Phylogenetic analysis of PHD3 proteins and the expression pattern of zebrafish phd3 in different tissues.A, alignment of PHD3 amino acid sequences of Homo sapiens (Hs) and Danio rerio (Dr). B, the phylogenetic tree of PHD3 proteins from 12 species was constructed using the neighbor-joining method. Homo sapiens (Hs), NM_022073.4; Mus musculus (Mm), NM_028133.2; Gallus gallus (Gg), XM_421233.6; Apteryx rowi (Ar), XM_026085427.1; Latimeria chalumnae (Lc), XM_005989196.2; Xenopus tropicalis (Xt), NM_001127012.1; Danio rerio (Dr), NM_213310.1; Oryzias latipes (Ol), XM_023951862.1; Takifugu rubripes (Tr), XM_029833019.1; Pseudonajia textilis (Pt), XM_026710948.1; Erpetoichthys calabaricus (Ec), XM_028821616.1; and Drosophila melanogaster (Dm), NM_001300242.1. C, the predicted protein structure of zebrafish phd3 by AlphaFold in Ensembl database (https://www.asia.ensembl.org/index.html). D, quantitative real-time PCR (qPCR) analysis of phd3 mRNA levels in different tissues of adult zebrafish (5 months post-fertilization, mpf).

Zebrafish phd3 is induced under hypoxia.A, qPCR analysis of the prolyl hydroxylase genes (PHDs), including phd1, phd2a, phd2b, and phd3 mRNA in zebrafish larvae (3 days post-fertilization, dpf) under normoxia (21% O₂) or hypoxia (2% O₂) for 5 h. B, qPCR analysis of hypoxia-responsive genes in zebrafish larvae (3 dpf), including ldha, cited2, and vegfaa under normoxia (21% O₂) or hypoxia (2% O₂) for 5 h. C, qPCR analysis of the PHD genes in ZFL cells, including phd1, phd2a, phd2b, and phd3 under normoxia (21% O₂) or hypoxia (1% O₂) for 24 h. D, qPCR analysis of hypoxia-responsive genes in ZFL cells, including ldha, cited2, and vegfaa under normoxia (21% O₂) or hypoxia (1% O₂) for 24 h. E–G, qPCR analysis of phd3 (E), ldha (F), and vegfaa (G) in ZFL cells transfected with Flag empty, Flag-hif1αa, Flag-hif1αb, Flag-hif2αa, or Flag-hif2αb for 24 h. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, using unpaired Student's t test; Data are representative of three independent experiments (mean ± SD of three technical replicates).

Generation of phd3-null zebrafish and phenotype analysis.A, schematic of the sequence information of phd3-null zebrafish. 8-bp nucleotides (5′-GGAGAGTT-3′) were deleted in exon 1 of phd3-null mutant, resulting in a reading frame shift. B, the predicted protein products in the mutants (70 aa) and their wildtype siblings (243 aa). aa, amino acids. C, verification of CRISPR/Cas9-mediated zebrafish phd3 disruption by heteroduplex mobility assay (HMA). D, qPCR analysis of phd3 mRNA in phd3-null zebrafish larvae and their wildtype siblings (phd3^−/− or phd3^+/+) (3 dpf). E, photographs of phd3-null zebrafish larvae and their wildtype siblings (phd3^−/− or phd3^+/+) (3 dpf or 6 dpf). Scale bar = 1 mm. F, measurement of body weight of phd3-null adult zebrafish and their wildtype siblings (phd3^−/− or phd3^+/+) (4 mpf). G, measurement of body length of phd3-null adult zebrafish and their wildtype siblings (phd3^−/− or phd3^+/+) (4 mpf). ns, not significant; ∗∗∗∗p < 0.0001, using unpaired Student's t test; data are representative of three independent experiments (mean ± SD of three technical replicates).

Disruption of phd3 in zebrafish enhances the expression of hypoxia-responsive genes and increase the number of erythrocytes in response to hypoxia.A–E, qPCR analysis of ldha (A), cited2 (B), vegfaa (C), epoa (D), and il11a (E) mRNA levels in phd3-null zebrafish larvae and their wildtype siblings (phd3^−/− or phd3^+/+) (3 dpf) under normoxia (21% O₂) or hypoxia (2% O₂) for 5 h. F and G, O-dianisidine staining of erythrocytes in phd3-null zebrafish larvae and their wildtype siblings (phd3^−/− or phd3^+/+) (6 dpf) under normoxia (21% O₂) or hypoxia (10% O₂) for 12 h. The quantitation of erythrocytes in (F) was determined by normalizing the intensities of O-dianisidine stained cells. H and I, O-dianisidine staining of erythrocytes in phd3-null zebrafish larvae and their wildtype siblings (phd3^−/− or phd3^+/+) (6 dpf) treated with FG4592 (20 μM) or DMSO as control for 12 h. The quantitation of erythrocytes in (H) was determined by normalizing the intensities of O-dianisidine stained cells. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, using unpaired Student's t test; data are representative of three independent experiments (mean ± SD of three technical replicates).

Disruption of phd3 in zebrafish facilitates hypoxia tolerance.A, photographs of phd3-null zebrafish larvae and their wildtype siblings (phd3^−/− or phd3^+/+) (3 dpf) under normoxia (21% O₂) or hypoxia (2% O₂) for 15 h. Red arrows indicate dead zebrafish. B, survival curve of phd3-null zebrafish larvae and their wildtype siblings (phd3^−/− or phd3^+/+) (3 dpf) under hypoxia (2% O₂). C, photographs of phd3-null adult zebrafish and their wild-type siblings (phd3^−/− or phd3^+/+) (4 mpf) under hypoxia (5% O₂) for 0 h, 2 h, and 4 h. Red arrows indicate dead zebrafish. D, survival curve of phd3-null adult zebrafish and their wildtype siblings (phd3^−/− or phd3^+/+) (4 mpf) under hypoxia (5% O₂). ns, not significant; ∗p < 0.05, ∗∗∗∗p < 0.0001, using log-rank test analysis; data are representative of three independent experiments.

Zebrafish phd3 suppresses HIF transcriptional activation.A, luciferase activity of hypoxia-responsive element (HRE) reporter in EPC cells transfected with Myc-phd3 or Myc empty vector (control) under normoxia (21% O₂) or hypoxia (1% O₂) for 24 h. B, luciferase activity of the HRE reporter in EPC cells co-transfected with Flag-hif1αa or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O₂) for 24 h. C, luciferase activity of the HRE reporter in EPC cells co-transfected with Flag-hif1αb or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O₂) for 24 h. D, luciferase activity of the HRE reporter in EPC cells co-transfected with Flag-hif2αa or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O₂) for 24 h. E, luciferase activity of the HRE reporter in EPC cells co-transfected with Flag-hif2αb or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O₂) for 24 h. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, using two-way ANOVA analysis; data are representative of three independent experiments (mean ± SD of three technical replicates).

Zebrafish phd3 induces the degradation of hif-1/2α proteins.A, Western blot analysis of hif1αa protein in HEK293T cells transfected with Flag-hif1αa together with Myc-phd3 or Myc empty vector (control) for 24 h. B and C, Western blot analysis of hif1αa protein in HEK293T cells transfected with Flag-hif1αa together with Myc-phd3 or Myc empty vector (control) for 20 h, followed by treatment with cycloheximide (CHX) (50 μg/ml) for the indicated time. The relative intensities of hif1αa in (B) were determined by normalizing the intensities of hif1αa to the intensities of β-actin. D, Western blot analysis of hif1αb protein in HEK293T cells transfected with Flag-hif1αb together with Myc-phd3 or Myc empty vector (control) for 24 h. E and F, Western blot analysis of hif1αb protein in HEK293T cells transfected with Flag-hif1αb together with Myc-phd3 or Myc empty vector (control) for 20 h, followed by treatment with CHX (50 μg/ml) for the indicated time. The relative intensities of hif1αb in (E) were determined by normalizing the intensities of hif1αb to the intensities of β-actin. G, Western blot analysis of hif2αa protein in HEK293T cells transfected with Flag-hif2αa together with Myc-phd3 or Myc empty vector (control) for 24 h. H and I, Western blot analysis of hif2αa protein in HEK293T cells transfected with Flag-hif2αa together with Myc-phd3 or Myc empty vector (control) for 20 h, followed by treatment with CHX (50 μg/ml) for the indicated time. The relative intensities of hif2αa in (H) were determined by normalizing the intensities of hif2αa to the intensities of β-actin. J, Western blot analysis of hif2αb protein in HEK293T cells transfected with Flag-hif2αb together with Myc-phd3 or Myc empty vector (control) for 24 h. K and L, Western blot analysis of hif2αb protein in HEK293T cells transfected with Flag-hif2αb together with Myc-phd3 or Myc empty vector (control) for 20 h, followed by treatment with CHX (50 μg/ml) for the indicated time. The relative intensities of hif2αb in (K) were determined by normalizing the intensities of hif2αb to the intensities of β-actin.

Zebrafish phd3 promotes polyubiquitination of hif-1/2α proteins.A, the ubiquitination of hif1αa protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h. The relative intensities of hif1αa ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif1αa in the Ni²⁺-pulldown to the intensities of hif1αa in the total cell lysates (TCL). B, the ubiquitination of hif1αb protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h. The relative intensities of hif1αb ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif1αb in the Ni²⁺-pulldown to the intensities of hif1αb in the TCL. C, the ubiquitination of hif2αa protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h. The relative intensities of hif2αa ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif2αa in the Ni²⁺-pulldown to the intensities of hif2αa in the TCL. D, the ubiquitination of hif2αb protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h. The relative intensities of hif2αb ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif2αb in the Ni²⁺-pulldown to the intensities of hif2αb in the TCL.