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Heart development involves the complex structural remodelling of a linear heart tube into an asymmetrically looped and ballooned organ. Previous studies have associated regional expansion of extracellular matrix (ECM) space with tissue morphogenesis during development. We have developed morphoHeart, a 3D tissue segmentation and morphometry software with a user-friendly graphical interface (GUI) that delivers the first integrated 3D visualisation and multiparametric analysis of both heart and ECM morphology in live embryos. morphoHeart reveals that the ECM undergoes regional dynamic expansion and reduction during cardiac development, concomitant with chamber-specific morphological maturation. We use morphoHeart to demonstrate that regionalised ECM expansion driven by the ECM crosslinker Hapln1a promotes atrial lumen expansion during heart development. Finally, morphoHeart's GUI expands its use beyond that of cardiac tissue, allowing its segmentation and morphometric analysis tools to be applied to z-stack images of any fluorescently labelled tissue. Tools to analyse early heart morphogenesis in detail are limited and morphometric tissue analyses often cannot handle the complexities of the heart. This study develops a computational software called morphoHeart that allows for integrated, three-dimensional analysis of both heart and extracellular matrix morphology in live zebrafish embryos. [ABSTRACT FROM AUTHOR]