G6f-like is an ITAM-containing collagen receptor in thrombocytes

Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution

Studies on Zebrafish Thrombocyte Function

Thrombocytes are important players in hemostasis. There is still much to be explored regarding the molecular basis of the thrombocyte function. In our previous microarray analysis data, we found IFT122 (an intraflagellar transport protein known to be involved in cilia formation) transcripts in zebrafish thrombocytes. Given recent discoveries of non-ciliary roles for IFTs, we examined the possibility that IFT122 affects thrombocyte function. We studied the role of IFT122 in thrombocyte function. We also found that IFT122 plays a central role in thrombocyte activation initiated by the agonists ADP, collagen, PAR-1 peptide and epinephrine. Although the receptors for ADP, PAR-1 peptide and epinephrine are present in the zebrafish genome, the collagen receptor GPVI was missing. In this study, we identified G6fL as a collagen receptor in zebrafish thrombocytes. Furthermore, IFT knockdown results in reduction in Wnt signaling. The Wnt signaling has been shown to be involved in megakaryocyte proliferation and proplatelets production. Therefore, defects in IFT could lead to thrombocytopenia. Splenectomy is performed in humans to treat such conditions. Therefore, in this study we developed a survival surgery protocol for splenectomy. We have shown that number of thrombocytes and their microparticles increase following splenectomy in zebrafish. Thus overall the studies on thrombocyte function in zebrafish could enhance fundamental knowledge on hemostasis and may provide future target candidates for therapies

Abstract 3251: Zebrafish: Is it a viable model for prostate cancer

Abstract Since its introduction as a vertebrate model organism for developmental studies, the Zebrafish model has continued to improve in its useful applications for studying human disease and more recently cancer. However, because there has been no evidence for the presence of prostate glands in fish, this model has not been exploited to study cancer of the prostate gland. In this abstract, we present evidence that fish carry prostate-like cells and, therefore, we postulate that zebrafish could be used as a model for prostate cancer research. We hypothesized that even though a well-defined prostate gland is not present in fish, there may be a few cells similar to thyroid gland. As a first approach, we developed an experimental design for detection of Prostate Specific Antigen (PSA) gene and protein expression, as well as PSA gene mapping. PSA is a serine protease that binds in seminal plasma to protein C inhibitor, which is involved in the clotting cascade in humans. When we analyzed the zebrafish genome, we found a PSA gene that is syntenic to human PSA and at the protein level zebrafish PSA is 70% homologous to human PSA. We also found PSA mRNA by RT-PCR analysis of the total testes RNA. In situ hybridization of the 4 day old zebrafish larva revealed expression of PSA in gonad. Treatment of zebrafish larvae with the anti-androgen, hydroxyl-flutamide, showed a PSA response profile similar to that in mammalian models in which there is a reduction in PSA mRNA at low doses and an increase at high doses with a return to baseline levels. Based on these findings we contend zebrafish have cells which are functionally equivalent to the prostate gland. We further believe that this information supports the notion of using zebrafish as a model to generate prostate cancer -like symptoms and propose for future studies expression of SV40 large T antigen under the PSA promoter to be used to demonstrate proof of principle. Such a model would be useful to explore not only early detection of prostate cancer but also to provide the opportunity for large scale studies of chemical suppression of cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3251.</jats:p

Splenectomy in zebrafish: a new model for immune thrombocytopenia

In humans, splenectomy is performed to treat many clinical disorders, including immune thrombocytopenia. However, the incidence of splenectomies for immune thrombocytopenia as a therapeutic has significantly declined over the past decade due to the availability of new therapies. Infection and sepsis as a result of splenectomies are well documented, but other long-term effects are not well characterized. Evidence suggests that persons who have had a prior splenectomy may be at an increased risk of vascular conditions. Also, elevated levels of cell-derived microparticles appear to contribute to an increased risk of thrombosis and cardiovascular disease. However, in vivo studies on the increased levels of microparticles following splenectomy are limited. In order to understand the effects of splenectomies, we developed a protocol for splenectomy in adult zebrafish. After anesthesia, the spleen was removed under a stereomicroscope after making an incision on the ventral side of the fish. The spleen was removed by pulling with forceps. The incision was closed by Vetbond tissue glue. Blood collected from both splenectomized zebrafish and those that underwent sham surgeries was immunolabeled with polyclonal antisera against αIIb, followed by flow cytometry. We observed elevated levels of thrombocytes and their microparticles in splenectomized zebrafish. Finally, by injecting αIIb antibody intravenously into zebrafish, we found the thrombocyte counts decreased, suggesting the fish developed immune thrombocytopenia like conditions, which were then reversed by splenectomy. In summary, the model developed here should be useful to study molecular changes due to splenectomy. Also, the zebrafish will be useful in modeling treatment of immune thrombocytopenia like conditions

Abstract 4377: Proper migration of mammalian prostate stem cells to the area of the prostate gland in zebrafish

Abstract Androgen independent prostate cancer contributes to the maximum mortality in prostate cancer patients. One school of thought is that these cancer cells arise in metastatic sites where the cancer cells take on stem cell behavior similar to the stem cell behavior observed in normal prostate gland. Thus, understanding prostate gland stem cells and their migration pattern may provide clues to their biology which may have applications in their migration during metastasis. To understand the migration of mammalian prostate stem cells we used zebrafish as a model. We have previously established zebrafish as a model to study prostate cancer because we found the presence of prostate-like- cells around the zebrafish testes. In this abstract we further characterized location of normal prostate-like cells. We found that they are present in the anterior and posterior aspects of testes. Furthermore, we cloned 2 kb prostate specific antigen promoter into pDsRed 2.1 plasmid and injected this plasmid DNA into single cell-stage zebrafish embryo with I-Sce1 to enhance transgenesis frequency and germline transmission. Larvae injected with PSA promoter-DSRed 2 constructs were screened three days post fertilization (dpf) for the expression of red fluorescence, which was observed in bilaterally arranged spots in gonads. We then used 5 dpf zebrafish larvae and IV-injected cells from four different cell lines expressing either GFP or RFP: two with stem cell phenotype RWPE-1-shDAB2IP (GFP), PZ-HPV7-shDAB2IP (GFP), and two as controls RWPE-1-shCon (GFP) and PC-3 (RFP). After two days, microscopy for green or red fluorescence signal revealed RWPE-1-shDAB2IP and PZ-HPV7-shDAB2IP cells bilaterally migrated to the yolk, i.e. the similar area where PSA promoter expression was found. This specific migration was not observed with RWPE-1-shCon and PC-3 cells lines. These same fluorescence reporter experiments were performed in parallel in 48 hr and 72 hr post fertilization (hpf) dechorinated larvae. We observed similar results in the 72 hpf larvae as were found in 5 dpf larvae but not in the 48 hpf larvae. Taken together, these results suggest that RWPE-1-shDAB2IP and PZ-HPV7-shDAB2IP cells have the homing receptors necessary for migrating to the same area in gonads. Understanding these homing mechanisms may be relevant for controlling the abnormal migration in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4377. doi:10.1158/1538-7445.AM2011-4377</jats:p

Intraflagellar transport proteins are involved in thrombocyte filopodia formation and secretion

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function

Zebrafish G6fL is required for haemostasis.

<p>A) A novel α-zebrafish G6fL antibody was used to determine knock-down of G6fL in morpholino (MO) treated larvae. 48 hrs post injection, 3 dpf larvae were lyophilised and lysed. Proteins were separated by SDS-PAGE and Western blotted with the antibody. The membrane was subsequently stripped and reprobed with an α-actin antibody to confirm equal loading. B) Control and G6fL MO treated adult fish were bled, and the blood used for a plate tilt aggregation assay. Time to aggregation (TTA) was measured following addition of either ADP (20 µM) or collagen (100 µg/ml). C) Control and G6fL MO treated larvae were immobilised on a microscope stage and then injured with a laser in the vessel wall of either veins or arteries (D). Time to occlusion (TTO) of vessels was measured for up to 2 min at which time the experiment was ceased. Statistical significance was calculated with a Student’s t-test (*P>0.05).</p