Harnessing zebrafish for the study of white blood cell development and its perturbation  Sara M.N. Onnebo, Simon H.S. Yoong, Alister C. Ward  Experimental Hematology  Volume 32, Issue 9, Pages 789-796 (September 2004) DOI: 10.1016/j.exphem.2004.04.012 Copyright © 2004 International Society for Experimental Hematology Terms and Conditions

Figure 1 Zebrafish blood cell markers. Schematic representation of blood cell development in zebrafish, indicating specific gene markers for each lineage (italicized). Examples of embryonic expression patterns obtained with some of these markers are shown, with the marker name and developmental time point (hpf) indicated. Experimental Hematology 2004 32, 789-796DOI: (10.1016/j.exphem.2004.04.012) Copyright © 2004 International Society for Experimental Hematology Terms and Conditions

Figure 2 Manipulation of zebrafish embryos. Specific genes (wild-type or mutant) can be expressed using either in vitro–transcribed RNA or linearized DNA constructs. Alternatively, genes can be inhibited using either antisense morpholino oligonucleotides or, possibly, siRNAs. Using a stereo dissecting microscope, 1–4 cell embryos are held in place with a micromanipulator and pierced with a finely drawn capillary. A small volume (1–10 nL) of sample solution is then injected by a pulse of pressurized air. Perturbations of development are then monitored via a range of microscopic and molecular techniques. Experimental Hematology 2004 32, 789-796DOI: (10.1016/j.exphem.2004.04.012) Copyright © 2004 International Society for Experimental Hematology Terms and Conditions

Figure 3 Mutagenesis screening in zebrafish. Classical “F3 phenotypic screen” for identifying phenotypic mutants in zebrafish. In this case, wild-type male fish are mutagenized and crossed with wild-type females to produce F1 progeny that carry a unique set of mutations at up to 100 loci. Individual F1 females are then crossed to wild-type males to produce F2 families which contain a mix of wild-type (+/+) and carrier (+/−) offspring. One quarter of random matings between individuals within the same F2 family will occur between carriers (+/− × +/−). These crosses will yield F3 progeny, one quarter of which will be homozygous for mutations (−/−) that are scored phenotypically. Identified carriers can then be used for mutation propagation and mapping. Coarse mapping is achieved via simple sequence length polymorphisms (SSLPs) and fine mapping by single nucleotide polymorphisms (SNPs). Ultimately candidate genes are sequenced to reveal the molecular basis of the mutation. Experimental Hematology 2004 32, 789-796DOI: (10.1016/j.exphem.2004.04.012) Copyright © 2004 International Society for Experimental Hematology Terms and Conditions

Figure 4 Leukemia screening in zebrafish. Zebrafish with a marked white blood cell compartment, such as spi1:GFP transgenic fish [44] are subjected to ENU-mediated mutagenesis followed by screening to identify mutants with an expanded fluorescent cell compartment. These mutants will be mapped to facilitate the isolation of the causal genetic lesion. Experimental Hematology 2004 32, 789-796DOI: (10.1016/j.exphem.2004.04.012) Copyright © 2004 International Society for Experimental Hematology Terms and Conditions