miR-27b controls venous specification and tip cell fate.

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR-27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets.

Transplantable tumor lines generated in clonal zebrafish.

Transplantable zebrafish tumors are a novel and very promising model in cancer research. However, further progress in this field has been contained by a lack of true inbred lines in zebrafish. To overcome this problem, we generated two lines of homozygous diploid clonal zebrafish lines (i.e., CB1 and CW1), which allowed us to carry out transplantation of any tissue, including tumors, from one fish to another within a line without rejection of the graft. The primary tumors in CB1 fish were induced by N-nitrosodiethylamine (DEN). The histologic analysis of these tumors revealed different types of hepatocellular carcinomas, hepatoblastomas, hepatoma, cholangiocarcinoma, and pancreatic carcinoma. Four spontaneous acinar cell carcinomas of pancreas were also found in 10- to 18-month-old CB1 fish. Small pieces of tissue or cell suspensions of either DEN-induced or spontaneous tumors were serially transplanted into the peritoneal cavity of syngeneic fish at different stages of development from 5-day-old larvae to adult fish. The development of grossly visible tumors occurred from 2 weeks to 3 months after tumor grafting and grew either as solitary smooth nodules or as an amorphous jelly-like mass infiltrating abdominal organs. The majority of tumors were also successfully transplanted to isogeneic (F1 generation from crossing CB1 x CW1) fish. At the present time, 19 transplantable zebrafish tumor lines have been generated and maintained for as long as 3 to 25 passages. This model provides a novel tool for studying experimental tumor biology and therapy and will become a cost effective system for high throughput screening of anticancer drugs.

Carcinogenic effect of N-nitrosodimethylamine on diploid and triploid zebrafish (Danio rerio).

Viability of polyploid organisms in lower vertebrates including fish provides an additional tool to investigate genetic mechanisms of neoplastic transformation caused by carcinogens. Here we present data on differential sensitivity of diploid and triploid zebrafish (Danio rerio) to N-nitrosodimethylamine (NDMA) induced hepatocarcinogenesis. The effect of the carcinogen was studied in 100 diploid and 120 triploid zebrafish. Zebrafish, age 5-6 weeks, were exposed to 50 ppm NDMA for 8 weeks and then were transferred into fresh carcinogen-free water until necropsy. At the necropsy performed 24 weeks after beginning the treatment, cholangiolar tumors (cholangiocarcinomas and cholangiomas) were essentially observed in diploid zebrafish only, while the incidence of hepatocellular tumors (hepatocellular carcinomas and adenomas) was similar in diploid and triploid zebrafish, 7.7% and 9.5%, respectively. By contrast, 36 weeks after beginning the treatment, the incidence of hepatocellular tumors was significantly lower in diploid animals as compared to triploid ones, 10.3% and 33.8%, respectively. The incidence of cholangiolar tumors in diploid and triploid zebrafish was not significantly different, 10.3% and 14.9%, respectively. Therefore, the increase of ploidy appeared to have a differential effect on the induction of these 2 types of liver tumors in zebrafish. This finding suggests a difference in genetic mechanisms of the tumor development revealed by utilization of triploid animals in this study. However, triploid zebrafish demonstrated an overall increase in latency period in the development of both types of hepatic tumors, a finding that can be interpreted as an increased resistance of triploid animals to the carcinogenic effect of NDMA.