A great deal of information on the effects of retinoids on heart development has been gathered from ectopic application of excess retinoids to embryos (6
,31 ,32) but physiologically more relevant are the experimental approaches that diminish or eliminate vitamin A function. Maternal insufficiency of vitamin A during pregnancy results in fetal death or severe abnormalities in the offspring, including abnormal heart development; many of the heart defects have been recapitulated in fetuses generated from various combinations of retinoid receptor knockouts. Heart abnormalities obtained include a thin-walled dilated heart cavity, abnormalities in ventricular chambers and defects in the outflow tract (3 ,4 ,6 ,18 19 20 ,22 ,25 ,31) . Important information has also been obtained by molecular analysis (9 ,18 19 20) , but the primary retinoid targets have not been identified. The phenotypes obtained may reflect secondary lesions and abnormalities due to RXR functions with other nuclear receptors (4 ,9) . In contrast, the quail embryo retinoid ligand knockout model allows an analysis of developmental regulation solely attributable to vitamin A and reveals the full function of this vitamin. The model has an advantage for studies of vitamin A function during early precardiac and subsequent heart-forming stages by "rescue" manipulations to restore normal gene expression and cardiogenesis. The VAD cardiac phenotype in this avian model is highly reproducible and, together with molecular analysis, allows us to draw certain conclusions about the initial cardiogenic events regulated by vitamin A (3 ,6 ,21 22 23) . The developing heart is a grossly abnormal, thin-walled, dilated and distended structure, without chambers, but it contracts until the embryo dies. This is not surprising because the expression of early cardiogenic genes that regulate heart precursor cell differentiation into cardiomyocytes is not affected by the lack of vitamin A (22) . In contrast, exogenous treatments with RA induce differentiation of precursor cells into cardiomyocytes (31) .
The concept of a specific retinoid function in axial specification has a long history, originating with the identification of RAR responsive elements in some of the evolutionarily conserved axial patterning genes (33) . It has been perpetuated by results from numerous studies in which exogenous RA applied to embryos of various species at various stages of development affects the specification of body axes, heart asymmetry and limb patterning (5 ,8 ,31 32 33) . These studies suggested a specific role for RA in heart asymmetry determination (23 ,31 ,32) . However, recent evidence points to vitamin A having a general rather than a specific role, i.e., it appears that vitamin A is required to provide a proper environment for the expression of adequate levels of heart asymmetry genes (23) . This concept is supported by evidence from the literature that the generation and distribution of RA in the embryo as well as the expression patterns of vitamin A metabolism enzymes and retinoid receptors are symmetric (7 ,11 12 13 14 ,21) .
A major developmental defect that may be linked directly to the early embryolethality of the VAD quail embryo is the absence of a cardiac inflow tract (3 ,6 ,22) , i.e., the VAD heart has no opening at its caudal end where the extraembryonal blood vessels converge into vitelline veins to deliver blood to the embryonic heart for distribution to the embryo. This defect has not been reported in any of the mammalian in vivo models addressing vitamin A function during embryogenesis. The formation of the cardiac inflow tract may be linked to the expression of the retinoid-regulated cardiac transcription factor GATA-4 in the posterior heart–forming area where this gene is involved in a BMP2 pathway that specifies the endodermal structures of the heart and the underlying foregut primordia, and where apoptosis is observed in the VAD embryo (34) . In these embryos, the extraembryonal vascular networks are sparse and fail to converge at the level of the cardiac inflow tract (35 ; Fig. 1 ). Defects in vitelline vessel formation have also been observed in cultured mouse embryos when the transfer of retinol from the yolk-sac to the embryo is prevented (36) . Anomalies in vasculogenesis have not been reported in retinoid receptor knockout mice, but may have contributed to embryolethality.
Figure 1. Vascular development in the 36–38 h normal and vitamin A–deficient (VAD) quail embryo. In the normal embryo, well-formed vascular networks (arrowhead) converge into vitelline veins at the cardiac inflow tract (black arrows); the VAD embryo has sparse, disorganized vascular networks and poorly developed vitelline veins. Heart (large arrows) has begun to loop in the normal embryo but is abnormal in the VAD embryo. Note the distorted head and short body in the VAD embryo. All views are ventral
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