Simple and Rapid Quantification of Thrombocytes in Zebrafish Larvae

Platelets are a critical component of hemostasis, with disorders of number or function resulting in coagulation disturbances. Insights into these processes have primarily been realized through studies using mammalian models or tissues. Increasingly, zebrafish embryos and larvae have been used to study the protein and cellular components of hemostasis and thrombosis, including the thrombocyte, a nucleated platelet analog. However, investigations of thrombocytes have been somewhat limited due to lack of a robust and simple methodology for quantitation, an important component of platelet studies in mammals. Using video capture, we have devised an assay that produces a rapid, reproducible, and precise measurement of thrombocyte number in zebrafish larvae by counting fluorescently tagged cells. Averaging 1000 frames, we were able to subtract background fluorescence, thus limiting assessment to circulating thrombocytes. This method facilitated rapid assessment of relative thrombocyte counts in a population of 372 zebrafish larvae by a single operator in less than 3 days. This technique requires basic microscopy equipment and rudimentary programming, lends itself to high throughput analysis, and will enhance future studies of thrombopoiesis in the zebrafish.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140302/1/zeb.2014.1079.pd

Emicizumab prophylaxis to facilitate anticoagulant therapy for management of intra‐atrial thrombosis in severe haemophilia with an inhibitor

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149546/1/hae13721.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149546/2/hae13721_am.pd

Differential Regulation of Primitive Myelopoiesis in the Zebrafish by Spi-1/Pu.1 and C/ebp1

The zebrafish has become a powerful tool for analysis of vertebrate hematopoiesis. Zebrafish, unlike mammals, have a robust primitive myeloid pathway that generates both granulocytes and macrophages. It is not clear how this unique primitive myeloid pathway relates to mammalian definitive hematopoiesis. In this study, we show that the two myeloid subsets can be distinguished using RNA in situ hybridization. Using a morpholino-antisense gene knockdown approach, we have characterized the hematopoietic defects resulting from knockdown of the myeloid transcription factor gene pu.1 and the unique zebrafish gene c/ebp1. Severe reduction of pu.1 resulted in complete loss of primitive macrophage development, with effects on granulocyte development only with maximal knockdown. Reduction of c/ebp1 did not ablate initial macrophage or granulocyte development, but resulted in loss of expression of the secondary granule gene lys C. These data reveal strong functional conservation of pu.1 between zebrafish primitive myelopoiesis and mammalian definitive myelopoiesis. Further, these results are consistent with a conserved role between c/ebp1 and mammalian C/EBPE, whose ortholog in zebrafish has not been identified. These studies validate the examination of zebrafish primitive myeloid development as a model for human myelopoiesis, and form a framework for identification and analysis of myeloid mutants.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63190/1/zeb.2007.0505.pd

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/1/jth14391.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/2/jth14391-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/3/jth14391_am.pd

A precursor‐inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154358/1/fsb2030005012.pd

Genome‐wide linkage analysis and whole‐exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150531/1/bjh15961_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150531/2/bjh15961.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150531/3/bjh15961-sup-0001-DataS1.pd

Characterization of Zebrafish von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage

von Willebrand disease (VWD) is the most common inherited human bleeding disorder and is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWF is a secreted glycoprotein that circulates as large multimers. While reduced VWF is associated with bleeding, elevations in overall level or multimer size are implicated in thrombosis. The zebrafish is a powerful genetic model in which the hemostatic system is well conserved with mammals. The ability of this organism to generate thousands of offspring and its optical transparency make it unique and complementary to mammalian models of hemostasis. Previously, partial clones of zebrafish vwf have been identified, and some functional conservation has been demonstrated. In this paper we clone the complete zebrafish vwf cDNA and show that there is conservation of domain structure. Recombinant zebrafish Vwf forms large multimers and pseudo-Weibel-Palade bodies (WPBs) in cell culture. Larval expression is in the pharyngeal arches, yolk sac, and intestinal epithelium. These results provide a foundation for continued study of zebrafish Vwf that may further our understanding of the mechanisms of VWD

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer