The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

Abstract

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 ( terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms.

Author Summary

By performing genetic mutant screens using senescence-associated biomarkers, we show that the zebrafish is a tractable model system for the study of aging. In vertebrate organisms, it has not previously been possible to carry out systematic screens for genes that are important for stress responses and aging in an unbiased way. However, such vertebrate models are of considerable importance, given the provocative evidence of common biochemical and functional pathways modulating stress responses and lifespan as well as aging in a wide range of organisms. Our present study has successfully employed a colorimetric high-throughput method using a senescence-associated β-galactosidase-based assay to screen for mutations that alter the stress responses in zebrafish embryos, in the hope that these might represent potential aging mutants. Subsequently, the mutations identified by embryonic senescence have indeed displayed adult aging-related phenotypes in zebrafish. Hence, our method for the identification of mutant zebrafish has the immediate potential to accelerate the discovery of novel genes and new functions relevant for our understanding of aging processes in vertebrates. Such knowledge will be essential for the ultimate development of pharmacological, nutritional, and behavioral interventions for the amelioration of oxidative stress- and age-associated diseases and disabilities in humans.