NG2-cells are not the cell of origin for murine neurofibromatosis-1 (Nf1) optic glioma

Abstract

Low-grade glial neoplasms (astrocytomas) represent one of the most common brain tumors in the pediatric population. These tumors frequently form in the optic pathway (optic pathway gliomas, OPGs), especially in children with the neurofibromatosis type 1 (NF1)-inherited tumor predisposition syndrome. To model these tumors in mice, we have previously developed several Nf1 genetically-engineered mouse strains that form optic gliomas. However, there are three distinct macroglial cell populations in the optic nerve (astrocytes, NG2+ (nerve/glial antigen 2) cells and oligodendrocytes). The presence of NG2+ cells in the optic nerve raises the intriguing possibility that these cells could be the tumor-initiating cells, as has been suggested for adult glioma. In this report, we used a combination of complementary in vitro and novel genetically-engineered mouse strains in vivo to determine whether NG2+ cells could give rise to Nf1 optic glioma. First, we show that Nf1 inactivation results in a cell-autonomous increase in glial fibrillary acidic protein+ (GFAP+), but not in NG2+, cell proliferation in vitro. Second, similar to the GFAP-Cre transgenic strain that drives Nf1 optic gliomagenesis, NG2-expressing cells also give rise to all three macroglial lineages in vivo. Third, in contrast to the GFAP-Cre strain, Nf1 gene inactivation in NG2+ cells is not sufficient for optic gliomagenesis in vivo. Collectively, these data demonstrate that NG2+ cells are not the cell of origin for mouse optic glioma, and support a model in which gliomagenesis requires Nf1 loss in specific neuroglial progenitors during embryogenesis.

Figure 1. Optic nerve astroglial cell populations in wild-type and Nf1 OPG mice

(A) Nf1^flox/mut; GFAP-Cre (OPG) mice develop optic nerve gliomas. Representative images of the optic nerves from 3-month old wild-type (WT) and OPG mice are shown. The arrow denotes an enlarged optic nerve and chiasm in one representative OPG mouse, not seen in WT mice. Histological comparison of cell type-specific markers demonstrates that ~30% of the cells are APC+, 11% of the cells are NG2+ and 7% of the cells are GFAP+. Increased numbers of GFAP-positive astroglial cells are found in the optic nerves of OPG mice compared to WT mice. Each error bar represents mean ± SEM. (B) Double-labeling of 3-month-old WT and OPG nerves shows that these three glial cell populations are distinct. APC/NG2 double-positive cells account for fewer than 5% of the cells in the optic nerve. Representative images are shown. Scale bar, 50μm. DAPI (blue) was used as a counterstain to identify all cells in the sections. (C) NG2 double-labeling experiments revealed that 68% and 11% of the NG2 cells are SMAα+ or PDGFRβ+, respectively (pericyte markers), whereas 26% and 54% of the NG2+ cells are Olig2+ and PDGFRα+, respectively (oligodendroglial lineage markers).

Figure 2. Neurofibromin loss results in increased NG2 and GFAP cell proliferation in vivo

(A) Proliferation was measured by Ki-67 labeling. Nf1 inactivation results in a three-fold increase in the percent of Ki-67 positive cells (labeling index) in the optic nerves of OPG mice compared to WT mice at 3 months of age. Representative images are shown. Arrows denote representative Ki-67-immunopositive cells. Scale bar, 100μm. (B) The percentages of GFAP/Ki-67, NG2/Ki-67 and APC/Ki-67 cells in the optic nerves of OPG mice at 3 months of age are shown. The images above each bar in the graph depict representative GFAP/Ki-67 double-positive, NG2/Ki-67 double-positive, or APC-positive/Ki-67-negative cells, respectively. Each error bar represents mean ± SEM. Asterisks denote statistically significant differences (*) p= 0.0482.

Figure 3. Neurofibromin loss results in increased optic nerve GFAP+ cell proliferation in vitro

(A) Double-labeling immunocytochemistry reveals that 30% of the cells in optic nerve glial cell cultures are GFAP+, while 70% of the cells are NG2+. There are no GFAP/NG2 double-positive cells. Following Nf1 inactivation by Ad5-Cre expression, loss of neurofibromin expression was observed (B) coincident with an increase in proliferating (BrdU+) cells (C). α-tubulin is used as an internal control for protein loading. (D) Following Nf1 inactivation by Ad5-Cre expression, only the GFAP+ population of cells exhibited increased proliferation. Representative BrdU+ proliferating cells (green) and DAPI+ nuclei (blue) are shown. The percentage of BrdU+/NG2+ and BrdU+/GFAP+ cells are represented as the mean ± SEM. Scale bar, 100μm. N. S. = not significant, (**) p= 0.0055, (*) p= 0.0129. (E) FISH revealed comparable levels of Nf1 mRNA expression (green particles; inset) in GFAP+ and NG2+ cells. The number of signals is the average of 10 counted cells per area and is displayed in the graph. Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar, 50μm.

Figure 4. Characterization of NG2+ optic nerve cells in vitro

(A) 64% of the NG2 cells are double-positive for SMAα and almost all are PDGFRβ+ (pericyte markers). None of the GFAP-positive cells are SMAα-positive or PDGFRβ-positive. In contrast, 40% and 67% of the NG2+ cells are A2B5+ or PDGFRα+, respectively (oligodendroglial lineage markers). (B) Following NG2 antibody immunopanning and replating, PN1 optic nerve NG2+ cells grown for 1, 3 and 7 days do not differentiate into either GFAP+ or O4+ cells in vitro. Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar, 100μm.

Figure 5. NG2-Cre cells give rise to NG2+, ALDH1L1+, and APC+ glial cell populations in vivo

Optic nerve sections from Rosa-GREEN × NG2-Cre mice reveal EGFP+/NG2+ cells, EGFP+/APC+ (oligodendrocytes) and EGFP+/ALDH1L1+ (astrocytes). Scale bar, 50μm. Representative images are shown with insets of immunopositive cells.

Figure 6. Nf1^flox/mut; NG2-Cre mice do not develop optic glioma

Representative images of the optic nerves from all three genotypes are shown. Nf1^flox/mut; NG2-Cre mice optic nerve volumes are slightly increased relative to control (NG2-Cre and Nf1^flox/wt; NG2-Cre) mice. However, no increases in the Ki67 labeling index, percent of Iba1+ microglia, or percent of GFAP+ or NG2+ cells were found in Nf1^flox/mut; NG2-Cre mice relative to control mice. Scale bar, 100μm; 50μm NG2 panel. Error bars represent mean ± SEM. Asterisks denote statistically significant differences (*) p= 0.0119, (**) p< 0.0052.

Figure 7. Nf1 inactivation is observed in Nf1^flox/mut; NG2-Cre mice

(A) Recombination PCR demonstrates Cre-mediated Nf1 gene recombination (R) in both Nf1^flox/mut; GFAP-Cre mice and Nf1^flox/mut; NG2-Cre mice. (B) The efficiency of Cre-mediated recombination was determined following intercrossing of GFAP-Cre and NG2-Cre with Rosa-GREEN reporter mice. The percent of EGFP+ cells relative to the total number of cells in Rosa-GREEN × GFAP-Cre mice (13%) is similar to that observed in Rosa-GREEN × NG2-Cre mice (20%). (C) FISH analysis reveals similar reductions in Nf1 mRNA (red) expression in the optic nerves of Nf1^flox/mut; GFAP-Cre mice (67%) and Nf1^flox/mut; NG2-Cre mice (42%) compared to WT mice. Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar, 50μm. (D) Nf1 deletion in Nf1^flox/mut; NG2-Cre mice was determined by GFAP/neurofibromin and NG2/neurofibromin double-labeling using Nf1^flox/flox mice as controls. Similar to Nf1^flox/mut; GFAP-Cre mice with optic glioma, Nf1^flox/mut; NG2-Cre mouse optic nerves exhibit a 30% reduction in the percentage of GFAP/neurofibromin double-positive cells. No change in the percent of NG2/neurofibromin double-positive was observed in Nf1^flox/mut; NG2-Cre mice or Nf1^flox/mut; GFAP-Cre mouse optic nerves. Each error bar represents mean ± SEM. Asterisks denote statistically significant differences (*) p= 0.044.

Figure 8. NG2-Cre is first expressed at E14.5 in cells in the third and lateral ventricles

(A) NG2-Cre- and (B) GFAP-Cre-mediated EGFP expression is first detected by embryonic day E14.5 in the lateral (lv-SVZ; dotted line) and third ventricle (TVZ; dotted line) of (A) Rosa-GREEN × NG2-Cre and (B) Rosa-GREEN × GFAP-Cre mice, respectively. Nestin/EGFP double-positive cells in Rosa-GREEN × NG2-Cre mice are randomly scattered in the areas around the lv-SVZ and TVZ, whereas nestin/EGFP double-positive cells in Rosa-GREEN × GFAP-Cre mice are preferentially localized to the TVZ, but not to the lv-SVZ, periventricular region. Representative images are shown with insets of immunopositive cells. Scale bar, 100μm. EB = eye ball, ON = optic nerve.