Distribution of Aldh1L1-CreERT2 Recombination in Astrocytes Versus Neural Stem Cells in the Neurogenic Niches of the Adult Mouse Brain

Abstract

In the adult central nervous system, neural stem cells (NSCs) reside in two discrete niches: the subependymal zone (SEZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Here, NSCs represent a population of highly specialized astrocytes that are able to proliferate and give rise to neuronal and glial progeny. This process, termed adult neurogenesis, is extrinsically regulated by other niche cells such as non-stem cell astrocytes. Studying these non-stem cell niche astrocytes and their role during adult neuro- and gliogenesis has been hampered by the lack of genetic tools to discriminate between transcriptionally similar NSCs and niche astrocytes. Recently, Aldh1L1 has been shown to be a pan-astrocyte marker and that its promoter can be used to specifically target astrocytes using the Cre-loxP system. In this study we explored whether the recently described Aldh1L1-CreER^T2 mouse line (Winchenbach et al., 2016) can serve to specifically target niche astrocytes without inducing recombination in NSCs in adult neurogenic niches. Using short- and long-term tamoxifen protocols we revealed high recombination efficiency and specificity in non-stem cell astrocytes and little to no recombination in NSCs of the adult DG. However, in the SEZ we observed recombination in ependymal cells, astrocytes, and NSCs, the latter giving rise to neuronal progeny of the rostral migratory stream and olfactory bulb. Thus, we recommend the here described Aldh1L1-CreER^T2 mouse line for predominantly studying the functions of non-stem cell astrocytes in the DG under physiological and pathological conditions.

Keywords: Aldh1L1; Aldh1L1-CreERT2; astrocytes; dentate gyrus; neural stem cells; neurogenic niche; subependymal zone.

FIGURE 1

Aldh1L1 expression in adult neurogenic niches.(A,B) Immunohistochemical co-staining of Aldh1L1 and GFAP in the adult mouse neurogenic niches (DAPI in blue, GFAP in red, Aldh1L1 in white). (A) Aldh1L1/GFAP double positive astrocytes reside in all DG layers (yellow arrows). Green arrowheads highlight GFAP-positive/Aldh1L1-negative radial processes of adult NSCs in the DG. DG compartment indicated on the left: oML, outer molecular layer; iML, inner molecular layer; GZ, granular zone; SGZ, subgranular zone; scale bar = 20 μm. (B) Aldh1L1/GFAP double positive cells residing in the SEZ (yellow arrows). Green arrowheads point toward GFAP-negative/Aldh1L1-positive cells in the SEZ (scale bar = 20 μm). (C) Crossing scheme showing the genotype of parental mice used to generate experimental Aldh1L1-CreER^T2; GFP mice. (D) In order to achieve sufficient recombination, we used different tamoxifen pulses in Aldh1L1-CreER^T2; GFP transgenic mice ranging from one to nine injections every 12 h (12 h) and analyzed recombination 2 h post-injection (2 h pi). (E–H) With increasing numbers of tamoxifen injections we observed an increase in the number of recombined astrocytes in the DG of adult Aldh1L1-CreER^T2; GFP mice (DAPI in blue, GFP in green, GFAP in red; scale bar = 50 μm).

FIGURE 2

Recombination in Aldh1L1-CreER^T2; GFP mice efficiently and specifically targets astrocytes but not neural stem cells in the adult dentate gyrus. (A) Schematic drawing showing the short-term tamoxifen pulse. Here, Aldh1L1-CreER^T2; GFP mice were injected with tamoxifen every 12 h (12 h) for five consecutive days and killed 2 h post-injection (2 h pi). (B) Representative pictures illustrate an overview of an immunohistochemically stained DG of adult Aldh1L1-CreER^T2; GFP mice. Arrows point toward GFP/GFAP/Aldh1L1 triple positive cells while arrowheads highlight non-recombined GFAP⁺/Aldh1L1⁺ astrocytes. The asterisk marks a non-recombined GFAP⁺ radial process of an adult NSC (DAPI in blue, GFP in green, GFAP in red, Aldh1L1 in white; scale bar = 20 μm). (C,D) The graphs show recombination efficiency in adult DG astrocytes and adult DG astrocyte subtypes (mean + SEM). (E,F) The graphs show recombination specificity in adult DG astrocytes and adult DG astrocyte subtypes (mean + SEM). (G) Overview image of an immunohistochemically stained Aldh1L1-CreER^T2, GFP transgenic mouse DG using antibodies against GFP (green), S100β (red), and Nestin (white) 2 h pi; DAPI is depicted in blue, scale bar = 20 μm. (H–K) Representative pictures of astrocytes of each DG layer in [(G); boxed areas] were chosen for magnification. Arrows point toward recombined GFP⁺ astrocytes (green) expressing S100β (red) in the molecular layer (H), the hilus (I), the granular zone (J), and the subgranular zone [SGZ; (K)] (scale bars = 20 μm). (L) Schematic drawing showing the long-term tamoxifen pulse. Here, Aldh1L1-CreER^T2; GFP mice were injected with tamoxifen every 12 h (12 h) for five consecutive days and killed 28 days post-injection (dpi). (M,N) Representative pictures of immunohistochemical staining against GFP (green) and DCX (white) and NeuN (red), respectively. Arrows point toward recombined GFP⁺ cells, which do not co-express either DCX or NeuN; DAPI is depicted in blue; scale bar = 20 μm.

FIGURE 3

Recombination in Aldh1L1-CreER^T2; GFP mice targets astrocytes and neural stem cells in the subependymal zone. (A) Schematic drawing showing the short-term tamoxifen pulse. Here, Aldh1L1-CreER^T2; GFP mice were injected with tamoxifen every 12 h (12 h) for five consecutive days and killed 2 h post-injection (2 h pi). (B) Representative pictures illustrate an overview of the immunohistochemically stained SEZ of adult Aldh1L1-CreER^T2; GFP mice. Arrows point toward GFP/GFAP/Aldh1L1 triple positive cells while arrowheads highlight non-recombined GFAP⁺/Aldh1L1⁺ cells (DAPI in blue, GFP in green, GFAP in red, Aldh1L1 in white; scale bar = 20 μm). (C,D) Representative images show an immunohistochemically stained SEZ of Aldh1L1-CreER^T2, GFP transgenic mice using antibodies directed against GFP (green), S100β (red), and Nestin (white). The arrows point toward a GFP/S100β double positive astrocyte in the SEZ (C), or a triple positive radial glia-like NSC (D) DAPI is depicted in blue, scale bar = 20 μm. (E) Images show an immunohistochemically stained SEZ of Aldh1L1-CreER^T2, GFP transgenic mice using antibodies directed against GFP (green), FoxJ1 (red), and Aldh1L1 (white). The arrows point toward a triple positive ependymal cell. DAPI is depicted in blue, scale bar = 20 μm. (F,G) Efficiency and specificity of recombination are expressed as the ratio of Aldh1L1/GFP double positive cells over all Aldh1L1⁺ or GFP⁺ cells, respectively (mean + SEM; 2 h pi). (H) Graph showing the ratio of S100β/GFP double positive cells over all recombined (GFP⁺) cells (mean + SEM; 2 h pi). (I) Graph showing the ratio of Nestin/GFP double positive NSCs over all recombined (GFP⁺) cells (mean + SEM; 2 h pi). (J–N) Using the long-term tamoxifen protocol (28 dpi) and immunohistochemical analysis with antibodies against GFP (green), DCX (white) and NeuN (red) we assessed the generation of neuronal progeny from recombined NSCs in the SEZ (L), the RMS (M) and the OB (N). Data in panels (J,K) represents mean + SEM. Arrows point toward GFP⁺ cells co-expressing either DCX (L,M) or NeuN (N). Asterisk in panel (N) highlights a recombined astrocyte with protoplasmic morphology in the OB (DAPI is depicted in blue, scale bar = 20 μm).