Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb

Abstract

The subventricular zone (SVZ) produces different subclasses of olfactory bulb (OB) interneurons throughout life. Little is known about the molecular mechanisms controlling the production of different types of interneurons. Here we show that most proliferating adult SVZ progenitors express the transcription factor Pax6, but only a small subpopulation of migrating neuroblasts and new OB interneurons derived from these progenitors retains Pax6 expression. To elucidate the cell-autonomous role of Pax6 in OB neurogenesis, we transplanted green fluorescent protein-expressing embryonic forebrain progenitors of the dorsal lateral ganglionic eminence from Pax6 mutant Small Eye (Pax6(Sey/Sey)) mice into the SVZ of adult wild-type mice. Pax6(Sey/Sey) progenitors produce neuroblasts capable of migrating into the OB but fail to generate dopaminergic periglomerular and superficial granule cells. Interestingly, superficial granule neurons also express mRNA for tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Our data show that SVZ neuroblasts are heterogeneous and that Pax6 is required in a cell-autonomous manner for the production of cells in the dopaminergic lineage.

Figure 1.

Dynamics of Pax6 expression during adult SVZ neurogenesis. a, Quantification of BrdU-Pax6 double-labeled cells among total BrdU⁺ cells counted at 1 h (n = 431 cells from 3 mice), 5 d (n = 1274 cells from 3 mice), 15 d (n = 1701 cells from 3 mice), and 45 d (n = 2125 cells from 3 mice) after a single BrdU injection. b, c, Representative pictures of BrdU-Pax6 labeling at 1 h and 45 d after BrdU injection. The box in the schematic on the left indicates the region from which the pictures were taken. Arrows indicate examples of double-labeled cells. c, Inset, High magnification of one BrdU-Pax6 double-labeled cell in the OB. d, Mash1 (red) and Pax6 (green) double-staining in the SVZ. GL, Glomerular layer. Scale bars: b, 50 μm; c, 100 μm; c, inset, 10 μm; d, 20 μm. Error bars indicate SEM.

Figure 2.

Migrating neuroblasts from the SVZ are molecularly heterogeneous. a, Here we show in the SVZ a chain of several migrating neuroblasts with elongated nuclei. Pax6 is expressed by a subset of these cells. The schematic shows the region of the SVZ in a. b, b′, Two examples of chains of migrating neuroblasts from an SVZ explant showing heterogeneity among migrating neuroblasts with respect to Pax6 expression. The early neuronal marker β III tubulin appears green, and Pax6 appears red. LV, Lateral ventricle. Scale bar (in a): a-b′, 20 μm.

Figure 3.

Distribution of Pax6^Sey/Sey and wild-type graft-derived cells in the OB 40 d after grafting. Note the similar distributions of GFP⁺ cells found in all layers of the OB between wild-type and Pax6^Sey/Sey grafts, indicating that Pax6^Sey/Sey-derived cells can migrate both tangentially and radially in the OB. For the Pax6^Sey/Sey graft, n = 1491 cells (3 mice). For the wild-type graft, n = 2511 cells (3 mice). Error bars indicate SEM.

Figure 4.

Loss of dopaminergic PGCs from Pax6^Sey/Sey graft. a, Quantification of the percentage of TH⁺ cells from a graft among total graft-derived PGCs at 40 d of survival; wild type, n = 202 cells (5 mice); Pax6^Sey/+, n = 68 cells (4 mice); Pax6^Sey/Sey, n = 96 cells (6 mice). b, Example of a GFP-TH double-labeled cell. Note identical morphology, confirming colabeling of the same cell. c, Example of a TH-negative GFP cell. d, Representative picture of a graft-derived cell from a Pax6^Sey/Sey graft coexpressing a mature neuronal marker, NeuN. e, The GABAergic neuronal marker, Gad67. Scale bars: (in b″) b-c″, 50 μm; (in d″)d-e″, 20 μm. Statistical analysis with χ² test; p < 0.001.

Figure 5.

Superficial GCs are lost from the Pax6^Sey/Sey graft. a, GFP⁺ cells from a wild-type graft are distributed uniformly throughout the GCL (2× magnification). b, GFP⁺ cells from a Pax6^Sey/+ graft show an intermediate phenotype between wild type and Pax6^Sey/Sey (2× magnification). c, GFP⁺ cells from a Pax6^Sey/Sey graft are predominantly localized to the deep GCL (2× magnification). Scale bar (in a) a-c′, 1 mm. d, Quantification of percentage of superficial GCs among total GFP⁺ GCs at 40 d of survival: wildtype, n = 4268 (5 mice); Pax6^Sey/+, n = 1246 (4 mice); Pax6^Sey/Sey, n = 2168 (5 mice). Statistical analysis with χ² test; p < 0.001. Dendrites from wild-type graft-derived cells arborize uniformly throughout the EPL (a′) but extend to only the deep EPL from Pax6^Sey/Sey graft-derived cells (c′). Scale bar (in a′) a′-c′, 200 μm. Representative images of GFP⁺ cells double-labeled with a marker for mature neurons, NeuN, and a marker for GABAergic neurons, Gad67, are shown. Note that GFP⁺ cells are in the deep GCL and not in the core of the OB where neuroblasts are still migrating tangentially. dGCL, Deep GCL; sGCL, superficial GCL; GL, glomerular layer; dEPL, deep EPL; sEPL, superficial EPL; sGC, superficial GC; wt, wild type.

Figure 6.

TH expression in the adult OB. a, TH protein expression localized predominantly in the OB glomerular layer. a′, TH mRNA is present in the superficial GCL in addition to the glomerular layer. b, b′, TH mRNA-expressing cells coexpress the mature neuronal marker NeuN. c′, Double-labeled TH mRNA and NeuN protein by fluorescence in situ hybridization and immunohistochemistry. Scale bars: b, 100 μm; b′, 30 μm; c″, 20 μm.