Ploidy manipulation and induction of alternate cleavage patterns through inhibition of centrosome duplication in the early zebrafish embryo.

Abstract

Whole genome duplication is a useful genetic tool because it allows immediate and complete genetic homozygosity in gynogenetic offspring. A whole genome duplication method in zebrafish, Heat Shock, involves a heat pulse in the period 13-15 min postfertilization (mpf) to inhibit cytokinesis of the first mitotic cycle. However, Heat Shock produces a relatively low yield of gynogenotes. A heat pulse at a later time point during the first cell cycle (22 mpf, HS2) results in a high (>80%) frequency of embryos exhibiting a precise one-cell division stall during the second cell cycle, inducing whole genome duplication. Coupled with haploid production, HS2 generates viable gynogenetic diploids with yields up to 4 times higher than those achieved through standard Heat Shock. The cell cycle delay also causes blastomere cleavage pattern variations, supporting a role for cytokinesis in spindle orientation during the following cell cycle. Our studies provide a new tool for whole genome duplication, induced gynogenesis, and cleavage pattern alteration in zebrafish, based on a time period before the initiation of cell division that is sensitive to temperature-mediated interference with centrosome duplication. Targeting of this period may also facilitate genetic and developmental manipulations in other organisms.