BMPR1a signaling determines numbers of oligodendrocytes and calbindin-expressing interneurons in the cortex

Abstract

Progenitor cells that express the transcription factor olig1 generate several neural cell types including oligodendrocytes and GABAergic interneurons in the dorsal cortex. The fate of these progenitor cells is regulated by a number of signals including bone morphogenetic proteins (BMPs) secreted in the dorsal forebrain. BMPs signal by binding to heteromeric serine-threonine kinase receptors formed by type I (BMPR1a, BMPR1b, Alk2) and type II (BMPRII) subunits. To determine the specific role of the BMPR1a subunit in lineage commitment by olig1-expressing cells, we used a cre/loxP genetic approach to ablate BMPR1a in these cells while leaving signaling from other subunits intact. There was a reduction in numbers of immature oligodendrocytes in the BMPR1a-null mutant brains at birth. However, by postnatal day 20, the BMPR1a-null mice had a significant increase in the number of mature and immature oligodendrocytes compared with wild-type littermates. There was also an increase in the proportion of calbindin-positive interneurons in the dorsomedial cortex of BMPR1a-null mice at birth without any change in the number of parvalbumin- or calretinin-positive cells. These effects were attributable, at least in part, to a decrease in the length of the cell cycle in subventricular zone progenitor cells. Thus, our findings indicate that BMPR1a mediates the suppressive effects of BMP signaling on oligodendrocyte lineage commitment and on the specification of calbindin-positive interneurons in the dorsomedial cortex.

Figure 1.

Ablation of BMPR1a mediated by Olig1-cre results in postnatal lethality. A, B, Olig1^cre/+;BMPR1a^+/− mice were mated with BMPR1a^fx/fx (A), and recombination was confirmed in the brain by extracting genomic DNA followed by PCR genotyping (B). The BMPR1a-null allele (null) and Cre recombinase (cre) expression were both present. Because whole brain contained a mixture of Olig1-Cre-expressing cells and nonexpressing cells, both an unrecombined floxed band (fx) and a recombined BMPR1a band (rec.) were detected. C, Cre-dependent recombination was detected by RT-PCR done with mRNA extracted from P21 whole brain. All of the mice that carried Olig1-cre showed deletion of BMPR1a exon2. D, Expression of mRNA for Olig2 was analyzed by quantitative RT-PCR performed with mRNA extracted from whole brains of P0 WT and KO mice. BMPR1a mutation did not change the levels of Olig2 transcripts.

Figure 2.

BMPR1a KO mice have increased numbers of mature oligodendrocytes. A–J, Immunohistochemistry was done for MBP (A–D) and CNPase (E–H) in brain sections from P0 and P20 mice, and cell counts were done using Volocity (I, J). K, L, Sagittal sections were used to analyze the ventral diencephalon of P0 brains (K), and coronal sections were used to analyze the cortex of P20 brains (L). The black bar in K denotes the position of coronal sections, and the red boxes are the areas in which cell counts were done. At P0, the KO mice have lower numbers of CNPase+ cells (E, F, J), whereas at P20, the numbers of CNPase+ and MBP+ cells are significantly increased (A–D, I). *p = 0.02, t test, n = 5; **p = 0.002, t test, n = 5. Error bars represent SEM. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Figure 3.

Smad1/5/8 signaling persists in the BMPR1a-null cells. Sections of P20 brains were immunostained for cre and pSmad1/5/8. A, B, Virtually all cells expressing cre recombinase (A) show nuclear pSmad1/5/8 (B). Cytoplasmic staining for pSmad1/5/8 was also present in some cells. A, C, Cre staining was cytoplasmic as expected. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Figure 4.

BMPR1a-null mutant mice have no change in the number of neurons or astrocytes. A–H, Immunohistochemistry was done for GFAP (A–D), S100β (E, F), and NeuN (G, H) in brain sections from P0 and P20 mice. Expression of markers was analyzed in the dorsal forebrain, except for that of GFAP and S100β in P0 mice, in which the analysis was done in the ventral diencephalon. The KO and WT brains had similar numbers of astrocytes and neurons. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Figure 5.

Olig1-expressing neural stem cells produce GABAergic interneurons. A–C, E, F, Immunohistochemistry in P4 Olig1-cre/R26 mice shows colocalization of β-gal with Olig1 (A–C), cre recombinase (E), and GABA (F) in cortical cells. D, β-Galactosidase staining of brain sections from P4 Olig1-cre/R26 mice shows labeling in the dorsal cortex. G–I, The cortex of P0 WT mice has cells coexpressing Olig1 and NeuN. The mouse anti-β-gal antibody shows punctate staining (E, F), whereas the chick anti-β-gal antibody shows uniform cytoplasmic labeling (A, C). DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Figure 6.

BMPR1a mutation does not regulate the total number of GABAergic interneurons. A, B, The nuclei in the P0 dorsomedial cortex (inset in E shows the area examined) are stained with DAPI to show the cortical layers. C–F, Immunohistochemistry for GABA shows no change in the number of GABAergic interneurons of BMPR1a mutant mice compared with wild-type littermates. A higher magnification of layer V is shown in E and F. n = 5. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Figure 7.

Ablation of BMPR1a leads to an increase in calbindin-expressing interneurons. A–E, Immunohistochemistry for expression of calbindin in the dorsomedial cortex (A–D) of P0 mice shows an increase in the number of calbindin-positive interneurons in the cortex (E) of KO mice compared with WT littermates, but the difference in their numbers is smaller at P21. C, D, Higher-magnification view of layers V/VI. *p = 0.04, t test; n = 5. Error bars represent SEM. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Figure 8.

BMPR1a mutation has no effect on the number of calretinin- or parvalbumin-expressing interneurons. A–D, Immunohistochemistry for calretinin in coronal sections of P0 mice shows no change in the number of calretinin-positive interneurons in the dorsomedial cortex of BMPR1a mutant mice compared with wild-type littermates. A higher magnification of layer VI is shown in C and D, and layer I is shown in A and B. E, F, Immunohistochemistry for parvalbumin of P0 mice also shows no change in the number of parvalbumin-positive interneurons in the dorsomedial cortex of mutant mice compared with wild-type littermates (n = 5). DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Figure 9.

Subventricular zone cells in the BMPR1a-null mutant mice have a shortened cell-cycle length. A–D, F, Immunohistochemistry was done for PCNA (A, B, F) and Ki67 (C, D, F) in coronal brain sections from P0 mice, and cell counts were done using Volocity. Expression was analyzed in the lateral subventricular zone marked by the box in E. F, The BMPR1a mutant brain shows a significant reduction in Ki67-positive cells. The proportion of Ki67-positive progenitor cells that are labeled by PCNA gives an estimate of the cell-cycle length. The BMPR1a mutant brains have a significantly higher PCNA/Ki67 ratio (F), indicating a reduction in cell-cycle length. *p = 0.02, t test, n = 5; **p = 0.003, n = 5. Error bars represent SEM. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride; LV, lateral ventricle.

Figure 10.

BMPR1a-null mutant mice have more GSH2-positive cells undergoing cell cycle in the subventricular zone. Immunohistochemistry was done at P0 and P21 for GSH2-positive cells in the subventricular zone double labeled with PCNA and Ki67. A, D, There was no difference in total numbers of GSH2-expressing cells between WT and KO. B, C, E–H, The percentage of GSH2-positive cells double labeled with PCNA is not changed in the KO mice at P0 or P21 (B, E, G); however, the KO mice have a greater percentage of GSH2-expressing cells labeled with Ki67 at P0 (C, F, H). *p = 0.019, t test, n = 3. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride; LV, lateral ventricle.

Figure 11.

BMPR1a mutation does not alter apoptosis in the ventral forebrain. A, B, Immunohistochemistry was done for cleaved caspase 3 (Cl. Caspase 3) in the ventral forebrain of P0 BMPR1a mutant and wild-type mice. Inset, Area of ventral forebrain in which cell count was done. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.