), showing coexpression of Ebf1 and Islet1 in corridor cells (arrowheads). (G) Calretinin-expressing TCAs navigate through the MGE corridor formed by Islet1-expressing cells. The corridor is just superficial to the route used by Calbindin-expressing interneurons to migrate toward the cortex. The triple staining image was composed from immediate adjacent sections using Adobe Photoshop software. (H) Higher magnification of the corridor, showing that Calretinin-expressing TCAs (open arrowheads) navigate through the superficial part of the MGE corridor, in close contact with Islet1-expressing cells (solid arrowheads). The bracket indicates the width of the corridor. (I) Schema summarizing gene expression during TCA pathfinding in the ventral telencephalon. NCx, neocortex; LGE, lateral ganglionic eminence. Scale bars = 300 μm (A, B, C, E, and G) and 50 μm (D, F, and H).

Figure 2. MGE Corridor Cells Derive from the LGE and Are Permissive for TCAs Outgrowth

(A–D) Coronal sections through the telencephalon of E11.5 (A and C) and E12.5 (B and D) embryos showing the expression pattern of Ebf1 (A and B) and double immunohistochemistry Islet1 and βIII-tubulin (C and D). (E) Experimental paradigm used to test the origin of corridor cells. (F) GFP immunohistochemistry showing LGE-derived cells in the striatum (solid arrowhead), neocortex (NCx), and MGE mantle (open arrowhead). (G) Higher magnification of LGE-derived GFP cells forming a stream superficial to the globus pallidus (GP). (H and I) Migratory morphology of GFP cells at the MGE corridor (H). Most of them express Islet1 (I, open arrowheads). (J) Experimental paradigm used to block cell migration between the LGE and MGE. (K and L) Expression of Islet1 in control (K) and experimental slices (L). Note that the membrane (delineated by arrows) does not affect Islet1-positive cells in the POa. Arrows in (K) indicate the location of the control incision. (L') Double immunohistochemistry for Islet1 and Nkx2-1 in the same slice shown in (L). (M) Experimental paradigm used to test the growth of E13.5 GFP dorsal thalamic (dTh) in the MGE. (N) Bright-field image of a slice with a GFP dTh explant in the POa after 72 hr in culture. (O and P) Islet1 and GFP immunohistochemistry showing that TCAs grow preferentially through the MGE Islet1-positive corridor (open arrowhead, bracket in [P]) before fanning out in the striatum (Str; solid arrowheads). VZ/SVZ, ventricular/subventricular zone. Scale bars = 100 μm (A, C, and O), 200 μm (B, D, K, L, L', and N), 300 μm (F), 60 μm (G), 20 μm (H and I), and 70 μm (P).

Figure 3. The Corridor Is a Permissive Territory Necessary and Sufficient for TCA Pathfinding

(A) Experimental paradigm used to test whether the medial ganglionic eminence (MGE) corridor is necessary for TCAs extension. (B and C) GFP immunohistochemistry showing the behavior of TCAs in control (B) and experimental slices (C). (D and D') Higher magnification of TCAs in (C) showing the presence of Islet1-positive corridor cells. (E) Experimental paradigm used to test the requirement of LGE to MGE migration for TCA guidance. (F) GFP immunohistochemistry showing that a control incision (left hemisphere) does not affect TCAs growth toward the neocortex (NCx, open arrowheads), whereas insertion of a membrane (asterisk, right hemisphere) impairs the growth of TCAs. (G and H) A higher magnification of control (G) and a membrane inserted (dashed line in H) slices showing GFP and Islet1 immunohistochemistry. TCAs outgrowth correlates with the presence of Islet1-expressing cells (open arrowhead in [G]). (I and J) Ebf1 mRNA expression at E14.5 shows that MGE corridor formation (solid arrowhead in [I]) is impaired in Mash1 mutant embryos (J). Open arrowheads mark the LGE/MGE boundary, and a red dashed line delineates the pallium/subpallium boundary (P/Sp). (K and L) DiI labeling of TCAs (open arrowheads) in coronal sections through E14.5 brains in control (K) and Mash1 mutant embryos (L). (M) Experimental paradigm used to test the ability of LGE-derived MGE corridor cells to restore TCAs growth in the Mash1 mutant telencephalon. (N) DiI-labeled TCAs do not grow toward the neocortex in Mash1 mutant slices, although they can ectopically invade the piriform cortex (PCx; arrow). (O) GFP expression showing that a graft of GFP-LGE VZ/SVZ (dotted circle) into the LGE of Mash1 mutant slices generates cells that migrate tangentially into the MGE (solid arrowheads), forming a corridor used by DiI-labeled TCAs (open arrowhead) to extend toward the NCx. (P) GFP and Islet1 immunohistochemistry showing that wild-type GFP-expressing neurons migrate from the LGE into the MGE of Mash1 mutant slices. GP, globus pallidus; Str, striatum; vTh, ventral thalamus; VZ/SVZ, ventricular/subventricular zone. Scale bars = 200 μm (B, C, and F), 100 μm (D, D', G, and H), 300 μm (I–L), 150 μm (N and O), and 100 μm (P).

Figure 4. CRD-Nrg1 Is Expressed in MGE Corridor Cells and Contributes to TCA Pathfinding

Serial coronal sections through the telencephalon of E13.5 embryos. (A) CRD-Nrg1 expression in the striatum (Str) and in cells forming the medial ganglionic eminence (MGE) corridor (arrowhead). (B) Islet1 expression in the Str and in cells forming the MGE corridor (arrowhead, bracket). (C) Islet 1 and CRD-Nrg1 in MGE corridor cells (arrowhead, bracket). Double in situ image was composed from immediate adjacent sections using Adobe Photoshop software. (D) ErbB4 expression in the dorsal thalamus (dTh). (E) Experimental paradigm used to analyze the response of TCAs to CRD-Nrg1-transfected COS cell aggregates in slice cultures. (F and I) DiI-labeled TCAs traveled normally through the telencephalon toward the neocortex (NCx) in controls (F) but derailed from their normal path (arrowhead in [I]) when they contact a COS cell aggregate expressing CRD-NRG1. Asterisks mark DiI placements in the dorsal thalamus (dTh). (G and J) Higher magnifications of the images shown in (F) and (I), respectively. (H and K) Schematic representation of the pathway taken by TCAs in response to control and CRD-Nrg1 transfected COS cell aggregates. (L and P) Nuclear counterstain of CRD-Nrg1 heterozygous (L) and CRD-Nrg1 mutant (P) E14.5 coronal sections shows that TCAs abnormally defasciculate in the MGE corridor of mutants (open arrowheads and brackets in [P] and [Q]) compared to controls (arrowheads and brackets in [L] and [M]). (M and Q) High magnifications of DiI-labeled axons in E14.5 CRD-Nrg1 heterozygous (M) and CRD-Nrg1 mutant (Q) showing that the MGE corridor is wider and more disorganized in CRD-Nrg1 mutants (arrowheads) than in control brains. (N and R) High magnification of L1-labeled axons observed in the NCx of control (N) and CRD-Nrg1 mutants (R) at E14.5. (O and S) Schematic representation of the pathway taken by TCAs in control and CRD-Nrg1 mutants. H, hippocampus; Hb, habenula; Hyp, hypothalamus; LGE, lateral ganglionic eminence; GP, globus pallidus; PCx, piriform cortex. Scale bars = 200 μm (A–D, H, L, J, and P), 1 μm (F and I), 300 μm (G and J), and 100 μm (M, N, Q, and R).

Figure 5. Ig-NRG1 Controls the Oriented Outgrowth of TCAs

(A) Ig-Nrg1 mRNA expression in the developing cortex at E13.5. (B and C) βIII-Tubulin immunohistochemistry showing dorsal thalamic (dTh) explants from E13.5 embryos after 96 hr in culture adjacent to mock transfected (B) or Ig-Nrg1 transfected (C) COS cell aggregates (dotted lines). Insets show GFP expression in transfected COS cells. (D) Quantification of axonal length in the experiments shown in (B) and (C). Additional quantifications are displayed in Figure S4. (E) Experimental paradigm used to test the effect of ventricular zone ablations in the angle region on the growth of GFP-positive dTh axons in E13.5 telencephalic slices. (F and G) GFP expression showing that TCAs (open arrowheads) extend through the medial ganglionic eminence (MGE), lateral ganglionic eminence (LGE), and neocortex (NCx) in control slices but fail to do so in angle ablation slices (asterisk in [G]). (H) Qualification of the experiments shown in (F) and (G). (I) Experimental paradigm used to test the effect of control or Ig-Nrg1 transfected COS cell aggregates on the growth of GFP-positive dTh axons in E13.5 angle-ablated telencephalic slices. (J–L) Nuclear staining and dsRed expression in angle-ablated telencephalic slices with control (J) or Ig-Nrg1 transfected (K and L) COS cell aggregates. The dotted lines delineate COS cell aggregates, whereas dashed lines delineate dTh explants. (J'–L') GFP expression in the same slices (J–L), showing that TCAs (open arrowheads) fail to extend toward the cortex in control slices (J') but do so in angle-ablated slices containing Ig-Nrg1 transfected COS cell aggregates (K' and L'). Scale bar = 500 μm (A–C) and 200 μm (F, G, J, J', K, K', L, and L').

Figure 6. Abnormal Development of TCAs in the Absence of All Nrg1 Isoforms

(A and E) Coronal sections through E14 control (A) and Nrg1 mutant (E) embryos showing nuclear staining and DiI labeling after dorsal thalamic (dTh) injections. (B, C, F, and G) Higher magnifications of the internal capsule region (arrows) in control (B and C) and Nrg1 mutant (F and G) embryos. (D and H) Schematic representation of the pathway taken by TCAs in control and Nrg1 mutant brains. GP, globus pallidus; Hyp, hypothalamus; Str, striatum. Scale bar = 300 μm (A and E) and 200 μm (B, C, F, and G).

Figure 7. Loss of ErbB4 Function Perturbs TCA Guidance

(A and D) Coronal sections through E13.5 ErbB4 heterozygous (A) and ErbB4 mutant (D) brains showing nuclear staining and DiI labeling (arrowheads in D) after dorsal thalamic (dTh) injections. (B and E) Higher magnifications of the images shown in (A) and (E), respectively. (C and F) Schematic representation of the pathway taken by TCAs in a control situation (C) or in the absence of ErbB4 function (F). (G) Experimental paradigm used to analyze the effect of a dominant-negative form of ErbB4 (dnErbB4) in the guidance of dTh axons. (H and I) GFP immunohistochemistry showing TCAs as they extend through the striatum (Str) toward the neocortex (NCx) in control (H) and Gfp + dnErbB4 electroporated slices (I). (J) Experimental paradigm used to test the growth of E13.5 wild-type or ErbB4^−/− dTh explants in wild-type telencephalic slices. (K and L) DiI labeling and nuclear staining showing wild-type (K) and ErbB4^−/− (L) TCAs as they extend through wild-type telencephalic slices. GP, globus pallidus; Hyp, hypothalamus; PCx, piriform cortex. Scale bars = 1 mm (A, D, H, I, K, and L) and 200 μm (B and E).

Figure 8. Tangential Migration and Axon Guidance in the Central Nervous System

(A) A model of TCAs guidance by tangential migration of corridor cells and NRG1 expression. GABAergic neurons migrate tangentially from the lateral ganglionic eminence (LGE) to form a corridor in the medial ganglionic eminence (MGE) around E12.5 (blue line) prior to the entrance of TCAs in the telencephalon. At this early stage, the MGE territory is not permissive for TCAs (dark purple area). LGE-derived neurons colonize the MGE mantle around E13.5, forming a permissive corridor for TCAs in this region. CRD-NRG1 expression by corridor cells contributes to the guidance of TCAs through this region, which also requires secreted Ig-NRG1 from the pallium (green gradient). Tangential migration and axon guidance in the developing neural tube. (B) Radial glia provides structural support for radial migration, a process that results in the generation of different nuclei topographically organized in relation to their place of origin. (B') Tangential migration is independent of radial glia processes and therefore does not respect topographical references. As a result, tangential migration produces an increase in the cellular complexity of neural circuits by providing cell types distinct from those locally generated and represents a novel mechanism for presenting cues to navigating axons.