EGF-induced expansion of migratory cells in the rostral migratory stream

Abstract

The presence of neural stem cells in the adult brain is currently widely accepted and efforts are made to harness the regenerative potential of these cells. The dentate gyrus of the hippocampal formation, and the subventricular zone (SVZ) of the anterior lateral ventricles, are considered the main loci of adult neurogenesis. The rostral migratory stream (RMS) is the structure funneling SVZ progenitor cells through the forebrain to their final destination in the olfactory bulb. Moreover, extensive proliferation occurs in the RMS. Some evidence suggest the presence of stem cells in the RMS, but these cells are few and possibly of limited differentiation potential. We have recently demonstrated the specific expression of the cytoskeleton linker protein radixin in neuroblasts in the RMS and in oligodendrocyte progenitors throughout the brain. These cell populations are greatly altered after intracerebroventricular infusion of epidermal growth factor (EGF). In the current study we investigate the effect of EGF infusion on the rat RMS. We describe a specific increase of radixin(+)/Olig2(+) cells in the RMS. Negative for NG2 and CNPase, these radixin(+)/Olig2(+) cells are distinct from typical oligodendrocyte progenitors. The expanded Olig2(+) population responds rapidly to EGF and proliferates after only 24 hours along the entire RMS, suggesting local activation by EGF throughout the RMS rather than migration from the SVZ. In addition, the radixin(+)/Olig2(+) progenitors assemble in chains in vivo and migrate in chains in explant cultures, suggesting that they possess migratory properties within the RMS. In summary, these results provide insight into the adaptive capacity of the RMS and point to an additional stem cell source for future brain repair strategies.

Figure 1. Cell density and size of the rostral migratory stream after EGF infusion.

To assess EGF-induced structural changes in the rostral migratory stream (RMS) cell density and cross-sectional area were quantified at a proximal and a distal position relative to the SVZ. (A, C) Cell density was determined by counting cell nuclei and expressed as the number of cells per mm² ± SEM. (B, D) The size of the RMS was determined by tracing the border of the RMS on coronal sections. Data are presented as average cross-sectional area per section expressed in mm² ±SEM. For both analyses 1–4 coronal sections per animal were used. Statistical significance was assumed at * p<0.05 and ** p<0.01 using Student's t-test.

Figure 2. mRNA expression of doublecortin and radixin in the SVZ and olfactory bulb after EGF infusion.

mRNA expression of the neuroblast-associated marker doublecortin (DCX) and radixin in the subventricular zone (SVZ) and olfactory bulb (OB) after 14 days of EGF infusion were determined by qPCR. Relative mRNA levels of DCX (A, B) and radixin (C, D) are expressed as fold-change compared to control ± SEM using the Delta-Delta Ct method. Statistical significance was assumed at * p<0.05, ** p<0.01 and *** p<0.001 using Student's t-test. n = 5 animals for the aCSF and the EGF group.

Figure 3. Radixin and doublecortin protein expression in the rostral migratory stream.

Immunofluorescence staining illustrating radixin (Rdx) and doublecortin (DCX) expression in coronal sections of the rostral migratory stream (RMS) after 7 days of aCSF or EGF infusion. (A, B) Color separation for radixin, (C, D) color separation for DCX, (E, F) color composite images of radixin (green), DCX (red) and the nuclear counterstain ToPro3 (blue). The high degree of DCX/radixin colabeling in controls (yellow labeling in E) is notably reduced in EGF infused animals. Scale bar in A = 50 µm.

Figure 4. High magnification images of radixin⁺ cells.

Immunofluorescence staining of cells coexpressing radixin(Rdx) and doublecortin(DCX) (A) or radixin and Olig2 (B) under control conditions. In (A) Radixin(green), DCX(red), and nuclear counterstain YoPro1(blue). In (B) Radixin(green), Olig2(red), and YoPro1(blue). Scale bar in A and B = 10 µm.

Figure 5. Olig2 and doublecortin expression in radixin⁺ cells.

Graphs visualizing the percentage of cells (±SEM) expressing doublecortin (DCX) or Olig2 in radixin⁺ cells within the rostral migratory stream (RMS) (A, C) after 7 days and (B, D) after 14 days of EGF infusion. Statistical significance was assumed at ** p<0.01 and *** p<0.001 using Student's t-test.

Figure 6. Radixin and Olig2 expression in the rostral migratory stream.

Immunofluorescence staining illustrating radixin (Rdx) and Olig2 expression in coronal sections of the rostral migratory stream (RMS) after 7 days of aCSF or EGF infusion. (A, B) color separation for radixin, (C, D) color separation for Olig2, (E, F) color composite images of radixin (green), Olig2 (red) and the nuclear counterstain ToPro3 (blue). A notable increase in Olig2 positive cells is visible under EGF stimulation (D) compared to aCSF (C). The increased population of Olig2 positive cells expresses radixin (F). Scale bar in A = 50 µm.

Figure 7. Phosphorylation of radixin in EGF-induced Olig2⁺ cells.

Radixin (Rdx) activation was determined using an antibody that recognizes the phosphorylated form of ERM (ezrin, radixin, and moesin) proteins (pERM). (A, D) In the control rostral migratory stream (RMS), coexpression of radixin (red) and pERM (green) was predominantly observed in Olig2-negative neuroblasts. In the EGF-treated RMS the majority of radixin⁺/pERM⁺ cells was also Olig2⁺ (blue) cells. (B, E) No pERM expression was found in the cortex of either control or EGF-treated animals. (C, F) In the striatum, EGF infusion induced the presence of pERM⁺/radixin⁺/Olig2⁺ cells, which was not observed under control conditions (C). Scale bar in A = 50 µm.

Figure 8. Expression of oligodendrocyte, astrocyte, and microglia markers in the rostral migratory stream.

(A, B) Immunofluorescence staining against CNPase and NG2 show expression of oligodendrocyte lineage markers in the aCSF and EGF-treated rostral migratory stream (RMS). NG2⁺/Olig2⁺ cells were occasionally found under both conditions (arrows in B). (C) The appearance of the GFAP expressing cells was more reactive, with less regular and thicker processes, in the EGF-treated RMS compared to control. (D) Microglia were visualized based on Iba1 immunoreactivity. No differences between control and EGF-treated RMS were found in cell distribution or shape. Scale bar in A = 50 µm and B–D = 20 µm.

Figure 9. Olig2/Sox2/radixin co-expression in the rostral migratory stream.

(A, B) Increased Olig2 immunoreactivity was observed after EGF infusion. (C, D) Olig2⁻/Sox2^high cells (red arrows) were found in both control and EGF-treated rostral migratory stream (RMS) (A, C, E) In the control RMS, Olig2 positive cells are few and either Sox2 negative (green arrows) or express Sox2^high (B, D, F) The expanded radixin⁺/Olig2⁺ population in the EGF-treated RMS express Sox2^high (yellow arrows). (E, F) Olig2⁺ cells negative for Sox2 were observed under both conditions (green arrows). Scale bar in A = 50 µm.

Figure 10. Proliferative response of Olig2⁺ cells in the rostral migratory stream to EGF infusion.

(A, B) Quantification of BrdU/Olig2 co-labeled cells reveals an increase in the fraction of Olig2⁺ cells among the newly generated cells after 1 day (A) and 7 days (B) of EGF infusion. (C) Schematic illustration of the proximal (p) and distal (d) rostral migratory stream (RMS) coordinates used for analysis. (D) Quantification of the proximal and distal part of the RMS reveals a rapid response of Olig2-positive cells, at both locations, to EGF after 1 day of infusion. (E, F) Percentage of radixin (Rdx) expressing cells in newly generated cells (BrdU⁺) is close to 100% after 1 day (E) and 7 days (F) of aCSF and EGF infusion. Data are presented as percent of BrdU⁺ cells ±SEM. Statistical significance was assumed at * p<0.05 and ** p<0.01 using Student's t-test (A, B) or Mann-Whitney U-test (E, F). (D) n(control): proximal = 4, distal = 3; n(EGF): proximal = 5, distal = 4.

Figure 11. Alignment of Olig2⁺ cells in chains in vivo and in vitro explants.

(A) Olig2⁺ cells in the control rostral migratory stream are few and separated (arrows). (B) Radixin (Rdx) and Olig2 co-expressing cells in the EGF-treated RMS express phosphorylated (ezrin, radixin, and moesin) (pERM) and are associated in chains (arrows). (C) Extensive radixin (red) and Olig2 (blue) immunoreactivity of migratory cells in explant cultures. (D) Tightly associated radixin⁺/Olig2⁺ cells in explant cultures resemble the appearance of in vivo migratory chains and also express pERM. Scale bar in A = 50 µm, C = 200 µm and D = 50 µm.