A Model System for Visualization of Whole-Body Sdf-1 Expression During Hematopoietic Cell Transplant.

Abstract 3623

Poster Board III-559

Following hematopoietic cell transplantation (HCT), transplanted cells must extravasate from the circulation and migrate through the tissue in the recipient, to arrive at niche locations and proliferate. The chemokine SDF-1 and its receptor, CXCR4, have been strongly implicated in this cell migration process during HCT in mammals. SDF-1 has been suggested to recruit CXCR4-expressing transplanted cells to both the HSC niche in the bone marrow as well as to extramedullary sites, such as the injured heart. Reports of anatomical sites of SDF-1 expression in mammals have included HSC niche cells in the bone marrow, cells in the heart, lung, skin, and in pericytes along small vessels. The zebrafish danio rerio is a small vertebrate model organism ideal for direct visualization of cell localization during experimental HCT. However, it is currently unknown whether the cell recruitment roles and expression patterns of sdf-1 in mammals are conserved in the zebrafish. Therefore, we have generated a transgenic reporter zebrafish tg(sdf-1a:dsRed2) that has allowed, for the first time, in-vivo detection of endogenous sdf-1a expression throughout the whole body of the living organism. We report that sdf-1 expression in the adult zebrafish is upregulated in the hematopoietic tissue in response to preconditioning radiation, in a dose-dependent manner. In zebrafish, the kidney is the major site of hematopoiesis. qRT-PCR indicates this dose-dependent sdf-1 increase is greatest in irradiated kidney tissue that has been enriched in kidney tubules with a 4.8-fold increase at 1 day after 30 Gy and a 8.9-fold increase after 40 Gy of irradiation. Similarly, FACS analysis of tg(sdf-1a:dsRed2) shows ∼2-fold increased numbers of sdf-1a:dsRed-positive renal tubule cells in transgenics that have received doses of 40 or 50 Gy, compared to unirradiated controls. These data suggest that renal tubule cells in zebrafish are the primary source of increased sdf-1a expression in the irradiated kidney. To assess the functional relevance of this increased sdf-1a expression to HCT in the zebrafish, we pre-treated hematopoietic cells with AMD3100, a CXCR4 antagonist prior to transplant. A significant decrease in cells with lymphocyte and putative stem cell forward and side scatter properties was seen in HCT recipient kidney two days after transplant, consistent with the blockade of hematopoietic stem cell homing into the kidney microenvironment by the CXCR4 antagonist. Sdf-1a:dsRed2 expression in the adult transgenic, confirmed by in-situ hybridization for sdf-1a, shows that sources of sdf-1a in the zebrafish include both tubular and vascular cells in the hematopoietic tissue, cells in the gills, and organized structures in the skin surrounding superficial vessels. The transgenic also shows sdf-1a:dsRed2 expression in the adult heart. Notably, this system has additionally allowed facile in-vivo detection of sdf-1a-expressing pericytes and vascular cells prevalent along microvessels in several locations throughout the fish, including the hematopoietic tissue, skin, and fins. Experiments are on-going to identify the sdf-1/cxcr4-dependent homing and extramedullary cell migration events following HCT, and to assess in vivo interactions between transplanted hematopoietic cells and putative HSC niche cells. Ongoing studies are focused upon organism-wide in-vivo interrogation of the roles of sdf-1-expressing cells, including pericytes and putative mesenchymal stem cells, in directing the cell migration events that are crucial for successful HCT.