Identification of molecular markers distinguishing adult neural stem cells in the subventricular and subcallosal zones

Abstract

Neural stem cells (NSCs) in the adult subventricular zone (SVZ) are regionally specified and have distinct molecular gene expression signatures. Recently, we identified the subcallosal zone (SCZ) as a novel brain region where adult NSCs maintain and spontaneously produce neuroblasts. In an attempt to isolate genes specifically expressed in the SCZ or SVZ, microarray analyses of their differentially expressing transcripts were done. The comparison between neurospheres generated from SVZ and SCZ revealed differential expression >1.5-fold in two groups in only 83 genes, representing <0.03% of the genes examined, suggesting that these two populations are largely similar. The differential expression patterns SCZ and SVZ genes were confirmed by RT-PCR and Western blots. The selective expressions of two genes (CRBP1, HMGA1) in SVZ-NSCs were further confirmed by immunohistochemistry. These molecular markers could be useful for further molecular and cellular characterization of NSCs.

Keywords: CRBP1; HMGA1; Subventricular zone; ZIC2; adult neural stem cell; microarray; subcallosal zone.

Figure 1.

Microarray analysis of neurospheres from the SCZ and SVZ. (A) Experimental scheme for microarray analysis. Among 28,000 genes from the array chip, 83 displayed changes ≥1.5 fold. Hierarchical entry tree is shown at the bottom left. (B) Adult brain SCZ and SVZ were cultured in vitro as neurospheres. (C) Passage 2–5 neurospheres were gathered and used for microarray analysis. (D) A Volcano plot. The red dot denotes genes with 1.5-fold changes. (E) The list of SVZ-enriched or SCZ-enriched genes. The red line denotes 1.5-fold changes. Fifty-five and 28 genes were selected as SVZ- and SCZ-enriched genes, respectively.

Figure 2.

Confirmation of microarray analysis. (A) Confirmation of gene expression according to microarray data using RT-PCR. SVZ- or SCZ-enriched genes were selected randomly, and bar graphs show the ratio of mRNA expression difference between two aNSCs populations upon normalization with GAPDH (N = 4). (B) Gene candidates were selected and confirmed by Western blot (N = 2), and representative images were shown. (C) CRBP1 and (D) HMGA1 proteins were immunostained with Nestin, a marker for NSCs, in the SVZ and SCZ, respectively. Magnified image of the box region is shown as an inset. Hoechest33343 was used as a nuclear marker.

Figure 3.

Expression profile of CBRP1 and HMGA1 in the SVZ. (A,B) CRBP1 and HMGA1 proteins were stained with Nestin (marker for type B cell), Mash1 (marker for type C cell), and DCX (marker for type A cell). (C) Quantification of the percentage of type B, C, and A cells with CRBP1 or HMGA1 in the SVZ. Statistical significance of differences between the groups was evaluated by independent sample t tests. All the analyses were carried out with SPSS software, and all values are given as mean ± SEM. A p-value <0.05 was considered statistically significant. (D,E) Representative images of CRBP1 + GFAP+ and HMGA1 + GFAP+ cells. Hoechest33343 was used as a nuclear marker. Inset images were shown from magnified cells.