Origin and molecular specification of globus pallidus neurons

Abstract

The mechanisms controlling the assembly of brain nuclei are poorly understood. In the forebrain, it is typically assumed that the formation of nuclei follows a similar sequence of events that in the cortex. In this structure, projection neurons are generated sequentially from common progenitor cells and migrate radially to reach their final destination, whereas interneurons are generated remotely and arrive to the cortex through tangential migration. Using the globus pallidus as a model to study the formation of forebrain nuclei, we found that the development of this basal ganglia structure involves the generation of several distinct classes of projection neurons from relatively distant progenitor pools, which then assemble together through tangential migration. Our results thus suggest that tangential migration in the forebrain is not limited to interneurons, as previously thought, but also involves projection neurons and reveal that the assembly of forebrain nuclei is more complex than previously anticipated.

Figure 1.

Neuronal populations in the globus pallidus. A–D‴, Coronal sections through the telencephalon of P60 wild-type mice showing coexpression of PV (A, A″, D′, D‴), ChAT (B, B″, D, D‴), Npas1 (C, C″, D″, D‴), and the neuronal marker NeuN (A′, A″, B′, B″, C′, C″) in the GP. Open arrowheads point to examples of double-labeled cells. Note that PV, ChAT, and Npas1 are expressed by primarily nonoverlapping populations of neurons in the GP. E, Schematic representation of the three distinct neuronal populations present in the GP and the molecular markers used to identify them. Scale bar, 50 μm.

Figure 2.

Molecular profile and time of neurogenesis distinguish the two main classes of GABAergic neurons in the globus pallidus. A–B″, Coronal sections through the telencephalon of P60 wild-type mice showing coexpression of PV (A, B) with Nkx2-1 (A′, A″) and Er81 (B′, B″) in the GP. Open arrowheads point to examples of double-labeled cells, whereas filled arrowheads point to neurons that express only one of the markers. C, Quantification of the percentage of PV-expressing and Npas1-expressing GP neurons labeled with BrdU at different developmental stages. Histograms show average ± SEM. E10.5, 24.91 ± 5.34% (PV), 3.88 ± 0.09% (Npas1); E11.5, 42.91 ± 5.14% (PV), 29.23 ± 3.79% (Npas1); E12.5, 6.13 ± 1.61% (PV), 28.72 ± 3.00% (Npas1); E13.5, 1.59 ± 0.29% (PV), 6.65 ± 0.86% (Npas1). Scale bar, 50 μm.

Figure 3.

Fate-mapping analysis of the globus pallidus with Nkx2-1–Cre mice. A, Schematic of the experimental design, indicating the progenitor domain labeled in Nkx2-1–Cre mice. B–C″, Coronal sections through the telencephalon of P30 Nkx2-1–Cre;Rosa26R–YFP mice showing coexpression of PV (B, B″) and Npas1 (C, C″) with YFP (B′, B″, C′, C″) in the GP. Open arrowheads point to examples of double-labeled cells, whereas filled arrowheads point to neurons that express only one of the markers. H, Hippocampus; NCx, neocortex. Scale bar, 50 μm.

Figure 4.

The MGE gives rise to cells that migrate to the embryonic globus pallidus. A, B, Coronal (A) and horizontal (B) sections through the telencephalon of E13.5 wild-type embryos showing the expression of Er81 mRNA. The red dotted line in A indicates the plane of the section shown in B. The arrows indicate lateral (L) and rostral (R) orientations within the brain. C, Schematic of the experimental design. D–E′, A representative case of the distribution of GFP-expressing (D, E) and Nkx2-1-expressing (D′, E′) cells in a coronal section through the telencephalon of an E15.5 embryo in which the caudoventral MGE was electroporated at E12.5. The white arrowhead in D and D′ indicates the place of electroporation. Note the presence of many GFP⁺ cells in the ventral division of the GP (known as the ventral pallidum). E and E′ are high-magnification images of the GP, in a section caudal to that shown in D and D′ where this nucleus reaches its maximum area. F–G′, Images of representative cells found in the GP after electroporation in the caudoventral MGE. These cells typically stain for Nkx2-1 (F′, open arrowheads) and Er81 (G′, open arrowheads). However, some cells do not stain for either of the two markers (filled arrowheads). ac, Anterior commissure; Str, striatum. Scale bars: A, 200 μm; D, D′, 250 μm; E, E′, 100 μm; F–G′, 25 μm.

Figure 5.

The MGE is the origin of globus pallidus neurons. A, Schematic of the experimental design. GFP⁺ donor pregnant mice received a single injection of BrdU at E12.5. Twelve hours after BrdU injection, the MGE was dissected from embryos and dissociated. Donor MGE cells were then injected into the MGE of E12.5 host embryos, and host embryos were analyzed at P14. B–B‴, Coronal section through the GP of a transplanted P14 mouse showing the expression of PV (B′, B‴) and Npas1 (B″, B‴) in GFP⁺ neurons derived from the MGE (B, B‴). Open arrowheads and filled arrowheads indicate PV/GFP and Npas1/GFP double-labeled cells, respectively. C, Quantification of the percentage of GFP⁺ transplanted neurons expressing PV or Npas1 from five different experiments (T79g, T73n, T89f, T90d, T80b). Histograms show average ± SEM. T79g, 25.00 (PV), 25.00 (Npas1); T73n, 30.77 (PV), 15.38 (Npas1); T89f, 20.00 (PV); T90d, 20.51 (PV), 28.21 (Npas1); T80b, 16.00 (PV), 20.00 (Npas1); total, 22.46 ± 4.34 (PV), 17.72 ± 8.02 (Npas1). D, Quantification of the percentage of GFP⁺ transplanted neurons expressing PV/BrdU or Npas1/BrdU in all experiments. Histograms show average ± SEM. Total: PV, 6.97 ± 1.15%; Npas1, 3.76 ± 3.56%. Scale bar, 50 μm.

Figure 6.

Origin of GP neurons in the POA. A, Schematic of the experimental design. B–C′, A representative case of the distribution of GFP-expressing (B, C) and Nkx2-1-expressing (B′, C′) cells in a coronal section through the telencephalon of an E15.5 embryo in which the POA was electroporated at E12.5. The white arrowhead in B and B′ indicates the place of electroporation. C and C′ are high-magnification images of the GP from the boxed area shown in B and B′, respectively. D–D′, Images of representative cells found in the GP after electroporation in the POA. E, Schematic of the experimental design, indicating the progenitor domains labeled in Dbx1–Cre mice. Note that only a few progenitor cells within the ventral pallium (VP) and the POA express Dbx1. F–G″, Coronal sections through the telencephalon of P30 Dbx1–Cre;Rosa26R–YFP mice showing coexpression of PV (F, F″) and Npas1 (G, G″) with YFP (F′, F″, G′, G″) in the GP. Open arrowheads point to examples of double-labeled cells, whereas filled arrowheads point to neurons that express only one of the markers. Scale bars: B, B′, 250 μm; C, C′, 100 μm; D–D″, 25 μm; F–G″, 50 μm. Str, Striatum; H, hippocampus.

Figure 7.

Origin of GP neurons in the LGE. A, Schematic of the experimental design. B–C′, A representative case of the distribution of GFP-expressing (B, C) and Nkx2-1-expressing (B′, C′) cells in a coronal section through the telencephalon of an E15.5 embryo in which the LGE was electroporated at E12.5. The white arrowhead in B and B′ indicates the place of electroporation. Note that most cells derived from the LGE seem to migrate radially toward the developing striatum (arrow), but a few appear to turn ventrally toward the GP. C and C′ are high-magnification images of the GP from the boxed area shown in B and B′, respectively. D, D′, Images of representative cells found in the GP after electroporation in the LGE. E, Schematic of the experimental design, indicating the progenitor domains labeled in Pax6–Cre mice. F–G″, Coronal sections through the telencephalon of P30 Pax6–Cre;Rosa26R–YFP mice showing coexpression of PV (F, F″) and Npas1 (G, G″) with YFP (F′, F″, G′, G″) in the GP. Open arrowheads point to examples of double-labeled cells, whereas filled arrowheads point to neurons that express only one of the markers. Scale bars: B, B′, 250 μm; C, C′, 100 μm; D, D′, 25 μm; F–G″, 50 μm. Str, Striatum; NCx, neocortex.

Figure 8.

Npas1-expressing cells are present in the globus pallidus of Nkx2-1 mutants. A–D, Coronal sections through the telencephalon of E18.5 wild-type (A, B) and Nkx2-1 mutant (C, D) fetuses showing the distribution of Er81-expressing (A, C) and Npas1-expressing (B, D) cells in the GP. Scale bar, 250 μm. Str, Striatum; ic, internal capsule.

Figure 9.

Neuronal diversity in the globus pallidus emerges from different and distant progenitor pools. A, Schematic representation of a transversal hemisection through an E13.5 developing telencephalon, depicting the putative routes of migration of GP neurons from their origins to their final destination. B, Schematic representation of neuronal diversity in the GP, based on the molecular profile of its constituents and their differential origin. 1, The contribution of the Pax6-expressing territory (LGE proper and/or CGE) to the population of Npas1⁺ neurons is inferred from the fate-mapping analysis of Nkx2-1–Cre;Rosa26R–YFP mice, because expression of Cre in Pax6–Cre mice is mosaic and does not label all LGE progenitors. In addition, it should be noted that the POH also expresses Pax6 (supplemental Fig. S2, available at www.jneurosci.org as supplemental material), and therefore some Npas1⁺ neurons might derive from this region. 2, The contribution of the POA to the GP might be underestimated, because progenitors other than those expressing Dbx1 may give rise to GP neurons. If this were the case, then the contribution of the MGE to the GP would be overestimated. H, Hippocampus; CPu, caudoputamen nucleus; PCx, piriform cortex; Str, striatum.