Multipotent stem cells from the mouse basal forebrain contribute GABAergic neurons and oligodendrocytes to the cerebral cortex during embryogenesis

Abstract

During CNS development, cell migrations play an important role, adding to the cellular complexity of different regions. Earlier studies have shown a robust migration of cells from basal forebrain into the overlying dorsal forebrain during the embryonic period. These immigrant cells include GABAergic neurons that populate the cerebral cortex and hippocampus. In this study we have examined the fate of other basal forebrain cells that migrate into the dorsal forebrain, identifying basal cells using an antibody that recognizes both early (dlx1/2) and late (dlx 5/6) members of the dlx homeobox gene family. We found that a subpopulation of cortical and hippocampal oligodendrocytes are also ventral-derived. We traced the origin of these cells to basal multipotent stem cells capable of generating both GABAergic neurons and oligodendrocytes. A clonal analysis showed that basal forebrain stem cells produce significantly more GABAergic neurons than dorsal forebrain stem cells from the same embryonic age. Moreover, stem cell clones from basal forebrain are significantly more likely to contain both GABAergic neurons and oligodendrocytes than those from dorsal. This indicates that forebrain stem cells are regionally specified. Whereas dlx expression was not detected within basal stem cells growing in culture, these cells produced dlx-positive products that are capable of migration. These data indicate that the developing cerebral cortex incorporates both neuronal and glial products of basal forebrain and suggest that these immigrant cells arise from a common progenitor, a dlx-negative basal forebrain stem cell.

Fig. 1.

Dlx expression in E14 mouse brain.A, Dlx is predominantly expressed in cells in the LGE at E14. Streams of dlx-positive cells are visible leading from basal forebrain into the cerebral cortex (small arrows). A higher magnification of the boxed area inA is shown in B. Arrowsindicate examples of dlx-positive nuclei entering the intermediate zone of the cortex. CTX, Cerebral cortex; LGE, lateral ganglionic eminence. Scale: A, 1 cm = 182 μm; B, 1 cm = 60 μm.

Fig. 2.

Dlx expression in developing white matter regions of E18 mouse brain. Low-power micrograph of coronal sections from E18 mouse brain illustrating the major white matter tracts in anterior (A) and posterior (A′) forebrain. CC, Corpus callosum;CWM, subcortical white matter; FIM,fimbria; OC, optic chiasm. B, The number of dlx-positive cells in sections of embryonic white matter tracts as a percentage of total cells (examples of staining are shown inC–F′). There was a significant difference among the different areas of white matter (ANOVA; p < 0.01).C–F′, Examples of different areas of white matter in E18 brain sections stained for dlx (brown) (C–F) and counterstained with DAPI (blue) (C′–F′). Note that in dlx-positive nuclei, DAPI staining is not visible, hence the total cell number is calculated by adding the number of DAPI-positive and dlx-positive cells. Dlx-positive cells were found in the cortical white matter, corpus callosum, and fimbria, but not in the optic chiasm. Numbers of dlx-positive cells in different forebrain white matter areas are compared in B. Scale: 1 cm = 91 μm.

Fig. 3.

NG2-positive cells expressing dlx in E18 mouse corpus callosum. E18 mouse brain sections were stained with anti-dlx and with NG2, a marker expressed by oligodendrocyte progenitor cells. A, B, Low-power light micrograph of the same field of a section of E18 corpus callosum double-labeled for dlx inbrown (A) and for NG2 with a Cy3-labeled secondary antibody (B). C, D, High magnification of the boxed areas inA and B, respectively.Arrows indicate a cell double-labeled for dlx and NG2. Scale bars: A, 50 μm; C, 25 μm.

Fig. 4.

NG2-positive cells acutely isolated from E18 mouse forebrain express dlx. E18 cortical and striatal tissue was enzymatically dissociated with papain to single cells, which were fixed acutely and stained for NG2 and dlx. A–C, Cortical cells; D–F, striatal cells. A, D, Phase images of cells acutely isolated from E18 cortex and striatum.B, E, NG2 staining of cells in A andD. Arrows indicate NG2-positive cells (red). C, F, Double staining of cells inA and D for NG2 (yellow) and dlx (green).Arrows indicate cells double-labeled for NG2 and dlx.Arrowhead in F indicates a cell that is dlx-positive but NG2-negative. Scale: 1 cm = 46.7 μm.

Fig. 5.

Dlx expression in O1-immunopanned cortical oligodendrocytes. Postmitotic oligodendrocytes (O1-positive) were immunoselected from P5 rat cerebral cortex. The cells were fixed acutely and stained with O4 antibody to confirm their oligodendrocyte identity and dlx. A, Phase image of two O1-immunoselected P5 cortical cells. B, O4 staining of cells in A. C, The oligodendrocyte on theleft is dlx-negative, whereas the one on theright is dlx-positive, with typical nuclear staining.D, The percentage of dlx-positive cells in O1-immunopanned P5 cortical cells was 38%. In contrast, no dlx-positive cells were found in oligodendrocytes isolated from the P5 optic nerve. Scale: 1 cm = 48 μm.

Fig. 6.

Oligodendroglia generating progenitor cells derived from E11.5 and E13 basal forebrain. Progenitor cells were isolated from basal forebrain and plated at clonal density and followed in culture for 5–12 d before staining for NG2 or O4 and β-tubulin III. Note that progenitor cells giving purely glial progeny were very rare at young ages and increased with development from E11.5 to E13. Hence, at early ages most oligodendrocytes arise from stem cells that also make neurons. G, Pure glial clones;SC, stem cell clones.

Fig. 7.

A clonal analysis of stem cells from dorsal and basal E13 forebrain reveals differences in their production of GABAergic neurons and oligodendrocytes. A, Progenitor cells were isolated from three forebrain regions at E13: cortex (CTX), lateral ganglionic eminence (LGE), and medial ganglionic eminence (MGE). The cells were cultured for 12 d and stained for GAD, O4, and β-tubulin III. Stem cell (SC) clones that contained both GABAergic neurons and oligodendrocytes were found in all three regions, as illustrated in A. β-tubulin III-positive neurons are shown in green, O4-positive oligodendrocytes in yellow, and GAD-positive neurons inbrown. Scale: 1 cm = 58 μm.B, The number of GAD-positive neurons expressed as a percentage of total neurons produced by progenitor cells from cortex, LGE, and MGE. There is a significant difference between the percentage of GAD-positive neurons made by cortical progenitors and the percentage made by LGE or MGE progenitor cells (ANOVA analysis;p < 0.001). tub, β-tubulin III.C, The number of GAD-positive neurons expressed as a percentage of total neurons produced within stem cell clones was calculated. MGE stem cells generated significantly more GAD-positive neurons than LGE or cortical stem cells (ANOVA analysis;p < 0.05). D, The percentage of stem cell clones containing both GABAergic neurons and oligodendrocytes was calculated for the three forebrain regions. LGE and MGE produced significantly more stem cell clones containing both these cell types than did cortex (ANOVA analysis; p < 0.05).GABA-N, GABAergic neuron; oligo, oligodendrocyte.

Fig. 8.

Dlx expression in a developing stem cell clone. Single cells from E13 forebrain were dissociated and plated at clonal density. Growing clones were mapped and followed every day for 5 d. Stem cell clones were stained for NG2 to identify glial progeny and β-tubulin III to identify neurons and dlx. A, Phase micrograph of a developing stem cell clone at 5 d in vitro. B, After staining for dlx, three cells are positive, as indicated by arrows. C,Membranous NG2 staining of the same clone (small arrowpoints to an NG2-positive cell seen in phase in A and inred fluorescence in C). D,β-tubulin III staining of the same clone (arrowsindicate that the three dlx-positive cells seen in B are also β-tubulin III-positive). Scale: 1 cm = 50 μm.