The early embryonic heart is a multi-layered tube consisting of (1) an outer myocardial tube; (2) an inner endocardial tube; and (3) an extracellular matrix layer interposed between the myocardium and endocardium, called \"cardiac jelly\" (CJ). During the past decades, research on CJ has mainly focused on its molecular and cellular biological aspects. This review focuses on the morphological and biomechanical aspects of CJ. Special attention is given to (1) the spatial distribution and fiber architecture of CJ; (2) the morphological dynamics of CJ during the cardiac cycle; and (3) the removal/remodeling of CJ during advanced heart looping stages, which leads to the formation of ventricular trabeculations and endocardial cushions. CJ acts as a hydraulic skeleton, displaying striking structural and functional similarities with the mesoglea of jellyfish. CJ not only represents a filler substance, facilitating end-systolic occlusion of the embryonic heart lumen. Its elastic components antagonize the systolic deformations of the heart wall and thereby power the refilling phase of the ventricular tube. Non-uniform spatial distribution of CJ generates non-circular cross sections of the opened endocardial tube (initially elliptic, later deltoid), which seem to be advantageous for valveless pumping. Endocardial cushions/ridges are cellularized remnants of non-removed CJ.

The molecular mechanism essential for the formation of heart valves involves complex interactions of signaling molecules and transcription factors. The Mediator Complex (MC) functions as multi-subunit machinery to orchestrate gene transcription, especially for tissue-specific fine-tuning of transcriptional processes during development, also in the heart. Here, we analyzed the role of the MC subunit Med12 during atrioventricular canal (AVC) development and endocardial cushion formation, using the Med12-deficient zebrafish mutant trapped (tpd). Whereas primary heart formation was only slightly affected in tpd, we identified defects in AVC development and cardiac jelly formation. We found that although misexpression of bmp4 and versican in tpd hearts can be restored by overexpression of a modified version of the Sox9b transcription factor (harboring VP16 transactivation domain) that functions independent of its co-activator Med12, endocardial cushion development in tpd was not reconstituted. Interestingly, expression of tbx2b and its target hyaluronan synthase 2 (has2) - the synthase of hyaluronan (HA) in the heart - was absent in both uninjected and Sox9b-VP16 overexpressing tpd hearts. HA is a major ECM component of the cardiac jelly and required for endocardial cushion formation. Furthermore, we found secreted phosphoprotein 1 (spp1), an endocardial marker of activated AV endocardial cells, completely absent in tpd hearts, suggesting that crucial steps of the transformation of AV endocardial cells into endocardial cushions is blocked. We demonstrate that Med12 controls cardiac jelly formation Sox9-independently by regulating tbx2b and has2 expression and therefore the production of the glycosaminoglycan HA at the AVC to guarantee proper endocardial cushion development.

Atrial natriuretic peptide (nppa/anf) and brain natriuretic peptide (nppb/bnp) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy; however, their genomic location in cis has impeded formal analysis. Using genome editing, we have generated mutants for nppa and nppb, and found that single mutants were indistinguishable from wild type, whereas nppa/nppb double mutants displayed heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4, tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirmed cardiac jelly expansion in nppa/nppb double mutants. Finally, bmp4 knockdown rescued the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber.

Embryonic development is an exceptionally dynamic process, requiring a provisional extracellular matrix that is amenable to rapid remodeling, and proteolytic or non-proteolytic mechanisms that can remodel the major components of this matrix. Versican is a chondroitin-sulfate proteoglycan that forms highly hydrated complexes with hyaluronan and is widely distributed in the provisional matrix of mammalian embryos. It has been extensively studied in the context of cardiovascular morphogenesis, neural crest cell migration and skeletal development. Analysis of Vcan transgenic mice has established the requirement for versican in cardiac development and its role in skeletogenesis. The ADAMTS family includes several versican-degrading proteases that are active during remodeling of the embryonic provisional matrix, especially during sculpting of versican-rich tissues. Versican is cleaved at specific peptide bonds by ADAMTS proteases, and the cleavage products are detectable by neo-epitope antibodies. Myocardial compaction, closure of the secondary palate (in which neural crest derived cells participate), endocardial cushion remodeling, myogenesis and interdigital web regression are developmental contexts in which ADAMTS-mediated versican proteolysis has been identified as a crucial requirement. ADAMTS proteases are expressed coordinately and function cooperatively in many of these contexts. In addition to versican clearance, ADAMTS proteases generate a bioactive versican fragment containing the N-terminal G1 domain, which we have named versikine. This review promotes the view that the embryonic extracellular matrix has evolved not only to provide a permissive environment for embryo growth and morphogenesis, but through its dissolution to influence and regulate cellular processes.