Abstract
Hematopoiesis is a tightly regulated process that requires the coordinated efforts of many tissue-specific transcription factors. Lateral mesoderm tissue undergoes a series of restricted proliferation and differentiation that result in the production of the hematopoietic stem cells. These cells then differentiate to populate the different cell lineages. While some factors essential for this process have been defined, many remain unknown. Zebrafish offer a powerful model system in which it is possible to conduct forward phenotype-driven screening. We have undertaken a chemical mutagenesis screen using N-nitroso-N-ethylurea (ENU) in the zebrafish to uncover mutants in myelopoiesis and early hematopoiesis. Adult male fish were exposed to ENU in a protocol designed to mutagenize spermatagonia with a mutation frequency of approximately 1:1000. Mutagenized males were crossed to wild-type females to generate F1 females. Haploid embryos from F1 females were screened at 27 hours of development by RNA in situ hybridization using the myeloid-specific marker, leukocyte-specific plastin (l-plastin), and the stem cell marker, core binding factor b (cbfb). One mutant line identified from this screen, mummy, showed reduced cbfb expression and lack of l-plastin expression. Additional in situ staining has confirmed that mummy has a myeloid defect as other myeloid markers such as Lys.C, Mpo, and C/ebp1 were also significantly reduced. Mummy mutant also have a degreased expression of gata1 and bE1 globin (RBC markers) as well as the stem cell marker scl. These results suggested that the defect in mummy is at or up-stream of the HSC level. The mummy mutant embryos also suffer from widespread cell death and die around 36 hours post fertilization. Genetic mapping and positional cloning has identified the mutated gene as the zebrafish homolog of DHX8, which encodes an RNA helicase. The yeast homolog of dhx8, prp22, functions during mRNA splicing and RNA processing. Morpholino injection into wild-type embryos has confirmed that reduced expression of dhx8 can produce a similar phenotype. The wild-type zebrafish dhx8 cDNA but not the mutated dhx8 cDNA identified in mummy was able to partially rescue the mummy phenotype. Preliminary data from RT-PCR and microarray analysis suggest that mummy mutants have defects in the production of spliced transcripts of some genes but not others. Genes with decreased spliced transcripts in the mummy mutant include scl, cbfb, and gata1, which may explain the hematpoietic defects observed. Our results demonstrate that mutations in an RNA helicase involved in splicing could result in specific blockage of hematopoiesis.
Neoadjuvant chemotherapy with gemcitabine for pancreatic cancer increases in situ expression of the apoptosis marker M30 and stem cell marker CD44
