HIF-1α Signaling Upstream of NKX2.5 Is Required for Cardiac Development in Xenopus

Kaori Nagao,Yoshiaki Taniyama,Thomas Kietzmann,Takefumi Doi,Issei Komuro,Ryuichi Morishita

doi:10.1074/jbc.m702563200

Kaori Nagao, Yoshiaki Taniyama + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m702563200

Copy DOI

Abstract

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.

Highlights

Xenopus embryos at 8-cell stage were microinjected with hif-1␣ morpholino oligonucleotides
Frequency of cardia bifida in embryos injected with antisense morpholino against HIF-1␣
Xenopus embryos at 8-cell stage were microinjected with morpholino oligonucleotides (CMO, n ϭ 34; hif-1␣ MO1, n ϭ 75; hif-1␣ MO2, n ϭ 68)

Summary

EXPERIMENTAL PROCEDURES

Construction of Plasmids and Mutagenesis—Xenopus cDNAs of cTnI (L25721), nkx2.5 (L25600), gata[4] (BC071107), tbx[5] (AF133036), hand[1] (Z95080), hand[2] (AAF67131), and bmp[4] (AJ005076) were cloned into pCS2ϩ (11) or pBluescript SK(Ϫ) (Stratagene) vectors. The synthetic mRNA encoding hif-1␣ (P558G) (750 pg) was microinjected into one of the two vegetal-dorsal blastomeres of the selected embryos at the 8-cell stage, leaving the other side as control. Immunoprecipitation of Endogenous HIF-1␣—Total cell lysates were prepared from Xenopus embryos microinjected with hif-1␣ MO in one of two vegetal-dorsal blastomeres at the 8-cell stage. At stage 20, 10 embryos were lysed in 800 ␮l of immunoprecipitation buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol and protease inhibitor mixture (Roche)). The pGL4.10 –9kb Nkx2.5 promoter (Nkx2.5 promoter-Luc) (100 pg) and pRL-null (2 pg) (an internal control) were injected into one side of the vegetal-dorsal blastomeres at the 8-cell stage. The Nkx2.5 promoter-Venus (100 pg) was injected into one of the two vegetal-dorsal blastomeres with CMO or hif-1␣ MO1. Differences with a p value less than 0.05 were considered significant

RESULTS

Cardia bifida

Hybridization probe

DISCUSSION