Differential modulation of BMP signaling promotes the elaboration of cerebral cortical GABAergic neurons or oligodendrocytes from a common sonic hedgehog-responsive ventral forebrain progenitor species

Abstract

During cerebral cortical development, excitatory glutamatergic projection neurons are generated from neural stem cells intrinsic to the early embryonic cortical ventricular zone by a process of radial migration, whereas most inhibitory gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes (OLs) appear to be elaborated from ventral forebrain stem cells that initially undergo tangential cortical migration before terminal lineage maturation. In contrast to the more compartmentalized developmental organization of the spinal cord, the generation of neurons and OLs from a common ventral forebrain stem cell would expose these cells to the sequential actions of ventral and dorsal gradient morphogens [sonic hedgehog (Shh) and bone morphogenetic proteins (BMPs)] that normally mediate opposing developmental programs. Here we report that Shh promotes GABAergic neuronalOL lineage restriction of forebrain stem cells, in part, by activation of the basic helix-loop-helix transcription factors, Olig2 and Mash1. In mutant mice with a generalized defect in tangential cortical migration (Dlx12--), there is a profound and selective reduction in the elaboration of both cortical GABAergic neurons and OLs. Our studies further demonstrate that the sequential elaboration of cortical GABAergic neurons and OLs from common Shh-responsive ventral forebrain progenitors requires the spatial and temporal modulation of cortical BMP signaling by BMP ligands and the BMP antagonist, noggin, respectively. These findings suggest an integrative model for cerebral cortical GABAergic neuronal and OL lineage maturation that would incorporate the sequential contributions of the ventral and dorsal forebrain, and the potential role of regional developmental cues in modulating transcriptional codes within evolving neural lineage species.

Fig 1.

Before the stage of tangential cortical migration, individual MGE and LGE progenitors from the ventral forebrain but not the cerebral cortex give rise to both GABAergic neurons and OLs that populate the postmigratory stage cerebral cortex. (A) The proportion of total clones containing GABAergic neurons, OL species, or GABAergic neurons and OL species generated after propagation of primary E12.5 MGE and LGE and E12.5 and E16.5 cerebral cortical (Ctx) progenitors for 6 DIV. All data points represent the proportion of target clones with its 95% confidence interval. (B) The proportion of clones containing GABAergic neurons and OL species generated after propagation of primary E12.5 Dlx1/2+/+, Dlx1/2+/−, and Dlx1/2−/− MGE and LGE progenitors for 6 DIV. Statistical analysis was calculated by using χ² analysis: *, P < 0.001 (compared with the E12.5 Dlx1/2+/+ MGE clonal condition); +, P < 0.001 (compared with the E12.5 Dlx1/2+/+ LGE clonal condition). (C) The proportion of total clones containing both GABAergic neurons and OL species or glutamatergic neurons generated after propagation of primary E16.5 Dlx1/2+/+, Dlx1/2+/−, and Dlx1/2−/− cerebral cortical progenitors for 6 DIV. Statistical analysis was calculated by using χ² analysis: *, P < 0.001 (compared with the Dlx1/2+/+ GABA/O4 clonal condition); +, P < 0.001 (compared with the Dlx1/2+/+ glutamate clonal condition).

Fig 2.

Shh promotes neuronal/OL lineage restriction of early embryonic forebrain stem cells and the selective induction of transcription factors involved in GABAergic neuronal and OL lineage specification and maturation. (A and B) Immunofluorescence micrographs of individual differentiated clones derived from E12.5 cerebral cortical cells after clonal expansion under control (bFGF, 10 ng/ml, A) and N-Shh (bFGF+N-Shh, 50 ng/ml, B) conditions for 3 DIV, and subsequent plating as intact clonal populations under differentiating conditions for an additional 6 DIV. Note that FGF-responsive clones are multipotent (neurons: β-tubulin, FITC; OLs: O4, cascade blue; astrocytes: glial fibrillary acidic protein, tetramethylrhodamine B isothiocyanate), whereas FGF+Shh-responsive clones are comprised of more lineage-restricted progeny (neurons: β-tubulin, FITC; OLs:O4, tetramethylrhodamine B isothiocyanate). (C) Comparative clonal lineage profiles of bFGF- and bFGF+Shh-responsive E12.5 cortical progenitor species plated and propagated under culture conditions as noted above. All data points represent the proportion of target clones with its 95% confidence interval. Statistical analysis was calculated by using χ² analysis: *, P < 0.001 (compared with individual clonal lineage profiles in control condition). (D) Comparative proportion of individual clones containing Mash1, Neurogenin1, Nkx2.2, GABA, and O4 derived from primary E12.5 cortical progenitors in the absence and presence of increasing concentrations of Shh after 6 DIV. Statistical analysis was calculated by using χ² analysis: P < 0.001 [for all data points relative to their control (Ngn1), or Shh, 1 ng/ml (Mash1, Nkx2.2, GABA, O4) conditions].

Fig 3.

Shh-mediated forebrain GABAergic neuronal/OL lineage restriction requires Olig2 and Mash1. (A) The comparative proportion of clones containing GABAergic neuronal/OL species and glutamatergic neurons generated from E12.5 FGF-responsive cortical progenitors after clonal expansion (2 DIV) in the absence or the presence of ectopic expression of Olig2 (Ad-O2) without or with concurrent ablation of Mash1 expression (Mash1as): Ad-GFP (adenoviral vector expressing GFP alone), Ad-O2 (adenoviral vector expressing Olig2 and GFP), Mash1ss (Mash1 sense oligonucleotides), and Mash1as (Mash1 antisense oligonucleotides). After clonal expansion in control and experimental conditions, intact clonal populations were plated under differentiating conditions for an additional 6 DIV. All data points represent the proportion of target clones and its 95% confidence interval. Statistical significance was calculated by using χ² analysis: *, P < 0.001 (compared with GABA/OL clones in the bFGF+Ad-O2 clonal condition); +, P < 0.001 (compared with glutamatergic clones in the bFGF condition). (B) The comparative proportion of clones containing GABAergic neuronal/OL species and glutamatergic neurons generated from E12.5 FGF+Shh-responsive cortical progenitors in the absence or presence of ablation of Olig2 expression (Olig2as) without or with concurrent ablation of Mash1 expression (Mash1as). O2ss, Olig2 sense oligonucleotides; O2as, Olig2 antisense oligonucleotides. Control and experimental conditions were subjected to clonal expansion and differentiating conditions as described above. (C) The comparative proportion of clones containing GABAergic neurons or OL species generated under clonal culture paradigms identical to those described in A. Statistical significance was calculated by using χ² analysis: *, P < 0.001 (compared with the bFGF+Ad-O2 GABA clonal condition); +, P < 0.001 (compared with the bFGF+Ad-O2 O4 clonal condition). (D) The comparative proportion of clones containing GABAergic neurons or OL species generated under clonal culture paradigms identical to those described in B.

Fig 4.

Developmental modulation of BMP signaling promotes the differential elaboration of GABAergic neurons and oligodendrocytes from Shh-responsive forebrain progenitors. (A and B) The comparative proportion of clones containing GABAergic neurons (white bars), OL (O4) species (black bars), or GABAergic neurons and OL species (striped bars) generated from primary E12.5 MGE (A) or LGE (B) progenitors plated in the absence (control) or presence of BMP2 or noggin and propagated for 6 DIV. All data point represents the proportion of target clones and its 95% confidence interval. Statistical significance was calculated by using χ² analysis: *, P < 0.001 (compared with GABA clonal control conditions); +, P < 0.05 (compared with O4 clonal control condition); †, P < 0.001 (compared with the GABA/O4 clonal control condition). (C) The comparative proportion of clones containing GABAergic neurons (white bars), OL species (black bars), or GABAergic neurons and OL species (striped bars) generated from primary E12.5 cortical (Ctx) progenitors plated in the absence or presence of N-Shh and in the absence or presence of BMP2 or noggin and propagated for 6 DIV. Statistical significance was calculated by using χ² analysis: *, P < 0.001 (compared with GABA [N-Shh] clonal conditions); +, P < 0.001 (compared with the O4 [N-Shh] clonal condition); †, P < 0.001 (compared with the GABA/O4 [N-Shh] clonal condition).