Figure 5

( A–D) Confocal imaging of  vmhc:mCherry-NTR ; klf2a:H2B-GFP hearts shows that  klf2a:H2B-GFP expression is activated in ( B)  wild-type ( wt) ventricle-ablated hearts at 24 hpt (6 dpf) compared to ( A) control hearts; however, this activation is reduced in ( D)  trpv4^-/- ventricle-ablated hearts. ( F–H) Confocal imaging of  vmhc:mCherry-NTR ; Tp1:d2GFP hearts further reveals that  Tp1:d2GFP is decreased in ( G)  klf2a^-/- and ( H)  trpv4^-/- ventricle-ablated hearts at 24 hpt (6 dpf) when compared to ( F) wild-type ( wt) hearts. ( E, I) Quantitation of the relative average fluorescence intensity confirms reduced injury-induced ( E)  klf2a:H2B-GFP activation in  trpv4^-/- ventricle-ablated hearts, and ( I)  Tp1:d2GFP activation in  trpv4^-/- and  klf2a^-/- ventricle-ablated hearts when compared to wild-type ventricle-ablated hearts ( klf2a:H2B-GFP n = 9 control  wt; 7 control  trpv4^-/-; 9 MTZ  wt; 10 MTZ  trpv4^-/-. Tp1:d2GFP n = 15 control  wt; 18 control  trpv4^-/-; 20 control  klf2a^-/-; 22 MTZ  wt; 21 MTZ  trpv4^-/-; 18 MTZ  klf2a^-/). ( J–L) Whole-mount in situ hybridizations show that  notch1b is decreased in ventricle-ablated ( K)  klf2a^-/- (n = 2/11) and ( L)  trpv4^-/- hearts (n = 6/15) at 24 hpt (6 dpf) when compared to ( J) wild-type hearts (n = 16/18). ( M–O) Confocal microscopy performed on  vmhc:mCherry-NTR ventricle-ablated hearts reveals that ( N)  klf2a^-/- and ( O)  trpv4^-/-ventricle-ablated hearts display reduced CM proliferative response when compared to ( M) wild-type ( wt) ventricle-ablated hearts at 48 hpt (7 dpf). White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. ( P) Quantitation of anti-phospho-histone H3⁺ CMs in these hearts confirms that  klf2a^-/- and  trpv4^-/- hearts fail to increase CM proliferation after ventricle-injury (n = 15 each condition). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. ( Q) Quantitation of the percentage of  vmhc:mCherry-NTR ventricle-ablated hearts that display recovered ventricular tissue and contractility (black bars) at 96 hpt (9 dpf) supports that  klf2a^-/- and  trpv4^-/- mutants exhibit impaired heart regeneration. The number of fish analyzed for each condition is indicated above each column. ( R–T) Confocal microscopy imaging of  vmhc:mCherry-NTR;  amhc:CreERT2;  β-actin2:RSGhearts at 72 hpt (8 dpf) shows that ( S)  klf2a^-/-and ( T)  trpv4^-/- ventricle-ablated hearts exhibit reduced contribution of genetically labeled atrial CMs (c-aGFP⁺) to the regenerating injured ventricle when compared to ( R) wild-type ventricle-ablated hearts. Green channel – ( R’–T’) genetically labeled c-aGFP⁺ atrial CMs. ( U) Quantitation of the percentage of ventricular area covered with c-aGFP⁺ CMs confirms that  klf2a^-/-or  trpv4^-/- hearts display reduced capacity to undergo injury-induced atrial-to-ventricular trans-differentiation during injury and regeneration (n = 13  wt; 8  klf2a^-/-; 10  trpv4^-/-). 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. Bars: 50 μm. ( E, I, P) Mean + s.e.m. ANOVA; ( Q) Total numbers, Binomial test (versus wild-type); ( U) Mean + s.d. ANOVA; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 5.