Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis.

Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, mir-7148, let-7b, and mir-223 in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after mir-7148, let-7b, and mir-223 knockdowns. These results suggested mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. We then explored miRWalk database for let-7b downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following let-7b knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, irf5, tgif1, irf8, cebpa, and rorca that showed thrombocytosis and one gene, ikzf1 that showed thrombocytopenia. Furthermore, we tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. We also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a differential expression profile in let-7b and mir-223 knockdowns but resulted in thrombocytopenia in ikzf1 knockdown in both adults and larvae we also studied an ikzf1 mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.

Characterization of zebrafish gp1ba mutant and modelling Bernard Soulier syndrome.

The aim of this study is to model classical Bernard Soulier Syndrome in the zebrafish by targeting Gp1ba. We obtained gp1ba mutant embryos from Zebrafish International Resource Center and grew them to adulthood. The tail clips from these fish were used to prepare DNA and sequenced to identify heterozygotes. They were then bred to obtain homozygotes. The mutation was confirmed by DNA sequencing as a termination codon UAA in place of AAA codon at position 886 in the gp1ba transcript. Thus, at the Pro-295, the Gp1ba protein could be terminated. The blood from gp1ba homozygous and heterozygous mutants showed decreased ristocetin-mediated agglutination in the whole blood agglutination assay. The gp1ba heterozygous and homozygous larvae were subjected to a laser-assisted arterial thrombosis assay, and the results showed the prolonged occlusion in the caudal artery. These results suggested that the gp1ba mutant had a bleeding phenotype. The blood smears from the adult gp1ba, heterozygous and homozygous mutants, showed macrothrombocytes, similar to the human GP1BA deficiency that showed giant platelets. The bleeding assay on these heterozygous and homozygous mutants showed greater bleeding than wildtype, confirming the above findings. Taken together, the characterization of gp1ba zebrafish mutant suggested an autosomal dominant mode of inheritance. The zebrafish gp1ba mutant models classical Bernard Soulier Syndrome and could be used for reversing this phenotype to identify novel factors by the genome-wide piggyback knockdown method.

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.

Role of ribosomal RNA released from red cells in blood coagulation in zebrafish and humans.

Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. It has previously been shown that the RNA released from damaged blood cells activates clotting. However, the nature of the RNA released from hemolysis is still elusive. We found that after hemolysis, red blood cells from both zebrafish and humans released RNA that contained mostly 5.8S ribosomal RNA (5.8S rRNA), This RNA activated coagulation in zebrafish and human plasmas. By using both natural and synthetic 5.8S rRNA and its truncated fragments, we found that the 3'-end 26-nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor, blocked 3'-26 RNA-mediated coagulation activation in the plasma of both zebrafish and humans. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activated normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via an FXII-like protein. Because zebrafish have no FXII and because hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from this knockdown fish does not respond to 3'-26 RNA. To summarize, we identified that an rRNA released in hemolysis activates clotting in human and zebrafish plasma. Furthermore, we showed that fish Hgfac plays a role in rRNA-mediated activation of coagulation.

Knockdown screening of chromatin binding and regulatory proteins in zebrafish identified Suz12b as a regulator of tfpia and an antithrombotic drug target.

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa in the coagulation cascade. It has been shown that forkhead box P3 protein is a TFPI transcriptional repressor. However, there are no studies on chromatin remodeling that control TFPI expression. We hypothesized that the genome-wide knockdowns of the chromatin binding and regulatory proteins (CBRPs) in zebrafish could identify novel tfpia gene regulators. As an initial step, we selected 69 CBRP genes from the list of zebrafish thrombocyte-expressed genes. We then performed a 3-gene piggyback knockdown screen of these 69 genes, followed by quantification of tfpia mRNA levels. The results revealed that knockdown of brd7, ing2, ing3, ing4, and suz12b increased tfpia mRNA levels. The simultaneous knockdown of these 5 genes also increased tfpia mRNA levels. We also performed individual gene and simultaneous 5-gene knockdowns on the 5 genes in zebrafish larvae. We found that after laser injury, it took a longer time for the formation of the thrombus to occlude the caudal vessel compared to the control larvae. We then treated the larvae and adults with a chemical UNC6852 known to proteolytically degrade polycomb repressor complex 2, where SUZ12 is a member, and observed prolongation of time to occlude (TTO) the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. In summary, our results have identified novel epigenetic regulators for tfpia and exploited this information to discover a drug that enhances tfpia mRNA levels and prolongation of TTO. This discovery provides the basis for testing whether UNC6852 could be used as an antithrombotic drug. This approach could be used to study the regulation of other plasma proteins, including coagulant and anticoagulant factors.

Identification of zebrafish ortholog for human coagulation factor IX and its age-dependent expression.

BACKGROUND: Coagulation factor IX (FIX) is a serine protease zymogen involved in the intrinsic blood coagulation pathway, and its deficiency causes hemophilia B. Zebrafish has three f9 genes, and the ortholog to human F9 is unknown. OBJECTIVE: To identify the zebrafish ortholog to F9 using sequence analysis and piggyback knockdown technology. METHODS: Gene and protein sequence analysis for three f9 genes, f9a, f9b, and f9l, present in the zebrafish genome was performed. In vivo and in vitro assays after knockdown of each gene and immunodepletion using specific antibodies were carried out. RESULTS: Sequence analysis revealed that f9a and f9b are similar to human F9, whereas f9l is similar to human F10. RNA analysis showed an age-dependent increase in expression of all three genes. Zebrafish f9a gene knockdown and Fixa immunodepletion prolonged kinetic partial thromboplastin time (kPTT), whereas f9l knockdown and Fixl immunodepletion prolonged kPTT, kinetic prothrombin time, and kinetic Russell viper venom activation time. Laser-assisted venous thrombosis increased time to occlusion after f9a and f9l knockdown and antibody inhibition of Fixa and Fixl. Further, analysis of plasma proteins by mass spectrometry and immunohistochemistry detected all three proteins. CONCLUSIONS: Our findings suggest that zebrafish f9a has functional activity similar to human F9. Fixl is functionally similar to Fx. The age-dependent increases of these factors are comparable to those observed in mice and humans. Thus, the zebrafish model could be used to study factors involved in increasing f9a expression during aging. It could also be used to test whether normal human Factor IX and Factor IX Leyden promoter work in zebrafish background.