Downstream targets of hif1α are temporally regulated to modulate HSC induction to match nutrient availability. (A) Western blot analysis of whole-embryo homogenates showed increased hif1α levels (top band) following exposure to glucose or pyruvate compared with untreated controls. β-actin is shown as a loading control (bottom band). Normalized quantification is below. (B) Analysis of temporal variation of gene expression from 18 to 36 hpf in glucose-treated embryos normalized pairwise to controls at each time point revealed enhanced induction of erythropoietic genes at early time points, and later induction of genes involved in definitive HSC development in response to glucose (t test vs 18 hpf, *P < .05, n = 3). (C) qPCR analysis indicated that functional hif1a is required to mediate the full effect of glucose exposure on hematopoietic-relevant targets (t test, * vs control, ** vs glucose, P < .05, n = 3). (D-E) Epistasis analysis demonstrated that (D) exogenous VEGF (10μM) could partially rescue a hif1a-MO knockdown on HSCs. (E) Elevations in runx1/cmyb expression mediated by loss of the negative hif1α regulator vhl by MO knockdown can be partially blocked if VEGF signaling is inhibited using SU1498 (10μM, n > 15/tx). (F) qPCR analysis following exposure to increasing glucose concentrations (0.5%-4%) in the fish water revealed a dose-dependent increase in genes involved in vascular and erythroid formation (ANOVA, P < .05, n = 3) at 24 hpf. (G) qPCR at 36 hpf following exposure to increasing glucose concentrations demonstrated increased expression of runx1, cmyb, and hif1α targets associated with definitive hematopoiesis (nos2, igf2, pdgf; ANOVA, P < .05, n = 3).