Figure 3.
BLBP+ cells include a long-term neurogenic population. (A): Blbp::CreERT2 construct driving expression of TAM-inducible CreERT2-recombinase from the mouse Blbp regulatory elements including promoter exons I, II, III, IV, introns, the endogenous 3-′untranslated region, and polyA. (B): Blbp::CreERT2 expressing cells were lineage traced by electroporation of the Blbp::CreERT2 and Blbp::mCherry constructs into the cells of LV wall of postnatal day 1 Rosa-CAG::GFP mice followed by TAM induction after 20 days and analysis 1 and 45 days later. BrdU was administered in the drinking water for 1 week before analysis at day 45. (C): Immediately after TAM administration (d1) sparse recombined (rGFP+) cells appeared in the V-SVZ and many had a radial glial morphology and expressed Blbp::mCherry. (D): Some rGFP+Blbp::mCherry+ cells in the V-SVZ had the polarized radial morphology typical of adult neural stem cells (NSCs) and expressed GFAP within their radial process (arrows) at day 1. (E): Some rGFP+Blbp::mCherry+ cells in the V-SVZ did not express GFAP but showed a radial morphology (arrowheads) at day 1. (F): 45 days after TAM induction, Blbp::CreERT2-derived rGFP+ cells had generated progeny that dispersed into the neuronal layers of the OB (arrows). (G): Quantification of Blbp::CreERT2 progeny (rGFP+) in the V-SVZ and OB depicted as the number of rGFP+ cells per section at day 1 and day 45 after TAM induction. The rGFP+ population expanded in the V-SVZ and accumulated in the OB, indicating that the initially targeted BLBP+ cells generated multiple progeny over time. (H, I): Blbp::CreERT2 cells generated neuroblasts (H, Dcx+) and neurons (I, NeuN+) in the OB 45 days after TAM induction. (J): Quantification of the phenotype of rGFP+ cells in the OB 45 days (d45) after TAM injection. Blbp::CreERT2 derived rGFP+ cells in the OB were neuroblasts (Dcx+), differentiating neurons (NeuN+Dcx+), or mature neurons (NeuN+). (K): Blbp::CreERT2-derived cells (rGFP+) remained mitotically active in the V-SVZ 45 days after TAM induction (BrdU+, arrowheads) and generated new neuroblasts (Dcx+BrdU+, arrows). (L): Quantification of proliferating progenitors (BrdU+), proliferating neuroblasts (Dcx+BrdU+), and neuroblasts (Dcx+) among rGFP+ cells in the V-SVZ 45 days after TAM induction. (M): Quantification of the phenotype of rGFP+ cells in the V-SVZ 1 day (black bars) or 45 days (red bars) after TAM injection. At day 1, most of rGFP+ cells coexpressed Blbp::mCherry+ and GFAP or were Blbp::mCherry+ but GFAP−. At day 45, the proportion of BLBP+ cells decreased while that of their progeny (“other,” transient amplifying progenitors and neuroblasts) proportionally increased. Dashed lines mark the lining of the lateral ventricle. All quantifications are show as mean ±SD. Scale bars =10 μm; in (C, F) =50 μm. Abbreviations: BrdU, 5-bromo deoxyuridine; BLBP, brain lipid binding protein; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; LV, lateral ventricle; OB, olfactory bulb; polyA, polyadenylation signal; SVZ, subventricular zone; TAM, Tamoxifen.