Embryonic Ethanol Exposure Dysregulates BMP and Notch Signaling, Leading to Persistent Atrio-Ventricular Valve Defects in Zebrafish

Abstract

Fetal alcohol spectrum disorder (FASD), birth defects associated with ethanol exposure in utero, includes a wide spectrum of congenital heart defects (CHDs), the most prevalent of which are septal and conotruncal defects. Zebrafish FASD model was used to dissect the mechanisms underlying FASD-associated CHDs. Embryonic ethanol exposure (3–24 hours post fertilization) led to defects in atrio-ventricular (AV) valvulogenesis beginning around 37 hpf, a morphogenetic event that arises long after ethanol withdrawal. Valve leaflets of the control embryos comprised two layers of cells confined at the compact atrio-ventricular canal (AVC). Ethanol treated embryos had extended AVC and valve forming cells were found either as rows of cells spanning the AVC or as unorganized clusters near the AV boundary. Ethanol exposure reduced valve precursors at the AVC, but some ventricular cells in ethanol treated embryos exhibited few characteristics of valve precursors. Late staged larvae and juvenile fish exposed to ethanol during embryonic development had faulty AV valves. Examination of AVC morphogenesis regulatory networks revealed that early ethanol exposure disrupted the Bmp signaling gradient in the heart during valve formation. Bmp signaling was prominent at the AVC in controls, but ethanol-exposed embryos displayed active Bmp signaling throughout the ventricle. Ethanol exposure also led to mislocalization of Notch signaling cells in endocardium during AV valve formation. Normally, highly active Notch signaling cells were organized at the AVC. In ethanol-exposed embryos, highly active Notch signaling cells were dispersed throughout the ventricle. At later stages, ethanol-exposed embryos exhibited reduced Wnt/β-catenin activity at the AVC. We conclude that early embryonic ethanol exposure alters Bmp, Notch and other signaling activities during AVC differentiation leading to faulty valve morphogenesis and valve defects persist in juvenile fish.

Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function

“…Developmental timing of chemical exposure can be tightly controlled to examine stage-specific exposure effects; a simple dissecting microscope can provide information on cardiac edema; transparent embryos with transgene expression in the heart that allows for the use of stereomicroscope to quickly evaluate heart formation defects; and advanced imaging facilitates detail analysis at the cellular level (Figure 1A–I). Studies have recapitulated cardiac defects in zebrafish similar to those seen in human patients due to prenatal exposure of various teratogens [34,35,36]. High fecundity, rapid development outside the mother’s body, and well-characterized cardiogenesis stages combined with transparency enabling non-invasive, whole animal imaging make the zebrafish embryo an ideal model system for cardiac teratogenicity screens.…”

“…Ethanol withdrawal long before endocardial cushion formation led to valve defects. Following this study, Sarmah and colleagues combined state-of-the-art zebrafish transgenic lines that label myocardium and endocardium with various cardiac regulatory signaling reporter lines including Bmp, Notch, and Wnt signaling reporters, marker staining, and advanced microscopy to gain mechanistic insight into the etiology of ethanol-induced atrioventricular valve defects [36]. This study showed that ethanol exposure reduced Bmp signaling during early heart developmental stages.…”

“…Those ethanol-exposed embryos showed aberrant valve formation during development and defective valves at the juvenile stage (Figure 2C–F). Valve leaflets were smaller, irregularly shaped, and did not closely correspond with each other to allow complete closure of the atrioventricular junction (Figure 2E,F) [36]. …”

“…Chronic alcohol exposure until heart tube formation not only led to a malformed heart but also led to minor tachycardia, suggesting altered heart function due to prenatal alcohol exposure [36]. …”

Asymmetry at cell-cell interfaces direct cell sorting, boundary formation, and tissue morphogenesis

“…Along with bone morphogenic protein signaling, proper localization of Notch signaling at the atrio-ventricular canal is necessary for cardiac valve morphogenesis. Changes in the distribution of Notch within this tissue compartment causes faulty valve development [79]. …”