Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

Abstract

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.

Figure 1

Candidate gene selection and validation. Candidate genes were selected from a list of 75 MK-specific genes encoding transmembrane proteins. (A) Overlap analysis of MK-specific, present in HUVECs and transmembrane protein encoding genes. Of the 279 MK-specific genes, 143 are also present in HUVECs, and 35 of these encode transmembrane proteins. (B) Heatmap of log2 transformed, normalized intensity values for the 6 MK genes used in this study, showing increased expression of 4 in MKs and HUVECs. (C) Domain architecture of BAMBI, DCBLD2, ESAM, and LRRC32 as predicted using the Eukaryotic Linear Motif resource. The length of the black line represents the number of amino acids with domain positions to scale. Domain acronyms: CUB indicates complement C1r/C1s, Uegf, Bmp1; F5_F8, coagulation factor 5/8 type; IgSF, immunoglobulin fold; LCCL, Limulus factor C, Coch-5b2 and Lgl1; LRR_1, leucine rich repeats; LRRNT, leucine rich repeat N-terminal domain; SigP, signal peptide; TM, transmembrane domain.

Figure 2

Expression of novel transmembrane proteins in human platelets. (A-D) Flow cytometric detection of the novel transmembrane proteins was carried out in platelet-rich plasma (i) or in permeabilized platelets (ii). Binding of individual antibodies to human platelets measured as fluorescence intensity is indicated by solid lines and matched preimmune serum by dotted lines. Expression of each protein in human platelet lysates was analyzed by Western blot. Molecular weight markers are indicated on each blot in kilodaltons. Bands of 67, 27, 85, and 40/43 kDa were detected, corresponding to ANTXR2, BAMBI, DCBLD2, and ESAM proteins, respectively. The presence of LRRC32 in platelets using similar detection procedures has already been reported.

Figure 3

Thrombus formation in GPIIb and FVIII MO-injected fish in a laser-induced arterial injury model. (A) Representative phenotypes of 3 dpf control siblings (i) and fish injected with 2 ng GPIIb MO (itga2b atg1, ii), 4 ng FVIII MO (f8 sp2, iii), or coinjected with both MOs at 2 ng and 8 ng, respectively (iv). An intracranial hemorrhage, as observed in 5% of larvae, is indicated by arrowhead (iv). (B) Representative images of thrombus formation (arrows) 2 minutes after injury in 3-dpf wild-type larvae (i) or larvae injected with 2 ng GPIIb (ii) or 4 ng FVIII MOs (iii). CA indicates caudal artery; CV, caudal vein. White lines represent each thrombus as carried out in ImageJ for determining TSA. (C) Representative images of thrombus formation in 4-dpf CD41-GFP fish controls (i), and siblings injected with 2 ng GPIIb (ii) or 4 ng FVIII (iii) MOs showing thrombocyte-rich thrombi. (D) TTA and (E) TSA (2 minutes after injury) were measured and compared with uninjected sibling controls. Statistical analyses were performed using an unpaired Student t test (*P < .05; ***P < .001).

Figure 4

Effects of antisense knockdown of ANTXR2, BAMBI, DCBLD2, ESAM, and LRRC32 zebrafish orthologs on thrombus formation. TTA (A) and TSA (B) in the MO-injected fish were compared with that of their matched sibling controls. Each experiment is representative of more than 4 experiments, including similar numbers for both groups, using 2 nonoverlapping MOs. Zebrafish larvae were injected with the following MOs: 2 ng ANTXR2 atg1, 2 ng ANTXR2 sp1, 2 ng BAMBI atg1, 1 ng BAMBI sp1, 4 ng DCBLD2 atg1, 0.5 ng DCBLD2 sp1, 1 ng ESAM sp1, 12 ng ESAM sp2, 4 ng LRRC32 atg1, or 2 ng LRRC32 sp1. Statistical analyses were performed using an unpaired Student t test (*P < .05; **P < .005). (C) Results from all experiments and for each of the 2 MOs tested per gene were combined for TTA (□) and TSA () using Cox regression and linear mixed modeling, respectively, as described in “Statistical analysis of the thrombosis model” (*P < .05; **P < .005; ***P < .001). †TTA data for each DCBLD2 MO were significantly different and therefore were not combined. ††Results for TSA are applicable for esam sp1 only. Detailed P values for the combined and individual MO analysis are presented in Table 3.