Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

Abstract

The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor, fibroblast growth factor receptor-3, in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro. Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo. Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence. Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro. Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain, namely the substantia nigra, compact part, dorsal tier, substantia nigra and reticular part, but was not detected in the forebrain comprising the caudate putamen and striatum. Unusual results were obtained in retrosplenial locations of adult rat brain. We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses. We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8, a secretory factor. Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells. In contrast, addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons. Our study may help delineate the important roles of fibroblast growth factor-8 in brain activities and neural stem/progenitor cell differentiation.

Keywords: dopaminergic neurons; fibroblast growth factor receptor-3; fibroblast growth factor-8; midbrain; neural regeneration; neural stem/progenitor cell differentiation.

Figure 1

Distribution pattern of fibroblast growth factor-8 (FGF-8) in adult rat brain, as detected by immunohistochemistry. Observed under a Leica DMIRE2 inverted microscope. Scale bars, A-i, B-i, C-i: 50 μm. n = 3. The localization of fibroblast growth factor-8 in the brain is shown in A-ii, B-ii, C-ii and D. (A) Negative (−); (B) positive (+); C: Unusual (±). Positive staining of fibroblast growth factor-8 on the plasma membrane and in the cytoplasm is indicated by black and red arrows, respectively.

Figure 2

Immunofluorescence analyses of fibroblast growth factor-8 (FGF-8) and fibroblast growth factor receptor-3 (FGFR-3) localization in cells during neural stem/progenitor cells (NSCs/NPCs) differentiation (confocal microscope). (A–D) FGF-8⁺ (green, FITC); (E-H) FGFR-3⁺ (green, FITC). Green: FITC, FGF-8⁺ or FGFR-3⁺; red: TRITC, nestin⁺; blue: DAPI, nuclei; multicolor: merged. (C, D, G, H) Triangle arrows indicate positive staining in the cytoplasm in permeable treatment groups. Thin arrows indicate negative staining. (B, F) Large arrows indicate positive staining on the plasma membranes of differentiated cells in NSCs/NPCs in non-permeable treatment groups. Scale bars: (A, E) 50 μm; (B, C, F, G) 100 μm; (D, H) 20 μm. n = 4. Non-PTG: non-permeable treatment group; PTG: permeable treatment group. FITC: Fluorescein isothiocyanate; TRITC: tetraethylrhodamine isothiocyanate.

Figure 3

Effects of an anti-fibroblast growth factor-8 (FGF-8) antibody on neural stem/progenitor cell (NSC/NPC) differentiation and expansion. (A, B) i–iii: differentiation statuses in the 3^rd, 10^th and 20^th day stages by light microscope observation; iv–vi: the corresponding detection results of percentages of nestin⁺ cells vs. total. Observed under a Leica inverted microscope and evaluated by cells positive for nestin by immunofluorescence and flow cytometric analyses. (A) Treatment group: the effects of adding anti-FGF-8 antibodies to the culture medium at 10 and 20 days during NSC/NPC differentiation stages. (B) Control group: the results of normal NSC/NPC differentiation under differentiation conditions. Scale bars: 100 μm.

Figure 4

Promotion of neural stem/progenitor cell differentiation into neurons including dopaminergic neurons by addition of fibroblast growth factor-8 (FGF-8) (confocal microscope). (A) Blue (DAPI): nuclei; green (FITC): TH⁺; red (TRITC): NSE⁺; multicolor: merged. Scale bars: 200 μm. (B) An annexin-V FITC & propidium iodide (PI) apoptosis assay at 20 days of differentiation showed the count of annexin-V-positive apoptotic cells, and the count of PI-stained necrotic cells. TH: tyrosine hydroxylase; NSE: neuron specific enolase; SR: serum replacement.