The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

<div><p>Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.</p> </div

Transgenic markers are expressed by specific NSC niche cell types.

<p>(A) A schematic of the adult mouse forebrain in the coronal plane and cellular components of the neural stem cell (NSC) niche. Ependymal cells in <i>FoxJ1-Cre;R26^YFP/+</i> mice express YFP. NSCs, transit-amplifying progenitors (TAPs), and astrocytes in <i>Gli1^CreER/+;hGFAP-GFP;R26^tdTomato/+</i> mice express GFP/tdTomato, only tdTomato, and only GFP, respectively. Vascular endothelial cells in <i>Tie2-GFP</i> mice express GFP. LV: lateral ventricle, SVZ: subventricular zone. (B) A schema represents the induction of transgene in <i>Gli1^CreER/+;hGFAP-GFP;R26^tdTomato/+</i> mice by tamoxifen treatment. Three weeks after the treatment, SVZs were dissected, dissociated, and subjected to FACS or analyzed for the <i>in vivo</i> transgene expression. <i>FoxJ1-Cre;R26^YFP/+</i> or <i>Tie2-GFP</i> mice were also used for FACS or the <i>in vivo</i> transgene expression analysis. (C and D) A coronal section of the SVZ of <i>Gli1^CreER/+;hGFAP-GFP;R26^tdTomato/+</i> mouse. <i>GFP</i> expressing cells (green) are localized in the SVZ as well as in the striatum (St). <i>Gli1</i> lineage cells (tdTomato+, red) are located predominantly in the SVZ. Dashed line indicates the border between the lumen of the LV and the SVZ. (C) Immunofluorescent staining for GFAP (magenta) labels NSCs (GFP+/tdTomato+, arrow) and astrocytes (GFP+/tdTomato−, arrow head) in the striatum. TAPs (GFP−/tdTomato+, open arrow head) are not stained by GFAP. (D) Immunofluorescent staining for Sox2 (magenta) labels NSCs (GFP+/tdTomato+, arrow) and TAPs (GFP−/tdTomato+, open arrow head). (E) A coronal section of the SVZ of <i>FoxJ1-Cre;R26^YFP/+</i> mouse. YFP expressing <i>FoxJ1</i> lineage cells were stained by anti-GFP antibody to enhance fluorescence signal (green). S100β (red) labels GFP+ ependymal cells in the ventricular wall of the LV. (F) A coronal section of the SVZ of <i>Tie2-GFP</i> mouse. CD31 (red) labels GFP+ endothelial cells (green) in the SVZ. Scale bars: 20 µm (C,F), 10 µm (D,E). Nuclei were counterstained with Hoechst 33258 (blue).</p

Secretory molecule expression profiles (SMEP) of niche cells from the adult mouse SVZ.

<p>Secretory molecule expression profiles (SMEP) of niche cells from the adult mouse SVZ.</p

The self-renewal capacity and multipotency of GFP+/tdTomato+ cells.

<p>(A) FACS isolated GFP+/tdTomato+ cells from <i>Gli1^CreER/+;hGFAP-GFP;R26^tdTomato/+</i> mice formed neurospheres and express NSC markers Sox2 and Nestin. (B) Neurospheres or neurosphere-derived dissociated cells (insets) cultured in the differentiation medium differentiated into neurons (TuJ1+), astrocytes (GFAP+) and oligodendrocytes (O4+, inset: CNPase+). Immunofluorescent staining results were pseudocolored (green) and nuclei were stained with Hoechst 33258 (blue).</p

Confirmation of genes in SMEP by qRT-PCR.

<p>A heat map represents the expression levels for 29 selected genes using qRT-PCR from 151 SST-REX identified genes and 81 potential NSC SMEP by microarray. Each colored grid in the heat map represents the relative abundance of the transcript compared to the <i>Gapdh</i> level. Each gene was identified by either single or multiple niche cell type SMEP (parentheses on the left column). N: NSCs, T: TAPs, A: astrocytes, Ep: ependymal cells, C: choroid plexus, En: endothelial cells. * indicates the SMEP from microarray experiment.</p

Functional validation of identified molecules <i>in vitro</i> neurosphere culture.

<p>(A) The number of neurospheres. In the presence of Ttr and CPE, fewer neurospheres formed, while Enpp2 and Sparcl1 treatment did not show significant differences (Ttr: p = 0.012, n = 4; CPE: p<0.0001, n = 3). (B) The size of neurospheres determined by the neurosphere diameter. In the presence of Ttr and CPE, smaller neurospheres were formed, while Enpp2 and Sparcl1 treatment did not show significant differences (Ttr: p = 0.0072, n = 4; CPE: p = 0.031, n = 3). (C) XTT cell proliferation assay. Ttr and CPE treatment resulted in less number of total cells in culture wells compared to the control, while Enpp2 and Sparcl1 treatment did not show significant differences (Ttr: p = 0.017, CPE: p = 0.020; n = 3). Each symbol represents the mean of triplicates from each independent experiment. Bars are the mean of 3 or 4 such independent experiments. Symbols with the same color in the graph are the data set of each independent experiment.</p

FACS isolation of SVZ niche cells.

<p>(A–D) FACS plots of dissociated SVZ cells from <i>Gli1^CreER/+;hGFAP-GFP;R26^tdTomato/+</i> mice. (A) Gate was set using wild-type mice as a negative control. (B) Gate setting for GFP using <i>hGFAP-GFP</i> mice. (C) Gate setting for tdTomato using <i>Nestin-Cre;R26^tdTomato/+</i> mice. (D) FACS plot of the isolation of GFP+/tdTomato+ cells (NSCs), GFP−/tdTomato+ cells (TAPs), and GFP+/tdTomato− cells (astrocytes) from <i>Gli1^CreER/+;hGFAP-GFP;R26^tdTomato/+</i> mice. (E-F) FACS plots of dissociated SVZ cells from <i>FoxJ1-Cre;R26^YFP/+</i> mice. (E) Gate setting for a negative control using wild-type mice. (F) FACS plot of the isolation of YFP+ ependymal cells. (G-H) FACS plots of dissociated SVZ cells from <i>Tie2-GFP</i> mice. (G) Gate setting for a negative control using wild-type mice. (H) FACS plot of the isolation of GFP+ endothelial cells. (I-N) Validation of FACS-Isolated NSC niche cells by immunofluorescent staining. Nuclei were stained with Hoechst 33258 (blue). (I) GFAP, Sox2, and Nestin label NSCs (GFP+/tdTomato+). (J) Mash1 and Sox2 label TAPs (GFP−/tdTomato+). (K) GFAP and S100β label astrocytes (GFP+/tdTomato−). (L) S100β and CD24 label ependymal cells (YFP+). (M) CD31 labels endothelial cells (GFP+). (N) Quantification of immunocytochemical validation. Data represent the ratio of total antibody marker expressing cells to transgenic marker expressing cells. The results are sum of at least 2 independent experiments.</p

Top enriched annotation terms for secretory molecule expression profile (SMEP) from subventricular zone niche cells.

<p>Top enriched annotation terms for secretory molecule expression profile (SMEP) from subventricular zone niche cells.</p