VE-cadherin is essential for endothelial cell-cell interaction and barrier integrity in vitro. Less is known about the in vivo role of this protein in cardiac and vascular development. Mice deficient in VE-cadherin manifest defective cardiac development, but it is unclear whether these defects are primarily due to the loss of VE-cadherin, or secondary to the concomitant severe vascular defects. In this study, we find that VE-cadherin knockdown does not directly affect vessel development, yet leads to profound structural and functional cardiac defects in zebrafish embryos.
Knockdown of VE-cadherin using a splice-blocking morpholino has been recently demonstrated 
, however no specific analysis of the effects on cardiac development was documented. In zebrafish, VE-cadherin is identified by 12 hpf in the anterolateral mesoderm as the vasculature begins to form, and in the heart field by 16 hpf 
. Despite this early expression, our analysis of VE-cadherin knockdown using both translation and splice-site morpholinos, noted no cardiac or vascular developmental differences when compared to controls until 32 hpf. This discrepancy is consistent with the observation that loss or truncation of the intracytoplasmic domain of VE-cadherin in mouse also demonstrated normal primary cardiac formation 
. Recently, Bussmann used a VE-cad in situ
hybridization to characterize the origin and migration of endocardial precursors as separate from nkx2.5
labeled myocardial precursors 
. Our results, coupled with the mouse knockout data, demonstrate that while VE-cadherin is present in this cell population, it is not required for endocardial cell differentiation and migration or cardiac tube formation.
Mouse knockout of VE-cadherin demonstrates normal cardiac chamber differentiation despite endocardial detachment at embryo day 8.5–8.75 
. The role of VE-cadherin in modulating further cardiac development is unclear however, as looping fails in the setting of endocardial detachment, vascular collapse and absent flow. In zebrafish, despite appropriate vascular sprouting and initial blood flow, the loss of VE-cad still profoundly impairs cardiac looping and function. The decreased electron density of the cardiac jelly after VE-cad MO injection, coupled with the defective endocardial barrier may allow equilibration of water and solutes between the cardiac jelly and endovascular compartment. These changes may decrease the effectiveness of myocardial contraction in driving circulation, and may explain the persistent, prominent separation of two layers seen from 32 hpf to beyond 96 hpf. Increased endothelial permeability due to the loss of VE-cad may also be responsible for the onset of pericardial edema, further impeding effective circulation.
Zebrafish carrying mutant alleles of several atrial, ventricular and structural proteins also show normal early cardiac formation but develop structural abnormalities by 48 hpf, similar to our VE-cad knockdown. Weak atrium
) mutants are deficient in the atrial myosin heavy chain (amhc
), but despite minimal atrial contraction, develop normally past 36 hours 
. By 48 hours however, embryos demonstrate atrial dilation and blood pooling in the sinus venosus. In contrast to VE-Cad knockdown embryos, some of these are capable of growth to maturity, and lack massive pericardial edema. Morpholino knockdown of the cardiac-restricted Leucine Rich Repeat Containing Protein 10 (LRRC 10) produces normal development until 2 dpf, when cardiac looping failure and pericardial edema were noted 
. Also similar to loss of VE-cad, circulation progressively decreased in LRRC 10 knockdown embryos, assessed by intersegmental artery perfusion. The loss of VE-cad decreases cardiac proliferation; however, as demonstrated by electron microscopy, the contractile bundles of the myocardial layer are preserved. This further supports the argument that altered fluid dynamics across the endocardium and cardiac jelly, not a specific developmental failure of myocardial elements, may impede cardiac looping and function. Previous work by Hove et al. demonstrated that changes in fluid forces alone profoundly affect cardiac looping and lead to arrest in zebrafish, also supporting our proposed mechanism 
In addition to this potential role in cardiac chamber looping, VE-cadherin is prominent in the region of the endocardial cushions in the developing zebrafish 
. This is not surprising, given the endocardial cell role in the epithelial-mesenchymal transformation (EMT) required for valve formation 
. After knockdown of VE-cad, no differences were noted in the gross structure or histology of the AV valve. Despite the circulatory failure produced by knockdown, the inter-chamber toggling of erythrocytes typical of embryos with valvular defects such as the jekyll
are not seen. Our results demonstrating that VE-cadherin is not necessary for early valve formation are consistent with in vitro
data showing that Jagged1/Notch induced EMT in cultured human endothelial cells produces a down-regulation of VE-cad 
In conclusion, the knockdown of VE-Cadherin using an antisense oligonucleotide produces early circulatory arrest and cardiac looping failure in the embryonic zebrafish due to increased endocardial permeability, without affecting gross peripheral vascular development. This robust, reproducible system has potential to yield insight into the complex signaling role of VE-Cadherin in early cardiovascular development.