Estrogen defines the dorsal-ventral limit of VEGF regulation to specify the location of the hemogenic endothelial niche.

Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17ß-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.

Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models.

Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34(+) hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34(+) MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.

Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish.

NRAS mutations are a common oncogenic event in skin cancer, occurring frequently in congenital nevi and malignant melanoma. To study the role of NRAS in zebrafish, a transgenic approach was applied to generate fish that express human oncogenic NRAS(Q61K) under the control of the melanocyte-restricted mitfa promoter. By screening the progeny of the injected animals, two strains stably expressing the NRAS transgene were identified: Tg(mitfa:EGFP:NRAS(Q61K))(1) and Tg(mitfa:EGFP:NRAS(Q61K))(2). Stable expression of this transgene results in hyperpigmented fish displaying a complete ablation of the normal pigment pattern. Although oncogenic NRAS expression alone was found to be insufficient to promote tumor formation, loss of functional p53 was found to collaborate with NRAS expression in the genesis of melanoma. The tumors derived from these animals are variably pigmented and closely resemble human melanoma. Underscoring the pathological similarities between these tumors and human disease and suggesting that common pathways are similar in these models and human disease, gene set enrichment analysis performed on microarray data found that the upregulated genes from zebrafish melanomas are highly enriched in human tumor samples. This work characterizes two zebrafish melanoma models that will be useful tools for the study of melanoma pathogenesis.

Defining cancer stem cells by xenotransplantation in zebrafish.

The zebrafish (Danio rerio) has become an increasingly utilized and relevant model organism in the study of cancer. The use of transgenic and reverse genetic approaches has yielded several strains that model a variety of human neoplasms. In addition to modeling human disease, these strains provide a platform for the analysis of tumor stem cells. Here we describe the basic technique for the isolation and transplantation of tumor tissue in the zebrafish. This technique was designed to study metastasis and invasive potential of zebrafish tumor cells. Additionally, the basic protocol can be modified in order to describe cancer stem cell characteristics, including proliferative capacity, self-renewal, and the minimum number of tumor cells required for engraftment.

Topoisomerase II alpha is required for embryonic development and liver regeneration in zebrafish.

Topoisomerases solve the topological problems encountered by DNA throughout the lifetime of a cell. Topoisomerase II alpha, which is highly conserved among eukaryotes, untangles replicated chromosomes during mitosis and is absolutely required for cell viability. A homozygous lethal mutant, can4, was identified in a screen to identify genes important for cell proliferation in zebrafish by utilizing an antibody against a mitosis-specific marker, phospho-histone H3. Mutant embryos have a decrease in the number of proliferating cells and display increases in DNA content and apoptosis, as well as mitotic spindle defects. Positional cloning revealed that the genetic defect underlying these phenotypes was the result of a mutation in the zebrafish topoisomerase II alpha (top2a) gene. top2a was found to be required for decatenation but not for condensation in embryonic mitoses. In addition to being required for development, top2a was found to be a haploinsufficient regulator of adult liver regrowth in zebrafish. Regeneration analysis of other adult tissues, including fins, revealed no heterozygous phenotype. Our results confirm a conserved role for TOP2A in vertebrates as well as a dose-sensitive requirement for top2a in adults.

Transparent adult zebrafish as a tool for in vivo transplantation analysis.

The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.