Dissecting the Links between Morphology and ECM Dynamics in the Developing Zebrafish Heart

During development, the heart tube comprises two cellular layers, an outer-myocardium and an inner-endocardium, between which lies a layer of extracellular matrix (ECM). This cardiac ECM creates specialised extracellular environments that mediate the chemical and biomechanical signals that shape the heart tube into a three-dimensional organ. Visualising and linking ECM and tissue morphology is challenging since sample processing may impact matrix hydration and tissue structure. To understand the spatiotemporal dynamics of the ECM and its role in cardiac morphogenesis, I developed morphoHeart, an image analysis tool that allows the 3D segmentation of the heart layers -including the ECM- from live embryos. Using morphoHeart, I identified an early left-right axis of ECM-regionalisation during development. To understand if defects in heart tube lateralisation result in abnormal ECM-regionalisation and disrupted morphogenesis, I investigated zebrafish hearts in which spaw (zebrafish homolog of Nodal responsible for left-right laterality) is disrupted. Analysis of cardiac ECM distribution in spaw mutants identified that irrespective of looping direction, the ECM asymmetry in the tube translates into a highly regionalised ECM in the atrial outer-curvature at looping stages, suggesting a possible link between the axis of ECM asymmetry and the direction of looping and chamber ballooning. In situ hybridisation and immunofluorescence assays identified regionalised expression/deposition of the hyaluronan and proteoglycan link protein 1a (hapln1a) in zebrafish at tube stage, overlapping the ECM expansion and suggesting Hapln1a protein may be involved in its modulation. Analysis of hapln1a promoter mutants, which lack hapln1a transcript, revealed that despite the defects in atrial ECM expansion, the left-sided atrial ECM asymmetry is reduced yet maintained, suggesting hapln1a is not the only component involved in its establishment. Together I propose a model whereby laterality cues orient an early asymmetric cardiac ECM that, mediated by Hapln1a‘s protective cross-linking properties, intrinsically sets an axis for chamber growth and looping morphogenesis to occur, ensuring robust dextral cardiac morphogenesis.