Park et al., 2020 - Zebrafish B cell acute lymphoblastic leukemia: new findings in an old model. Oncotarget   11:1292-1305 Full text @ Oncotarget

Figure 1 B-ALL in <italic>hMYC</italic> zebrafish.

(A) Curves displaying B- and T-ALL incidence by 184 dpf, as determined by fluorescence microscopy. (B) Fluorescence microscopy images of fish with B-ALL (left), both B- and T-ALL (center), or T-ALL (right). Flow cytometry plots of ALL samples from these fish are shown beneath them. (C) B-ALL appearance in fish with rag2:hMYC and different transgenic markers: cd79a:GFP (left), cd79b:GFP (center), or lck:GFP (right). WT cd79a:GFP or cd79b:GFP fish without B-ALL are shown beneath for comparison. Images are representative of > 50 animals examined for each genotype.

Figure 2 B-ALL regress with DXM or IR treatments.

(A) B-ALL in rag2:hMYC fish treated with DXM for 9 days (n = 19). Post-treatment and relapsed images were obtained on days 9 and 28, respectively. Every (19/19) B-ALL regressed after DXM, but 11/19 (58%) B-ALL relapsed by day 28 (19 days after DXM withdrawal). (B) B-ALL in rag2:hMYC fish treated with IR (n = 13). Post-treatment and relapsed images were obtained on days 7 (i. e., 2 days after the 2nd IR dose) and 23, respectively. Every B-ALL (13/13) regressed after IR, but 100% recurred within 3 weeks of the 2nd IR dose. Upon relapse, flow cytometric testing (See Figure 1B) was used to verify that each relapse was GFPlo B-ALL. Images of rag2:hMYC, lck:GFP fish (top 5 animals) have had brightness enhanced to facilitate visualization of dim B-ALL. Images of the rag2:hMYC, cd79a:GFP fish (lowest animal) are unmodified.

Figure 4 Gene expression in individual <italic>hMYC</italic> B- and T-ALL cells.

(A) rag2:hMYC;lck: GFP fish with dim (left) and bright (center) cancers. B-ALL is only visible with 1.5 s exposure (upper left); T-ALL is visible with either 1.5 s (upper middle) or 200 ms (lower middle) exposures. Control lck:GFP fish images are shown at right, where only the thymus is visible. (B) Flow cytometry plots of the same ALL samples demonstrate GFPlo B-ALL and GFPhi T-ALL specimens. (C) Gene expression in single B- and T-ALL cells of the same cancers (n = 10 cells for each), as determined by Fluidigm™ Biomark qRT-PCR. B-ALL cells express B cell, but not T cell, genes; T-ALL show the opposite pattern. Expression of eef1a1l1 (homologue of human eukaryotic translation elongation factor 1 alpha 1, aka EF1A) was used as a threshold control for the presence of RNA in each well. CT values were converted to Log2Ex for visualization (scale bar at left).

Figure 5 Alignment of human and murine MYC proteins.

Transgenic human and mouse MYC are 435 and 439 amino acids, respectively (last four mMyc residues not shown). Proteins show > 91% identity (399 residues, shaded light blue) and > 94% similarity (410 residues; 11 additional similar residues shaded darker blue).

Figure 6 Differentially-expressed genes in <italic>hMYC</italic> versus <italic>mMyc</italic> B-ALL.

(A) Heatmap depicting 62 differentially-expressed mRNA, including genes of the Gene Ontology (GO) RNA binding pathway (blue bar), D. rerio fos/jun-family members (green bar), endogenous myc-family members (red bar), and other myc-related proteins, including several max heterodimeric partners (purple bar). Samples hMYC-1, -3, -4, and -5 are four distinct hMYC B-ALL; 9.2A/B and 10.2A/B pairs are RNA-seq technical replicates of two mMyc B-ALL [57, 60]. Genes not meeting differential expression testing thresholds or other filtering criteria are marked by asterisks (FDR < 0.05, absolute fold-change ≥ 1.5). Read counts are shown as z-scores (scale bar at left). (B) Top ten biologic pathways up-regulated in hMYC (top) or mMyc B-ALL (bottom; FDR < 0.05). Pathways are ordered according to the number of genes detected in the gene set (x-axis) and colored based on FDR q-value. Data point sizes correspond to the percentage of genes over-expressed in each pathway.

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