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The adhesion molecule TAG-1 mediates the migration of cortical interneurons from the ganglionic eminence along the corticofugal fiber system

¹ Department of Basic Science, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, PO Box 1527, 711 10 Heraklion, Greece
² Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK
³ Zentrum für Molekulare Neurobiologie, Universitaet Hamburg, Martinistrasse 52, D-20246, Hamburg, Germany

View larger version (80K): [in a new window]   Fig. 1. TAG-1 is expressed by the developing corticofugal fibers. (A,B) Immunohistochemistry for TAG-1 in coronal sections of E12.5 (A) or E14.5 (B) mouse cortex. (A) Labeled cells in the PP project to the ventral telencephalon in the region of the IC. (B) Labeled axons are oriented tangentially in the MZ and IZ, and radially in the CP. (C) In situ hybridization for TAG-1 mRNA in coronal sections of E14.5 mouse cortex. The signal is detected predominantly in the IZ and MZ. (D) Western blot analysis of total lysates of E14.5 mouse spinal cord (control, lane 1) and cortex (lane 2) reveals a single band of 135 kDa, as expected of the TAG-1 antigen (arrowhead). The molecular weight standards shown (lane 3) are 111 and 83 kDa (arrows). (E) DiI crystal placement into the dorsal cerebral cortex of E14.5 mouse, labels corticofugal fibers descending via the IZ into the IC. (F-K) Immunohistochemistry for TAG-1 in coronal sections of E14.5 mouse cortex, after placement of DiI in the cortex of the whole brain, reveals numerous DiI- labeled corticofugal fibers that are immunopositive for TAG-1. DiI labeling (red, G,J) and immunohistochemistry for TAG-1 (green, F,I) in the IZ and CP of E14.5 cortical slices (F,G), as well as in the area of the IC (I,J). (H,K) Superimposed images of F,G and I,J, respectively. Arrows in H indicate DiI-labeled-TAG-1-immunopositive corticofugal fibers, radially oriented in the CP, cortical plate. IC, internal capsule; IZ, intermediate zone; MZ, marginal zone; PP, preplate. Scale bars: 100 µm in A,E,I-K; 50 µm in B,F-H; 60 µm in C.

View larger version (75K): [in a new window]   Fig. 2. Migratory routes of cortical interneurons along the TAG-1 immunopositive corticofugal fibers. CP, cortical plate; IC, internal capsule; IZ, intermediate zone; LGE, lateral ganglionic eminence; MZ, marginal zone. The broken line demarcates the ventricle. (A) Tangential migration of neurons to different layers of the developing cerebral cortex in cortical slice cultures of an E16 rat embryo, after placement of DiI into the ganglionic eminence. (B) Double immunohistochemistry against GABA (red) and TAG-1 (green) in coronal sections of E12.5 mouse cortex, shows the juxtaposition of TAG-1 immunoreactive fibers and GABAergic cells at the IC. (C,D) DiI tracing for tangentially migrating neurons (red) and immunohistochemistry against TAG-1 (green), in slice cultures of E15 (C) or E16 (D) rat cortex. (C) Tangentially migrating neurons in close association with TAG-1 immunoreactive fibers in the IZ. (D) DiI-labeled migrating neuron in the CP, with the leading process oriented perpendicular to the pial surface and in close apposition to the radially arranged bundles of the TAG-1 immunopositive axons. (E,F) Double immunohistochemistry against GABA (red) and TAG-1 (green), in slice cultures of E15 (E) or E16 (F) rat cortex. (E) Tangentially oriented GABAergic neurons are found dispersed between TAG-1-labeled axons, in the IZ. (F) GABAergic neurons and fibers in the IZ, CP and MZ. In IZ and MZ, cells show tangential orientation, whereas, in the CP, they tend to be radially organized in apposition to the TAG-1 positive axons. (G) Double immunohistochemistry with RC2 antibody (green) and anti-GABA (red) in slice cultures of E14.5 mouse cortex. Arrows point to migrating neurons in the CP with the leading process perpendicular to the pial surface and in close apposition to the radial glial fibers. (H) Double immunohistochemistry with RC2 antibody (green) and anti-TAG-1 (red) in coronal sections of E14.5 mouse cortex. TAG-1 and RC2 are not co-localized in the CP. Inset: higher magnification of the red box. Scale bars: 100 µm in A,B; 40 µm in C,F; 25 µm in G,H; 10 µm in inset.

View larger version (80K): [in a new window]   Fig. 3. TAG-1 mediates the tangential migration of MGE neurons along the fibers of the corticofugal system. CP, cortical plate; LGE, lateral ganglionic eminence. The broken line demarcates the ventricle and the edge of the slice. The migration of the MGE cells is examined in cortical slice cultures by placing a crystal of DiI in the LGE (A,B), as well as by using immunohistochemistry against GABA (C-F). Cortical slices were cultured for 2 days in the presence of monoclonal or Fab fragments of polyclonal antibodies against TAG-1 (A,C, respectively) or control antibodies (same isotype, Fab fragments of control antisera, see Materials and Methods) (B,D, respectively). Very few DiI-labeled migrating neurons (A) and GABAergic neurons (C) are detected in the presence of antibodies against TAG-1 in E15 rat (A) or E13.5 mouse (C), when compared with the controls in the cortex of E15 rat (B) or E13.5 mouse (D). A marked decrease in the number of GABAergic neurons in E16 rat slice cultures is observed in the presence of TAG-1-Fc protein (E). No effect is observed when control soluble Fc protein (MUC18) is added in the culture medium (F). Antigen depleted antibodies do not reduce the number or affect the density of GABA-containing neurons in the cortex (G). No differences in the expression pattern of TAG-1 is detected in cortical slices cultured in the presence of antibodies against TAG-1 and incubated with the secondary antibody alone (H). Quantification of the blocking effect of antibodies to TAG-1 (I) or TAG-1-Fc (J) protein on the migration of GABAergic neurons. Densitometry of GABA-expressing cells in slice cultures in the presence of Fab antibodies against TAG-1 (I) and control Fab fragments or TAG-1 Fc protein (J) and control MUC18 Fc protein (see also Materials and Methods). Values represent the ratio of the area where pixel densities were measured to the total area measured (i.e. all of neocortex). Value 1.00 would represent the entire length of the neocortex. Error bars represent the s.d.; *P<0.05, one-factor ANOVA. Values were collected from independent slice cultures (A, n=9; B, n=7) Scale bars: 100 µm in A-H.

View larger version (53K): [in a new window]   Fig. 4. L1 and TAG-1 immunopositive axons are both found in the intermediate zone. Double immunostaining against TAG-1 (green, A) and L1 (red, B) in cryostat sections of E14.5 mouse brain. (C) Superimposed image of A and B. No differences in the expression pattern of L1 are detected in cortical slices cultured in the presence of antibodies against TAG-1 (E) compared with control ones (D). Scale bar: 100 µm.

View larger version (77K): [in a new window]   Fig. 5. L1 is not involved in the migration of cortical GABAergic interneurons. The broken line demarcates the ventricle. Arrows point to streams of GABA-immunopositive cells. The migration of MGE cells was examined in cortical slice cultures in the presence (A) or absence (B) of L1 Fab fragments. Immunostaining against TAG-1 in cryostat sections of E14.5 wild-type (C) and L1 mutant (D) mouse brains. Immunostaining against GABA in cryostat sections of E14.5 wild-type (E,G) and L1 mutant (F,H) mouse brains. Scale bars: 100 µm in A-D; 110 µm in E,F; 55 µm in G,H.

View larger version (26K): [in a new window]   Fig. 6. Proposed mechanism for the migration of cortical interneurons from the MGE. The TAG-1 immunopositive fibers are depicted in green, migrating interneurons in red, radial glial fibers in yellow and Slit1 mRNA in blue. MGE cells migrate away from the GE probably owing to a chemorepulsive effect of Slit1. They then use the TAG-1 immunopositive axons arranged tangentially in the MZ and IZ to migrate into the neocortex. They reach their positions in the CP by using the radially arranged bundles of efferent axons or radial glial fibers.

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