FGF8-FGFR1 signaling regulates human GnRH neuron differentiation in a time- and dose-dependent manner

Abstract

Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time- and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10 days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25 ng/ml, 50 ng/ml or 100 ng/ml) for 10 days or by treatment with 100 ng/ml FGF8 for different durations (2, 4, 6 or 10 days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5 days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (P<0.05, Rs=0.49). However, cells treated with 100 ng/ml FGF8 for 2 days induced the expression of genes, such as FOXG1, ETV5 and SPRY2, and continued FGF8 treatment induced the dynamic expression of several other genes. Moreover, during exposure to FGF8, FGFR1 localized to the cell surface and its specific inhibition with the FGFR1 inhibitor PD166866 reduced expression of GNRH1 (P<0.05). In neurons, FGFR1 also localized to the nucleus. Our results suggest that dose- and time-dependent FGF8 signaling via FGFR1 is indispensable for human GnRH neuron ontogeny. This article has an associated First Person interview with the first author of the paper.

Keywords: FGF8; FGFR1; GnRH neuron; Transcriptome; hPSCs.

Fig. 1.

Schematic of the 25-day GnRH neuron differentiation protocol and dose–time-series experiments. (A,B) In both experimental set-ups, the differentiation protocol starts with 10 days of dual-SMAD inhibition. In set-up A, this was followed on day 11 by treatment with 25 ng/ml, 50 ng/ml or 100 ng/ml FGF8 for 10 days (conditions A, B or C, respectively). In set-up B, dual-SMAD inhibition was followed on day 11 by treatment with 100 ng/ml FGF8 for different durations, i.e. for 2, 4, 6 or 10 days (conditions D,E, F or G, respectively). After treatment with FGF8, cells were matured by addition of the Notch inhibitor DAPT for 5 days (Lund et al., 2016). Black asterisk, cell splitting; red asterisk, end of experiment.

Fig. 2.

FGF8 dose and time effect on GNRH1 expression. (A) Relative expression of GNRH1 at day 25 of the differentiation protocol in response to increasing FGF8 concentrations between day 11 and day 21 (see Fig. 1, conditions D-G in Fig. 1). One-way ANOVA indicated a **P≤0.01 between sample sets. Differentiation experiments were repeated three times (n=3). Error bars indicate +standard error of the mean (+s.e.m.). (B) Relationship between the duration of treatment with FGF8 (at 100 ng/ml) and the relative expression of GNRH1 at day 25 of the differentiation experiment (Spearman’s rank correlation coefficient R_S=0.49; P=0.01). Each dot represents the relative expression of GNRH1 from a single experiment (n=4, for 2 and 4 days of treatment with FGF8; n=9 for 6 and 10 days of treatment with FGF8.). Treatment with 100 ng/ml FGF8 for 2 days induced significant expression of GNRH1 (P<0.05). Horizontal bars indicate median values.

Fig. 3.

Cellular expression of FGFR1 during differentiation to GnRH neurons. (A) Localization of FGFR1 in an FGFR1-GFP reporter cell line on day 11 of the differentiation protocol. Green, FGFR1-GFP fluorescence; blue, NucBlue™ Live ReadyProbes ™ cell-permeant dye used to counterstain nuclei. Scale bars: 100 μm. (B) Immunostaining of FGFR1 (green) on day 17 (6 days after treatment with FGF8 at 100 ng/ml) of the differentiation experiment, indicating its localization to the cell membrane. Nuclei are shown in blue (DAPI). Scale bars: 20 μm. (C,D) Immunostaining for FGFR1 (red) on day 25 of the differentiation, showing its nuclear localization in neurons, including GnRH neurons (green) is shown C. The boxed area is shown magnified in D, demonstrating the nuclear localization of FGFR1 in a GNRH1-expressing neuron. Nuclei are shown in blue (DAPI). Scale bars: 10 μm (C), 2 μm (D).

Fig. 4.

Effect of FGFR1 inhibition on the relative gene expression of GNRH1. (A) Schematic of the differentiation protocol when cells were treated with 100 ng/ml FGF8 alone or with FGF8 and 10 µM of the FGFR1 inhibitor PD166866 (PD); black asterisk, cell splitting; red asterisk, end of differentiation. (B) Bar graphs show the relative levels of GNRH1 RNA obtained from cells at day 25 of the differentiation protocol, after treatment with FGF8 alone or FGF8+PD compared to undifferentiated hPS control cells. (n=4, *P<0.05; error bars indicate the +standard error of the mean (+s.e.m.).

Fig. 5.

RNA sequencing strategy and over-representation analysis. (A) Schematic illustrating the five bulk RNA-Seq time points; dual-SMAD inhibition and FGF8-treated (100 ng/ml) samples after 2, 4, 6 and 10 days, starting at day 11 of the differentiation protocol. Asterisk indicates cell splitting; red double arrow indicates all the RNA-Seq sample timepoints. (B) Results of over-representation analysis (ORA) of upregulated genes after 2 days of FGF8 (d13), represented as a bar chart showing the most-enriched KEGG pathways (red) and the most-depleted ones (blue) on the y-axis, and their respective P-values on the x-axis.

Fig. 6.

Early transcriptomic changes after 2 days of FGF8. (A) Heatmap of the top 50 differentially expressed genes after 2 days of treatment with 100 ng/ml FGF8 (d13) in comparison with dual-SMAD inhibition (d10). (B) Heatmap, showing the differential expression of FGF8 synexpression group genes upon treatment with FGF8. In contrast to their relatively low expression after dual-SMAD inhibition (d10), all four genes were substantially upregulated after treatment with 100 ng/ml FGF8 for 2 or 10 days (d13 or d21, respectively). Colour range: blue (low expression) to red (high expression), numbers indicate the expression scale.

Fig. 7.

Analysis of the FGF8–FGFR1 mechanistic network. (A) Venn diagram indicating the 461 differentially expressed genes (DEGs; encircled in red) on day 13 that belong to FGF8–FGFR1 mechanistic networks as identified by IPA. (B) Venn diagram indicating the DEGs (red circle) across all FGF8 treatment time points that belong to FGF8–FGFR1 mechanistic networks as identified by IPA.