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Aims Vertebrate heart development requires the complex morphogenesis of a linear tube to form the mature organ, a process essential for correct cardiac form and function, requiring coordination of embryonic laterality, cardiac growth, and regionalized cellular changes. While previous studies have demonstrated broad requirements for extracellular matrix (ECM) components in cardiac morphogenesis, we hypothesized that ECM regionalization may fine tune cardiac shape during heart development. Methods and results Using live in vivo light sheet imaging of zebrafish embryos, we describe a left-sided expansion of the ECM between the myocardium and endocardium prior to the onset of heart looping and chamber ballooning. Analysis using an ECM sensor revealed the cardiac ECM is further regionalized along the atrioventricular axis. Spatial transcriptomic analysis of gene expression in the heart tube identified candidate genes that may drive ECM expansion. This approach identified regionalized expression of hapln1a, encoding an ECM cross-linking protein. Validation of transcriptomic data by in situ hybridization confirmed regionalized hapln1a expression in the heart, with highest levels of expression in the future atrium and on the left side of the tube, overlapping with the observed ECM expansion. Analysis of CRISPR-Cas9-generated hapln1a mutants revealed a reduction in atrial size and reduced chamber ballooning. Loss-of-function analysis demonstrated that ECM expansion is dependent upon Hapln1a, together supporting a role for Hapln1a in regionalized ECM modulation and cardiac morphogenesis. Analysis of hapln1a expression in zebrafish mutants with randomized or absent embryonic left–right asymmetry revealed that laterality cues position hapln1a-expressing cells asymmetrically in the left side of the heart tube. Conclusion We identify a regionalized ECM expansion in the heart tube which promotes correct heart development, and propose a novel model whereby embryonic laterality cues orient the axis of ECM asymmetry in the heart, suggesting these two pathways interact to promote robust cardiac morphogenesis., Graphical Abstract

This work was supported by BHF project grants (09/287/28051 and 12/12/ 29433) awarded to T.J.A.C. F.J.M.E. was supported by EC-FP7 HEALTHF4-2010-242048. MRC Centre Grant (G0700091) awarded to Professor PW Ingham provided a clinical training fellowship to OJW. Funding for Open Access was provided by he research was funded by the British Heart Foundation. The contribution of blood flow to angiogenesis is incompletely understood. We examined the effect of blood flow on Notch signalling in the vasculature of zebrafish embryos, and whether blood flow regulates angiogenesis in zebrafish with constitutively up-regulated hypoxic signalling. Developing zebrafish (Danio rerio) embryos survive via diffusion in the absence of circulation induced by knockdown of cardiac troponin T2 or chemical cardiac cessation. The absence of blood flow increased vascular Notch signalling in 48 h post-fertilization old embryos via up-regulation of the Notch ligand dll4. Despite this, patterning of the intersegmental vessels is not affected by absent blood flow. We therefore examined homozygous vhl mutant zebrafish that have constitutively up-regulated hypoxic signalling. These display excessive and aberrant angiogenesis from 72 h post-fertilization, with significantly increased endothelial number, vessel diameter, and length. The absence of blood flow abolished these effects, though normal vessel patterning was preserved. We show that blood flow suppresses vascular Notch signalling via down-regulation of dll4. We have also shown that blood flow is required for angiogenesis in response to hypoxic signalling but is not required for normal vessel patterning. These data indicate important differences in hypoxia-driven vs. developmental angiogenesis. Publisher PDF