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. Because 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 defects of cardiogenesis. Whole mount in situ hybridization for cardiac troponin I (cTnI) 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 cTnI 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 the Nkx2.5 promoter showed that knockdown of HIF-1alpha decreased its promoter activity. The cardiac abnormality in the 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.