A study of zebrafish hematopoiesis based on chemical screening and gene knock-down by morpholino with particular reference to ADP-ribosylation factor like 4 (ARL4)

Abstract

Zebrafish has emerged as an important vertebrate model for studying hematopoiesis and its genetic and chemical modifiers. The zebrafish embryos are unique in their optical transparency, ease of maintenance and high fecundity. They are also amendable to genetic and pharmacological perturbation at high throughput. As a result, the embryos are suitable for various experimental techniques and have a high efficiency in large-scale drug screening. Recently, zebrafish has also emerged as a model for the study of human disease.

In this model organism, primitive hematopoiesis is transitory and it occurs in the intermediate cells mass and comprises primarily erythroid cells. Definitive hematopoiesis arises from the ventral wall of dorsal aorta and moves to the caudal hematopoietic tissues, thence the kidney, where life-long and multi-lineage differentiation occurs.

The chemical screening platform comprises O-dianisidine staining for hemoglobin containing cells (erythroid) during primitive hematopoiesis. Positive hits were validated based on flow cytometry of dissociated transgenic Tg(gata1:GFP) embryos and whole-mount in-situ hybridization (WISH) for hematopoietic genes. Gene knock-down was conducted by morpholinos (MO) injected into zebrafish embryos at 1-4 cell stage and the effects on hematopoietic development evaluated by WISH and quantitative real-time PCR.

Chemical screening of 74 compounds has been performed. These compounds were obtained from a chemical library comprising 879 compounds from NIH (National Institutes of Health) and pre-screened by their effects on cancer cell lines. Four compounds (Tin(IV), chlorotriphenyl [1-(4-ethoxyphenyl)- 3-cyanoureato]-hydrogen,triethylamine, Nogamycin, N,N-Dibenzyldaunorubicin hydrochloride and Allyl 2,3,4-tri-O-benzyl-6-O-(tert-butyldimethylsilyl)-α- D-gluco-Pyranoside) which significantly reduced O-dianisidine staining were identified of which one (Allyl 2,3,4-tri-O-benzyl-6-O-(tert-butyldimethylsilyl) -α-D-glucopyranoside was shown to reduce GFP+ population in Tg(gata1:GFP) population Another chemical (2-Propanol,1,1'-[(1-methylethylidene)bis(4, 1-phenyleneoxy)]bis[3-[(1,1,3,3-tetramethylbutyl)amino]-,dihydrochloride]) was shown to reduce c-myb (marker of definitive hematopoiesis) expression in the ventral wall of dorsal aorta.

I also attempted gene knock in zebrafish embryos based on anti-sense morpholino microinjection. A gene encoding for arl4ab was examined, as it was shown to be expressed in hematopoietic tissue in zebrafish embryos but its function is entirely unknown. Knock-down of arl4ab significantly reduced c-myb and runx1 expression in the ventral wall of dorsal aorta and it can be reversed by co-injecting arl4ab mRNA. scl and gata1 expression as well as GFP expression in transgenic Tg(flk1:GFP) embryos that represented vascular development was unaffected.

In summary, a zebrafish platform for the study of chemical and genetic modifiers was established. The results have provided important leads for further study into the mechanisms whereby these modifiers regulate hematopoiesis in the zebrafish embryos.