FIGURE SUMMARY
Title

A chemical genetic screen in zebrafish for pathways interacting with cdx4 in primitive hematopoiesis

Authors
Paik, E.J., de Jong, J.L., Pugach, E., Opara, P., and Zon, L.I.
Source
Full text @ Zebrafish

Screen design. cdx4 heterozygous adult fish were incrossed weekly to generate embryos for the chemical screen. Chemicals from the Institute of Chemistry and Cell Biology library were transferred by robotics from 384-well plates to 48-well plates. Each plate contained negative (1% DMSO) and positive (20 μM DEAB) control wells. About 20 synchronized embryos at 50% epiboly stage were placed manually into each well of the 48-well plates. Once the embryos reached the 10-somite stage, they were fixed and whole-mount in situ hybrization was performed with both gata1 and cdx4 riboprobes. Each well was examined and scored manually. A posterior view of representative embryos is shown, with anterior to the left and posterior to the right. cdx4-mutant embryos were identified by lack of cdx4 expression. In most wells, cdx4-mutant embryos had little or no gata1 expression. The wells with increased gata1 expression in the cdx4-mutant embryos were scored as a “rescue.” DMSO, dimethyl sulfoxide; DEAB, 4-diethylamino benzaldehyde; WT, wild type. Color images available online at www.liebertonline.com/zeb.

Scoring system for rescued embryos. The level of gata1 rescue was determined by the percentage of rescued mutant embryos in each well and by the intensity of gata1 expression in these embryos in comparison to the negative and positive control wells for each individual plate. DMSO vehicle was the negative control, and 20 μM DEAB was the positive control. Representative mutant embryos are shown after in situ hybridization for gata1 and cdx4 expression. Color images available online at www.liebertonline.com/zeb.

Bergapten increases gata1 and pu.1 in cdx4-mutant embryos. All embryos are displayed with anterior to the left and posterior to the right. (A) cdx4 heterozygous adults were incrossed, and the resulting embryos were treated with varying doses of bergapten. Bergapten rescues gata1 expression (level 3) in the cdx4-mutant embryos, and this rescue is most effective at 33 μM. Whole-mount embryos at the 10-somite stage are displayed from a representative experiment. (B) cdx4 heterozygous adults were incrossed, and the resulting embryos were treated with DMSO vehicle control, 20 μM DEAB, or 30 μM bergapten. Flatmounted embryos at the 10-somite stage are shown. WT embryos and cdx4 mutants stained with pu.1 were confirmed by genotyping. Bergapten does not change the expression pattern of mesodermal genes fli1, scl, and pu.1 in WT embryos. In cdx4-mutant embryos, however, pu.1 expression is rescued in the posterior lateral mesoderm when either DEAB or bergapten is applied. Ber, bergapten. Color images available online at www.liebertonline.com/zeb.

Bergapten and other psoralen compounds affect anterior–posterior patterning. (A) Chemical structures of the compounds tested. (B) Whole-mount in situ hybridization of WT AB embryos with krox-20 and myoD riboprobes. Whole-mount 10-somite stage embryos are shown from a representative experiment. The average distance between krox-20 and myoD in control embryos is 138.6 μm (±7.6 μm). The bergapten-treated embryos lose the gap between krox-20 and myoD, similar to DEAB-treated embryos. 8-MOP also affects the distance, but to a lesser degree (85.8 ± 4 μm). In contrast, PAP-1 does not affect this distance. (C) cdx4 heterozygous adults were incrossed, and the resulting embryos were treated with DMSO vehicle control, 20 μM DEAB, 33 μM 8-MOP, or 33 μM PAP-1. Whole-mount in situ hybridization was done with gata1 and cdx4 riboprobes at the 10-somite stage. 8-MOP robustly rescues gata1 expression in the cdx4-mutant embryos, whereas PAP-1 does not rescue. ss, somite stage; PAP-1, 5-(4-phenoxybutoxy)psoralen; 8-MOP, 8-methoxypsoralen. Color images available online at www.liebertonline.com/zeb.

The effect of 8-MOP on fli1, scl, and pu.1 expression in the WT and cdx4-mutant embryos. 8-MOP does not change the expression pattern of mesodermal genes fli1, scl, and pu.1 in WT embryos. In cdx4-mutant embryos, however, pu.1 expression is rescued in the posterior lateral mesoderm when 8-MOP is applied. WT, wild type; 8-MOP, 8-methoxypsoralen.

krox20–myoD expression in the WT and cdx4-mutant embryos. DEAB- and bergapten-treated embryos lose the gap between krox-20 and myoD regardless of their genotype. DEAB, 4-diethylamino benzaldehyde; Ber, bergapten.

Bergapten does not affect the expression pattern of hoxa9a and hoxb7a in the cdx4-mutant embryos.

Acknowledgments
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