In mammals stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and

In mammals stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and migration. and distal renal tubules and collecting ducts. Importantly transplant of hematopoietic cells into myelosuppressed recipients indicated migration of hematopoietic cells to expression and function in the adult zebrafish have important similarities to mammals and this transgenic vertebrate will be useful in characterizing the hematopoietic cell niche and its interactions with hematopoietic cells. Introduction The zebrafish expression is a characteristic of the zebrafish HSC niche. Although mammals have a single gene with multiple splice variants zebrafish have 2 genes and share approximately 45% and 47% nucleotide identity respectively with the human gene.22 The zebrafish and genes are located VS-5584 on different chromosomes and share approximately 75% amino acid identity.22 23 We report that this anatomic locations of expression in the zebrafish are similar to those patterns previously reported in mammals. Moreover is expressed in high levels in renal tubule cells which compose a majority of the marrow in the adult zebrafish and may form the HSPC niche. is up-regulated in a dose-dependent fashion after radiation in the zebrafish and acts as a chemoattractant to adult zebrafish hematopoietic cells. Because previous studies22 as well as our own experiments indicated predominant expression of might play a predominant role in recruiting HSPCs. Therefore we created a transgenic fluorescence reporter of expression to achieve simultaneous assessment of donor-derived hematopoietic cell VS-5584 migration and expression patterns in HSPC transplant recipients. Using this transgenic reporter VS-5584 putative hematopoietic niche-defining cells in the kidney were localized as kidney tubule cells. Sdf-1a-expressing cells also were found in regions throughout the skin of the transplant recipients. Cumulatively our findings suggest that SDF-1 may guideline hematopoietic cell migration after HCT RAB11B in the adult zebrafish to both medullary and extramedullary sites. Methods Zebrafish strains and fish husbandry Fish were maintained by the University of Minnesota Zebrafish Core Facility according to standardized procedures24 and with the approval of the Institutional Animal Care and Use Committee University of Minnesota. Wild-type fish were obtained from Segrest Farms and bred in-house. Other fish strains used include AB mutant.28 Transgenic construction VS-5584 VS-5584 The 4.3-kb promoter region of was cloned upstream of the fluorophore in the Tol2 backbone and injected along with transposase mRNA to facilitate integration into early zebrafish embryos.29 Embryos were screened for fluorescence raised to maturity and bred to identify founders. Two impartial transgenic lines each generated from a separate founder fish were used for this work. Fish irradiation Radiation was delivered through one of 2 systems With a J.L. Shepherd Mark 1 Model 30 Irradiator with a Cs137 source. Radiation chambers were constructed from Petri dishes radially divided with Plexiglas into 16 wedge-shaped compartments filled with fish water each compartment made up of a single unanesthetized zebrafish. Radiation chambers were placed on a rotating turntable in front of the source. Fish were irradiated with a single unfractionated dose at a rate of 6.55 Gy/min. The mean lethal dose identified for this cesium system was 30 Gy and this dose was used for cesium radiation preconditioning for most transplant experiments. With a X-Rad320 irradiator (Precision X-Ray Inc). Unanesthetized fish were VS-5584 placed in a Petri dish filled with fish water and placed below the source. Fish were irradiated with a single unfractionated dose at a rate of 2.7 Gy/min. The mean lethal dose identified for this x-ray system was 20 Gy and this dose was used for x-ray preconditioning for most transplant experiments. The studies were started with the cesium-based source but this was decommissioned partway through the experiments. Subsequent studies were performed around the x-ray-based source and there were no biologic differences found in the ability of the 2 2 radiation systems to up-regulate or myeloablate recipients. Kaplan-Meier survival graphs documenting survival after radiation doses were prepared for each of these radiation delivery methods.