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 regionalised cellular changes. While previous studies have demonstrated broad requirements for extracellular matrix (ECM) components in cardiac morphogenesis, we hypothesised that ECM regionalisation 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 regionalised 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 regionalised expression of hapln1a, encoding an ECM cross-linking protein. Validation of transcriptomic data by in situ hybridisation confirmed regionalised 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 regionalised ECM modulation and cardiac morphogenesis. Analysis of hapln1a expression in zebrafish mutants with randomised or absent embryonic left-right asymmetry revealed that laterality cues position hapln1a-expressing cells asymmetrically in the left side of the heart tube. Conclusions We identify a regionalised 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. Translational Perspective This study reveals that the cardiac ECM exhibits regional specialisation required for heart morphogenesis, and sheds light on how embryonic left-right asymmetry acts in concert with ECM regionalisation to fine tune heart shape. This work can help us understand the origins of congenital heart defects, and in particular the nature of morphological heart abnormalities in patients with heterotaxia-associated heart malformations. Furthermore, recent studies suggest the ECM is a key regulator of regenerative potential in the heart, thus defining how distinct ECM composition impacts upon heart form and function has implications for developing regenerative therapies in the future.

16The mature vertebrate heart develops from a simple linear cardiac tube during early 17 development through a series of highly asymmetric morphogenetic processes including cardiac 18 looping and chamber ballooning. While the directionality of heart morphogenesis is partly 19 controlled by embryonic laterality signals, previous studies have suggested that these extrinsic 20 laterality cues interact with tissue-intrinsic signals in the heart to ensure robust asymmetric 21 cardiac morphogenesis. Using live in vivo imaging of zebrafish embryos we describe a left-22 sided, chamber-specific expansion of the extracellular matrix (ECM) between the myocardium 23 and endocardium at early stages of heart morphogenesis. We use Tomo-seq, a spatial 24 transcriptomic approach, to identify transient and regionalised expression of hyaluronan and 25 2 proteoglycan link protein 1a (hapln1a), encoding an ECM cross-linking protein, in the heart 26 tube prior to cardiac looping overlapping with regionalised ECM expansion. Loss-and gain-27 of-function experiments demonstrate that regionalised Hapln1a promotes heart morphogenesis 28 through regional modulation of ECM thickness in the heart tube. Finally, we show that while 29 induction of asymmetric hapln1a expression is independent of embryonic left-right 30 asymmetry, these laterality cues are required to orient the hapln1a-expressing cells 31 asymmetrically along the left-right axis of the heart tube. 32Together, we propose a model whereby laterality cues position hapln1a expression on the left 33 of the heart tube, and this asymmetric Hapln1a deposition drives ECM asymmetry and 34 subsequently promotes robust asymmetric cardiac morphogenesis. 35 36 in driving rightward looping of the linear heart tube in multiple organisms (Levin et al. 1995; 51 Lowe et al. 1996;Long 2003;Brennan et al. 2002;Toyoizumi et al. 2005). However, while 52 embryos with defective asymmetric Nodal signalling display disrupted directionality of heart 53 looping, the heart still undergoes looping morphogenesis (Noël et al. 2013; Brennan et al. 54 2002). This indicates that while extrinsic asymmetric cues provide directional information to 55 the heart, regionalised intrinsic signals help to promote asymmetric morphogenesis. Supporting 56

During heart development, the embryonic ventricle becomes enveloped by the epicardium, a layer of mesothelium which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall, projecting into the cardiac lumen to begin building the muscle mass necessary for adult cardiac function. Lethal(2) giant larvae homolog 1 (Llgl1) regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation, including trabeculation and epicardial establishment. We found thatllgl1mutant zebrafish embryos exhibit aberrantly positioned cardiomyocytes during early trabeculation, some of which extrude apically into the pericardial space. While investigating apical cardiomyocyte extrusion we identified a basal to apical shift in laminin deposition in the ventricular wall. Initially laminin deposition occurs on the luminal (basal) surface of the heart but concomitant with the onset of trabeculation basal laminin is removed and is instead deposited on the exterior (apical) surface of the ventricle. We find that epicardial cells express several laminin subunits as they adhere to the ventricular wall, and show that the epicardium is required for laminin deposition on the ventricular surface. Inllgl1mutants the timing of the basal-apical laminin shift is delayed, in line with a delay in establishment of the epicardial layer. Analysis of earlier epicardial development reveals that while both Llgl1 and laminin are not required for specification of the proepicardial organ, they are instead required for dissemination of epicardial cells to the ventricular surface. Together our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the myocardial wall during early trabeculation.