Early, nonlethal ploidy and genome size quantification using confocal microscopy in zebrafish embryos

Abstract

Ploidy transitions through whole genome duplication have shaped evolution by allowing the sub‐ and neo‐functionalization of redundant copies of highly conserved genes to express novel traits. The nuclear:cytoplasmic (n:c) ratio is maintained in polyploid vertebrates resulting in larger cells, but body size is maintained by a concomitant reduction in cell number. Ploidy can be manipulated easily in most teleosts, and the zebrafish, already well established as a model system for biomedical research, is therefore an excellent system in which to study the effects of increased cell size and reduced cell numbers in polyploids on development and physiology. Here we describe a novel technique using confocal microscopy to measure genome size and determine ploidy non‐lethally at 48 h post‐fertilization (hpf) in transgenic zebrafish expressing fluorescent histones. Volumetric analysis of myofiber nuclei using open‐source software can reliably distinguish diploids and triploids from a mixed‐ploidy pool of embryos for subsequent experimentation. We present an example of this by comparing heart rate between confirmed diploid and triploid embryos at 54 hpf.

Interacting Effects of Cell Size and Temperature on Gene Expression, Growth, Development and Swimming Performance in Larval Zebrafish

“…This general pattern of triploid zebrafish larvae having larger but fewer cells need not necessarily apply to all cell types, as we only measured erythrocyte volume directly, but increases in cell volume in polyploid fish have been shown in a range of tissues, including muscle ( Suresh and Sheehan, 1998 ; Vargas et al, 2015 ), brain and liver (reviewed by Benfey, 1999 ). Furthermore, triploid zebrafish larvae have been found to be very similar to their diploid counterparts under non-demanding conditions ( van de Pol et al, 2020 ; Small et al, 2021 ), which makes triploid zebrafish a good model to study the effects of cell size. For this study, we tested whether the consequences of cell size are temperature dependent by rearing and testing the zebrafish at different temperatures.…”