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Injury-activated endocardium plays structural and signalling roles in zebrafish heart regeneration

ABSTRACT: The zebrafish heart remarkably regenerates after a severe ventricular damage followed by inflammation, fibrotic tissue deposition and removal concomitant with cardiac muscle replacement. We have investigated the role of the endocardium in this regeneration process. 3D-whole mount imaging in injured hearts revealed that GFP-labelled endocardial cells in ET33mi-60A transgenic fish become rapidly activated and highly proliferative at 3 days post cryoinjury (dpci). Endocardial cells extensively expand within the injury site and organize to form a coherent structure at 9 dpci that persists throughout the regeneration process. Upon injury, endocardial cells strongly up-regulate the Notch pathway ligand delta like4 (dll4) and the Notch receptors notch1b, notch2 and notch3. Expression profiling showed that Notch signalling inhibition affects endocardial gene expression and genes related to extracellular matrix remodelling and inflammation. Gain- and loss-of-function experiments revealed that Notch is required for the organization of the endocardium, attenuation of the inflammatory response and cardiomyocyte proliferation. These results demonstrate a novel structural and signalling role for the endocardium during heart regeneration.

ORGANISM(S): Danio rerio

PROVIDER: GSE68650 | GEO | 2017/03/31

SECONDARY ACCESSION(S): PRJNA283297

REPOSITORIES: GEO

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Project description:Ventricular chambers are essential for the rhythmic contraction and relaxation that occurs in every hearbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is still poorly understood. Here we show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation and later coordinates ventricular patterning and compaction with coronary vessel development to give rise to the mature chamber via a temporal sequence of ligand signalling determined by the glycosyltransferase Manic Fringe (Mfng). The early endocardial expression of Mfng favours Dll4-Notch1 signalling, Which induces trabeculation in the developing ventricle.Ventricular maturation and compaction in turn require Mfng and Dll4 downregulation in the endocardium, Which allows myocardial Jag1- And Jag2- Signalling to Notch1 in this tissue.Timely and spatial perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies. Dll4 and Notch1 conditional KOs using Nfact1 and/or Tie2 driven Cre expression: RNA was isolated from pooled whole hearts of 8 (Nfact1) or 9 (Tie2) E9.5 embryos per replicate. Dll4flox;Nfatc1-Cre and WT siblings (4 KO and 4 WT replicates), Notch1flox;Nfatc1-Cre and WT siblings (3 KO and 2 WT replicates), Dll4flox;Tie2-Cre and WT siblings (3 KO and 3 WT replicates). Jag1, Jag2 and Jag1Jag2 conditional KOs using cTnT driven Cre expression: RNA was isolated from pooled heart ventricles of 4 E15.5 embryos per replicate. Jag1flox;cTnT-Cre and WT siblings (3 KO and 3 WT replicates), Jag2flox;cTnT-Cre and WT siblings (3 KO and 2 WT replicates). Jag1flox;jag2flox;cTnT-Cre and WT siblings (3 KO and 2 WT replicates). MFng Gain Of Function using Tie2 driven Cre expression: RNA was isolated from pooled heart ventricles of 4 E15.5 embryos per replicate. MFng;Tie2-Cre and WT siblings (4 GOF and 4 WT replicates). For Dll4, Noth1 and Jag1 KOs, libraries were prepared using the standard Illumina TrueSeq RNASeq library preparation kit and sequenced in a GAIIx Illumina sequencer using a 75bp single end elongation protocol. For Jag2 and Jag1Jag2 KOs and MFng GOF libraries were prepared prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina and sequenced in a HiSeq2500 Illumina sequencer using a 61bp single end elongation protocol

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Injury-activated endocardium plays structural and signalling roles in zebrafish heart regeneration

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