Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.
In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.
O'Connor
Marie N
MN
Department of Haematology, University of Cambridge, Cambridge, United Kingdom.
Salles
Isabelle I
II
Cvejic
Ana
A
Watkins
Nicholas A
NA
Walker
Adam
A
Garner
Stephen F
SF
Jones
Chris I
CI
Macaulay
Iain C
IC
Steward
Michael
M
Zwaginga
Jaap-Jan
JJ
Bray
Sarah L
SL
Dudbridge
Frank
F
de Bono
Bernard
B
Goodall
Alison H
AH
Deckmyn
Hans
H
Stemple
Derek L
DL
Ouwehand
Willem H
WH
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D023281:Genomics; D006801:Humans; D007834:Lasers; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010641:Phenotype; D010974:Platelet Aggregation; D013927:Thrombosis / Q000209:etiology / Q000378:metabolism*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D029961:Zebrafish Proteins