FIGURE SUMMARY
Title

Hemodynamic-mediated endocardial signaling controls in vivo myocardial reprogramming

Authors
Gálvez-Santisteban, M., Chen, D., Zhang, R., Serrano, R., Nguyen, C., Zhao, L., Nerb, L., Masutani, E.M., Vermot, J., Burns, C.G., Burns, C.E., Del Álamo, J.C., Chi, N.C.
Source
Full text @ Elife
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.

EXPRESSION / LABELING:
Gene:
Fish:
Conditions:
Anatomical Terms:
Stage: Days 7-13
PHENOTYPE:
Fish:
Conditions:
Observed In:
Stage: Days 7-13
Figure 1—figure supplement 1.

Metronidazole-ablated <italic>vmhc:</italic>mCherry-NTR ventricles display initial cardiomyocyte death, loss of ventricular tissue and impairment of contractile function but later exhibit cardiomyocyte proliferation and recovery of ventricular tissue and contractile function.

Following ventricular ablation, ventricular and atrial cardiomyocyte proliferation leads to cardiac recovery at 96 hpt. (A, B) Confocal maximum intensity projections of Tg(vmhc:mCherry-NTR) fish show extensive cell death as detected by TUNEL staining in the ventricles of (B) MTZ-treated fish when compared with (A) DMSO-treated controls at 24 hpt. (C) Quantitation of TUNEL-positive CMs in the ventricle of control (n = 15) and ablated (n = 13) fish hearts at 24 hpt confirms cell death is induced by MTZ treatment. (D, E) Confocal maximum intensity projections of Tg(cmlc2:GFP; vmhc:mCherry-NTR) fish show increased proliferation of CMs as detected by EdU+/cmlc2:GFP+ double positive staining (white arrows) in the ventricles and atria of (E) MTZ-treated fish when compared with (D) DMSO-treated controls at 48 hpt. Yellow arrows point to EdU+ proliferating non-cardiomyocytes. (D’, E’) Single confocal sections show EdU+ proliferating cells. (F) Quantitation of EdU+/cmlc2:GFP+ double positive CMs in the ventricle or atrium of control (n = 12) and ablated (n = 12) fish hearts at 48 hpt confirms increased proliferation in ventricle-ablated hearts. (G) Quantitation of ventricular area shows loss of myocardial ventricular tissue in Tg(vmhc:mCherry-NTR) MTZ-treated fish at 24 hpt (n = 12) when compared to control non-ablated fish (n = 7). At 96 hpt, Tg(vmhc:mCherry-NTR) MTZ-ablated hearts show recovery of ventricular area (n = 8, control; n = 9, ablated). (H) Quantitation of ventricular fractional area change (FAC) reveals decreased ventricular function in Tg(vmhc:mCherry-NTR) MTZ-treated fish at 24 hpt (n = 9) when compared to control non-ablated fish (n = 6). At 96 hpt, Tg(vmhc:mCherry-NTR) MTZ-ablated hearts (n = 5) dispaly comparable ventricular function to that of control hearts (n = 6). V, ventricle; A, atrium; dpf, days post-fertilization. Dashed lines outline the heart. Bars: 50 μm. (C) Mean + s.d. (F–H) Mean + s.e.m. Student’s t-test, ***, p<0.001; ns, p>0.05.
Figure 1—figure supplement 2.

Reprogramming of atrial cardiomyocytes into ventricular cardiomyocytes during development.

(A) Schematic representation of the transgenic lines utilized in lineage tracing experiments. Treatment with 4-hydroxytamoxifen (4-OHT) activates the amhc:CreERT2 protein, which causes the recombination of β-actin2:RSG into β-actin2:GFP, thus permanently labeling amhc expressing cells in green. (B) vmhc:mCherry-NTR; amhc:CreERT2; β-actin2:RSG hearts were exposed to tamoxifen at (C) 3, (D) 4 or (E) 5 dpf in order to genetically label atrial CMs with GFP (c-aGFP), and then confocal imaged at 7 dpf. These hearts that were genetically labeled at (C) 3 (n = 21), (D) 4 (n = 14) or (E) 5 (n = 22) dpf show varying contributions of c-aGFP+ CMs to the ventricles. Green channel – (C’–E’) genetically labeled c-aGFP+ atrial CMs. (F) Quantitation of the percentage of ventricular area covered with c-aGFP+ CMs reveals that although uninjured hearts genetically labeled at 3 and 4 dpf exhibit a small contribution of c-aGFP+ CMs to the ventricle, uninjured hearts labeled at 5 dpf do not. All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization. Dashed lines outline the heart. Bars: 50 μm. Mean + s.d. ANOVA test, ***, p<0.001.
Figure 1—figure supplement 3.

Endocardial Notch signaling is transiently activated after myocardial injury.

Confocal microscopy imaging was performed on (A) Tp1:eGFP; vmhc:mCherry-NTR control or (B) ventricle-ablated hearts at 24 hr post-treatment/hpt (6 dpf). Green channel – (A’, B’) Tp1:eGFP. (C, D) Confocal microscopy imaging was performed on (C) Tp1:d2GFP; kdrl:ras-mCherry control or (D) Tp1:d2GFP; kdrl:ras-mCherry; vmhc:mCherry-NTR ventricle-ablated hearts at 24 hr post-treatment/hpt (6 dpf). Green channel – (C’, D’) Tp1:d2GFP. (E, F) Whole-mount in situ hybridizations show that notch1b expression is increased in (F) Tp1:d2GFP; vmhc:mCherry-NTR ventricle-ablated hearts (n = 16/18) at 24 hpt (6 dpf) compared to (E) control hearts (n = 0/20). (G) Quantitation of the average fluorescence intensity of the Tp1:d2GFP signal analyzed in control (black lines) or ventricle-ablated (gray lines) Tp1:d2GFP; vmhc:mCherry-NTR hearts shows that Tp1:d2GFP expression peaks at 24 hpt and gradually decreases as the ventricle regenerates (n = 10 control 0–96 hpt; 12 MTZ 0–96 hpt). 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. Dashed lines outline the heart. Bars: 50 μm. Mean + s.e.m. Student’s t test,*, **, p<0.05 and p<0.01.
Figure 1—figure supplement 4.

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.
Figure 2

Ventricle-ablated hearts display altered oscillatory blood flow and Klf2a activation.

( A, B) High-speed confocal imaging was performed on ( A)  gata1:DsRed control hearts and ( B)  vmhc:mCherry-NTR ; gata1:DsRed ventricle-ablated hearts at 24 hpt (six dpf). Arrows represent particle image velocimetry (PIV) generated vectors from blood flow at different stages of the heart cycle: ( A, B) atrial diastole, ( A’, B’) atrial systole, ( A’’, B’’) ventricular diastole, ( A’’’, B’’’) ventricular systole. ( C, D) Schematic representation indicates where flow rate ( Q) was calculated in ( C’– C’’’) control and ( D’– D’’’) ventricle-ablated hearts (dotted lines). Graphs show the calculated flow rate (mm/s) in the ( C’, D’) ventricle, ( C’’, D’’) atrio-ventricular canal (AVC) and ( C’’’, D’’’) atrium over time (t[s]). Black dots – flow rate ( Q) in each experimental replicate. Red lines – average blood flow rate ( Q). Experimental replicates = 5 control ventricle; 4 control AVC; 4 control atrium; 5 ablated ventricle; 3 ablated AVC; 3 ablated atrium. Colored horizontal bars on the bottom of each graph represent the cardiac cycle period: pink  – atrial diastole; magenta  – atrial systole; light blue – ventricular diastole; dark blue – ventricular systole. ( E) Fundamental harmonic index (FHI = Q1/ Q0) of the flow rate ( Q) in the ventricle, AVC and atrium of control and ventricle-ablated hearts at 24 hpt (6 dpf). n = 3 hearts each condition. ( F, G) Whole-mount in situ hybridizations show that  klf2aexpression is increased in ( G)  vmhc:mCherry-NTR ventricle-ablated hearts (n = 12/12) compared to ( F) control hearts at 24 hpt (n = 0/16) (6 dpf). ( H, I) Confocal imaging was performed on  klf2a:H2B-GFP ; vmhc:mCherry-NTR ( H) control and ( I) ventricle-ablated hearts at 24 hpt (6 dpf) (n = 20 each condition). ( J) Quantitation of the relative average fluorescence intensity of  klf2a:H2B-GFP in  klf2a:H2B-GFP;  vmhc:mCherry-NTR hearts at 24 hpt (6 dpf) confirms that  klf2a:H2B-GFP is increased in ventricle-ablated hearts (n = 20 each condition). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; AVC, atrio-ventricular canal; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment. Dashed lines outline the heart. Bars: 50 μm. ( E, J) Mean + s.e.m. Student’s  t-test, *, **, ***, p<0.05, p<0.01, p<0.001. The following figure supplements are available for Figure 2.

Figure 2—figure supplement 2.

Post-injury Notch signaling is activated in Klf2a-positive endocardial cells.

(A, B) Confocal imaging performed on (A) control and (B) ventricle-ablated klf2a:H2B-GFP; vmhc:mCherry-NTR; Tp1:nls-mCherry hearts at 24 hpt (6 dpf) shows co-expression of Tp1:nls-mCherry (red) in klf2a:H2B-GFP (green) activated cells (maximum intensity projections showed). (A’–A’’’’, B’–B’’’’) Single stacks of the projections in (A, B) show (A’’–B’’) co-expression of Tp1:nls-mCherry and klf2a:H2B-GFP (merged). (A’’-A’’’’-B’’, B’’’’) are magnifications of the area in the yellow dashed box in (A’, B’). (A’’, B’’) merge; (A’’’, B’’’) mCherry – Red or (A’’’’, B’’’’) GFP – Green. (C) Quantitation of the number of cells that display Tp1:nls-mCherry only (Tp1+/Klf2a-), Tp1:nls-mCherry and klf2a:H2B-GFP (Tp1+/Klf2a+) or total Tp1:nls-mCherry (Tp1+/Klf2a- and Tp1+/Klf2a+) in A, B) reveals that ventricle-ablated hearts have significantly more Tp1:nls-mCherry/klf2a:H2B-GFP-positive cells than control sham-ablated hearts at 24 hpt (6 dpf) (n = 10 each condition). 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. Mean + s.e.m. Student’s t test, **, p<0.01.
Figure 3

Reduced hemodynamic forces affect endocardial Notch and Klf2a post-injury activation.

Confocal imaging performed on ( A–D)  klf2a:H2B-GFP ; vmhc:mCherry-NTR or ( J–M)  Tp1:d2GFP;  vmhc:mCherry-NTR ventricle-ablated hearts at 24 hpt (6 dpf) reveals that ( D, M)  gata2a-/- mutant hearts as well as wild-type hearts treated with ( B, K) blebbistatin (Blebb) or ( C, L) tricaine (Tric) exhibit reduced  klf2a:H2B-GFP or  Tp1:d2GFP expression when compared to ( A, J) ethanol (EtOH) sham-treated hearts. Quantitation of the relative average fluorescence intensity in control (black bars) and ventricle-ablated (gray bars) hearts confirms reduced ( E)  klf2a:H2B-GFP or ( N)  Tp1:d2GFP expression in  gata2a-/- mutant hearts as well as wild-type hearts treated with blebbistatin or tricaine ( klf2a:H2B-GFP n = 12 control EtOh; 5 control Blebb; 7 control Tric; 9 control  gata2a-/-; 11 MTZ EtOH; 6 MTZ Blebb; 6 MTZ Tric; 9 MTZ  gata2a-/-. Tp1:d2GFP n = 12 control EtOh; 6 control Blebb; 7 control Tric; 9 control  gata2a-/-; 11 MTZ EtOH; 6 MTZ Blebb; 6 MTZ Tric; 9 MTZ  gata2a-/-). Whole-mount in situ hybridizations at 24 hpt (6 dpf) show that ( F–I)  klf2a and ( O–R)  notch1b expression is decreased in  vmhc:mCherry-NTR ventricle-ablated hearts treated with ( G, P) blebbistatin (n = 0/8  klf2a; n = 2/12  notch1b) or ( H, Q) tricaine (n = 0/7  klf2a; n = 0/10  notch1b) as well as in ( I, R)  gata2a-/- ventricle-ablated hearts (n = 3/9  klf2a; n = 5/12  notch1b) when compared to ( F, O) hearts sham-treated with ethanol (n = 7/7  klf2a; n = 10/10  notch1b). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ treatment. Dashed lines outline the heart. Bars: 50 μm. ( E, N) Mean + s.e.m. 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 3.

EXPRESSION / LABELING:
Genes:
Fish:
Conditions:
Anatomical Term:
Stage: Day 6
PHENOTYPE:
Fish:
Conditions:
Observed In:
Stage: Day 6
Figure 3—figure supplement 1.

Inhibiting hemodynamic flow leads to reduced cardiac Klf2a and Notch signaling.

( A–D) Confocal imaging performed on  klf2a:H2B-GFP ; vmhc:mCherry-NTR uninjured hearts at 24 hpt (6 dpf) reveals decreased  klf2a:H2B-GFP expression in ( B) blebbistatin (Blebb) and ( C) tricaine (Tric) treated hearts as well as in ( D)  gata2a-/- mutant hearts when compared to ( A) ethanol (EtOH)-treated control hearts (n = 12 control EtOh; 6 control Blebb; 7 control Tric; 9 control  gata2a-/-). ( E–H) Confocal imaging performed on  Tp1:d2GFP ; vmhc:mCherry-NTR uninjured hearts at 24 hpt (6 dpf) shows decreased  Tp1:d2GFP Notch reporter expression in hearts treated with ( F) blebbistatin or ( G) tricaine when compared to ( E) ethanol-treated control hearts; however, ( H)  gata2a-/-hearts do not display significant changes as quantitatively assessed in  Figure 3N(n = 12 control EtOh; 5 control Blebb; 7 control Tric; 9 control  gata2a-/-). ( I–L) Whole-mount in situ hybridizations reveal decreased  notch1b expression in hearts treated with ( J) blebbistatin (n = 0/9) or ( K) tricaine (n = 0/5) and in ( L)  gata2a-/- hearts (n = 4/12) when compared to ( I) ethanol-treated control hearts (n = 10/10) at 24 hpt (6 dpf). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-DMSO treatment. Dashed lines outline the heart. Bars: 50 μm.

Figure 4

Hemodynamic forces control regeneration and myocardial reprogramming.

( A–D) Confocal microscopy imaging performed on  vmhc:mCherry-NTR ventricle-ablated hearts reveals that ( B) blebbistatin or ( C) tricaine treatment as well as ( D) the  gata2a-/- mutant allele inhibit CM proliferation at 48 hpt (7 dpf) when compared to CM proliferation of ( A) ethanol-treated control hearts. White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. ( E) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that blebbistatin or tricaine treatment, and  gata2a-/- defects block CM proliferation in injured hearts (n = 23, control EtOH; 21 MTZ EtOH; 9 control Blebb; 9 MTZ Blebb; 10 control Tric; 10 MTZ Tric; 9 control  gata2a-/-; 16 MTZ  gata2a-/-). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. ( F) 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) shows that inhibiting contractility by blebbistatin or tricaine treatment as well as decreasing blood viscosity using  gata2a-/- mutants impair heart regeneration. The number of fish analyzed for each condition is indicated above each column. ( G–J) Confocal microscopy imaging of  vmhc:mCherry-NTR;  amhc:CreERT2;  β-actin2:RSG hearts at 60 hpt (7.5 dpf) reveals that ( H) blebbistatin-treated, ( I) tricaine-treated, or ( J)  gata2a-/- ventricle-ablated hearts exhibit reduced ability of genetically labeled atrial CMs (c-aGFP+) to contribute to the regenerating injured ventricle when compared to ( G) ethanol sham-treated hearts. ( G’–J’) Green channel – genetically-labeled c-aGFP+ atrial CMs. ( K) Quantitation of the percentage of ventricular area covered with c-aGFP+ CMs confirms the reduced atrial CM contribution to the regenerating injured ventricle in  gata2a-/- mutant hearts as well as wild-type hearts treated with blebbistatin or tricaine (n = 12 ethanol; 7 blebbistatin; 7 tricaine; 9  gata2a-/-). 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) Mean + s.e.m. ANOVA; ( K) Mean + s.d. ANOVA; ( F) Total numbers, Binomial test (versus EtOH); ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 4.

Figure 4—figure supplement 1.

Inhibiting hemodynamic flow perturbs post-injury re-activation of cardiac factors.

Whole-mount in situ hybridizations show increased (A) gata4 (n = 9/11), (E) hand2 (n = 10/10) and (I) nkx2.5 (n = 7/8) expression in vmhc:mCherry-NTR ventricle-ablated (ethanol/EtOH-treated) control hearts at 48 hpt (7 dpf). However, these cardiogenesis factors fail to increase after ventricle-ablation in (B, F, J) blebbistatin (Blebb) (n = 0/13 gata4; n = 1/10 hand2; n = 0/10 nkx2.5) or (C, G, K) tricaine (Tric) (n = 0/14 gata4; n = 2/10 hand2; n = 0/6 nkx2.5) treated hearts as well as in (D, H, L) gata2a-/- hearts (n = 5/17 gata4; n = 4/11 hand2; n = 2/7 nkx2.5). V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-DMSO treatment. Dashed lines outline the heart. Bars: 50 μm.
Figure 5

The mechanosensitive channel Trpv4 regulates endocardial Notch activation and myocardial regeneration through Klf2a.

( 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.

EXPRESSION / LABELING:
Genes:
Fish:
Condition:
Anatomical Terms:
Stage: Day 6
PHENOTYPE:
Fish:
Condition:
Observed In:
Stage Range: Day 6 to Days 7-13
Figure 5—figure supplement 1.

<italic>klf2a</italic> and <italic>trpv4</italic> mutants display reduced endocardial Notch signaling.

(A–C) Confocal microscopy imaging performed on vmhc:mCherry-NTR, Tp1:d2GFP control hearts reveals reduced Tp1:d2GFP activation in (B) klf2a-/- and (C) trpv4-/- hearts at 24 hpt (6 dpf) when compared to (A) wild-type (wt) hearts (n = 15 control wt; 18 control trpv4-/-; 20 control klf2a-/-; 22 MTZ wt; 21 MTZ trpv4-/-; 18 MTZ klf2a-/-). (D–F) Whole-mount in situ hybridizations reveal reduced gene expression of notch1b in (E) klf2a-/- (n = 4/19) and (F) trpv4-/- (n = 14/29) vmhc:mCherry-NTR control hearts at 24 hpt (6 dpf) when compared to (D) wild-type (wt) hearts (n = 22/25). 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.
Figure 5—figure supplement 2.

<italic>klf2a</italic> and <italic>trpv4</italic> mutants show impaired post-injury re-activation of cardiogenesis transcription factors.

(A–R) Whole-mount in situ hybridizations show increased (B) gata4 (n = 21/24), (H) hand2 (n = 13/15) and (N) nkx2.5 (n = 12/13) expression in vmhc:mCherry-NTR ventricle-ablated wild-type (wt) hearts at 48 hpt (7 dpf) when compared to (A, G, M) sham-ablated control hearts. However, these cardiogenesis transcription factors fail to increase in (D, J, P) klf2a-/- (n = 7/21 gata4; n = 4/9 hand2; n = 5/14 nkx2.5) and (F, L, R) trpv4-/- (n = 7/17 gata4; n = 5/11 hand2; n = 5/12 nkx2.5) ventricle-ablated hearts. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment. Dashed lines outline the heart. Bars: 50 μm.
Figure 6

Erbb2 and BMP signaling regulate cardiac regeneration and atrial-to-ventricular trans-differentiation.

( A–D) Confocal microscopy performed on  vmhc:mCherry-NTR ventricle-ablated hearts reveals that ( B) dorsomorphin (DM) and ( C) AG1478-treated as well as ( D)  erbb2 loss-of-function mutation ( erbb2-/-) ventricle-ablated hearts exhibit reduced CM proliferative response when compared to ( A) DMSO-treated ventricle-ablated hearts (control) at 48 hpt (7 dpf). White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. ( E) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that dorsomorphin and AG1478 treatments as well as loss of  erbb2 function ( erbb2-/-) prevent CMs from proliferating in injured hearts (n = 15 each condition). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. ( F–I) Confocal imaging of  vmhc:mCherry-NTR;  amhc:CreERT2;  β-actin2:RSGventricle-ablated hearts shows that ( G) dorsomorphin (DM) or ( H) AG1478 treatment as well as ( I)  erbb2-/- blocks the contribution of genetically-labeled atrial CMs (c-aGFP+) to the regenerating ventricle-ablated hearts when compared to ( F) ventricle-ablated DMSO-treated control hearts at 72 hpt (8 dpf). Green channel – ( F’–I’) genetically labeled c-aGFP+ atrial CMs. ( J) Quantitation of the percentage of ventricular area covered with c-aGFP+ atrial CMs confirms that dorsomorphin or AG1478 treatments as well as loss of  erbb2 function ( erbb2-/-) prevent atrial CMs from regenerating the injured ventricle (n = 11 DMSO; 6 dorsomorphin; 7 AG1478;  7 erbb2-/-). ( K) 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) confirms that inhibiting BMP signaling with dorsomorphin or Erbb2 signaling with AG1478 impairs heart regeneration. The number of fish analyzed for each condition is indicated above each column. 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) Mean + s.e.m. ANOVA; ( J) Mean + s.d. ANOVA; ( K) Total numbers, Binomial test (versus DMSO); ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 6.

Figure 6—figure supplement 1.

Inhibiting BMP or Erbb2 signaling impairs reactivation of early cardiogenesis transcription factors.

Whole-mount in situ hybridizations show increased (B) gata4 (n = 19/25), (J) hand2 (n = 10/12) and (R) nkx2.5 (n = 12/13) expression in vmhc:mCherry-NTR ventricle-ablated hearts at 48 hpt (7 dpf) when compared to (A, I, Q) sham-ablated control hearts. However, these cardiogenesis transcription factors fail to increase in (D, L, T) dorsomorphin (DM) (n = 4/21 gata4; n = 3/12 hand2; n = 4/16 nkx2.5) and (F, N, V) AG1478-treated ventricle-ablated hearts (n = 5/20 gata4; n = 1/8 hand2; n = 3/10 nkx2.5) as well as in (H, P, X) erbb2 loss-of-function mutation (erbb2-/-) (n = 4/18 gata4; n = 5/24 hand2; n = 3/15 nkx2.5). V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment. Dashed lines outline the heart.
Figure 7

Blood flow regulates myocardial Erbb2 and BMP signaling through endocardial Notch. (A–F) Confocal imaging of BRE:d2GFP; vmhc:mCherry-NTR hearts at 48 hpt (7 dpf) shows that (B) BRE:d2GFP is activated after ventricular ablation (n = 21/21) when compared to (A) uninjured control hearts (n = 0/29); however, (D) blebbistatin (Blebb) (n = 0/11) or (F) DAPT (n = 0/14) treatment inhibits this BRE:d2GFP injury-induced activation. (G–R) Whole-mount in situ hybridizations reveal that bmp10 and nrg1 expression are increased in vmhc:mCherry–NTR (H, N) ventricle-ablated hearts (n = 13/14 bmp10; 7/8 nrg1) at 48 hpt (7 dpf) when compared to (G, M) control uninjured hearts (n = 0/18 bmp10; 0/13 nrg1), while treatment with (J, P) blebbistatin (n = 2/13 bmp10; 0/6 nrg1) or (L, R) DAPT (n = 4/16 bmp10; 0/7 nrg1) inhibits the injury-induced activation of bmp10 and nrg1. 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. The following figure supplements are available for Figure 7.
Figure 7—figure supplement 1.

Myocardial specific activation of BMP signaling.

(A–F) Confocal single-stacks corresponding to the projections shown in Figure 7A–F show (B–B’’’) BRE:d2GFP is activated in the myocardium of ablated hearts at 48 hpt (seven dpf) (n = 21/21), in contrast to (A–A’’’) uninjured controls (n = 0/29). (B’–B’’’) Magnifications of the boxed area in (B) show (B’) co-localization (arrowheads) of BRE:d2GFP and vmhc:mCherry-NTR. However, (D–D’’’) blebbistatin (Blebb) (n = 0/11) or (F–F’’’) DAPT (n = 0/14) treatment inhibits this myocardial BRE:d2GFP injury-induced activation. (A’–F’) merge; (A’’–F’’) Red channel - vmhc:mCherry-NTR; (A’’’–F’’’) Green channel-BRE:d2GFP. V, ventricle; A, atrium. hpt, hours post-MTZ/DMSO treatment.
Figure 7—figure supplement 2.

Endocardial specific inhibition of Notch signaling blocks post-injury activation of <italic>bmp10</italic> and <italic>nrg1.</italic>

Whole-mount in situ hybridizations of heat-shocked (HS) (A, B, E, F) vmhc:mCherry-NTR; hsp70l:RS-dnM or (C, D, G, H) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM hearts show that dnMAML endocardial expression can block (D, H) bmp10 and nrg1 (n = 3/19 bmp10; 0/8 nrg1) activation after ventricle-injury at 48 hpt (7 dpf) when compared to (B, F) ventricle-ablated control hearts (n = 21/21 bmp10; n = 18/19 nrg1). V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment. Dashed lines outline the heart.
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