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Genetic inhibition of endocardial Notch signaling impairs myocardial reprogramming and regeneration.(A–L) Whole-mount in situ hybridizations of heat-shocked (A, B, E, F, I, J) vmhc:mCherry-NTR or (C, D, G, H, K, L) vmhc:mCherry-NTR; hsp70l:dnM hearts show that dnMAML (dnM) Notch inhibition decreases (D) gata4 (n = 3/15), (H) hand2 (n = 1/6) and (L) nkx2.5 (n = 2/8) reactivation in ventricle-ablated hearts at 48 hpt (seven dpf) when compared to (B, F, J) control ventricle-ablated (no dnM) hearts (n = 12/12, gata4; n = 5/5, hand2; n = 7/7, nkx2.5). (M) Schematic representation of the transgenic lines utilized in endocardial Notch inhibition studies for Figure 1L–Q and Figure 1—figure supplement 4N–Z´). Heat-shock induction of kdrl:Cre; hsp70l:RS-dnM allows for conditional expression of dnM specifically in endothelial/endocardial cells. (N–O) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM were heat-shocked at 5 dpf and sham-ablated (control) or MTZ-ablated at 5.5 dpf. Confocal imaging was performed on vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM (N) control and (O) ventricle-ablated hearts at 24 hpt (6.5 dpf). (N’–N’’’, O’– O’’’) Insets are magnification of boxes in (N and O) and highlight endocardial expression of dnMAML-GFP in the ventricle. Yellow dashed lines indicate boundary between myocardium (myo) and endocardium (end). Green channel – (N’’, O’’) kdrl:Cre recombined hsp70l:RS-dnM; Red channel – (N’’’, O’’’) vmhc:mCherry-NTR; Merge – (N, N’, O, O’). (P–Z’) Whole-mount in situ hybridizations of heat-shocked (P, Q, T, U, X, Y) vmhc:mCherry-NTR or (R, S, V, W, Z, Z’) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM hearts show that endocardial expression of dnM decreases (S) gata4 (n = 2/16), (W) hand2 (n = 0/5) and (Z’) nkx2.5 (n = 1/7) reactivation in ventricle-ablated hearts at 48 hpt (7 dpf) when compared to (Q, U, Y) control ventricle-ablated (no dnM) hearts (n = 16/18, gata4; n = 8/8, hand2; n = 10/10, nkx2.5). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment; dpt, days post-MTZ treatment. White dashed lines outline the heart. Bars: 50 μm.
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