vhl mutants display a systemic hypoxic response. (A) From 7 dpf onward, vhl mutants display a significantly increased heart rate. Cardiac output (stroke volume × heart rate) is significally up-regulated 3.2-fold at 4 dpf (139 ± 7.6 vs 43.5 ± 4.1 nL/min) to 15-fold (137.3 ± 17.5 vs 9.4 ± 1.5 nL/min) in 10 dpf vhl^−/− larvae. (B) vhl mutants develop progressive cardiac dilation, as shown by cardiomegaly at 8 dpf (TG(cmlc2:egfp background)). (C) Sectioning (7 μm) through the heart reveals vhl^−/− cardiomyocytes to be stretched (arrow), suggestive of dilated cardiomyopathy. Sibling and mutant hearts are presented with the same magnification (×20). Blood cells are stained with o-dianisidine (brown). (D) As determined by microarray expression profiling of 7 dpf vhl mutants compared with siblings, loss of vhl leads to a general overexpression of hypoxia response genes, which was confirmed by whole-mount in situ hybridizations. (E) This includes the up-regulation of genes involved in oxygen sensing (phd3) and anaerobic metabolism (ndufa4, ldha1) as early as 1 dpf. (F) As shown at 7.5 dpf, glut1 (liver), vegfa (glomerulus, pancreatic islet, brain, and PHT), and its receptors kdr-like and kdr (liver, glomerulus, and blood vessels) are up-regulated. (G) Furthermore, epo (liver and glomerulus) and its receptor epor are highly up-regulated in 7.5 dpf vhl mutants. (H) Western blot analysis of STAT5/phosphoSTAT5 levels at 2 and 4 dpf. At both time points, phosphoSTAT5 levels are highly increased in vhl mutants, whereas STAT5 levels remain comparable with siblings, indicating increased EPO signaling. One lane represents 1.5 embryo equivalents. β-Actin is used as a loading control. Original magnifications, ×5 (B,D-F), ×20 (C). L indicates liver; G, glomerulus; PI, pancreatic islet; and H&E, hematoxylin and eosin.