Figure 3.
Tail transection stimulates localized Padi2-dependent histone H4 citrullination.(A) Representative Western blot of padi2−/− and WT whole 2 dpf larvae lysates showing protein levels of citrullinated Histone4 (H4cit3), total Histone4 (H4), total Padi2 (zPadi2), and total actin (Actin; representative of two replicates). (B) Representative images of H4cit3 antibody staining in tail transected fins at 20× magnification. Merge images with bright-field are shown for orientation. Box denotes region imaged at 60× magnification in C showing H4cit3 immunolabel alone. (B and C) Representative images from three independent replicates. (D–F) Representative multiphoton microscopy enface (x, y view) and orthogonal (x, z view is below; y, z view is to the right) sections of 6 hpw WT caudal fins labeled with H4cit3 immunofluorescence (green) in conjunction with either SHG (D; white) or DAPI-labeled nuclei (E; blue) and rhodamine-phallodin–labeled actin (magenta) or DAPI-labeled nuclei (F; blue) and (Tg(krt-4:EGFP))-expressing epithelium (magenta). Note in F, fluorophore tag for epithelium crosses into H4cit3 antibody channel at the setting needed to detect the antibody signal. Arrow indicates nucleus with citrullinated histones in the notochord region, while arrowhead points to one example of a nucleus with citrullinated histones in the notochord bead. For image presentation, section thickness shown is 2 µm for both x and y, 10 µm in z. Representative images from two independent replicates. Videos 1, 2, 3, 4, 5, and 6 present z-stacks and 3D reconstructions of data in D-F. (G) Representative images of H4cit3 immunostaining in 24 hpw WT cousin (left) and padi2−/− (right). (H) Quantification of H4cit3 signal area at the notochord at 24 hpw and 3 dpf (no wound control). (I) Quantification of H4cit3 integrated density in 24 hpw larvae normalized to the average of 3 dpf for each genotype. (J) Quantification of the notochord bead area at 24 hpw. All quantifications are from three pooled independent replicates, have the lsmeans (±) SEM reported and P values calculated by ANOVA. (H–J) 24 hpw, n = 38 +/+, 41 −/−; 3 dpf n = 25 +/+, 25 −/−. Scale bars, 100 µm in B and G; 50 µm in C; 20 µm in D–F.

Tail transection stimulates localized Padi2-dependent histone H4 citrullination. (A) Representative Western blot of padi2−/− and WT whole 2 dpf larvae lysates showing protein levels of citrullinated Histone4 (H4cit3), total Histone4 (H4), total Padi2 (zPadi2), and total actin (Actin; representative of two replicates). (B) Representative images of H4cit3 antibody staining in tail transected fins at 20× magnification. Merge images with bright-field are shown for orientation. Box denotes region imaged at 60× magnification in C showing H4cit3 immunolabel alone. (B and C) Representative images from three independent replicates. (D–F) Representative multiphoton microscopy enface (x, y view) and orthogonal (x, z view is below; y, z view is to the right) sections of 6 hpw WT caudal fins labeled with H4cit3 immunofluorescence (green) in conjunction with either SHG (D; white) or DAPI-labeled nuclei (E; blue) and rhodamine-phallodin–labeled actin (magenta) or DAPI-labeled nuclei (F; blue) and (Tg(krt-4:EGFP))-expressing epithelium (magenta). Note in F, fluorophore tag for epithelium crosses into H4cit3 antibody channel at the setting needed to detect the antibody signal. Arrow indicates nucleus with citrullinated histones in the notochord region, while arrowhead points to one example of a nucleus with citrullinated histones in the notochord bead. For image presentation, section thickness shown is 2 µm for both x and y, 10 µm in z. Representative images from two independent replicates. Videos 1, 2, 3, 4, 5, and 6 present z-stacks and 3D reconstructions of data in D-F. (G) Representative images of H4cit3 immunostaining in 24 hpw WT cousin (left) and padi2−/− (right). (H) Quantification of H4cit3 signal area at the notochord at 24 hpw and 3 dpf (no wound control). (I) Quantification of H4cit3 integrated density in 24 hpw larvae normalized to the average of 3 dpf for each genotype. (J) Quantification of the notochord bead area at 24 hpw. All quantifications are from three pooled independent replicates, have the lsmeans (±) SEM reported and P values calculated by ANOVA. (H–J) 24 hpw, n = 38 +/+, 41 −/−; 3 dpf n = 25 +/+, 25 −/−. Scale bars, 100 µm in B and G; 50 µm in C; 20 µm in D–F.

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