HIF-1alpha signaling upstream of Nkx2.5 is required for cardiac development in xenopus

HIF-1alpha is originally identified as a transcription factor that activates gene expression in response to hypoxia. In metazoans, HIF-1alpha functions as a master regulator of oxygen homeostasis and regulates adaptive responses to change in oxygen tension during embryogenesis, tissue ischemia, and tumorigenesis. Since HIF-1alpha-deficient mice exhibit a number of developmental defects, the precise role of HIF-1alpha in early cardiac morphogenesis has been uncertain. Therefore, to clarify the role of HIF-1alpha in heart development, we investigated the effect of knockdown of HIF-1alpha in Xenopus embryos using antisense morpholino oligonucleotide microinjection techniques. Knockdown of HIF-1alpha resulted in defect of cardiogenesis. Whole-mount in situ hybridization for cardiac troponin I (TnIc) showed the two separated populations of cardiomyocytes, which is indicative of cardia bifida, in HIF-1alpha-depleted embryos. Furthermore, the depletion of HIF-1alpha led to the reduction in TnIc expression, suggesting the correlation between HIF-1alpha and cardiac differentiation. We further examined the expression of several heart markers, Nkx2.5, GATA4, Tbx5, BMP4, HAND1 and HAND2 in HIF-1alpha-depleted embryos. Among them, the expression of Nkx2.5 was significantly reduced. Luciferase reporter assay using Nkx2.5 promoter showed that knockdown of HIF-1alpha decreased its promoter activity. The cardiac abnormality in HIF-1alpha-depleted embryo was restored with co-injection of Nkx2.5 mRNA. Collectively, these findings reveal that HIF-1alpha-regulated Nkx2.5 expression is required for heart development in Xenopus.