Figure 1. Zebrafish coronary vessels develop during juvenile stages and respond to increased heart size.

Confocal images of whole larval, juvenile and adult double transgenic hearts, Tg(fli1a:EGFP; myl7:DsRed2-NLS (A–D) and Tg(fli1a:EGFP; kdrl:mCherry) (EG). flila:EGFP-positive endothelial (endocardial) cells are visible only on the inside of the myocardium (labeled with myl7:DsRed2-NLS) and bulbous arteriosus after hatching (A and inset 14 dpf and B, 36 dpf). Five to seven weeks later, endothelial cells are found on the surface of the heart (C, 49 dpf, arrowhead and inset), forming a vessel connection between the gills and AV-boundary after one month (D, 56 dpf, arrowheads and inset). The major vessel over the bulbous arteriosus expresses high levels of kdrl:mCherry (marked by arrowhead in G). Branches from these early vessels appear to progressively cover and interconnect over the juvenile heart into adulthood; however, the majority express low kdrl:mCherry levels (E, 70 dpf to G, 133 dpf, insets). The emergence and coverage of these vessels is stochastic, but correlates strongly with age and the area of the ventricle (H). Endocardial cells prior to the emergence of vessels express arterial marker dll4:EGFP (I). Emerged endothelial cells on the ventricle surface also have an arterial identity (J, arrowhead and inset), which is selectively down-regulated in some vessels as they form (K, arrow). A subset of vessels maintain dll4:EGFP (L, arrowhead) and express flt1:tdTomato confirming they are arteries (M, arrowhead). Vessels which down-regulate arterial makers appear to not progress to full venous identity as flt4:YFP expression is not observed at significant levels (N). a, atrium; av, AV canal; v, ventricle. Zebrafish age listed as days post fertilization (dpf) or italicized total body lengths with equivalent age in brackets when zebrafish were raised under non-standard conditions. Asterisk denotes artery. Scale bars, 50 µm except for that in inset A, which is 10 µm.