The Delta-Notch signaling pathway plays a central role in the development of most vertebrate organs. The Hey family of bHLH transcription factors are direct targets of Notch signaling. Loss of Hey2 in the mouse leads to cardiac defects with high postnatal lethality. We have now generated a mouse Hey1 knockout that has no apparent phenotypic defect. The combined loss of Hey1 and Hey2, however, results in embryonic death after embryonic day 9.5 (E9.5) with a global lack of vascular remodeling and massive hemorrhage. Initial vasculogenesis appears unaffected, but all subsequently developing major vessels in the embryo and yolk sac are either small or absent. Furthermore, the placental labyrinth completely lacks embryonic blood vessels. Similar vascular defects are observed in Jagged1 and Notch1 knockout mice. In the latter we found Hey1 and Hey2 expression in yolk sacs to be strongly reduced. Remaining large arteries in both Notch1 and Hey1/Hey2 knockout mice fail to express the arterial endothelial markers CD44, neuropilin1, and ephrin-B2. This indicates that Hey1/Hey2 are essential transducers of Notch signals in cardiovascular development that may mediate arterial cell fate decision.[Keywords: Notch pathway; angiogenesis; vasculogenesis; arteriogenesis; Hey1; Hey2] Supplemental material is available at http://www.genesdev.org.

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The Hey basic helix-loop-helix transcription factors are downstream effectors of Notch signaling in the cardiovascular system. Mice lacking Hey2 develop cardiac hypertrophy, often associated with congenital heart defects, whereas combined Hey1/Hey2 deficiency leads to severe vascular defects and embryonic lethality around embryonic day E9.5. The molecular basis of these disorders is poorly understood, however, since target genes of Hey transcription factors in the affected tissues remain elusive. To identify genes regulated by Hey factors we have generated a conditional Hey1 knockout mouse. This strain was used to generate paired Hey2-and Hey1/2-deficient embryonic stem cell lines. Comparison of these cell lines by microarray analysis identified GATA4 and GATA6 as differentially expressed genes. Loss of Hey1/2 leads to elevated GATA4/6 and ANF mRNA levels in embryoid bodies, while forced expression of Hey factors strongly represses expression of the GATA4 and GATA6 promoter in various cell lines. In addition, the promoter activity of the GATA4/6 target gene ANF was inhibited by Hey1, Hey2, and HeyL. Protein interaction and mutation analyses suggest that repression is due to direct binding of Hey proteins to GATA4 and GATA6, blocking their transcriptional activity. In Hey2-deficient fetal hearts we observed elevated mRNA levels of ANF and CARP. Expression of ANF and Hey2 is normally restricted to the trabecular and compact myocardial layer, respectively. Intriguingly, loss of Hey2 leads to ectopic ANF expression in the compact layer, suggesting a direct role for Hey2 in limiting ANF expression in this cardiac compartment.

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Abstract.To define the role of protein kinase C (PKC) in oxygen-dependent production of erythropoietin (EPO) in the liver, we have determined EPO messenger ribonucleic acid (mRNA) expression in primary cultures of juvenile rat hepatocytes incubated at different oxygen tensions in the absence and presence of phorbol esters, vasopressin, and structurally different kinase inhibitors. Upon reduction of oxygen concentrations from 40% to 3% EPO mRNA in cultured hepatocytes increased markedly within 1.25 h, reached maximal values after 2.5 h and remained elevated for up to 72 h. Treatment of hepatocytes during 1.25-5 h of hypoxic exposure with phorbol 12-myristate-13 acetate (PMA) attenuated hypoxiainduced EPO mRNA levels dose-dependently by a maximum of approximately 50%. This inhibitory effect of PMA disappeared upon treatment for more than 5 h and was completely lost after incubation for 9 and 18 h in the presence of 10 -6 M and 10 -7 M PMA, respectively. Phorbol 12,13-dibutyrate and vasopressin also inhibited EPO mRNA accumulation, whereas 4 alpha-phorbol 12,13-didecanoate was ineffective. Western blot analysis of PKC isozymes revealed the presence of PKC alpha, beta II, delta, epsilon and zeta and provided no evidence that the PMA-induced inhibition of EPO expression was associated with depletion of any of these isozymes. Conversely, PMA-induced inhibition of EPO mRNA accumulation was paralleled by translocation of PKC alpha from cytosol to membranes and the time-and dose-dependent attenuation of the inhibitory effect of PMA on EPO mRNA levels was paralleled by down-regulation of PKC alpha. A dose-dependent inhibition of EPO mRNA formation, independent of effects on total RNA synthesis, as determined by [3H]uridine incorporation, was also found in the presence of the kinase inhibitor staurosporine (EDso --2 • 10 s M) and three structurally related derivatives with increased selectivity for PKC (RO 317549, EDso --1 • -6 M; RO 318220, EDso --lX

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