Establishment of a Bernard-Soulier syndrome model in zebrafish.

Platelets play an essential role in thrombosis and hemostasis. Abnormal hemostasis can cause spontaneous or severe post-traumatic bleeding. Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder caused by a complete quantitative deficiency in the GPIb-IX-V complex. Multiple mutations in GP9 lead to the clinical manifestations of BSS. Understanding the roles and underlying mechanisms of GP9 in thrombopoiesis and establishing a proper animal model of BSS would be valuable to understand the disease pathogenesis and to improve its medical management. Here, by using CRISPR-Cas9 technology, we created a zebrafish gp9SMU15 mutant to model human BSS. Disruption of zebrafish gp9 led to thrombocytopenia and a pronounced bleeding tendency, as well as an abnormal expansion of progenitor cells. The gp9SMU15 zebrafish can be used as a BSS animal model as the roles of GP9 in thrombocytopoiesis are highly conserved from zebrafish to mammals. Utilizing the BSS model, we verified the clinical GP9 mutations by in vivo functional assay and tested clinical drugs for their ability to increase platelets. Thus, the inherited BSS zebrafish model could be of benefit for in vivo verification of patient-derived GP9 variants of uncertain significance and for the development of potential therapeutic strategies for BSS.

Famciclovir leads to failure of hematopoiesis, but may have the benefit of relieving myeloid expansion in MDS-like zebrafish.

Famciclovir (FCV) is an antiviral drug that is often utilized after bone marrow transplantation to prevent viral infection. Yet, its role in hematopoiesis is poorly understood. Here, by utilizing a zebrafish model, we found that FCV exposure led to hematopoietic failure by impairing the proliferation of hematopoietic stem and progenitor cell (HSPC) and inducing HSPC apoptosis. On the other hand, FCV treatment could effectively relieve myeloid malignancies in the c-mybhyper MDS-like fish model, and played a role not only in the embryonic stage but also in adult zebrafish. This study reveals that FCV functions as a double-edged sword, with hematotoxicity at a high level, but that appropriate FCV treatment may be beneficial for the treatment of MDS.

c-Myb acts in parallel and cooperatively with Cebp1 to regulate neutrophil maturation in zebrafish.

Neutrophils are the key effectors for generating innate immunity in response to pathogenic infection and tissue injury in vertebrates. Dysregulation of neutrophil development and function is known to associate with various human disorders. Yet, the genetic network that orchestrates lineage commitment, differentiation, and maturation of neutrophils remains incompletely defined. Here, we present an in vivo study to delineate the genetic program underlying neutrophil development during zebrafish embryonic myelopoiesis. We show that loss of c-Myb function has no effect on macrophages but severely impairs neutrophil terminal differentiation, resulting in the accumulation of neutrophils with unsegmented nuclei and scant granule. This neutrophilic defect, which resembles the neutrophil-specific granule deficiency (SGD) caused by the mutations in CCAAT/enhancer-binding protein ε (C/EBPε) in humans, is attributed, at least in part, to the downregulation of the granule protein transcription. Likewise, genetic inactivation of Cebp1, the zebrafish functional homolog of mammalian C/EBPε, also leads to a similar SGD-like phenotype in zebrafish. Genetic epistasis and biochemical analysis further reveals that c-Myb and Cebp1 act in parallel and cooperatively to control neutrophil differentiation by directly regulating granule protein gene transcription. Our study indicates that c-MYB is an intrinsic master regulator for neutrophil terminal differentiation and a potential target in SGD patients.

[Effects of peroxisome proliferator-activated receptors γ on the expression of insulin receptor substrate-4 gene in rat cortical neurons and mouse brain].

OBJECTIVE: To investigate the effect of peroxisome proliferator-activated receptors γ (PPARγ) on insulin receptor substrate-4 (IRS-4) gene expression in the brain. METHODS: Primarily cultured cortical neurons from E17-18 Sprague Dawley rats, after 1 week of plating, were exposed to 10 µmol/L PPARγ agonist rosiglitazone for 0, 1, 4 or 24 h. Adult C57BL/6J mice or conditional brain PPARγ knock-out mice (B-PPARγ-KO, BKO) received an intraperitoneal injection of rosiglitazone in 10% DMSO at 12 mg/kg or injection of the same volume of saline containing 10% DMSO. The effect of rosiglitazone on the survival of the neurons was examined by MTT assay. The expression of IRS-4 mRNA was analyzed by real-time quantitative PCR. RESULTS: The survival of the cortical neurons showed no significant difference between the agonist groups and the control group. The expression of IRS-4 mRNA was significantly up-regulated in the cortical tissues and neurons of the agonist groups compared with the control groups (P<0.05), but in BKO mice without treatment, IRS-4 mRNA expression was significantly down-regulated (P<0.05). CONCLUSION: PPARγ can enhance the expression of IRS-4 mRNA in the brain.