Nancy H. Hopkins
Nancy H. Hopkins
"Our laboratory uses zebrafish to study the genes essential for early development, longevity and predisposition to cancer. We have developed a powerful technique for generating and screening novel mutations in these organisms, from which we have isolated mutations in approximately 400 genes. 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."
Dr. Hopkins is an Amgen professor of biology at MIT, appointed to encourage research and education in the life sciences. She is an alumna of Radcliffe College and earned a Ph.D. from the Department of Molecular Biology and Biochemistry at Harvard University in 1971. Professor Hopkins joined MIT in 1973 as an assistant professor at the Center for Cancer Research. Her work has helped to identify the role genes play in longevity and cancer predisposition in adult fish. Dr. Hopkins chaired the committee that wrote the 1999 Report on the Status of Women Faculty in the School of Science at MIT and she is a contributing author to Becoming MIT: Moments of Decision, a joint project of MIT150 and the MIT Press, edited by MIT professor David Kaiser.
Large-scale forward genetic screens are a powerful approach to identifying the genetic basis of developmental processes. Such screens, first applied to invertebrate animals, have also been used in vertebrates, 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 genetic screens in zebrafish employed chemical mutagens or radiation to induce mutations. However, cloning genes mutated by these agents is tedious. Thus. Some years ago, our lab 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, proviral insertions are mutagenic, and the mutated genes can be cloned very rapidly using the viral tag. Using this technology, we carried out a large screen and identified 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, which lay in about ~400 different genes. We cloned the genes mutated in 375 of the mutants, and these included lesions in 275 different genes. Almost all the genes have human homologues. This mutant collection includes at least 25% of the genes that are genetically essential for development of the 5-day old fish. Thus there are only about 1600 embryonic and early larval lethal genes in zebrafish.
To identify genes that play essential roles in specific aspects of development we and other labs re-screened (called "shelf-screening") our mutant collection using specific assays. Specific screens in our lab included 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 yielded between a few up to 20 genes. In the case of kidney, the genes that were identified corresponded to some for human cystic kidney. Other labs have screened the collection to identify 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. 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 haploinsufficient tumor suppressor genes. These turned out to be genes that encode ribosomal proteins. Interestingly, the tumors they cause (malignant peripheral nerve sheath tumors, MPNST) and the latent period are the same as those caused in fish by mutations in the well-known tumor suppressor gene, p53. We have continued to study the mechanisms by which mutations in ribosomal protein genes predispose fish to cancer.
We found that fish MPNSTs, like human MPNST and many other human tumors, are highly aneuploid. Furthermore, the number of chromosomes varies from cell to cell within a single tumor. Certain chromosomes are preferentially over or under-represented within each tumor and the preferences are shared by many tumors. Focal amplifications also occur and some involve the same oncogenes involved in human cancers. The evolutionary distance between fish and humans suggests that one can use the highly anueploid fish MPNST model to identify genes that are drivers of human cancer.
Zhang, GJ., Hoersch, S., Amsterdam, A., Whitaker, C., Lees, JA., Hopkins, N. Highly aneuploid zebrafish malignant peripheral nerve sheath tumors have genetic alterations similar to human cancers. Proc. Natl. Acad. Sci. USA, 107:16940-5 (2010)
Amsterdam, A., Sadler, K.C., Lai, K., Farrington, S., Bronson, R.T., Lees, J.A., and Hopkins N. (2004). Many ribosomal protein genes are cancer genes in zebrafish. PloS Biol. 2: 690-698.
Amsterdam, A., Burgess, S., Golling, G., Chen, W., Sun, Z., Townsend, K., Farrington, S., Haldi, M., and Hopkins, N. A large scale insertional mutagenesis screen in zebrafish. Genes and Development, 13: 2413-2724 (1999).