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Shear stress activation of Notch signaling to promote trabeculation.
4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation
Paper
2016
The Journal of Clinical Investigation |
DOI
Background
This study integrates 4D selective plane illumination microscopy (SPIM) with a synchronization algorithm to image zebrafish cardiac morphogenesis and test how hemodynamic shear stress modulates trabeculation. Reducing blood viscosity with gata1a morpholino, arresting contraction with tnnt2a morpholino, or using weak atrium mutants lowered shear stress, downregulated Notch signaling, and attenuated trabeculation. Endocardial Notch activation was localized using Tg(flk:mCherry;tp1:gfp) fish. Neuregulin1 mRNA rescued trabeculation and contractile function. Pulsatile shear stress upregulated Notch signaling in endothelial cells, and an ADAM10 inhibitor blocked this response, linking shear stress to endocardial Notch-dependent trabeculation.
Key Findings
4D SPIM captured trabecular initiation in zebrafish from ~50 to 70 hpf. gata1a MO reduced hematopoiesis/viscosity, delayed trabeculation, and downregulated Dll4, Jag1, Jag2, Notch1b, Nrg1, and ErbB2. tnnt2a MO and wea mutants reduced shear stress, downregulated Notch-related genes, and attenuated or abolished trabeculation. clo mutants lacking endocardium developed nontrabeculated ventricles with reduced Notch signaling. Crossbreeding Tg(flk:mCherry) with Tg(tp1:gfp) localized shear-activated Notch to endocardium. Nrg1 mRNA coinjection restored trabeculation, upregulated Notch-related genes, and improved ventricular strain, fractional shortening, and ejection fraction. In HAEC, pulsatile shear stress upregulated Notch ligands and target genes, which was blocked by an ADAM10 inhibitor. EPO-augmented viscosity and isoproterenol did not increase trabeculation, while high-dose Nrg1 caused abnormal ventricular morphogenesis. Trabecular myocardium exhibited higher local strain, and loss of trabeculation reduced contractile function.
Contribution
Creative Biogene provided the zebrafish EPO cDNA/donor plasmid that was cloned into pCS2+ and used to synthesize EPO mRNA. This reagent enabled experiments increasing hematopoiesis and blood viscosity to test whether augmented shear stress alters Notch signaling and cardiac trabeculation.
This study integrates 4D selective plane illumination microscopy (SPIM) with a synchronization algorithm to image zebrafish cardiac morphogenesis and test how hemodynamic shear stress modulates trabeculation. Reducing blood viscosity with gata1a morpholino, arresting contraction with tnnt2a morpholino, or using weak atrium mutants lowered shear stress, downregulated Notch signaling, and attenuated trabeculation. Endocardial Notch activation was localized using Tg(flk:mCherry;tp1:gfp) fish. Neuregulin1 mRNA rescued trabeculation and contractile function. Pulsatile shear stress upregulated Notch signaling in endothelial cells, and an ADAM10 inhibitor blocked this response, linking shear stress to endocardial Notch-dependent trabeculation.
Key Findings
4D SPIM captured trabecular initiation in zebrafish from ~50 to 70 hpf. gata1a MO reduced hematopoiesis/viscosity, delayed trabeculation, and downregulated Dll4, Jag1, Jag2, Notch1b, Nrg1, and ErbB2. tnnt2a MO and wea mutants reduced shear stress, downregulated Notch-related genes, and attenuated or abolished trabeculation. clo mutants lacking endocardium developed nontrabeculated ventricles with reduced Notch signaling. Crossbreeding Tg(flk:mCherry) with Tg(tp1:gfp) localized shear-activated Notch to endocardium. Nrg1 mRNA coinjection restored trabeculation, upregulated Notch-related genes, and improved ventricular strain, fractional shortening, and ejection fraction. In HAEC, pulsatile shear stress upregulated Notch ligands and target genes, which was blocked by an ADAM10 inhibitor. EPO-augmented viscosity and isoproterenol did not increase trabeculation, while high-dose Nrg1 caused abnormal ventricular morphogenesis. Trabecular myocardium exhibited higher local strain, and loss of trabeculation reduced contractile function.
Contribution
Creative Biogene provided the zebrafish EPO cDNA/donor plasmid that was cloned into pCS2+ and used to synthesize EPO mRNA. This reagent enabled experiments increasing hematopoiesis and blood viscosity to test whether augmented shear stress alters Notch signaling and cardiac trabeculation.
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