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, 7 (22), 1688-94

Changes in Expression and Secretion Patterns of Fibroblast Growth Factor 8 and Sonic Hedgehog Signaling Pathway Molecules During Murine Neural Stem/Progenitor Cell Differentiation in Vitro

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Changes in Expression and Secretion Patterns of Fibroblast Growth Factor 8 and Sonic Hedgehog Signaling Pathway Molecules During Murine Neural Stem/Progenitor Cell Differentiation in Vitro

Jiang Lu et al. Neural Regen Res.

Abstract

In the present study, we investigated the dynamic expression of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway related factors in the process of in vitro hippocampal neural stem/progenitor cell differentiation from embryonic Sprague-Dawley rats or embryonic Kunming species mice, using fluorescent quantitative reverse transcription-PCR and western blot analyses. Results demonstrated that the dynamic expression of fibroblast growth factor 8 was similar to fibroblast growth factor receptor 1 expression but not to other fibroblast growth factor receptors. Enzyme-linked immunosorbent assay demonstrated that fibroblast growth factor 8 and Sonic Hedgehog signaling pathway protein factors were secreted by neural cells into the intercellular niche. Our experimental findings indicate that fibroblast growth factor 8 and Sonic Hedgehog expression may be related to the differentiation of neural stem/progenitor cells.

Keywords: Sonic Hedgehog; differentiation; dynamic; fibroblast growth factor 8; neural progenitor cells; neural regeneration; neural stem cells; neurons; secretion; signal pathway.

Conflict of interest statement

Conflicts of interest: None declared.

Figures

Figure 1
Figure 1
Dynamic expression of FGF8, FGFRs and Sonic Hedgehog signaling pathway molecule mRNA during neural stem/progenitor cells differentiation in vitro was measured on day, 10 and 20 by reverse transcription-PCR. aP < 0.05, vs. expression of the previous differentiation stage. The results were expressed as absorbance ratio of mRNA expression on day 10 or 20 to that of the neural stem/progenitor cell stage, which was assigned a value of 1 (mean ± SD, n = 4). FGF: Fibroblast growth factor; FGFR: fibroblast growth factor receptor; NSCs/NPCs: neural stem/progenitor cells.
Figure 2
Figure 2
Dynamic expression of FGF8, FGFR3 and Sonic Hedgehog signaling pathway molecule protein levels during neural stem/progenitor cell differentiation in vitro. aP < 0.05, vs. expression level of the previous differentiation stage (Duncan multiple comparison test). The western blot data are expressed as the absorbance ratio of target protein to GAPDH on day 10 or 20 to that of the neural stem/progenitor cell stage (mean ± SD, n = 4). FGF: Fibroblast growth factor; FGFR: fibroblast growth factor receptors; NSCs: neural stem cells.
Figure 3
Figure 3
Dynamic secretion of fibroblast growth factor 8 (FGF8) and Sonic Hedgehog (SHH) proteins during neural stem/progenitor cell differentiation in vitro by enzyme-linked immunosorbent assay. Tissue culture supernatant was used for enzyme-linked immunosorbent assay of FGF8 or SHH proteins everyday between day 1 and 22 (n = 4). Data are expressed as mean ± SD.
Figure 4
Figure 4
Immunofluorescence analysis of FGF8 expressions and distribution during neural stem/progenitor cell differentiation in vitro. (A–C) Immunofluorescence staining on days 3, 10 and 20 during NSC/NPC differentiation. Green (FITC): FGF8+; red (TRITC): Nestin+; multicolor: merged. Scale bars: A, 100 μm; B, 50 μm; C, 200 μm. (D) Relative intensity of FGF8+ (green) and Nestin+ (red) fluorescence signals, and the signal ratios of FGF8 to Nestin (green/red) on days 3, 10 or 20 during NSC/NPC differentiation (mean ± SD, n = 4). Nestin+ fluorescence signal intensity was used as a control. The ratio of FGF8+ fluorescence intensity to nestin+ fluorescence intensity reflected the relative expression level of FGF8 at each stage. aP < 0.05, vs. that of the prior differentiation stage (Duncan multiple comparison test). FGF8: Fibroblast growth factor 8; NSC/NPC: neural stem/progenitor cell.
Figure 5
Figure 5
The time course of neural stem/progenitor cell differentiation in vitro (Leica microscope or confocal microscope; scale bar, A–E: 100 μm; F: 20 μm). (A) Neural stem/progenitor cell. (B–E) Cultured neural stem/progenitor cells were observed by microscopy on days 3, 7, 10, and 20. (F) Hochest33258 staining was performed to observe apoptotic nuclei on day 20 after differentiation. Arrowheads indicate glial cell nuclei; thin arrows indicate non-glial cell nuclei.

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