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. 2021 May 26;10:e54894. doi: 10.7554/eLife.54894

Figure 3. Oep levels regulate Nodal ligand capture and signaling range.

(A–B) Oep overexpression increases sensitivity to Nodal ligands. (A) Upper panel: control transplant of GFP-marked Mvg1 sensor cells (yellow) to the margin of wild-type hosts. Nodal signaling activity was measured by α-pSmad2 immunostaining (magenta). In all panels, YSL boundaries are marked with dashed white curves, and sensor cells have been outlined in solid white in all α-pSmad2 panels. Lower panel: quantification of Nodal signaling in sensor (red) and host cells (blue) across replicate embryos. Sliding window averages are plotted as solid curves. Plot was derived from eight replicate embryos. (B) Upper panel: transplant of sensor cells from an Mvg1 donor injected with gfp and 110 pg oep mRNA at the one-cell stage to the margin of wild-type hosts. Sensor cells (yellow) exhibit enhanced Nodal signaling activity (magenta) compared to their host-derived neighbors. Lower panel; staining of host (blue) and sensor (red) cells was quantified as in (A). Plot was derived from nine replicate embryos. (C-D) Oep overexpression restricts Nodal spread. (C) Upper panel: sensor cell measurement of the Nodal gradient in MZsmad2 embryos. Mvg1 sensor cells were marked with GFP (yellow), and Nodal signaling activity was measured by α-pSmad2 immunostaining (magenta). Lower panel: quantification of Nodal signaling in sensor (red) and host cells (blue) was quantified as in (A). Plot was derived from nine replicate embryos. (D) Upper panel: Mvg1 sensor cell measurement of the Nodal signaling gradient in MZsmad2 hosts injected with 110 pg oep mRNA at the one-cell stage. Lower panel; gradients were quantified as in (A). Plot was derived from nine replicate embryos.

Figure 3—source data 1. In Figure 3A, sensor cell assay results were quantified by segmenting nuclei and classifying each nucleus as host- or donor-derived by GFP intensity.
The quantified fluorescence intensities are organized in this table. Each row corresponds to an individual nucleus. The following characteristics were quantified: average GFP pixel intensity (‘GFP’), average pixel intensity for α-pSmad2 staining (‘pSmad2Raw’), α-pSmad2 staining intensity normalized to background (‘pSmad2 Normalized’), average DAPI pixel intensity (‘DAPI’), distance from the embryonic margin in μm (‘marginDist’), GFP staining status (‘GFP_Flag’, 0 denotes a cell host nucleus, one denotes a sensor cell nucleus), and embryo replicate number (‘Embryo Number’). We note that normalized α-pSmad2 staining was used to generate the figure panel.
elife-54894-fig3-data1.xlsx (415.8KB, xlsx)
Figure 3—source data 2. In Figure 3B, sensor cell assay results were quantified by segmenting nuclei and classifying each nucleus as host- or donor-derived by GFP intensity.
The quantified fluorescence intensities are organized in this table. Each row corresponds to an individual nucleus. The following characteristics were quantified: average GFP pixel intensity (‘GFP’), average pixel intensity for α-pSmad2 staining (‘pSmad2Raw’), α-pSmad2 staining intensity normalized to background (‘pSmad2 Normalized’), average DAPI pixel intensity (‘DAPI’), distance from the embryonic margin in μm (‘marginDist’), GFP staining status (‘GFP_Flag’, 0 denotes a cell host nucleus, one denotes a sensor cell nucleus), and embryo replicate number (‘Embryo Number’). We note that normalized α-pSmad2 staining was used to generate the figure panel.
elife-54894-fig3-data2.xlsx (374.3KB, xlsx)
Figure 3—source data 3. In Figure 3C, sensor cell assay results were quantified by segmenting nuclei and classifying each nucleus as host- or donor-derived by GFP intensity.
The quantified fluorescence intensities are organized in this table. Each row corresponds to an individual nucleus. The following characteristics were quantified: average GFP pixel intensity (‘GFP’), average pixel intensity for α-pSmad2 staining (‘pSmad2Raw’), α-pSmad2 staining intensity normalized to background (‘pSmad2 Normalized’), average DAPI pixel intensity (‘DAPI’), distance from the embryonic margin in μm (‘marginDist’), GFP staining status (‘GFP_Flag’, 0 denotes a cell host nucleus, one denotes a sensor cell nucleus), and embryo replicate number (‘Embryo Number’). We note that normalized α-pSmad2 staining was used to generate the figure panel.
elife-54894-fig3-data3.xlsx (772.4KB, xlsx)
Figure 3—source data 4. In Figure 3D, sensor cell assay results were quantified by segmenting nuclei and classifying each nucleus as host- or donor-derived by GFP intensity.
The quantified fluorescence intensities are organized in this table. Each row corresponds to an individual nucleus. The following characteristics were quantified: average GFP pixel intensity (‘GFP’), average pixel intensity for α-pSmad2 staining (‘pSmad2Raw’), α-pSmad2 staining intensity normalized to background (‘pSmad2 Normalized’), average DAPI pixel intensity (‘DAPI’), distance from the embryonic margin in μm (‘marginDist’), GFP staining status (‘GFP_Flag’, 0 denotes a cell host nucleus, one denotes a sensor cell nucleus), and embryo replicate number (‘Embryo Number’). We note that normalized α-pSmad2 staining was used to generate the figure panel.
elife-54894-fig3-data4.xlsx (356.1KB, xlsx)

Figure 3.

Figure 3—figure supplement 1. Nodal ligand range is expanded in MZoep mutants.

Figure 3—figure supplement 1.

(A) Direct visualization of long-range spread of Nodal ligand was carried out using a modified sensor cell assay in host embryos producing Vg1-HaloTag and Squint in the YSL. Sensor cells from a wild-type donor embryo injected with 110 pg oep mRNA were transplanted to the animal pole of host embryos injected with vg1-halotag and squint mRNAs in the YSL. Sensor cells producing extra Oep were used to enhance the sensitivity of the assay, akin to previous morphotrap studies (Stapornwongkul et al., 2020; Almuedo-Castillo et al., 2018; Harmansa et al., 2017; Harmansa et al., 2015). The experiment compared wild-type hosts (left), MZoep hosts (middle), or MZoep hosts injected with 110 pg oep mRNA at the one-cell stage (right). HALO-tagged ligands were labeled by staining with Janelia Fluor 646 Halo ligand. Sensor cells were transplanted to the animal pole of labeled host embryos after 1 hr of staining (approximately sphere stage). Loss of oep results in increased accumulation of tagged ligand at the animal pole. (B) Quantification of ligand accumulation in sensor cells. Sensors were manually segmented and average Janelia Fluor 646 channel pixel intensities were calculated. Each point represents a single sensor cell. Violin plots depict the median (white circle) and 25–75 percentile ranges (grey line). Asterisks denote statistical significance of median comparisons between indicated samples (Wilcoxon rank-sum test).