Mechanically activated piezo channels modulate outflow tract valve development through the Yap1 and Klf2-Notch signaling axis

  1. Anne-Laure Duchemin
  2. Hélène Vignes
  3. Julien Vermot  Is a corresponding author
  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
  2. Centre National de la Recherche Scientifique, France
  3. Institut National de la Santé et de la Recherche Médicale, France
  4. Université de Strasbourg, France
11 figures, 2 videos and 1 additional file

Figures

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 …

https://doi.org/10.7554/eLife.44706.002
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 …

https://doi.org/10.7554/eLife.44706.003
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 …

https://doi.org/10.7554/eLife.44706.004
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 …

https://doi.org/10.7554/eLife.44706.005
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) …

https://doi.org/10.7554/eLife.44706.006
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 …

https://doi.org/10.7554/eLife.44706.007
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.

https://doi.org/10.7554/eLife.44706.008
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 …

https://doi.org/10.7554/eLife.44706.010
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.

https://doi.org/10.7554/eLife.44706.011
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 …

https://doi.org/10.7554/eLife.44706.013
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)…

https://doi.org/10.7554/eLife.44706.017
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 = …

https://doi.org/10.7554/eLife.44706.018
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…

https://doi.org/10.7554/eLife.44706.020
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 …

https://doi.org/10.7554/eLife.44706.021
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-/-

https://doi.org/10.7554/eLife.44706.022
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) …

https://doi.org/10.7554/eLife.44706.023
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 …

https://doi.org/10.7554/eLife.44706.025
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.44706.016

Additional files

Download links