Fluorescent images of Tg (gata1:eGFP) / hif3a^+/+ (top) and Tg (gata1:eGFP) / hif3a^-/- (bottom) indicated that hif3a^-/-  have fewer gata1-positive erythrocytes at 24 hpf and 48 hpf. (Image by IHB) 

The hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) are master regulators of the cellular response to O₂. To date, their function in hypoxia signaling is relatively clear. HIF-3α, another hypoxia-inducible factor, has some different isoforms. The molecular mechanism and biological function of HIF-3α in hypoxia signaling pathway is largely unknown.   

In a recent study published online in Development, the research group led by Prof. XIAO Wuhan from Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences revealed that HIF-3α facilities hypoxia tolerance by modulating erythropoiesis via gata-1 regulation. 

Using CRISPR/Cas9 technology, the researchers knocked out hif-3α in zebrafish. In the following experiments, they found that more hif-3α^-/- larvae were dead than hif-3α^+/+ larvae after exposing larvae to 2% O₂ for 12 hours. These data suggested that disruption of hif-3α attenuated hypoxia tolerance in zebrafish. 

When they routinely examined the hif-3α^+/+ and hif-3α^-/- larvae under a dissection microscope, the researchers noticed that the hif-3α^-/- larvae always had fewer blood cells compared to hif-3α^+/+ larvae. Using O-dianisidine staining to measure the red blood cells, they found fewer O-dianisidine-positive cells in the hif-3α^-/- larvae than in the hif-3α^+/+ larvae, indicating that knockout of hif-3α disrupted erythropoiesis in zebrafish. 

To determine whether the defective erythropoiesis displayed by the hif-3α^-/- was associated with erythroid maturation, the researchers analyzed the morphology of isolated red blood cells by using May-Grunwald-Giemsa staining. They found that hif-3α^-/- had a higher percentage of proerythroblasts and a lower percentage of mature erythroid precursors than the wild type, suggesting that the deletion of hif-3α might impede erythroid cell maturation. 

In addition, the researchers used the whole mount in situ hybridization to examine the expression of hematopoietic markers (such as gata1), and figured out the mechanisms of hif-3α on erythropoiesis - the disruption of zebrafish hif-3α abrogated the expression of hematopoietic marker genes, resulting in defects of erythropoiesis; and gata-1 might be the downstream effector mediating the function of hif-3α in erythropoiesis. 

Hypoxia-response element (HRE), a response element recognized by hypoxia-inducible factors, was located in the promoter of downstream targets of hypoxia-indecible factors. The researchers revealed that there is a potential HRE located at -105— -101 in the promoter of gata1, and confirmed that zebrafish hif-3α directly activated gata1 expression by recognizing the HRE site, thus modulating the erythropoiesis. 

This study demonstrated that zebrafish hif-3α facilitate hypoxia tolerance via regulation of gata1, and suggested that hif-3α might behave similar to hif-1α and hif-2α in hypoxia signaling pathway.