ZFIN ID: ZDB-FIG-171025-20
Mancias et al., 2015 - Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis. eLIFE   4 Full text @ Elife
EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Terms:
Stage Range: Prim-5 to Protruding-mouth
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage: Prim-15

Fig. 5

Knockdown of ncoa4 impacts erythropoiesis in zebrafish and tissue culture cells.

(A, B) Expression of HGBG (A), HGBA (B) determined by quantitative RT-PCR in K562 cells expressing a control (shGFP) or two independent shRNAs targeting NCOA4. Hemin treatment (25 μM, 72 hr) as indicated. (C) Immunoblot analysis of K562 cells as in (A) and (B) with indicated antibodies. Hemin treatment (25 μM, 72 hr) as indicated. (D) Appearance of K562 pellets as in (A, B, and C). Red coloration indicates appropriate hemoglobinization of cells after hemin differentiation. Brown coloration indicates accumulation of iron without hemoglobinization. (E) Expression of ncoa4 in circulating erythroid cells relative to sites of primitive hematopoiesis. Abbreviations: hpf = hours post-fertilization, ICM = intermediate cell mass, YS = yolk sac, H = heart, L = liver. (F) Circulating RBCs are visualized in globin-LCR:eGFP erythrocyte reporter zebrafish at 30 hpf. Morpholino-mediated knockdown of ncoa4 severely disrupts erythropoiesis (n > 30 each condition). Inset shows erythrocytes circulating in the caudal artery and caudal vein plexus. (G) FACS quantification of erythrocytes in globin-LCR:eGFP reporter zebrafish at 30 hpf following control or ncoa4-morpholino-mediated knockdown (ncoa4 MOa and MOb). ***p < 0.001 and **p < 0.002 by two-tailed unpaired t-test. (H) o-dianisidine staining (brown) for hemoglobinized red cells in zebrafish embryos. Embryos were grown from zygotes injected at the one- to two-cell stage with ncoa4-targeting morpholinos (MOa and MOb) or control injected zygotes (control). Ncoa4 MOa: 15/16 embryos with diminished staining in comparison to control. Ncoa4 MOb: 18/22 embryos with diminished staining in comparison to control.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
ncoa4 WT standard conditions Prim-5 intermediate cell mass of mesoderm ISH
Long-pec nucleate erythrocyte ISH
Protruding-mouth nucleate erythrocyte ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
cz3325Tg + MO1-ncoa4 standard conditions Prim-15 erythrocyte differentiation disrupted, abnormal
Prim-15 nucleate erythrocyte decreased amount, abnormal
Prim-15 nucleate erythrocyte hemoglobin complex decreased amount, abnormal
cz3325Tg + MO2-ncoa4 standard conditions Prim-15 erythrocyte differentiation disrupted, abnormal
Prim-15 nucleate erythrocyte decreased amount, abnormal
Prim-15 nucleate erythrocyte hemoglobin complex decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal eLIFE for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Elife