Gene expression analysis in zebrafish marrow niche cells reveals dpt as a modulator of hematopoietic cell homing. (A) Strategy for enriching marrow niche cells. The zebrafish marrow compartment contains hematopoietic cells, endothelial cells, renal tubules, and other supportive niche cells shown by hematoxylin and eosin staining. Tituration and filtration through a 40-μm filter allows for smaller HSPCs to be separated from larger niche cells. See supplemental Figure 1 for full image. Scale bar, 50 μm. (B) qRT-PCR of hematopoietic (red) and niche-related (blue) factors in marrow cells that were separated using a filter into 2 cell fractions. n = 3 to 6 animals per group (technical triplicates) in 2 independent experiments. P < .01 for each gene (niche vs hematopoietic). (C) Scatter plot of Affymetrix RNA array expression data from niche cells isolated 24 hours after zebrafish received 0-Gy or 30-Gy radiation. Axes give expression in log₂. The thin red lines demark a twofold change in gene expression; radiation-induced increased gene expression is indicated by red dots and decreased expression is indicated by green dots (n = 3 per condition). (D) Unsupervised cluster analysis of 144 niche-related genes induced after radiation. Red indicates genes that were expressed more highly (more than twofold) after 30 Gy. Green indicates genes with a lower expression level in irradiated animals (more than twofold). Scale bar indicates absolute log₂ expression scale (2.3-11.7). (E) Ingenuity Pathway Analysis showing the highest ranked network (score of 41) and its composition of 18 proteins. (F) qRT-PCR of Dpt expression in the marrow after 30-Gy radiation (n = 6 per group). (G) Immunofluorescence of Dpt in zebrafish 24 hours after radiation. Quantitation was performed using ImageJ; 24 images were quantified across 3 biologic replicates per condition. (H) Western blot of zebrafish WKM for Dpt (*) 24 hours after radiation. A human fibroblast line served as a positive control.