Zebrafish Cardiac Valve Development Is Affected By Overdose Nitrite Exposure Or Lack Of Endogenous Retinoic Acid Signaling

Cardiac valves, lining in outflow tract (OFT) and atrioventricular canal (AVC), prevent blood flow retrograde within the heart tube of vertebrates. Because of their complicated structures and corresponding long time to develop during embryogenesis, processes of valve development are very sensitive to teratogenetic factors, and morbidity of congenital valve disease occurs in a high level in newborns. Till now, only 20% of the diseases have been attributed to the known causes. Therefore, understanding the effect of environmental and genetic factors on valve morphogenesis is very helpful to prevent and cure congenital cardiac defects.Sodium nitrite, traditionally utilized as a food additive, exists widely not only in the environment, but also in our tissues and organs. In vivo, nitrite is involved in many physiological processes mediated by nitrate-nitrite-NO pathway. It has been revealed that overdose nitrite can lead to toxicological effects on human health; however, the function of nitrite in vertebrate heart valve morphogenesis remains unknown. Herein, we use zebrafish, with special advantages in the research of cardiovascular development, to study the effect of nitrite on early cardiac valve development during vertebrate embryogenesis. Exposed with nitrite, zebrafish embryos exhibited cardiac edema in a dose dependent way, and the major time window of nitrite affecting zebrafish cardaic development was after 36 hpf (hour post fertilization). Comprehensive analysis between 24 hpf to 48 hpf showed that hemodynamics factors such as heartbeat, blood circulation, cardiac function, myocardium and endocardium development were not affected in embryos exposed with overdose nitrite. But after 48 hpf, development of endocardial cells in AVC in nitrite exposure embryos was disrupted, which led to abnormal developmental defect of valve leaflets at 76 hpf Consistently, whole mount in siu hybridization revealed that overdose nitrite diminished expressions of valve progenitor markers including bmp4, has2, versican and notchlb as early as 43 hpf Furthermore, we found that cGMP level, downstream of NO signaling, was increased after overdose nitrite treatment and microinjection of soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) could partly rescue the cardiac valve malformation caused by nitrite. Taken together, our results show that overdose nitrite treatment could affect early valve leaflets formation by producing too much NO signaling.Retinoic acid (RA), an important morphogen, participates in early vertebrate embryogenesis in a gradient way. Previous studies have shown that RA signaling is critical to vertebrate cardiac development; however, physiological role of endogenous RA signaling, or the function of major RA synthesizing enzyme Aldhla2, in cardiac valve formation in AVC has not been explored. In this study, we used aldhla2 morphant to study the physio logical role of endogenous RA signaling in AVC valve morphogenesis. Knockdown of aldhla2 caused cardiac edema and blood cell accumulation at 50 hpf, and developmental defect of primary valve leaflets at 76 hpf in AVC. Comprehensive analysis of aldhla2 morphants between 24 hpf to 50 hpf showed that hemodynamics such as heartbeat, blood circulation, cardiac function, myocardium and endocardium formation was not affected. Whole mount in situ hybridization revealed that expressions of myocardial AVC markers were normal whereas the expressions of adjacent endocardial genes including notchlb and has2 were significantly reduced. Consistently, one of the downstream gene of Notch signaling, cadherin genes such as cdh5 (encoding VE-cadherin), exhibited ectopic expression over endocardial AVC in aldhla2 morphants. As a consequence, endocardial invagination was inactivated at 76 hpf. Time window assay showed the major time period of RA acting on valve formation was between 16 hpf to 26 hpf Addition of RA to aldhla2 morphants could partly rescue the gene expressions in endocardium. Taken together, our date reveals that endogenous RA signal regulates endocardial invagination in AVC and therefore influence valve morphogenesis by Notchl-VE-cadherin pathway.In summary, vertebrate AVC valves development is regulated by both environmental and genetic factors. Overdose nitrite treatment affects zebrafish cardiac valve development by yielding too much NO signaling, and endogenous RA signaling regulates AVC valves through Notchl-VE-cadherin pathway.