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figure 1

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ZDB-IMAGE-190723-1061
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Figures for Gálvez-Santisteban et al., 2019
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figure 1

Endocardial Notch signaling controls myocardial reprogramming and cardiac regeneration. (A–D, L–O) Confocal microscopy imaging of heat-shocked/HS (A, B, L, M) vmhc:mCherry-NTR, (C, D) vmhc:mCherry-NTR; hsp70l:dnM and (N, O) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM hearts reveals that (D) global or (O) endocardial-specific dnMAML (dnM) Notch inhibition inhibits CM proliferation in ventricle-ablated hearts at 48 hpt (7 dpf) when compared to (B, M) CM proliferation in control ventricle-ablated (no dnMAML) hearts. White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. (E, P) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that (E) global or (P) endocardial-specific dnM Notch inhibition prevents CMs from proliferating in injured hearts (n = 23, control dnM-; 31, MTZ dnM-; 10, control dnM+; 18, MTZ dnM+; 16, control RSdnM-; 16, MTZ RSdnM-; 15, Control RSdnM+; 15, MTZ RSdnM-). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. (F, Q) Quantitation of the percentage of heat-shocked vmhc:mCherry-NTR, vmhc:mCherry-NTR; hsp70l:dnM and vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM ventricle-ablated hearts that display recovered ventricular tissue and contractility (black bars) at 96 hpt (nine dpf) shows that (F) global or (Q) endocardial-specific Notch inhibition between 0 and 1 dpt leads to the greatest inhibitory effect on overall recovery from ventricle injury. The number of fish analyzed for each condition is indicated above each column. (G–K) To examine the effects of Notch signaling on cardiac reprogramming, (G, H) vmhc:mCherry-NTR; amhc:CreERT2; myl7:CSY and (I, J) vmhc:mCherry-NTR; amhc:CreERT2; myl7:CSY; hsp70l: dnMAML (dnM) hearts were exposed to tamoxifen at 5 dpf to genetically label atrial CMs with YFP (c-aYFP), then (G, I) sham-ablated (control) or (H, J) ventricle-ablated, and finally heat-shocked to (I, J) induce dnM expression. Confocal microscopy imaging at 72 hpt (8 dpf) reveals that (J) heat-shock induction of dnM inhibited the ability of genetically labeled c-aYFP+ atrial CMs to contribute to the regeneration of ventricle-ablated hearts when compared to (H) heat-shock control ventricle-ablated hearts. Yellow channel – (G’–J’) genetically labeled c-aYFP+ atrial CMs. (K) Quantitation of the percentage of ventricular area covered with c-aYFP+ CMs supports that dnMAML Notch inhibition prevents atrial CMs from regenerating the injured ventricle (n = 8 hsp70:dnM-, seven hsp70l:dnM+). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post MTZ/DMSO treatment. Dashed lines outline the heart. Bar: 50 μm. (E, P) Mean + s.e.m. ANOVA; (K) Mean + s.d. Student’s t-test; (F, Q) Total numbers, Binomial test (versus 0 dpt); ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 1.

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