Genetic Screens

The zebrafish provides a unique vertebrate model system for the analysis of developmental processes due to the transparency of the larvae, the short generation time and the ease with which mutations can be created and detected by various types of screens. Standard ethylnitrosourea (ENU)-based large-scale screens have identified a large number of mutants affecting early development and organogenesis. Gynogenetic diploid embryos can be obtained through Early Pressure (EP, see Fig.1), thus permitting the expeditious detection of heterozygous individuals. Zebrafish produce a large number of embryos and can be mated every week, facilitating the swift accumulation of meiotic recombinants for the purpose of positional cloning of mutant genes.

We employ a variety of standard and innovative approaches to adapt genetic screens with the aim of creating immunodeficiencies, autoimmunity and leukemia in mutant zebrafish. The centerpiece of these screens is a transgenic line of zebrafish we have created, where all T cells are marked with GFP. The screen consists of generating gynogenetic diploid embryos by EP treatment (see Fig. 2). This allows us to identify interesting mutations in heterozygous females, obviating the need for generation of large numbers of families to screen through. Gynecogenetic diploid embryos and adult individuals are subjected to fluorescent in-vivo imaging to identify absent (bona fide immunodeficiency), or increased (leukemia) numbers of fluorescent T cells, or their infiltration into tissues (autoimmunity) (see Fig.3). To overcome problems with tissue-autofluorescence we use a liquid crystal tunable filter (CRI Inc., Woburn, Mass.), which allows multispectral imaging and unmixing of the background, resulting in pure GFP signals. With this set-up we are able to identify GFP signals that arise from zebrafish internal organs, such as the pancreas.