A novel zebrafish jak2a(V581F) model shared features of human JAK2(V617F) polycythemia vera.
OBJECTIVE
The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV.


METHODS
Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.


RESULTS
Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019).


CONCLUSIONS
The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.
Ma
Alvin C H
AC
Department of Medicine, The University of Hong Kong, Hong Kong.
Fan
August
A
Ward
Alister C
AC
Liongue
Clifford
C
Lewis
Rowena S
RS
Cheng
Suk H
SH
Chan
P K
PK
Yip
Sze-Fai
SF
Liang
Raymond
R
Leung
Anskar Y H
AY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004195:Disease Models, Animal; D004625:Embryo, Nonmammalian / Q000201:enzymology / Q000378:metabolism; D004920:Erythropoiesis / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011087:Polycythemia Vera / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013449:Sulfonamides / Q000494:pharmacology; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000096926:Benzenesulfonamides
D004921:Erythropoietin; D053614:Janus Kinase 2; D011505:Protein-Tyrosine Kinases; D011743:Pyrimidines; D050799:STAT5 Transcription Factor; D013449:Sulfonamides; D029961:Zebrafish Proteins; C522865:TG101209; D000096926:Benzenesulfonamides; C494098:ihb394 protein, zebrafish; C507924:JAK2 protein, human; C534485:Jak2a protein, zebrafish