Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1. Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.

Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299) and the mouse lung adenocarcinoma (CL13). Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP) vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase) inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to measure cancer cell metastasis. This assay represents not only the useful test for patient diagnosis, but also has the potential for evaluating anti-cancer drugs treatment.

Personalized medicine holds tremendous promise for improving safety and efficacy of drug therapies by optimizing treatment regimens. Rapidly developed patient-derived xenografts (pdx) could be a helpful tool for analyzing the effect of drugs against an individual’s tumor by growing the tumor in an immunodeficient animal. Severe combined immunodeficiency (SCID) mice enable efficient in vivo expansion of vital tumor cells and generation of personalized xenografts. However, they are not amenable to large-scale rapid screening, which is critical in identifying new compounds from large compound libraries. The development of a zebrafish model suitable for pdx could facilitate large-scale screening of drugs targeted against specific malignancies. Here, we describe a novel strategy for establishing a zebrafish model for drug testing in leukemia xenografts. We used chronic myelogenous leukemia and acute myeloid leukemia for xenotransplantation into SCID zebrafish to evaluate drug screening protocols. We showed the in vivo efficacy of the ABL inhibitor imatinib, MEK inhibitor U0126, cytarabine, azacitidine and arsenic trioxide. We performed corresponding in vitro studies, demonstrating that combination of MEK- and FLT3-inhibitors exhibit an enhanced effect in vitro. We further evaluated the feasibility of zebrafish for transplantation of primary human hematopoietic cells that can survive at 15 day-post-fertilization. Our results provide critical insights to guide development of high-throughput platforms for evaluating leukemia.

Hematopoietic stem/progenitor cells (HSPC) in zebrafish emerge from the aortic hemogenic endothelium (HE) and migrate towards the caudal hematopoietic tissue (CHT), where they expand and differentiate during definitive hematopoiesis. Phospholipase C gamma 1 (Plcγ1) has been implicated for hematopoiesis in vivo and in vitro and is also required to drive arterial and HSPC formation. Genetic mutation in plcg1^−/− (y10 allele) completely disrupts the aortic blood flow, specification of arterial fate, and HSPC formation in zebrafish embryos. We previously demonstrated that ginger treatment promoted definitive hematopoiesis via Bmp signaling. In this paper, we focus on HSPC development in plcg1^−/− mutants and show that ginger/10-gingerol (10-G) can rescue the expression of arterial and HSPC markers in the HE and CHT in plcg1^−/− mutant embryos. We demonstrate that ginger can induce scl/runx1 expression, and that rescued HE fate is dependent on Bmp and Notch. Bmp and Notch are known to regulate nitric oxide (NO) production and NO can induce hematopoietic stem cell fate. We show that ginger produces a robust up-regulation of NO. Taken together, we suggest in this paper that Bmp, Notch and NO are potential players that mediate the effect of ginger/10-G for rescuing the genetic defects in blood vessel specification and HSPC formation in plcg1^−/− mutants. Understanding the molecular mechanisms of HSPC development in vivo is critical for understanding HSPC expansion, which will have a positive impact in regenerative medicine.