Mechanically activated piezo channels modulate outflow tract valve development through the Yap1 and Klf2-Notch signaling axis
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
- Centre National de la Recherche Scientifique, France
- Institut National de la Santé et de la Recherche Médicale, France
- Université de Strasbourg, France
Figures
Figure 1
The OFT develops from 56 hpf to form functional valves at 144 hpf.
(A) Top: Z-section of the double transgenic line Tg(kdrl:nls-mCherry; myl7:GFP) showing the overall structure of the heart. Bottom: Scheme of the zebrafish heart with the endocardium, myocardium and …
Figure 2 with 1 supplement
The endothelium contribution to emerging OFT valve leaflets.
(A) Experimental set-up for the photoconversion studies. Heart was stopped at 72 hpf, the region of interest exposed to 405 nm light to convert kaede from green to red (shown in magenta) fluorescent …
Figure 2—figure supplement 1
The endothelium contribution to emerging OFT versus AVC valves.
(A) Z-sections of the Tg(fli:gal4FF/UAS:Kaede) line just after photoconversion (72 hpf), and at 96 hpf and 120 hpf. The star highlights the photoconverted cell in the top, middle and bottom part of …
Figure 3 with 1 supplement
The OFT endothelium is surrounded by smooth muscle cell progenitors expressing fibronectin and elastin.
(A) Staining of Fibronectin1 (magenta) on Tg(myl7:GFP; fli1a:nls-mCherry), highlighting the myocardium (white) and the endothelium (green) at 72 hpf. Scale bar: 20 μm. V: ventricle. Fibronectin1 …
Figure 3—figure supplement 1
Smooth muscle identity is revealed by NO.
(A) Z-sections of the Tg(kdrl:nls-mCherry) line counterstained by DAF-FMDA (cyan) and Fn1 (magenta) at 72 hpf, 96 hpf and 120 hpf. Scale bar: 20 µm. (B) Co-localization of Fibronectin1 (magenta) …
Figure 4 with 1 supplement
Klf2a and Notch reporters are activated in the OFT endothelium Confocal z-section of the Tg(klf2a:H2B-GFP; fli:nls-mCherry).
(A) and Tg(tp1:dGFP) (B) at 72hpf. OFT: outflow tract, At: atrium, V: ventricle. Scale bar: 20 µm. (C) Confocal z-section of OFT valves expressing the Klf2a reporter and Notch reporter at 72 hpf, 96 …
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Figure 4—source data 1
Fluorescence intensity measurements.
- https://doi.org/10.7554/eLife.44706.009
Figure 4—figure supplement 1
Expression of the klf2a reporter Z-section of the Tg(klf2a:H2B-EGFP; kdrl:nls-mCherry) at 72 hpf, 96 hpf and 120 hpf used to quantify the reporter expression.
Scale bar: 20 µm. Results obtained from three independent experiments.
Figure 5 with 1 supplement
Hippo pathway effector Yap1 is active and Yap1 is essential for valve formation in the OFT.
(A) Confocal z-sections of the double transgenic line 4xGTIIC:d2GFP; kdrl:membrane-mCherry counterstained with the Elnb antibody and focused on the OFT. Scale bar: 20 µm. (B) Example of the valve …
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Figure 5—source data 1
Phenotypic quantifications.
- https://doi.org/10.7554/eLife.44706.012
Figure 5—figure supplement 1
Yap1 is expressed in the OFT Yap1 antibody staining on Tg(fli:lifeact-EGFP; kdrl:nls-mCherry) (A) and zoom on the OFT (B) at 72 hpf.
Asterisks show endothelial cells expressing Yap1. At: atrium, V: ventricle. Scale bar: 20 µm. V: ventricle, At: atrium. Resultsobtained from three independent experiments.
Figure 6
Klf2a and notch response, as well as the smooth muscle cell identity, are flow-dependent.
Quantification of the Klf2a and Notch reporter expressions in tnnt2a-morpholino injected embryos showing a ‘beating heart’ (p=0,27 and p=0,01 respectively). (A) and a ‘slow beating heart’ (p=0,7 and …
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Figure 6—source data 1
Phenotypic and fluorescence reporters quantifications.
- https://doi.org/10.7554/eLife.44706.014
Figure 7 with 1 supplement
Klf2a and notch are necessary for valve formation.
Quantification of the valve phenotypes at 72 hpf, 96 hpf and 120 hpf (normal, thick, delayed) in klf2a+/+(n = 11), and klf2a-/- (n = 12) using the Tg(fli:gal4FF/UAS:Kaede). (A) and notch1b+/+ (n = 7)…
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Figure 7—source data 1
Fluorescent intensity quantifications.
- https://doi.org/10.7554/eLife.44706.019
Figure 7—figure supplement 1
Klf2a regulates notch1b expression but Notch1b does not regulate klf2a expression.
(A) In situ hybridization of notch1b in wild-type embryos at 48 hpf and 72 hpf. In situ hybridization of notch1b in klf2a+/+ (n = 134 and n = 107 at 48 hpf and 72 hpf respectively) and klf2a-/- (n = …
Figure 8 with 3 supplements
Flow and mechanosensitive channels are necessary for proper OFT valve formation.
(A) Z-sections and quantifications of the valves phenotypes (normal, thick, delayed) of the Tg(fli:lifeaAct-EGFP; kdrl:nls-mCherry) at 72 hpf (B), 96 hpf (C) and 120 hpf (D) in trpp2+/+(n = 11, n = 1…
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Figure 8—source data 1
Phenotypic and fluorescence reporters quantifications.
- https://doi.org/10.7554/eLife.44706.024
Figure 8—figure supplement 1
Embryo phenotypes in controls and mutants.
(A) Relative fractional shortening in the atrium and ventricle in control, vlad tepes mutant, trpp2-/-, trpp2-/-; trpv4-/-, piezo1-/-, piezo1-/-; piezo2a-/-. (B) Flow profile in the OFT at 72 hpf …
Figure 8—figure supplement 2
Valve phenotypes in trpp2-/- and in piezo1-/-.
(A) Z-sections of the Tg(fli:lifeAct-EGFP: kdrl:nls-mCherry) in control (trpp2+/+ and trpp2+/-) and trpp2-/- obtained from three independent experiments and in control (piezo1+/+) and piezo1-/- …
Figure 8—figure supplement 3
Trpp2 and Piezo1 expression in the OFT.
(A) Z-projection of the RNAscope of kdrl and single Z-sections of the RNAscope of trpp2 and piezo1 on 72 hpf wild-type embryos in the head and the OFT. (B) Z-section of the Tg(piezo1:nls-Venus) …
Figure 9
Working model summarizing OFT valve morphogenesis in response to mechanical forces.
Two cell layers forming the OFT respond to piezo1 activity: the endothelium (green) and smooth muscle cells (magenta). Endothelial klf2a expression is repressed by Piezo1. In the smooth muscle …
Author response image 1
(A) Z-section of the kdrl:nls-mCherry and fli1a:lifeact-eGFP in the OFT (B) Z-projection of the RNAscope of kdrl in the head and in the OFT (n=6 embryos).
Author response image 2
(A) Pictures of the different mutants and their controls showing overall shape and heart shape.
Videos
Video 1
Bright field videomicroscopy of the typical heart function of a tnnt2a MO group1 embryo where the heart is still beating normally.
https://doi.org/10.7554/eLife.44706.015
Video 2
Bright field videomicroscopy of the typical heart function of a tnnt2a MO group2 embryo where the heart is still beating but at an abnormal slow rate (less than 2 Hz).
https://doi.org/10.7554/eLife.44706.016Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.44706.026
