Insertional Mutagenesis in Zebrafish

Large-scale forward genetic screens are a powerful approach to identifying the genetic basis of developmental processes. Such screens have long been central to studies of invertebrate animals, and more recently the approach has been applied to vertebrate animals, including zebrafish and mice. Forward screens are particularly suitable in the fish. This is because it is possible to breed and maintain large numbers of zebrafish in the lab, and because early developmental mutations are easy to identify in fish embryos since embryos develop outside the mother and are transparent for the first week of life.

Most screens in zebrafish have employed chemical mutagens or radiation to induce mutations. However, cloning genes mutated by these agents is tedious. Thus we developed a method of insertional mutagenesis for the zebrafish using mouse retroviral vectors. Retroviruses are excellent mutagens since when they infect cells, a DNA copy of their genome is inserted into the host cell genome at many different locations. If the DNA insertion occurs in a gene and disrupts it, the viral DNA serves as a tag for cloning the mutated gene. We found that mouse retroviral vectors can infect the fish germ line efficiently, that proviral insertions are mutagenic, and that the mutated genes can be cloned very rapidly using the viral tag. Using this technology, we carried out a large screen to identify mutants with developmental defects visible by 5 days post fertilization. By this time fish are already free-swimming larvae. Most mutations we identified are embryonic or larval lethals. About 1/3 of the mutants have relatively specific phenotypes, while about 2/3 have less specific defects that involve many cells in the embryo. The latter often result from mutations in genes required for cell viability, the former from genes required for the patterning, differentiation, or growth of specific organs and structures.

We isolated ~550 mutants. These represent mutations in ~400 different genes. We have cloned the genes mutated in 375 of the mutants, and these include lesions in 275 different genes. About 20% of the genes are novel and almost all have clearly identifiable human homologues. Compelling evidence indicates that this collection of mutants includes at least 25% of the genes essential for development of the 5-day old fish. Thus there are only about 1600 embryonic or larval lethals in zebrafish.

To identify genes that play essential roles in specific aspects of development we are re-screening (called “shelf-screening”) our mutant collection using specific assays. Specific screens include those for mutants with cystic kidneys, those with defects in development of the jaw and cartilage, hair cell function and lateral line, those with defects in forebrain patterning, cell cycle, nuclear coded mitochondrial genes, and liver growth. Each screen yields between a few up to 20 genes. In the case of kidney, the genes that were identified appear to comprise a pathway and this pathway appears to correspond to that for human cystic kidney. In collaboration with other labs, we are shelf screening the collection to identify the genes essential for about 20 other developmental processes.

In the course of maintaining the mutants, we noted that some lines display early mortality and develop tumors as heterozygous adults. In collaboration with the lab of Jackie Lees, we analyzed tumor spectrum and frequency in the colony as a whole. This allowed us to identify a set of lines that define a novel class of tumor suppressors. Together these studies demonstrate the power of forward insertional mutagenesis in zebrafish to identify genes important for vertebrate development and disease, and to assign function to many genes whose biological and biochemical functions were not previously known.

Future directions: We are focusing on mutants with defects in genes required for cell cycle and organ growth and genes that predispose to cancer. We are pursuing the mechanism of action of the novel tumor suppressor genes we have identified to date.