Abstract
Fibroblast growth factor 8 (FGF8) is a potent morphogen that regulates the embryonic development of hypothalamic neuroendocrine cells. Indeed, using Fgf8 hypomorphic mice, we showed that reduced Fgf8 mRNA expression completely eliminated the presence of gonadotropin-releasing hormone (GnRH) neurons. These findings suggest that FGF8 signaling is required during the embryonic development of mouse GnRH neurons. Additionally, in situ hybridization studies showed that the embryonic primordial birth place of GnRH neurons, the olfactory placode, is highly enriched for Fgf8 mRNA expression. Taken together these data underscore the importance of FGF8 signaling for GnRH emergence. However, an important question remains unanswered: How is Fgf8 gene expression regulated in the developing embryonic mouse brain? One major candidate is the androgen receptor (AR), which has been shown to upregulate Fgf8 mRNA in 60–70% of newly diagnosed prostate cancers. Therefore, we hypothesized that ARs may be involved in the regulation of Fgf8 transcription in the developing mouse brain. To test this hypothesis, we used chromatin-immunoprecipitation (ChIP) assays to elucidate whether ARs interact with the 5′UTR region upstream of the translational start site of the Fgf8 gene in immortalized mouse GnRH neurons (GT1-7) and nasal explants. Our data showed that while AR interacts with the Fgf8 promoter region, this interaction was androgen-independent, and that androgen treatment did not affect Fgf8 mRNA levels, indicating that androgen signaling does not induce Fgf8 transcription. In contrast, inhibition of DNA methyltransferases (DNMT) significantly upregulated Fgf8 mRNA levels indicating that Fgf8 transcriptional activity may be dependent on DNA methylation status.
Highlights
Fibroblast growth factor 8 (FGF8) has been studied in the context of a potent morphogen that is required for establishing morphogenetic centers in the developing mammalian neural tube (Crossley and Martin, 1995; Meyers et al, 1998; Sun et al, 1999)
Using ICC, we confirmed the presence of gonadotropin-releasing hormone (GnRH) peptide in these cells (Figures 1A,B) using PCR, we verified that our GT1-7 neurons express significant levels of GnRH, androgen receptor (AR), and Fgfr1 and Fgf8 mRNA (Figure 1C)
Student t-tests showed that ChIP with an AR-specific antibody was able to pull-down more 5′UTR Fgf8 DNA sequences that contain androgen response element (ARE) 1, ARE 2, and ARE 3, in contrast to control IgG (p < 0.05; Figures 2A–C)
Summary
Fibroblast growth factor 8 (FGF8) has been studied in the context of a potent morphogen that is required for establishing morphogenetic centers in the developing mammalian neural tube (Crossley and Martin, 1995; Meyers et al, 1998; Sun et al, 1999). Additional studies showed that GnRH neurons were already missing in the E11.5 Fgf hypomorphic olfactory placode (OP) (Wray et al, 1989; Chung et al, 2008) from which the majority of GnRH neurons emerge (Schwanzel-Fukuda and Pfaff, 1989; Wray et al, 1989). Together these data clearly support the supposition that FGF8 function is required for vertebrate GnRH neuron development (Chung et al, 2008; Tsai et al, 2011)
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