Embryonic CD133-Positive Ependymal Cells Possessed the Characteristics of NSCs
The SVZ and ependymal layer of LV-wall tissue were isolated, micro-dissected and dissociated into a single ependymal cell from the forebrain of embryonic day 12 (E12) mouse. Our results found that a very large percentage of cells (94.63 ± 2.09%) were CD133-positive ependymal cells when these cells were immunoreactive with CD133/Hoechst, revealing that CD133 was highly expressed in these primarily cultured ependymal cells (Fig. 1a).
Having been plated on the uncoated surfaces for 2 ~ 3 days, most CD133-positive ependymal cells showed an oval or tadpole-like morphology with short processes. There was approximately 3 ~ 5 neuro-spheres on the adherent surfaces at a low (Fig. 1c) or high magnification of visual field (Fig. 1d) and these neuro-spheres were immunoreactive with Nestin, demonstrating that CD133-positive ependymal cells possessed the properties of NSCs (Fig. 1e).
With the prolonged time in vitro primary culture, the soma of ependymal cells became much larger in diameter, meanwhile, their processes gradually grew ramifications vigorously and formed the neural networks. Having been seeded into the differentiation medium without bFGF, CD133-positive ependymal cells-derived neuro-spheres ultimately generated the bipolar or multipolar cells on DIV 9 (Fig. 1b).
RBP-J Interference Promoted the Proliferation and Differentiation of CD133-Positive Ependymal Cells in Vitro
To determine the effect of RBP-J inactivation on the neurogenesis of CD133-positive ependymal cells, CD133-positive ependymal cells were administrated with RBP-J-siRNA on DIV 3 and then were co-labelled with DCX, β-tubulin III, MAP2 and PCNA on DIV 5, 7 and 10. Our studies showed that CD133 was mainly enriched in the plasma membrane of ependymal cells and their projections. Under a fluorescent microscope, a cluster morphology-like DCX (Fig. 2a), as well as β-tubulin III (Fig. 2c) and MAP2 (Fig. 2e) were expressed in the prominent cytoplasm and neurite extension while PCNA was mainly present in the obvious nucleus (Fig. 2g).
As compared with the control, Lipo2000 and Control-siRNA + lipo2000 groups, RBP-J interference induced by RBP-J-siRNA significantly upregulated the ratio of CD133/DCX- (Fig. 2b; F(3.000, 16.64) = 692.4, W(3.000, 10.90) = 440.7, p<0.0001), CD133/MAP2- (Fig. 2f; F(3.000, 17.88)=559.8, W(3.000, 10.97)=640.1, p<0.0001) and CD133/PCNA-double positive cells (Fig. 2h; F(3.000, 7.230)=344.1, W(3.000, 9.773)=1952, p<0.0001) (Fig. 2i; F(3.000, 15.35)=691.4, W(3.000, 10.74)=452.9, p<0.0001) (Fig. 2j; F(3.000, 17.38)=557.9, W(3.000, 10.90)=525.2, p<0.0001) relative to the total CD133-positive ependymal cells, as well as the number of DCX- (Fig. 2b; F(3.000, 14.50)=76.89, W(3.000, 8.802)=63.53, p<0.0001), MAP2- (Fig. 2f; F(3.000, 8.288)=9.721, p=0.0044; W(3.000, 9.060)=201.4, p<0.0001) and PCNA-positive cells (Fig. 2h; F(3.000, 14.45)=97.87, W(3.000, 9.331)=70.24, p<0.0001) (Fig. 2i; F(3.000, 11.25)=77.57, W(3.000, 10.20)=41.78, p<0.0001) (Fig. 2j; F(3.000, 12.99)=95.65, W(3.000, 10.17)=54.79, p<0.0001) (normality and variance homogeneity, Brown-Forsythe and Welch ANOVA tests), except for the ratio of CD133/β-tubulin III double-positive cells (Fig. 2d; F(3, 20)=2.683, p=0.0743) or the number of β-tubulin III -positive cells (Fig. 2d; F(3, 20)=0.3696, p=0.7757) (normality and variance homogeneity, ordinary one-way ANOVA test) on DIV 5, 7 and 10.
There was a significant decrease in CD133-positive ependymal cells of RBP-J-siRNA + lipo2000 groups (Fig. 2b; F(3.000, 10.36) = 14.40, p<0.001; W(3.000, 10.25)=53.47, p<0.0001) (Fig. 2f; F(3.000, 14.22)=4.237, p=0.0247; W(3.000, 8.480)=10.48, p=0.0032), as compared with the control, Lipo2000 and Control-siRNA+lipo2000 groups (normality and variance homogeneity, Brown-Forsythe and Welch ANOVA tests). However, no increase or decrease existed in the number of CD133-positive ependymal cells (Fig. 2d; F(3, 20)=2.951, p=0.0575) (Fig. 2h; F(3, 20)=1.509, p=0.2428) (Fig. 2i; F(3, 20) = 2.536, p = 0.0857) (Fig. 2j; F(3, 20)=0.7938, p = 0.5116) (normality and variance homogeneity, ordinary one-way ANOVA test).
RBP-J Was Expressed in CD133-Positive Ependymal Cells in Vivo
To determine whether RBP-J was detected in CD133-positive ependymal cells, ROSA26-LacZ reporter mice were crossed with both CD133-CreER™ and RBP-J+/+ or RBP-J−/− mice. Since β-Gal-positive cell was regarded as an indicator of CD133-positive ependymal cell and its downstream lineage, we performed an immunofluorescent staining for β-Gal. Our present results indicated that the immunoreactivity for β-Gal was exclusively localized in the SVZ or ependymal layer, as well as the vicinity of LV, 3V and Aq, which was in accordance with the previous studies (Henry et al. 2009; Xie et al. 2017).
In CD133-CreERTM::RBP-J−/−::ROSA26-LacZ mice, RBP-J was expressed in the nuclear fraction of β-Gal-stained CD133-positive ependymal cells (Fig. 3e) after the mice have treated with TAM intraperitoneally for 5 consecutive days, moreover, approximately 90.26% of β-Gal-positive cells were co-expressed with RBP-J, revealing that RBP-J was active in the vast majority of CD133-positive ependymal cells. In contrast to the littermate controls, RBP-J expression was almost absent among the recombined cells of RBP-J CKO mice (Fig. 3f), leaving less than 5% of β-Gal-positive cells were co-labelled with RBP-J. These results indicated that RBP-J knockout was validated in CD133-positive ependymal cells of RBP-J CKO mice.
RBP-J Knockout Promoted the Proliferation and Differentiation of β-Gal-Labelled CD133-Positive Ependymal Cells in Vivo
Since CD133-positive ependymal cells could be labelled with β-Gal, we examined β-Gal-positive cells with a variety of cell markers including PCNA, DCX or β-tubulin III and NeuN to determine the proliferation and differentiation of adult CD133-positive ependymal cells following a conditional ablation of RBP-J by a double immunofluorescent staining. The fluorescent microscope showed that β-Gal- (Fig. 4a, c, e and g; Fig. 5a ~ h), DCX- (Fig. 4a, 5a, 5b), β-tubulin III- (Fig. 4c, 5c, 5d) and PCNA-expressing cells (Fig. 4g, 5g, 5h) were prevalent in the regions surrounding LV-, 3V- and Aq-SVZ or ependymal layer. Interestingly, most NeuN-positive cells were seen to be away from the SVZ or ependymal layer into the adjacent parenchyma (Fig. 4e, 5e, 5f). The expression of β-Gal, DCX, β-tubulin III, NeuN and PCNA in vivo were in accordance with those of CD133, DCX, β-tubulin III, MAP2 and PCNA in vitro.
In the LV-, 3V- and Aq-SVZ or ependymal layer, our statistical results revealed that RBP-J ablation in CD133-positive ependymal cells resulted in a dramatic increase in the number of DCX- (Fig. 4b; t(9.522) = 54.31, p<0.0001) (Fig. 5a’; t(7.816)=7.019, p<0.001) (Fig. 5b’; t(6.782)=10.11, p<0.0001), β-tubulin III- (Fig. 4d; t(5.416)=14.45, p<0.0001) (Fig. 5c’; t(9.690)=5.696, p\(=\)0.0002) (Fig. 5d’; t(6.265)=29.22, p<0.0001), NeuN- (Fig. 4f; t(6.791)=5.230, p=0.0013) (Fig. 5e’; t(9.317)=14.14, p<0.0001) (Fig. 5f’; t(7.179)=8.935, p<0.0001) and PCNA-positive cells (Fig. 4h; t(6.085)=10.31, p<0.0001) (Fig. 5g’; t(6.214)=6.492, p<0.001) (Fig. 5h’; t(9.764)=3.933, p = 0.0029), as compared with the control group (unpaired t test with Welch’s correction).
In comparison with the control group, RBP-J CKO mice had a significant increase in the fraction of β-Gal/DCX- (Fig. 4b; t(6.261) = 18.41, p<0.0001) (Fig. 5a’; t(8.482)=15.92, p<0.0001) (Fig. 5b’; t(9.779)=25.85, p<0.0001), β-Gal/β-tubulin III- (Fig. 4d; t(5.606)=10.50, p<0.0001) (Fig. 5c’; t(6.351)=10.27, p<0.0001) (Fig. 5d’; t(6.565)=18.84, p<0.0001), β-Gal/NeuN- (Fig. 4f; t(9.282)=7.223, p<0.0001) (Fig. 5e’; t(5.061)=17.61, p<0.0001) (Fig. 5f’; t(7.682)=7.014, p<0.001) and β-Gal/PCNA-double positive cells (Fig. 4h; t(5.725)=20.65, p<0.0001) (Fig. 5g’; t(9.981) = 17.91, p<0.0001) (Fig. 5h’; t(5.584) = 13.34, p<0.0001) relative to β-Gal-positive cells (unpaired t test with Welch’s correction). Strikingly, our results showed that β-Gal-positive cells differentiation into the immature neurons (DCX and β-tubulin III) were much higher than the mature neurons (NeuN), further confirming that RBP-J deletion easily made β-Gal-positive cells differentiate into the immature rather than the mature neurons.
In addition, our results revealed that there was almost lack of an obvious alteration in the number of most β-Gal-positive cells of RBP-J CKO mice (Fig. 4b; t(10) = 1.380, p = 0.1976) (Fig. 4h; t(10) = 0.6654, p = 0.5208) (Fig. 5a’; t(10) = 0.3270, p = 0.7504) (Fig. 5b’; t(10) = 0.9825, p = 0.3490) (Fig. 5c’; t(10) = 0.4066, p = 0.6929) (Fig. 5e’; t(10) = 2.143, p = 0.0577) (Fig. 5g’; t(10) = 1.857, p = 0.0930) except for some increased (Fig. 4d; t(5.778) = 7.203, p<0.001) (Fig. 5d’; t(5.079)=5.882, p=0.0019) (Fig. 5f’; t(5.332)=12.19, p<0.0001) (Fig. 5h’; t(6.132) = 11.37, p<0.0001) or decreased β-Gal-positive cells (Fig. 4f; t(9.389)=4.336, p = 0.0017) (unpaired two-tailed t tests and unpaired t test with Welch’s correction), which was in coincidence with those findings in the primarily cultured CD133-positive ependymal cells.
RBP-J Interference or Knockout Regulated the RBP-J-, Notch1- and Hes1-mRNA Expressions in Vitro or in Vivo
Notch1 is an upstream component that binds RBP-J while Hes1 is a downstream target that represses the expression of pro-neural genes, thus we examined whether the expression levels of Notch1- and Hes1-mRNA were altered following RBP-J inactivation in vitro and in vivo using real-time qPCR. As compared with the control, Lipo2000 and Control-siRNA + lipo2000 groups in the primarily cultured CD133-positive ependymal cells, our results indicated that the relative expression level of RBP-J-mRNA was significantly decreased following RBP-J interference (Fig. 6a; F(3.000, 14.76) = 3507, W(3.000, 10.14) = 5032, p<0.0001) (normality and variance homogeneity, Brown-Forsythe and Welch ANOVA tests). RBP-J knockout in CD133-positive ependymal cells also significantly decreased RBP-J mRNA expression level in the LV-wall tissue to about 85.18~91.5% of the control group (Fig. 6g; t(9.644)=36.82, p<0.0001) (unpaired t test with Welch’s correction), indicating that TAM-induced Cre-expression resulted in an efficient recombination of RBP-J.
Meanwhile, the relative expression level in RBP-J-siRNA + lipo2000 group of primarily cultured CD133-positive ependymal cells or RBP-J CKO mice had a significant increase in Notch1-mRNA (Fig. 6b: F(3.000, 5.690) = 194.4, W(3.000, 10.64) = 61.01, p<0.0001; Fig. 6h: t(5.008)=13.77, p<0.0001) and decrease in Hes1-mRNA (Fig. 6c: F(3.000, 16.50) = 149.5, W(3.000, 10.59) = 117.4, p<0.0001); Fig. 6i: t(5.480)=48.17, p<0.0001) (Brown-Forsythe and Welch ANOVA tests and unpaired t test with Welch’s correction). These results demonstrated that RBP-J deletion could upregulate and downregulate the relative expression levels of Notch1- and Hes1-mRNA, respectively.
RBP-J Interference or Knockout Regulated the RBP-J, Notch1 and Hes1 Protein Expressions in Vitro or in Vivo
Additionally, we examined the expression levels of RBP-J, Notch1 and Hes1 proteins using Western blot in order to further confirm whether these protein expressions were in accordance with their mRNA levels. Consistent with RBP-J-mRNA, the decreased relative expression level of RBP-J protein was more evident following RBP-J interference, as compared to the control, Lipo2000 and Control-siRNA + lipo2000 groups in the primarily cultured CD133-positive ependymal cells (Fig. 6d; F(3.000, 18.22) = 2363, W(3.000, 10.83) = 1861, p<0.0001) (normality and variance homogeneity, Brown-Forsythe and Welch ANOVA tests). Furthermore, RBP-J knockout in CD133-positive ependymal cells abruptly decreased RBP-J protein expression level in the LV-wall tissue to about 69.03~71.71% of the control group (Fig. 6j; t(5.233)=46.66, p<0.0001) (unpaired t test with Welch’s correction).
Our results demonstrated that the relative expression levels following RBP-J siRNA or knockout had a significant increase in Notch1 protein (Fig. 6e: F(3.000, 16.81) = 489.7, W(3.000, 10.83) = 338.3, p<0.0001; Fig. 6k: t(6.493)=24.23, p<0.0001) and an obvious reduction in Hes1 protein (Fig. 6f: F(3.000, 13.14)=720.0, W(3.000, 9.911)=1715, p<0.0001; Fig. 6l: t(5.472)=22.84, p<0.0001), which was in accordance with their mRNA expression levels. Taken together, our results suggested that Notch1 and Hes1 were specifically upregulated and downregulated in the absence of RBP-J by the transcriptional and translational mechanisms, which might account for RBP-J deletion-induced the proliferation and differentiation of CD133-positive ependymal cells.