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Interneuron Migration from Basal Forebrain to Neocortex: Dependence on Dlx Genes

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Science  17 Oct 1997:
Vol. 278, Issue 5337, pp. 474-476
DOI: 10.1126/science.278.5337.474

Abstract

Although previous analyses indicate that neocortical neurons originate from the cortical proliferative zone, evidence suggests that a subpopulation of neocortical interneurons originates within the subcortical telencephalon. For example, γ-aminobutyric acid (GABA)–expressing cells migrate in vitro from the subcortical telencephalon into the neocortex. The number of GABA-expressing cells in neocortical slices is reduced by separating the neocortex from the subcortical telencephalon. Finally, mice lacking the homeodomain proteins DLX-1 and DLX-2 show no detectable cell migration from the subcortical telencephalon to the neocortex and also have few GABA-expressing cells in the neocortex.

The primary subdivisions of the forebrain, including the neocortex and the basal ganglia, have distinct molecular and cellular properties (1,2). Previous evidence suggests that these subdivisions develop from separate proliferative zones that do not intermix (3). Here we show that cell migration occurs between the primordia of the basal ganglia and the cerebral cortex. Our results suggest that many neocortical interneurons are generated by the proliferative zone of the basal ganglia.

Neocortical neurons include two types: the excitatory pyramidal neurons and the inhibitory (GABA-containing) interneurons. During development, neocortical neurons were thought to derive from the proliferative zone of the neocortical primordium. However, studies of neuronal migration in vitro indicate that cells migrate from the lateral ganglionic eminence (LGE) (4), which is the primordium of the striatum (5), into the neocortex. Other evidence suggests that these cells might be interneurons. For example, clonally related GABA-containing cells tend to be more dispersed across the neocortex than are clones of pyramidal neurons (6); there are GABA-containing cells in the intermediate zone (IZ) at the transition between the LGE and the neocortex, which have a morphology of tangentially migrating cells (7); and interneurons migrate tangentially from the subventricular zone (SVZ) near the cortical-striatal junction into the olfactory bulb (8).

To investigate the migration of subcortically derived cells into the neocortex, we used a slice culture preparation (9). Crystals of 1,1′-dihexadecyl-3,3,3′-tetramethylindo-carbocyanine perchlorate (DiI) were placed into the LGE of embryonic day 12.5 (E12.5) mice; after 36 hours in culture, many labeled cells were detected in the neocortex (Fig. 1A). This migration begins on about E12.5, as only a few labeled cells reached the cortex from E11.5 slices that were grown in culture for 36 hours (10). Many of the DiI-containing cells in the neocortex look like tangentially migrating cells, with leading processes tipped by growth cones and a trailing process (Fig. 1, A and F).

Figure 1

Cell migration from the LGE to the neocortex (NCX) in slice cultures. (A) This slice was prepared from an E12.5 embryo and cultured for 36 hours. Many DiI-labeled cells are present in the NCX. The arrow indicates the cell shown in (F). Analysis of GABA- (Bthrough E) and calbindin- (G throughJ) expressing cells in slice cultures. Slices were prepared at E12.5 with DiI placed in the LGE (12). (B and G) Low-power views of GABA (B) and calbindin (G) immunofluorescence. The arrows indicate the positions of the cells shown in (C) and (H) [(C), GABA; (H), calbindin]; these cells also contain DiI [(D) and (I)]. [(E) and (J)] Double exposures show colabeling with immunofluorescence and DiI [(E), GABA; (J), calbindin]. Scale bars, 100 μm; except in (C) and (F), where bar = 15 μm.

Calbindin is present in cells resembling the tangentially oriented GABA-containing cells that are found in the IZ of the developing neocortex (7, 11). To determine whether cells migrating from the LGE into the neocortex express calbindin or GABA, DiI was inserted into the LGE of slices from E12.5 to E14.5 mice; the slices were then incubated for 30 hours and resectioned. GABA (Fig. 1, B through E) or calbindin (Fig. 1, G through J) immunofluorescence was present in about 20% of DiI-labeled neocortical cells (12).

To provide additional evidence for the migration of GABA- and calbindin-expressing cells from the subcortical telencephalon to the neocortex, we made slice cultures that were transected at the cortical/subcortical angle on one side. After 40 hours in vitro, the neocortical IZ on the transected sides had about 10 times fewer GABA- (13, 14) and calbindin-expressing cells (Fig.2, A through C) than on the intact sides. To study the molecular regulation of this process, we focused on the transcription factors Dlx-1 and Dlx-2, which are homeobox-containing genes with virtually identical patterns of expression in the developing forebrain (15). Although their expression in the telencephalon is initially restricted to the proliferative zone of the basal ganglia primordia (16), by E13.5 they are expressed in the neocortex (10,16). Like GABA and calbindin, DLX-1 expression is reduced in the neocortex of transected slice cultures (Fig. 2, D through F). Double labeling for DLX-1 and either GABA or calbindin revealed coexpression in tangentially oriented IZ cells (Fig. 2, G and H), which suggests that Dlx genes could be required for cell migration from the subcortical telencephalon to the neocortex.

Figure 2

Calbindin (A throughC), and DLX-1 (D through F) immunoreactivity in E12.5 slices cultured after transection between the NCX and LGE on the right side. Slices were then resectioned at 10 μm and processed for immunohistochemistry (13). (A) After 40 hours in culture, calbindin-positive cells are reduced in the transected (C) as compared with the intact (B) side of the slice. Boxed areas in (A) are shown at higher magnification in (B) and (C). (D) DLX-1 expression after 60 hours in culture; the expression boundary in the LGE is maintained (arrow), and DLX-1 positive cells are detectable in the NCX of the intact side but not on the transected side (F). Boxed areas in (D) are shown at higher magnification in (E) and (F). (G and H) DLX-1 (dark nuclear stain) is coexpressed with GABA (G) and calbindin (H) (brown cytoplasmic stain) in the neocortical primordium of the E13.5 embryo. Arrowheads, double-labeled cells; black arrows, DLX-1 only; white arrows, calbindin (G) or GABA (H) only. Scale bars in (A) and (D), 200 μm; in (G), 15 μm.

Analysis of mice with a mutation in both Dlx-1 andDlx-2 (Dlx-1/2) also suggests that these genes are required for the subcortical-to-cortical migration. HomozygousDlx-1/2 mutants have abnormal migration out of the LGE, resulting in an accumulation of partially differentiated neurons in the LGE, hypoplasia of the striatum, and a loss of normal olfactory bulb interneurons (9). These findings suggest that migration from the LGE to the neocortex might also be affected in theDlx-1/2 mutants. In the slice culture preparation, there is no detectable migration of cells from the LGE to the neocortex in E12.5 or E15.5 mutant embryos (Fig. 3).

Figure 3

Comparison of cell migration out of the LGE in slice cultures from wild-type (A andC) and Dlx-1/2 mutants (B andD). Slices were cultured for 36 hours. At E12.5 [(A) and (B)], cells in the wild-type slice (A) have migrated from the LGE into the NCX; this migration is absent in the mutant slice (B). [(C) and (D)] Slices from E15.5 animals. The DiI was photoconverted in diaminobenzidine. As at the earlier age, little or no migration into the neocortex occurred in the Dlx-1/2 mutant slice [(D)]. v, lateral ventricle. Scale bar, 200 μm.

Because migration from the LGE to the neocortex is not detectable in the Dlx-1/2 mutants, we predicted a reduction of GABA- and calbindin-expressing cells in their neocortexes. Indeed, early in corticogenesis and at the day of birth (P0) (when the mutants die), the number of neocortical GABA- (17) and calbindin-expressing cells (Fig. 4, A through D) is greatly reduced in the Dlx-1/2 mutants. The expression of the synthesizing enzyme for GABA, glutamic acid decarboxylase (GAD), was also reduced in the neocortex of these mutants (Fig. 4, E and F). The reduction of interneuron markers was present throughout the neocortex and the hippocampal CA fields. However, not all cortical areas were affected by the mutation, as the GAD immunoreactivity of the paleocortex marginal zone (MZ) appeared normal (arrow in Fig. 4F).

Figure 4

Analysis of the developing NCX in theDlx-1/2 mutants. Immunohistochemistry for calbindin (A through D) in coronal sections. (A) At E14.5, tangentially oriented cells are present in the IZ (arrow) of the wild-type NCX. (B) The number of these cells is markedly reduced in the mutant. [(C) and (D)] At P0, calbindin-expressing pyramidal neurons are present in layer V of the mutant neocortex (D), but fewer calbindin-positive interneurons [arrows in (C)] are detectable. (E and F) At P0, GAD immunoreactivity in layer I (arrowheads) is reduced in the mutant NCX (F). This reduction occurs abruptly in the region (arrow) between the NCX and paleocortex (PCX). Scale bars, 100 μm in (A), 50 μm in (C), and 500 μm in (E).

Although the number of GABA-reactive cells in layer I of theDlx-1/2 mutants was reduced (17), Cajal-Retzius neurons, which facilitate radial migration within the developing neocortex, were present, as indicated by the expression of reelin and calretinin (10, 18). The analysis of cortical lamination in the Dlx-1/2 mutants by Nissl stain and bromodeoxyuridine birth-dating (10), as well as the appearance of calbindin-reactive pyramidal neurons in layer V (Fig. 4D), suggest that the radial migration of neocortical projection neurons is unaffected in these mutants.

In summary, we have provided evidence for the migration of GABA-expressing cells from a subcortical source (the LGE) to the neocortex (19, 20). Because this migration is absent from the Dlx-1/2 mutants, which   also lack most GABA-expressing cells in the olfactory bulb (9), we propose that the subcortical SVZ produces interneuron precursors for both the neocortex and the olfactory bulb (21). The residual presence of neocortical GABA-expressing cells in the mutants suggests that neocortical interneurons are derived from multiple spatially distinct sources.

Although we have previously proposed that the LGE and neocortex are independent compartmentlike structures (1), the present study demonstrates that a more complex situation exists, in which there is cell mixing between the mantle zones of these domains. We suggest that the ventricular zones (VZs) of the neocortex and LGE correspond to compartments, where the regional identity of precursor cells are specified and there is clonal restriction of precursor cells to one compartment. As cells mature and leave the VZ, specific lineages (such as interneurons and projection neurons) follow distinct differentiation programs, which include tangential migration of some interneurons to different forebrain regions.

  • * To whom correspondence should be addressed. E-mail: jlrr{at}cgl.ucsf.edu

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