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doi: 10.1242/10.1242/dev.00417

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Directional guidance of interneuron migration to the cerebral cortex relies on subcortical Slit1/2-independent repulsion and cortical attraction

Oscar Marín^1,*, Andrew S. Plump^2,, Nuria Flames^1,*, Cristina Sánchez-Camacho^1,, Marc Tessier-Lavigne² and John L. R. Rubenstein^1,

¹ Department of Psychiatry, Nina Ireland Laboratory of Developmental Neurobiology, Langley Porter Psychiatric Institute, 401 Parnassus Avenue, University of California San Francisco, CA 94143, USA
² Department of Biological Sciences, Howard Hughes Medical Institute, 371 Serra Mall, Stanford University, Stanford, CA 94305, USA
^* Present address: Instituto de Neurociencias, CSIC-Universidad Miguel Hernández, 03550 San Juan, Alicante, Spain
Present address: Merck & Co., 126 East Lincoln Avenue, Rahway, NJ 07065, USA
Present address: Instituto de Neurobiología Ramón y Cajal, Avda. Doctor Arce, 37, 28002 Madrid, Spain

View larger version (69K): [in a new window]   Fig. 3. The cortex contains an attractive activity for tangentially migrating cells derived from the MGE. (A) Schematic of the experimental paradigm used to quantify the migration of MGE-derived cells towards an ectopic cortex. (B) Migration of DiI labeled cells from the MGE (asterisk) after 48 hours in culture. Most cells migrate towards the ectopic cortex. (C) The number of DiI-labeled cells that migrated into the ipsilateral side of the slice (ipsi) or into the ectopic cortex (eNCx). The number of cells in the ipsilateral side includes those in the cortex and those en route towards the cortex (MGE and LGE). Total number of cells (n=18): 1590 (ipsi), 2582 (eNCx). Histograms show averages ±s.d. *P<0.001. (D) Schematic of the slice transplantation paradigm used to analyze the migration of MGEGFP-derived cells when transplanted ectopically into the dorsal cortex. (E,F) Migration of MGEGFP-derived cells (asterisk) after 48 hours in culture, labeled with GFP (E), and schematic representation of the routes followed by the MGEGFP-derived cells (F). Note that cells fail to migrate into the more medial aspect of the embryonic hippocampus (i.e. the developing dentate gyrus and cortical hem; arrowhead in E, hatched region in F) (see also Polleux et al., 2002). (G) Schematic of the slice transplantation paradigm used to analyze the migration of MGEGFP-derived cells towards an inverted dorsal cortex. (H,I) Migration of MGEGFP-derived cells (asterisk) after 48 hours in culture, labeled with GFP (H), and schematic representation of the routes followed by the MGEGFP-derived cells (I). The boxes shown in H (m and l) indicate the regions from which cell counting was made. eNCx, ectopic neocortex; H, hippocampus; L, lateral region of the dorsal cortex; LGE, lateral ganglionic eminence; M, medial region of the dorsal cortex; MGE, medial ganglionic eminence; NCx, neocortex; PCx, piriform cortex; POa, preoptic area; Str, striatum. Scale bars: 300 µm.

View larger version (78K): [in a new window]   Fig. 1. The basal telencephalon contains a repulsive activity for tangentially migrating cells. (A) Schematic of the slice transplantation paradigm used to analyze the migration of MGEGFP-derived cells in the absence of cortex. (B,C) Migration of MGEGFP-derived cells (asterisk) after 48 hours in culture, labeled with GFP (B), and schematic representation of the routes followed by the MGEGFP-derived cells (C). Arrowheads point to cells in the pallial/subpallial boundary, dorsal to the striatum. (D) Schematic of the slice transplantation paradigm used to analyze the migratory behavior of MGEGFP-derived cells transplanted into dorsal (striatal; position 1), intermediate (pallidal; position 2) or ventral (preoptic; position 3) regions within the subpallium. (E,F) Migration of MGEGFP-derived cells after 48 hours in culture, labeled with GFP. Examples of migratory behavior of cells derived from transplants (asterisks) into intermediate (E) or ventral (F) regions the subpallium. Arrowheads point to cells migrating from the transplants. Note the limited number of cells migrating from the transplant in position 3. (G) Schematic of the slice transplantation paradigm used to analyze the behavior of MGEGFP-derived cells forced to migrate towards the preoptic area. (H) Migration of MGEGFP-derived cells (asterisk) after 48 hours in culture, labeled with GFP. Note that very few cells migrate into the MGE mantle (arrowhead) and virtually none into the POa. (I) Analysis of the number of cells that migrated in to the mantle of the LGE, MGE or POa [which contains the ventral midline (m)] in the experiments described in G. Total number of cells (n=25): 5240 (LGE), 711 (MGE), 123 (POa). Histograms show averages±s.d. Asterisks denote significant differences in migration between the LGE and MGE (*) and between the MGE and POa (**). P<0.001 in both cases. LGE, lateral ganglionic eminence; m, ventral midline; MGE, medial ganglionic eminence; NCx, neocortex; PCx, piriform cortex; POa: preoptic area; Str, striatum. Scale bars: 300 µm.

View larger version (109K): [in a new window]   Fig. 2. The preoptic area prevents tangential migration towards an ectopic cortex. (A) Schematic of the slice transplantation paradigm used to analyze the migration of MGEGFP-derived cells towards an ectopic cortex. (B) Migration of MGEGFP-derived cells (asterisk) after 48 hours in culture, labeled with GFP. Most cells migrate towards the ipsilateral cortex, whereas very few cells migrate medially towards the ectopic cortex (arrowhead). (C) Schematic of the slice transplantation paradigm used to analyze the migration of MGEGFP-derived cells towards an ectopic cortex in the absence of the preoptic area. (D) Migration of MGEGFP-derived cells (asterisk) after 48 hours in culture, labeled with GFP. A large number of cells migrate towards the ectopic cortex in the absence of the preoptic area (arrowheads). eNCx, ectopic neocortex; H, hippocampus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; POa, preoptic area; Str, striatum. Scale bar: 300 µm.

View larger version (46K): [in a new window]   Fig. 4. Chemoattractive activity of the neocortex on cells migrating from the MGE. (A) Schematic of the experimental paradigm used for E13.5 cells. (B) An example of the distribution of neurons originating in the MGE after 36 hours of co-culture with a neocortical explant. The presence of more MGE cells in the regions proximal to the cortex than distal to it indicates an attractive effect of the cortex on MGE cells. (C) Diagram of the scheme used to semiquantify the effects of the cortex on MGE migration [modified from Zhu et al. (Zhu et al., 1999)] and score of migration in co-culture experiments (with cortex) or in MGE alone experiments (control). MGE, medial ganglionic eminence; NCx, neocortex. Scale bar: 200 µm.

View larger version (103K): [in a new window]   Fig. 5. Tangential migration to the cortex of Slit1;Slit2 double mutants. Coronal sections through the telencephalon of E14.5 embryos showing expression of GAD67 (A,B) and Lhx6 (C,D) in wild-type (A,C) and Slit1;Slit2 mutant (B,D) mice. Arrowheads point to cells migrating in the marginal and intermediate zones of the cortex. H, hippocampus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; POa, preoptic area; Str, striatum. Scale bars: 500 µm.

View larger version (149K): [in a new window]   Fig. 6. Normal number of interneurons in the neocortex and hippocampus of Slit1;Slit2 double mutants. Coronal sections through intermediate (A-F) and caudal (G-L) levels of the telencephalon of E18.5 wild-type (A,B,G,H) and Slit1;Slit2 mutant (C,D,I,J) mice stained with anti-calbindin antibodies. (B,C) High magnification of the regions boxed in A and D, respectively. (E,F) Nissl staining of sections adjacent to B and C, respectively. (H,I) High magnification photographs of the regions boxed in G and J, respectively. (K,L) Nissl staining of sections adjacent to H and I, respectively. cp, cortical plate; dgc, dentate gyrus cell layer; fr, fasciculus retroflexus; GP, globus pallidus; H, hippocampusl hi, hilus; Hyp; hypothalamus; iz, intermediate zone of the cortex; LA, lateral nucleus of the amydgala; mz, marginal zone of the cortex; NCx, neocortex; PCx, piriform cortex; slm, stratum lacunosum moleculare; sp, stratum piramidale; sr, stratum radiatum; Str, striatum; vLGN, ventral lateral geniculate nucleus; 6, layer 6 of the cortex. Scale bars: 300 µm (A,D,G,J); 200 µm (B,C,E,F,H,I,K,L).

View larger version (50K): [in a new window]   Fig. 7. The repulsive activity present in the basal telencephalon is maintained in Slit1;Slit2 double mutants. (A) Schematic of the experimental paradigm used to analyze the migration of MGE-derived cells in the presence or absence of cortex. (B) Migration of DiI-labeled cells from the MGE (asterisks) after 24 hours in culture. Labeled cells (arrowheads) approach the pallial/subpallial boundary (dotted white line) at the same time in the side of the slice without cortex (left) as in the side with cortex (right). (C) Schematic of the slice transplantation paradigm used to analyze the migration of MGEGFP-derived cells in the absence of cortex in slices obtained from Slit1;Slit2 double mutants. (D) Migration of MGEGFP-derived cells (asterisk) revealed after 48 hours in culture, labeled with GFP. Arrowheads point to cells in the pallial/subpallial boundary, dorsal to the striatum. (E) Schematic of the slice transplantation paradigm used to analyze the behavior of MGEGFP-derived cells forced to migrate towards the preoptic area in slices obtained from Slit1;Slit2 double mutants. (F) Migration of MGEGFP-derived cells (asterisk) after 48 hours in culture, labeled with GFP. Note that very few cells migrate into the MGE mantle (arrowhead) and none into the preoptic area. H, hippocampus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; POa, preoptic area; Str, striatum. Scale bars: 300 µm.

View larger version (131K): [in a new window]   Fig. 8. Interneurons migrate to the cortex in Slit1;Ntn1 and Slit1;Slit2;Ntn1 mutants. Coronal sections through the telencephalon of E18.5 fetuses showing calbindin immunohistochemistry in wild-type (A,B) and Slit1;Ntn1 (C,D) mutant mice. (B,C) High magnification photographs of the regions boxed in A and D, respectively. (E,F) Nissl staining of from sections adjacent to B and C, respectively. (H-K) Coronal sections through the telencephalon of E18.5 embryos showing expression of GAD67 (H,J) and Lhx6 (I,K) in Slit1 (H,I) and Slit1;Slit2;Ntn1 (J,K) mutant mice. cc, corpus callosum; cp, cortical plate; GP, globus pallidus; H, hippocampus; hc, hippocampal commissure; iz, intermediate zone of the cortex; mz, marginal zone of the cortex; NCx, neocortex; PB, Prost bundle; PCx, piriform cortex; POa, preoptic area; S, septum; Str, striatum; Scale bars: 300 µm (A and D); 200 µm (B, C, E, and F); 500 µm (H-K).

View larger version (149K): [in a new window]   Fig. 9. Cholinergic neurons from the basal magnocellular complex are displaced in the midline of Slit1;Slit2 double mutants. Coronal sections through the telencephalon of newborn fetuses showing choline acetyltransferase (ChAT) immunohistochemistry in wild-type (A,C,D,G,H) and Slit1;Slit2 mutant (B,E,F,I,J) mice. (A,B) Distribution of cholinergic cells at the level of the anterior commissure in wild-type (A) and Slit1;Slit2 mutant (B) mice. In the preoptic area, note the collection of ectopic cholinergic neurons close to the midline, where some cells and fibers cross the midline in Slit1;Slit2 mutant mice (arrow in B). Ectopic cells appear to be continuous with other cholinergic neurons in the basal magnocellular complex (arrowhead). Additional ectopic cells are present in the anterior commissure (open arrowhead). (C,D,E,F,G,I) High magnification the boxed areas in A and B. (H,J) High magnification photographs of the basal magnocellular complex at caudal levels in wild-type (H) and Slit1;Slit2 mutants (J). Dashed lines in C and E indicate the midline. ac, anterior commissure; BMC, basal magnocellular complex; GP, globus pallidus; PCx, piriform cortex; POa, preoptic area; Str, striatum; Scale bars: 300 µm (A,B); 100 µm (C-J).

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