Dynamic expression of the pro-dopaminergic transcription factors Pax6 and Dlx2 during postnatal olfactory bulb neurogenesis

Abstract

Olfactory bulb (OB) neurogenesis generates neurons that use GABA or dopamine as their neurotransmitters throughout life. Regionalized stem cell populations in the periventricular zone (PVZ) of the lateral ventricles (LVs) have been shown to be at the basis of neuronal diversity in the system. For example dopaminergic neurons arise predominantly from neural stem cells (NSCs) residing in the dorsal PVZ and depend on the expression of the transcription factors Pax6 and Dlx2 for their specification. In addition, Dlx2 is required for neurogenesis in general. Using targeted in vivo electroporation combined with immuno-fluorescence imaging and microarray analysis, we provide here detailed spatial and temporal expression data with cellular resolution in this system. We find that all along the neurogenic process Pax6 expression remains restricted to the dorsal PVZ, whereas nearly all neuroblasts express Dlx2, including those of the dorsal lineage, which are switched on for Dlx2 when they enter the rostral migratory stream (RMS). These data allow to explain and precise the functions of these two genes in postnatal OB neurogenesis.

Keywords: Dlx2; Pax6; SVZ; in vivo electroporation; olfactory bulb; postnatal neurogenesis.

Figure 1

Pax6 expression is restricted dorsally in the periventricular region. (A) Low magnification view of a coronal section of the P1 forebrain labeled with Pax6 antibody (red). The signal is strong and widespread in the dorsal periventricular zone (dPVZ), weaker at the junction between the dorsal and lateral walls (horn and yellow) and faint and rare in the lateral wall (lPVZ). (B) PVZ coronal section 6 h after a dorsal (top) or a lateral (bottom) electroporation with a GFP expressing plasmid. Pax6 is labeled in red. (C and D) Pax6 expression (red) by GFP-expressing cells after GFP electroporation in the dorsal (upper panel) or lateral wall (lower panel). At 6 hpe (C), radial glial neural stem cells express Pax6 in the dorsal, but not lateral, PVZ. (D) Similarly, most amplifying precursors and neuroblasts of the dorsal but not lateral PVZ express Pax6 (arrowheads: GFP⁺/Pax6⁺ cells, arrows: GFP⁺/Pax6⁻ cells). (E) Histograms depicting in three regions of the PVZ (dorsal PVZ, horn, and lateral PVZ) the percentage of GFP electroporated cells co-expressing Pax6 6 h or 2 h days after dorsal or lateral electroporation (n = 3 animals per group, more than 200 cells per group, ^*p < 0.05, ^**p < 0.01). LV: lateral ventricle. Scale bars: 20 μm.

Figure 2

Pax6 remains preferentially expressed in the dorsal lineage in neuroblasts migrating in the RMS and in new neurons of the olfactory bulb. (A and B) Distribution of dorsally or laterally generated GFP⁺ cells in the RMS at 4 dpe (A) and in the olfactory bulb at 15 dpe (gl: glomerular layer, epl: external plexiform layer, sl: superficial layer of the granule cell layer, dl: deep layer of the granule cell layer) (B). (C) 3-D reconstructions of merged images of dorsally (upper panel) or laterally (lower panel) electroporated GFP⁺ cells (green) labeled with Pax6 antibody (red) at 4 or 15 dpe. These images show that dorsally but not laterally generated GFP⁺ cells in the RMS and in the granule cell layer (GCL) or glomerular layer (GL) of the OB express Pax6. (D) Histograms representing the percentage of GFP electroporated cells expressing Pax6 4 days after electroporation in the RMS and 15 days after electroporation either in the GCL or in the GL, after dorsal (dark gray bars) or lateral (light gray bars) electroporation (n = 3–5 mice per group). Scale bars: A, 50 μm; B, 200 μm; C, 20 μm.

Figure 3

Dlx2 expression in the PVZ is restricted to the non-radial glial progenitor cells of the lateral wall. (A) Low magnification of a P1 mouse brain coronal section labeled with Pax6 (red) and Dlx2 (blue) antibodies showing an overall complementary, non-overlapping expression of these two genes in the PVZ. (B–E) High magnification of dorsally or laterally GFP electroporated cells labeled with Pax6 and Dlx2 antibodies at 6 h (B,C) and two days (D,E) after electroporation. Note that the dorsally electroporated cells expressed Pax6 but not Dlx2 (B,D). As expected the laterally electroporated cells did not express Pax6 at any time. The lateral radial glial neural stem cells did not express Dlx2 at 6 hpe (C), while the transit-amplifying and neuroblast progeny did express Dlx2 at 2 dpe (E). Arrowheads: GFP⁺/marker⁺ cells, arrows: GFP⁺/marker⁻ cells. Scale bar: 10 μm.

Figure 4

Dlx2 gets activated in the dorsal lineage once cells have entered the RMS and is maintained in the OB. (A) Evolution of Dlx2 transcript expression over time in the dorsal (red) and lateral (blue) lineages obtained from microarray analysis performed on FAC-sorted GFP electroporated cells at successive time points. (B–D) High magnifications of dorsally generated GFP⁺ cells labeled with Pax6 (red) and Dlx2 (blue) antibodies in the RMS at 4 dpe (B), or in the granule cell layer (C) and glomerular layer (D) at 15 dpe. Arrowheads: GFP⁺/marker⁺ cells. Note that both in the RMS and the GCL and GL of the OB Dlx2 is co-expressed with Pax6 in dorsally generated GFP electroporated cells. (E) Evolution of Dlx2 expression in dorsal (red) or lateral (blue) GFP⁺ electroporated cells at successive time after electroporation (n = 3 mice per group). Scale bar: 10 μm.

Figure 5

The lateral PVZ lineage remains positive for Dlx2 in neuroblasts and mature neurons. High magnification of laterally electroporated GFP+ cells labeled with Pax6 (red) and Dlx2 (blue) antibodies at 4 or 15 days after electroporation. Note that both in the RMS at 4 dpe and in the GCL and GL of the OB at 15 dpe cells are negative for Pax6 as expected and remain positive for Dlx2.