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Transmembrane protein 59 (tmem59) is a gene that encodes a protein involved in autophagy and apoptosis in human. A previous study in zebrafish showed that tmem59 mRNA was several folds higher in thrombocytes than those found in red blood cells (RBCs). Therefore, we hypothesized that tmem59 has a role in thrombocytes function. We injected a hybrid of control vivo-morpholino (cVMO) and tmem59 specific antisense standard oligonucleotide (tmem59SO) into adult zebrafish to knockdown tmem59.This piggyback knockdown approach resulted in fish that had more bleeding in gill bleeding assay than the control fish. The thrombocytes fromtmem59 knockdown zebrafish aggregated faster with ADP and collagen agonists. Also, the number of blood cells was reduced after the knockdown of tmem59. We also found the effects of MS-222 anesthesia on hemostasis and found that the bleeding was reduced yielding less blood and the blood cell counts increased probably due to vasoconstriction of the blood vessels. In summary, we found tmem59 is a negative regulator of hemostasis and inferred that anesthesia should be avoided in hemostasis studies.

Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our results reveal a novel trypsin-initiated escape response in fish. These studies should enhance our understanding of fish communication in general and alarm behavior in particular. Furthermore, since pain receptors in other animals are also PAR2, our finding may be useful in exploring pathways of pain reception.

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.

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