FGFR-1 and FGFR-2 Were Expressed in the Fmbryonic Gonad and Were Readily Detectable in Highly Purified PGC RNA
To further explore whether bFGF acts directly on PGCs, we sought to determine whether PGCs express an FGF receptor. Four murine high-affinity tyrosine kinase receptor genes for FGFs have been identified to date. To investigate whether PGCs might express one of these receptors, 11.5 dpc genital ridge RNA was tested for expression for all four genes by RNase protection assays. FGFR-1 and FGFR-2 RNA were readily detected in the genital ridge RNA (Fig. 2). buy yasmin online
We used a degenerate oligonucleotide RT-PCR approach to determine whether FGF receptors were encoded in highly purified (more than 90% free of somatic cells) 11.5 dpc PGC RNA. In this method, degenerate primers recognizing conserved motifs in the kinase domains of all four FGF receptors are used to amplify a 341-bp band from all FGFR transcripts. The resulting PCR fragment is then digested with restriction enzymes diagnostic for each FGFR. FGFR sequences were readily detected in 11.5 dpc PGC RNA as well as in 11.5 dpc whole embryo, which served as a positive control. No amplification was detectable in control reactions lacking reverse transcriptase (Fig. 3A). PvuII digestion of the PGC band readily revealed 195/146-bp and 233/108-bp bands indicative of FGFR-1 and FGFR-2, respectively. The 175/166-bp bands indicative of FGFR-3 were not readily apparent following Psd digestion; 216/ 125-bp £coRI bands, indicative of FGFR-4, were faintly detectable (Fig. 3B). Thus, FGFR-1, FGFR-2, or potentially FGFR-4 may be expressed by proliferative PGCs.
FIG. 2. RNase protection analysis for FGF receptors. Five micrograms of 11.5 dpc genital ridge RNA was analyzed for the presence of each of the four known FGF receptor gene RNAs. A tRNA negative control was included in each assay. Each assay included an internal actin control for RNA quality and loading. The actin controls for each RNase protection assay are shown. The positive control RNAs are as follows: FGFR-1, NIH3T3 cells; FGFR-2, BM-wt cells; FGFR-3, adult brain; FGFR-4, adult liver.
FIG. 3. Degenerate oligonucleotide RT-PCR analysis of FGF receptor expression. A) FGF receptor sequences were amplified from RNA obtained from highly purified (> 90%) 11.5 dpc PGCs, or from whole 11.5 dpc embryos (WE) using degenerate primers that recognize conserved sequences in the kinase domains of all four FGF receptors. A 341-bp band is expected for all four receptors. The 396- and 344-bp molecular size markers are indicated. M, size marker lane; +/— indicate presence or absence of reverse transcriptase in the cDNA preparation step. B) The PGC band was purified and digested with PvuII (Pv), PstI (Ps), FcoRI (Ec), or was left uncut (Un). The sizes of expected fragments for each FGFR are FGFR-1, 195/146 in the PvuII digest; FGFR-2, 233/1 08 in the PvuII digest; FGFR-3, 1 75/1 66 in the Pst\ digest; FGFR4, 216/125 in the FcoRI digest.