2009 Southern Regional Meeting Abstracts

Purpose of Study: VE-Cadherin (VE-Cad) is a structural protein that has demonstrated significance in angiogenesis, although its precise role in vivo remains unclear. Our goal was to create a robust in vivo VE-Cad knockdown model using zebrafish, and to subsequently evaluate the development, function, and structure of the cardiovascular system in the absence of VE-Cad.
Methods Used: An antisense morpholino targeting the translational start site of VE-cad mRNA (VE-Cad MO) was synthesized, and 2 nL of 0.5 mM VE-cad MO or a standard control morpholino was injected into the yolk of single-cell stage embryos. After injection, embryos were observed to quantify cardiovascular phenotype. Measurements of circulation, pericardial edema, and cardiac folding were made. At 24 hour intervals, both heart rate and perfusion were measured. Number of erythrocytes passing through the two most distal patent arteries was counted to assess perfusion. To measure the endocardial/myocardial separation, injected fli1:GFP transgenic embryos were used (GFP+ endocardium, GFP- myocardium).
Summary of Results: Knockdown of VE-Cad results in failure for circulation to initialize, pericardial edema, and absent cardiac folding, resulting in cardiovascular failure and embryonic mortality between 72-96 hpf. There was also a statistically significant increase in separation between the endocardial and myocardial elements in the VE-cad MO injected fish. Neither heart tube formation, primitive hematopoiesis, nor vascular sprouting are affected by VE-cad knockdown.
Conclusions: In conclusion, the knockdown of VE-Cad produces early circulatory arrest, pericardial edema, and cardiac looping failure in embryonic zebrafish without affecting vascular development. This robust, reproducible system has potential to yield insight into the complex signaling role of VE-Cadherin in early cardiac development. The zebrafish knockdown model allows manipulation of VE-Cad in the living organism with reproducible, easily identifiable effects. Its potential applications include in vivo analysis of VE-Cad signaling, endocardial/myocardial interaction, structure/function assays, and screening for agents which affect VE-Cad signaling.