Figure 1

Generation of leukemia-derived G-CSFR truncation mutants. Schematic diagram of the G-CSFR showing the extracellular immunoglobulin domain (pink), cytokine receptor homology domain (orange) and fibronectin type III-like domains (purple), transmembrane region (green), and intracellular region (blue) containing Box 1-3 (black rectangles). The intracellular region is expanded to show tyrosine (Y) residues and a di-leucine motif (LL) in both human and zebrafish proteins, with the relative positions of truncation mutations found in various human leukemias or demonstrated to be leukemogenic in vitro shown above (A). Exons 14-16 of the zebrafish csf3r gene encoding the intracellular region presented as numbered boxes with connecting lines depicting introns (B). Sequence traces of the indicated part of csf3r showing homozygous wild-type (wt/wt) and mutant zebrafish, with nucleotide sequence above and encoded protein sequences below, in purple for native and orange for de novo sequence (C). The gRNA target site is shown above in blue, with deleted nucleotide sequences indicated with a brown dotted box and inserted sequences shown with black boxes. The csf3r^mdu26 (mdu26) allele represents a complex 1 bp insertion/8 bp deletion and the csf3r^mdu27 (mdu27) allele a 5 bp insertion both of which cause a frameshift resulting in translation from an alternative reading frame followed by a stop codon. Gene expression analysis of csf3r in pooled WT and mutant embryos at the indicated timepoints presented as fold-change (log₂) relative to actb (D), showing mean and SEM (not significant, n=3).