Fig. 4: Endogenously tagged Nanog exhibits different behavior compared to overexpressed Nanog. | Nature Communications

Fig. 4: Endogenously tagged Nanog exhibits different behavior compared to overexpressed Nanog.

From: Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo

Fig. 4

a Live imaging schematic: EGFP-NbALFA mRNA (75 pg) was injected into ALFA-nanogKI/KI embryos to visualize endogenous Nanog, or imaging was done directly from ALFA-nanogKI/KI; Tg(actb2:EGFP-NbALFA)/+ crosses. For exogenous Nanog, WT embryos were co-injected with ALFA-nanog mRNA (25 pg) and EGFP-NbALFA. b Max-projected stills comparing endogenous and exogenous ALFA-Nanog. Endogenous Nanog was visualized via EGFP-NbALFA mRNA injection (row 1) or transgene expression (row 2); exogenous Nanog via co-injection of ALFA-nanog and EGFP-NbALFA mRNAs into WT embryos (row 3). Stills show matched cell stages (E = early, L = late); brightness and contrast adjusted per image for visibility. Insets highlight Nanog foci (arrowheads). c Quantification of the number of visible Nanog foci during the 256-cell stage; mean ± SD of N = 3 embryos with n = 6 (endogenous) and 7 (exogenous) nuclei. d Quantification of Nanog foci from 50% to 75% of the 256-cell stage; n = 37 (endogenous) and 35 (exogenous) data points from N = 3 embryos. Box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum value. Mann–Whitney test, ∗∗∗∗ p < 0.0001 (p-value < 1 × 10−11). e Live imaging setup: mScarlet-i3-NbALFA and EGFP-fused Pol-II Ser2-P mintbody (recognizing Ser2P in C-terminal domain (CTD)) were co-injected into ALFA-nanogKI/KI embryos at the 1-cell stage to visualize endogenous Nanog and elongating RNA Pol II. f Stills and 3D rendering of a 512-cell stage nucleus show Nanog foci (magenta, white arrows) appearing before and near miR-430 Pol-II transcription sites (green, blue arrows). Example 3D distances: 0.56 and 2.80 μm³. g Quantification from (f); n = 15 Nanog/Pol-II measurements at 512- and 1k-cell stages from N = 3 embryos. Bright Nanog foci closely associate with two large Pol-II transcription foci (mean = 1.80 μm³). Box plots show median; limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum value in the data. Scale bars: 10 mm (b), 5 mm (f), and 2 mm (b and f insets). See also Fig. S7.

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