Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation

Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation The transcription factor Prdm14 is a critical regulator of pluripotency and germline development. This study shows that Prdm14 represses DNA methylation and FGF signaling, thereby promoting both germ cell fate and naive pluripotency in ESC.

con over con over con over con over con over con over   Figure 4) (A-C) qPCR validation of transcript levels for genes associated with early differentiation (A), pluripotency (B) or FGF pathway components (C) that were found to be differentially expressed by microarray analysis, as shown in Figure 3. Data shown represent the mean of six biological replicates and is relative to Gapdh. Error bars represent the standard error of the mean; statistical significance was determined by a t-test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Mouse strains and embryo dissections:
For whole-mount immunofluorescence, Blimp1-GFP mice [1], maintained on a mixed CBA and C57BL/6 background, or Stella-GFP-BAC mice [2], maintained on a C57BL/6 genetic background were used. Prdm14 tm1e(EUCOMM)wtsi/+ mice (Prdm14 -/+ ) were maintained on a pure C57BL/6 background unless crossed to Stella-GFP-BAC mice to label PGCs by GFP for FACS sorting. Embryos were staged according to Downs and Davies [3] before somitogenesis, while somite numbers were counted thereafter. Embryos were dissected out of uterus and decidua in PBS under a Leica Wild 3M dissecting microscope (Leica Microsystems) and washed in PBS with 0.2% polyvinylpyrrolidone (Sigma-Aldrich) before further processing by dissociation or fixation. Images were acquired on a Zeiss SV11 dissecting microscope equipped with a colour digital camera (Jenoptik ProgRes C14) using Openlab image software (PerkinElmer Improvision).

Histological methods:
For tissue-nonspecific alkaline phosphatase (TNAP) staining, dissected gonads from E12.5 embryos were fixed in 4 % paraformaldehyde (PFA) for 2 hours at 4 °C, washed in PBS, incubated in 70 % ethanol for 1 hour at 4 °C and then washed again in water. The embryos were then transferred into staining solution (3 ml water, 0.5 ml of 5 mg/ml 1,5-naphthalenedisulfonate Fast Red, 0.5 ml of 1mg/ml sodium 1-naphthyl phosphate, 0.5 ml of 0.6 % magnesium chloride and 0.5 ml of sodium 5,5-diethylbarbiturate; all from Sigma-Aldrich). The staining was carried out at room temperature and monitored through a dissection microscope. The staining procedure was stopped when the stain was sufficiently developed (approximately after 10 to 20 minutes) by washing the gonads in water to avoid background stain. Stained gonads were cleared in 70 % glycerol overnight at room temperature and mounted onto object slides for imaging. were mounted with Vectashield and the slides stored at 4 °C until imaging. Images were acquired as described above as described in the Whole-mount immunostaining section.

Immunofluorescence
For immunofluorescence of ES cells, cells were grown on fibronectin-coated glass-coverslips.
Cells were washed twice in 1xPBS, fixed in 4% PFA in PBS for 10-15 minutes at room temperature, followed by three washes in 1xPBS. Cells were subsequently permeabilized in 0.1% Tween-20 in PBS for 10-15 minutes at room temperature before incubation/blocking in blocking buffer (5% FBS, 0.1% Tween-20 in PBS) for 1 hour at room temperature. Cells were then incubated with primary antibody (anti-KLF4 (1:167, goat IgG, AF3158, R&D Systems); anti-NANOG (1:167, rabbit IgG, Abcam); anti-GFP (1:500, rat IgG)) diluted in blocking buffer overnight at 4 °C in a humidified chamber in the dark. The following day, cells were washed three times in blocking buffer before incubation with secondary antibodies (Molecular Probes, Life Technologies) diluted 1:500 in blocking buffer for 1 hour at room temperature. Finally, cells were washed three times in 1x PBS at room temperature, before mounting coverslips in Vectashield plus DAPI on microscope slides. Slides were sealed with nailpolish and stored at 4 °C until imaging. Images were acquired on a Leica SP5 confocal microscope. Fluorescence levels were quantified using Python Software.

Colony formation assay
For colony formation assays, ES cells were plated at a dilution of 600 cells per well onto gelatine-coated 6-well culture dishes (Nunc), with 3 wells per genotype, in standard serum/LIF (GMEM/10%FBS/1000U LIF/ml) conditions. Culture media was replaced on day 1 and day 8 after seeding cells, with media containing 500U/ml of LIF. Colonies were stained on day 12 using an Alkaline Phosphatase Kit (Sigma-Aldrich) to assess colony composition and plating efficiencies.
For Southern blot genomic DNA extracted from ES cell clones G10 or H10 was digested overnight with AatI or AflII restriction enzymes, precipitated with ethanol, separated on 0.8 % agarose gels and transferred onto a Hybond-N + membrane (Amersham) by capillary transfer. For detection of the different Prdm14 alleles, a 32 αP dCTP-labelled probe was generated using the Prime-a-gene labelling kit (Promega) and purified with Probe Quant G-50 micro columns (Amersham). The sequences of the primers used to generate the southern probe were: forward: CACCATTCCAGTTCCTCTTTG and reverse: TGCAGGTATATTTGCCTCAAT. Following overnight-incubation with the probe, the membrane was washed and exposed to a screen for 2 to 3 days before scanning on a phosphoimager (Fujifilm).