Figure 1.
Gene expression pattern of hif-1alpha, hif-2 alpha and hif-3 alpha in zebrafish embryos.
The expression patterns of hif1-alpha (A–D), hif2-alpha (E–H) and hif3-alpha (I–L) were performed with anti-sense probes by WISH in zebrafish embryos at 2–5 dpf. WISH, whole-mount in situ hybridization. dpf, days post-fertilization. lv, liver.
Figure 2.
Knockdown of hif2-alpha confers a small liver phenotype.
(A–J) Development of the embryonic liver was monitored by the expression pattern of the lfabp gene in wild-type embryos and hif2-alpha ATG-MO-injected embryos at 1–5 dpf by WISH, thereby revealing a small liver phenotype in hif2-alpha morphants. (K) Expression of the lfabp and fabp10a genes was investigated in wild-type embryos and hif2-alpha morphants at 5 dpf by qPCR (n = 30). Expression was normalized to β-actin. WT, wild type. The experiment was performed in triplicate, error bars indicate S.D. *, p<0.05, unpaired t-test.
Figure 3.
Hif2-alpha plays a major role in liver development.
The specificity of knockdown by MO was demonstrated by monitoring lfabp gene expression at 5 pdf by WISH in wild-type embryos (A) and embryos injected with hif2-alpha ATG-MO (B). The small liver phenotype resulting from the knockdown of hif2-alpha can be rescued by co-injection of hif2-alpha mRNA (C). ATG-MO of hif2-alpha but not hif1-alpha caused the small liver phenotype (E). ATG-MO of hif3-alpha also caused a slight effect on liver development (F).
Figure 4.
Hif2-alpha is not required for liver specification in zebrafish embryos.
Liver specification in hif2-alpha morphants was detected through the expression of the hhex and prox1 genes. The expression of embryonic liver specification genes, hhex (A, B, E, F) and prox1 (C, D, G, H), were examined at 30 hpf (A–D) and 55 hpf (E–H) in wild-type and hif2-alpha ATG-MO-injected embryos by WISH.
Figure 5.
Knockdown of hif2-alpha damages liver cell proliferation.
WT (Tg(lfabp:EGFP)) embryos (A) and hif2-alpha ATG-MO-injected embryos (B) were examined for liver cell proliferation using an anti-pH3 antibody at 4 dpf. Cell proliferation in hif2-alpha ATG-MO-injected embryos was reduced compared with WT embryos. (C) Quantification of pH3-positive cells in the liver (n = 11, p<0.05). (D) The EGFP-positive cells were counted by FACS. Quantification of pH3-positive cells in the trunk (E) and tail (F) (n = 4, p<0.05).
Figure 6.
Hif2-alpha is required for the expansion of the exocrine pancreas and the intestine.
The development of the embryonic pancreas and the intestine was monitored by examining the gene expression pattern of the endocrine pancreas (ins) (A, B), the exocrine pancreas (try) (C, D), and the intestine (ifabp) (E, F) in wild-type embryos compared with hif2-alpha ATG-MO-injected embryos at 85 hpf by WISH.
Figure 7.
Hif2-alpha knockdown affects lipid metabolism but not EPO production in zebrafish embryos.
The expression of EPO and the genes involved in lipid metabolism were investigated in wild-type embryos and hif2-alpha morphants at 5 dpf by qPCR (n = 30). Expression was normalized to β-actin. WT, wild type. The experiment was performed in triplicate, error bars indicate S.D. *, p<0.05, unpaired t-test.
Figure 8.
leg1 is required for hepatic outgrowth in zebrafish embryos.
The expression pattern of the lfabp gene was examined in Tg(lfabp:EGFP) embryos (A) and compared with leg1 ATG-MO-injected embryos (0.5 µM) (B) as well as embryos co-injected with leg1 ATG-MO (0.5 µM) and leg1 cRNA (C).
Figure 9.
Leg1 expression was down-regulated in hif2-alpha morphants.
leg1 gene expression was examined in wild-type embryos and compared with hif2-alpha ATG-MO-injected embryos at 3–5 dpf by qPCR (A) and WISH (B). Expression was normalized to β-actin.
Figure 10.
The small liver phenotype caused by hif2-alpha knockdown can be rescued by ectopic leg1 expression.
The expression pattern of the lfabp gene was examined in Tg(lfabp:EGFP) embryos (A) and compared with hif2-alpha ATG-MO-injected embryos (8 ng) (B) as well as embryos co-injected with hif2-alpha ATG-MO (8 ng) and leg1 cRNA (C). (D) lfabp mRNA expression was detected by qPCR in wild-type embryos and compared with hif2-alpha ATG-MO-injected embryos, hif2-alpha ATG-MO and leg1 cRNA co-injected embryos, as well as hif2-alpha ATG-MO and hif2-alpha cRNA co-injected embryos. Expression was normalized to β-actin.
Figure 11.
Hif2-alpha binds to leg1a and leg1b promoters.
The binding of hif2-alpha to the promoter region of the leg1a (A) and leg1b (B) genes was examined by ChIP-PCR. Seven and nine HRE-containing modules in the promoter regions of leg1a and leg1b, respectively, are amplified from the immunocomplexes obtained by ChIP assays performed using a polyclonal antibody against anti-Hif2-alpha or a preimmune serum (IgG) as controls. (C) Schematic representation of the leg1a and leg1b promoter regions. HRE (A/G-C-G-T-G) are annotated as dark lines. The positions of the modules analyzed in the ChIP-PCR assays are shown as grey boxes. The amplified fragments in ChIP assays, including modules 3, 4, 5 and 7 in the leg1a promoter and modules 1, 2 and 9 in the leg1b promoter, are outlined in red (A, B).
Figure 12.
The regulation of hepatic outgrowth by hif2-alpha in zebrafish embryos is not through the FGF, HGF, or Wnt pathways.
Gene expression of fgfr1, fgfr2, fgfr3, fgfr4, met, and epcam was examined in wild-type embryos and compared with hif2-alpha ATG-MO-injected embryos at 3–5 dpf by qPCR. Expression was normalized to β-actin.
Figure 13.
CoCl2 treatment increased the expression of the target genes of Hif1-alpha rather than those of Hif2-alpha.
(A) Gene expression of igfbp-1, leg1, birc5a, and birc5b mRNA without (control, in blue) or with 10 mM CoCl2 treatment (red) was examined in zebrafish embryos at 72 hpf by qPCR. Expression was normalized to β-actin. *indicates a significant difference (p<0.05) between control embryos and CoCl2-treated embryos by an unpaired t-test. The experiment was performed in triplicate, and error bars indicate the standard deviation. (B) The Flag-protein expression levels were performed by western blot and were normalized to tubulin.
Figure 14.
Distribution of HRE clustering.
The distances between HRE for the first neighbored HRE (A, B) and the second neighbored HRE (C, D) were measured, and the distributions of the distance are shown in 100 bps bins.
Table 1.
HRE cluster detection in the upstream sequences of animals.