An ongoing major effort in the lab is to use the zebrafish as an in vivo model to validate and study the biological effects of small molecules, which are being generated through our other project (HG000030-18) for the development of novel compounds that target CBF leukemia, those with fusion genes affecting CBF genes, i.e., RUNX1 and CBFB. Both of these genes are required for the establishment of definitive hematopoiesis, so compounds designed to interrupt their function in leukemia would be expected to affect definitive hematopoiesis during embryo development. The zebrafish also serve the function of toxicity studies for these chemicals. This assay system has turned out to be a sensitive, fast and reliable system to screen potential compounds before they are characterized further in other systems, such as mouse models. Some of the findings have been reported in our recently published paper (Cunningham et al., PNAS 109:14592, 2012). A few years ago we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in the zebrafish to identify novel genes involved in early hematopoiesis. The mutant fish line mummy (mmy) from the screen was investigated in detail because of its multiple hematopoietic defects. Homozygous mmy embryos lacked circulating blood cells and were dead by 30 hours post-fertilization (hpf). The mmy mutants did not express myeloid cell markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis. Through positional cloning, we identified a truncation mutation of the dhx8 gene in the mmy fish. The encoded dhx8 protein is the zebrafish ortholog of the yeast splicing factor prp22, which is a DEAH-box RNA helicase. Mmy mutants had splicing defects in many genes, including several hematopoietic genes. Mmy embryos also showed cell division defects as characterized by disorganized mitotic spindles and formation of multiple spindle poles in mitotic cells. These cell division defects were confirmed by DHX8 knockdown in HeLa cells. Together, our results confirm that dhx8 is involved in mRNA splicing and suggest that it is also important for cell division during mitosis. This is the first vertebrate model for dhx8, whose function is essential for primitive hematopoiesis in developing embryos. This work was finished last year and the results were published recently (English et al., Developmental Dynamics 241:879889, 2012). Despite detailed in vivo knowledge of glycolytic enolases and many bacterial non-enolase members of the superfamily, little is known about the in vivo function of vertebrate non-enolase enolase superfamily members (ENOSF1s). Results of previous studies suggest involvement of the βsplice form of ENOSF1 in breast and colon cancers. We therefore used the zebrafish (Danio rerio) as a vertebrate model to study ENOSF1βfunction. WISH showed that zebrafish ENOSF1β(enosf1b) is zygotic and expressed ubiquitously through the first 24 hpf. After 24 hpf, enosf1b expression is restricted to the notochord. Embryos injected with enosf1b-EGFP mRNA grew slower than EGFP mRNA-injected embryos but caught up to the EGFP-injected embryos by 48 hpf. Embryos injected with ATG or exon 10 enosf1b mRNA-targeting morpholinos had kinked notochords, shortened anterior-posterior axes, and circulatory edema. WISH for ntl or pax2a expression showed that embryos injected with either morpholino have deformed notochord and pronephros. TUNEL staining revealed increased apoptosis in the peri-notochord region. This study is the first report of ENOSF1 function in a vertebrate and shows that ENOSF1 is required for embryonic development. Increased apoptosis following enosf1b knockdown suggests a potential survival advantage for increased ENOSF1βexpression in human cancers. This study was finished last year and was published recently (Finckbeiner et al., Cell &Bioscience 1:32, 2011).
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