Zebrafish Myc-induced T-ALLs are arrested at the CD4⁺/CD8⁺ cortical thymocyte stage and reexpress stem cell genes. (A) Merged bright field and fluorescent images of Tg(rag2:dsRed) zebrafish (top left) or fluorescent leukemic fish. LPC frequency for each individual T-ALL is denoted in bottom left corner of each image, and clone name is denoted in the top left corner. The white arrowhead designates thymus. Bars, 2.5 mm. (B) May-Grunwald staining of representative T-ALL cells (left) and normal WKM (right; red blood cell, red arrow; myeloid cell, green arrow, and lymphocyte, blue arrow). Bars, 25 µm. (C) Kaplan-Meier analysis of leukemia onset in transplanted zebrafish. Number designations correspond to leukemias described by Blackburn et al. (2014). (D) Percentage of T cells that express CD4 and CD8 in normal rag2:dsRed⁺ thymic T cells and fluorescent-labeled T-ALLs. ***, P = 0.0001, by Fisher’s exact test. (E) Principal component analysis identified three distinct clusters of cells that include the following: mature T cells (Tg(lck:GFP) dark blue, n = 83 cells), thymocytes (Tg(rag2:dsRed) light blue, n = 77 cells from a single animal), and T-ALLs (pink, n = 250 cells, combined from three independent leukemias). (F) WGCNA of T-ALLs revealed that stem cell genes are reexpressed in T-ALLs and expressed together. Each row and column corresponds to a specific gene primer set. The heat map represents WCGNA adjacency dissimilarity values in the range between 0 (low topological overlap, light yellow) and 1 (high topological overlap, dark red). Red lettering denotes genes shown in G. (G) Violin plots show the distribution of transcript expression for genes involved in T cell differentiation and stem cells. ***, P = 0.0001, by Fisher’s exact test.