Antagonistic Activities of Vegfr3/Flt4 and Notch1b Fine-tune Mechanosensitive Signaling during Zebrafish Cardiac Valvulogenesis

Abstract

Highlights d Flt4 is suppressed by fluid shear stress within the atrioventricular valve leaflet d Fluid shear stress activates Notch and Klf2a signaling in an independent manner d The Klf2a and Notch pathways repress Flt4 d Notch and Flt4 work antagonistically to shape the atrioventricular valve

Mechanosensitive Notch-Dll4 and Klf2-Wnt9 signaling pathways intersect in guiding valvulogenesis in zebrafish

“…Also, we do not have a good understanding how this pathway is integrated with the mechanosensitive Klf2 signaling pathway, which is also regulated by blood flow during cardiac valve development ( Donat et al., 2018 ; Goddard et al., 2017 ; Steed et al., 2016 ; Vermot et al., 2009 ). Recent functional studies have demonstrated that Notch activity and Klf2 signaling within the atrioventricular endocardium are not contingent on each other ( Fontana et al., 2020 ). It remains unclear how Notch signaling and the mechanosensitive Klf2 signaling pathway are integrated.…”

“…First, we tested whether the process of Wnt pathway activation was dependent on Klf2. At 54 hpf, in double mutants of klf2a sh317 ( Novodvorsky et al., 2015 ) and klf2b pbb42 ( Fontana et al., 2020 ) (hereafter referred to as klf2 mutants), there was a complete loss of expression from the TCF reporter and ingression of atrioventricular endocardial cells into the cardiac jelly was abolished ( Figures 2 C, 2D, S2 C, S2C′, S2D, and S2D′) (100%, n = 13 embryos analyzed). Instead, the endocardium remained a luminal monolayered tissue.…”

“…Handling of zebrafish was done in compliance with German and Brandenburg state law, carefully monitored by the local authority for animal protection (LUGV, Brandenburg, Germany; Animal protocol #2347-18-2015). The following strains of zebrafish were maintained under standard conditions as previously described ( Westerfield et al., 1997 ): klf2a sh317 ( Novodvorsky et al., 2015 ), klf2b pbb42 ( Fontana et al., 2020 ), wnt9a sd49 ( Grainger et al., 2016 ), Tg( Dll4:Gal4 ) pbb65 (this study), Tg( 5xUAS:EGFP ) nkuasgfp1a ( Asakawa et al., 2008 ), Tg( 7xTCF-Xla.Siam:nls-mCherry ) ia5 ( Moro et al., 2012 ), Tg( EPV.Tp1-Mmu.Hbb:Venus-Mmu.Odc1 ) s940 [here referred to as Tg( TP1:VenusPEST ) s940 ] ( Ninov et al., 2012 ), Tg( kdrl:EGFP ) s843 ( Jin et al., 2005 ), Tg( klf2a:YFP ) mu107 ( Sugden et al., 2017 ), Tg( kdrl:Cre ) s898 ( Bertrand et al., 2010 ), Tg( hsp70l:loxP-STOP-loxP-EGFP-dntcf. ) mu200 ( Hübner et al., 2018 ), Tg( fli1a:Gal4FF ) ubs3 ( Herwig et al., 2011 ), Tg( UAS:klf2a ) ig1 ( Renz et al., 2015 ), TgBAC( nfatc1:GAL4ff ) mu286 ( Pestel et al., 2016 ), and Tg( 5xUAS-E1b:6xMYC-notch1a intra ) kca3 ( Scheer et al., 2002 ).…”

Strong as a Hippo’s Heart: Biomechanical Hippo Signaling During Zebrafish Cardiac Development

“…Trpp2 and Trpv4 play similar roles during the formation of zebrafish atrioventricular valves (Heckel et al, 2015). In the OFT region, blood flow affected the expression of the flow-sensitive Klf2a (Vermot et al, 2009;Heckel et al, 2015;Steed et al, 2016b) and Notch (Samsa et al, 2015;Fontana et al, 2020) signaling pathways, which were strongly active in regions of highest shear stress. Yap1 was expressed in both, endothelial and smooth muscle cells surrounding the OFT.…”

The physiological and pathological functions of VEGFR3 in cardiac and lymphatic development and related diseases

“…A role for Vegfr3 in valvulogenesis has recently been shown by Fontana et al . 141 , 142 during cardiac development in zebrafish. Fluid shear stress acting on endocardial cells lining the atrioventricular valve leaflet independently activates Notch or Klf2a (Krüppel-like Factor 2) signalling and spatial antagonism between the Notch and Vegfr3 pathways defines atrioventricular valve morphology.…”