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

Abstract

Background 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 minutes post-fertilization (mpf) to inhibit cytokinesis of the first mitotic cycle. However, Heat Shock produces a relatively low yield of gynogenotes. Results 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. Conclusions Our studies provide a new tool for whole genome duplication, induced gynogenesis and cleavage pattern alteration in zebrafish, based on a time period prior to 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.

Transient window of resilience during early development minimizes teratogenic effects of heat in zebrafish embryos

“…In addition to 1C and 2C stalls, early embryonic cytokinesis geometries also deviate from the norm after a heat shock (Fig. 1S–U and Heier et al, 2015). We hypothesized that the embryonic lethality typically associated with a transient heat shock in zebrafish embryos may be due to alterations in cytokinesis geometries, which may culminate in eventual morphological abnormalities and/or lethality.…”

“…We tested whether the phenomenon of teratogenic heat resilience during prometaphase/metaphase would hold true for subsequent mitosis in zebrafish by exposing zebrafish embryos to transient heat for 2 min between 40 and 52 mpf, which encompassed prophase to anaphase of mitosis‐II. However, early embryonic lethality was found to be ∼98% in all mitosis‐II heat shocks (data not shown and Heier et al, 2015). …”

“…2A and Heier et al, 2015). In both 1C and 2C stalls, we also found embryos that underwent normal cytokinesis geometries similar to control embryos.…”

“…1A–C,G–I,M–O,F,L,R,S and Heier et al, 2015). In addition to 1C and 2C stalls, early embryonic cytokinesis geometries also deviate from the norm after a heat shock (Fig.…”

“…A proportion of heat shocked embryos do not undergo cytokinesis stalls and were not analyzed further (Fig. 1S and Heier et al, 2015). …”