The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions. qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The beta-actin, EF1alpha and Rpl13alpha genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1alpha, Rpl13alpha and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent.

The zebrafish (Danio rerio) provides an excellent model for studying vertebrate development and human disease because of its ex utero, optically transparent embryogenesis and amenability to in vivo manipulation. The rapid embryonic developmental cycle, large clutch sizes and ease of maintenance at large numbers also add to the appeal of this species. Considerable genomic data has recently become publicly available that is aiding the construction of zebrafish microarrays, thus permitting global gene expression analysis. The zebrafish is also suitable for chemical genomics, in part as a result of the permeability of its embryos to small molecules and consequent avoidance of external confounding maternal effects. Finally, there is increasing characterization and analysis of zebrafish models of human disease. Thus, the zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effect of small molecules as well as for dissecting biological pathways.

The zebrafish has been the model of choice amongst developmental biologists for many years. This small freshwater species offers many advantages to the study of organ and tissue development that are not provided by other model systems. Against this background, modern molecular genetic approaches are being applied to expand the physical and genetic mapping of the zebrafish genome. These approaches complement the large-scale mutagenic screens that have led to the isolation of mutant phenotypes. Some of the phenotypes have been found to resemble human disease states, while mapping and sequencing data have revealed zebrafish genes with significant homology to human disease-causing genes. It is the realization that the zebrafish offers an amenable system for understanding disease, as opposed to development, that underpins this review. The adventitious identification of disease phenotypes amongst zebrafish mutants and the important area of deliberate disease modelling using transgenesis and gene targeting should lead to a better application of the zebrafish as a vertebrate model of human diseases.