Journal: Developmental biology

Article Title: A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice

doi: 10.1016/j.ydbio.2018.06.017

Figure Lengend Snippet: A-C. Representative images showing 5 μm maximum intensity projections of confocal optical sections of wild-type, Fgf4−/−, Fgfr1−/− and Fgfr2−/− embryos fixed and immunostained at sequential stages of pre-implantation development (A, 32–64 cell stage; B, 64–90 cell stage; C, 90–120 cell stage). Venus signal from the Spry4 locus was detected by immunostaining using an anti-GFP antibody and an AlexaFluor® 488 coupled secondary antibody. All embryos were stained for NANOG and GATA6 to identify the Epi, PrE and double positive (DP) populations. Color-coding is indicated. Total cell number (c) for each individual embryo shown is indicated. ICM magnifications for each channel are shown in grayscale. D-F. Boxplots showing relative Venus levels in indicated ICM lineages of wild-type, Fgf4−/−, Fgfr1−/− and Fgfr2−/− embryos (x-axis) at sequential stages of pre-implantation development. (D, 32–64 cell stage; E, 64–90 cell stage; F, 90–120 cell stage). Scattered points represent measurements in individual nuclei, color-coded for identity as indicated. In all boxplots, top and bottom edges of boxes represent third and first quartiles, respectively (interquartile range, IQR). Middle lines mark the median. Whiskers extend to 1.5 * IQR. N indicates number of embryos analyzed for each group. Scale bars, 20 μm.

Article Snippet: For activation of the FGF/ERK pathway in pre-implantation embryos, 1 μg/ml of recombinant human FGF4 (rhFGF4, R&D Systems) and 1 μg/ml of Heparin Sulfate were diluted in culture medium (KSOM-AA) and embryos cultured as described above.

Techniques: Immunostaining, Staining

Journal: Developmental biology

Article Title: A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice

doi: 10.1016/j.ydbio.2018.06.017

Figure Lengend Snippet: A. Brightfield (right panels) and Venus-fluorescence (middle) in Spry4H2B-Venus ESCs grown in serum/LIF medium (upper panels) or serum/LIF supplemented with 1 μM PD0325901 for 3 days (lower panels). Scale bar, 25 μm. B. Schematic diagram depicting experimental approach to determine ERK responsiveness of the reporter. Cells were cultured for 3 days in serum/LIF conditions supplemented with 1 μM PD0325901 to downregulate reporter expression, followed by transfer into subsaturating PD03 concentrations for one day. C. Flow cytometry analysis of Venus expression in Spry4H2B-Venus ESCs treated as indicated in (B). Uppermost histogram is from the Spry4 wild-type parental cell line. D. Quantification of results from (C). Expression of Venus in cells maintained in serum/LIF medium was normalized to 1, data points show average +/− SD from four independent experiments. E. Schematic of experimental approach to determine FGF responsiveness of the reporter. Wild-type and Fgf4 mutant cells were cultured in 2i/LIF conditions for three days followed by transfer into N2B27 medium supplemented with various doses of FGF4 or FGF2 for one day, or no FGF for parental, wild-type and mutant controls. F. Flow cytometry analysis of Venus expression in Fgf4 wild-type (wt) and Fgf4−/− mutant Spry4H2B-Venus ESCs treated as indicated in (E). The level of Venus expression in Fgf4 mutant cells is comparable to wt parental ESCs without the reporter, indicating that reporter expression in Fgf4 wt cells is mainly triggered by ESC-secreted FGF4. N = 3, one representative experiment shown. G. Total ERK and ppERK levels in Fgf4 mutant cells treated as indicated in (E) determined by immunoblotting. N = 3, one representative experiment shown. H. Relationship between ERK phosphorylation and Venus expression in Fgf4−/− mutant ESCs treated with different doses of FGF4 and FGF2 compiled from data in panels F and G. Data normalized to ppERK and Venus levels in cells treated with the highest dose of FGF4 (shown as 1). Data points indicate average +/− SD from two (Fgf4 mutant, 0 ng/ml FGF) or three (all other data points) independent experiments.

Article Snippet: For activation of the FGF/ERK pathway in pre-implantation embryos, 1 μg/ml of recombinant human FGF4 (rhFGF4, R&D Systems) and 1 μg/ml of Heparin Sulfate were diluted in culture medium (KSOM-AA) and embryos cultured as described above.

Techniques: Fluorescence, Cell Culture, Expressing, Flow Cytometry, Mutagenesis, Western Blot, Phospho-proteomics

Journal: Developmental biology

Article Title: A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice

doi: 10.1016/j.ydbio.2018.06.017

Figure Lengend Snippet: A. Schematic diagram showing the treatment regime with either 1000 μg/ml of FGF4 (+ 1 μg/ml Heparin), FGFRi (1 μM AZD4547) or MEKi (1 μM PD0325901), all diluted in KSOM culture media. Results of 48 hour (h) treatments from 8-cell stage are shown in panels B,C; 20h treatments from mid blastocyst (with FGFRi or MEKi only) are shown in panels (D-G), as indicated. B. Representative images showing 5 μm projections of confocal optical sections of immunostained embryos cultured from the 8-cell stage, for 48h, in the conditions indicated (see Materials and methods). All embryos were stained for NANOG and GATA6 to identify the Epi, PrE and double positive (DP) populations, as described [52, 55, 56] and with anti-GFP and an AF®488 coupled secondary antibody to detect H2B-Venus. Color-coding is indicated. Total cell number (c) for each individual embryo displayed is indicated. ICM magnifications for each channel are shown in grayscale. C. Boxplots showing Venus levels (as detected by immunostaining using anti-GFP + AF®488) in the ICM of Spry4H2B-Venus/+ embryos treated in each of the conditions indicated. Scattered dots represent fluorescence values of individual ICM cells from Spry4+/+ (wt) and Spry4H2B-Venus/+ (het), as indicated. Solid and dashed lines indicate the background signal in ICM cells of wild-type embryos stained only with secondary antibody, as shown in Fig. 3E (solid line, median value; dashed lines, inter-quartile range (IQR)). N indicates number of Spry4H2B-Venus/+ embryos analyzed for each group. D. Representative images showing 5 μm projections of confocal optical sections of immunostained embryos cultured as shown in panel A. All embryos were stained for NANOG and GATA6 to identify the Epi, PrE and double positive (DP) populations, as described [52, 55, 56] and with anti-GFP and an AlexaFluor®488-coupled secondary antibody to detect H2B-Venus. Color-coding is indicated. Total cell number (c) for each individual embryo displayed is indicated. ICM magnifications for each channel are shown in grayscale. E. Boxplots showing quantitation of Venus levels (detected by immunostaining using anti-GFP and an AlexaFluor® 488 coupled secondary antibody) in each condition, in ICM cells as well as TE cells, in which Spry4H2B-Venus is not expressed and signal represents Venus baseline levels as an internal negative control, in fixed and stained embryos shown in D. Scattered points represent measurements in individual nuclei. F. Upper panel is a schematic representation of the experimental procedure. Spry4H2B-Venus/+ embryos were isolated at the mid blastocyst stage (>64 cells) and cultured ex vivo either in control conditions or in the presence of small molecule inhibitors (as described in panel A) while being imaged by confocal microscopy for approximately 20 hours of development. Lower panels show still images of the ICM of embryos from the time-lapse microscopy in the conditions indicated. For each condition, top panel shows Venus signal overlaid on brightfield, bottom panel shows Venus signal alone in grayscale. G. Boxplots showing quantitation of Venus signal over time (shown in 4h bins) for the treatments indicated. Venus signal for each group and time point is normalized against the average of the corresponding control. Signal was not corrected for loss of fluorescence in the z-axis. In all boxplots, top and bottom edges of boxes represent third and first quartiles, respectively (interquartile range, IQR). Middle lines mark the median. Whiskers extend to 1.5 * IQR. Open circles represent outliers (values beyond 1.5 * IQR). Scale bars, 20 μm.

Article Snippet: For activation of the FGF/ERK pathway in pre-implantation embryos, 1 μg/ml of recombinant human FGF4 (rhFGF4, R&D Systems) and 1 μg/ml of Heparin Sulfate were diluted in culture medium (KSOM-AA) and embryos cultured as described above.

Techniques: Cell Culture, Staining, Immunostaining, Fluorescence, Quantitation Assay, Negative Control, Isolation, Ex Vivo, Control, Confocal Microscopy, Time-lapse Microscopy

Journal: Developmental biology

Article Title: A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice

doi: 10.1016/j.ydbio.2018.06.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For activation of the FGF/ERK pathway in pre-implantation embryos, 1 μg/ml of recombinant human FGF4 (rhFGF4, R&D Systems) and 1 μg/ml of Heparin Sulfate were diluted in culture medium (KSOM-AA) and embryos cultured as described above.

Techniques: Virus, Recombinant, Software