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Fig. 3 Visualizing the nanoscale organization of Nanog, nucleosomes, Pol II, and transcription. (A to D) Representative images showing Nanog and H3 staining at 4 hpf; (B) represents class 1 organization. (C) represents the white boxed region in (A) and shows class 2 organization. (D) represents the green boxed region in (A) and shows class 3 organization. n = 3 nuclei from 2 embryos. (E to G) Box plots quantifying the percentage of H3 particles in each class of Nanog–H3 interactions. The median is denoted by the orange line. n = 104,347 distances. (H to K) Representative images showing Nanog and Pol II pSer5 staining at 4 hpf, n = 7 nuclei from 2 embryos. (I) represents the green boxed region in (H) and shows class 1 organization; areas indicated with arrowheads are magnified to the right. (J) represents class 2 organization. (K) represents the magenta boxed region in (H) and shows class 3 organization; arrowheads indicate regions enriched for Nanog (open arrowhead) and Pol II pSer5 (closed arrowhead). (L) Density plot of the distance to nearest neighbor for Nanog and Pol II pSer5 particles; n = 169,866 distances. (M) Box plots quantifying the number of Pol II pSer5 particles in class 1 (n = 36) and class 2 (n = 5682) strings. (N) Box plots quantifying the number of Nanog particles in class 1 and class 2 Pol II pSer5 strings. (O) Box plots quantifying the length of Pol II pSer5 strings in class 1 and class 2. (P and Q) ChromExM images showing Pol II pSer5 and miR-430 RNA detected by hybridization chain reaction RNA FISH at 4 hpf. (Q) represents the boxed region in (P). (R to U) Representative images showing nascent RNA and Pol II pSer5 at 4 hpf. n = 7 nuclei from 3 embryos. The closed arrowhead in (R) indicates a central Pol II pSer5 hub with an emanating string of individual Pol II pSer5 particles associated with nascent transcripts (open arrowhead). Open arrowheads in (S) indicate individual Pol II pSer5 particles.