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.

Spinster (Spin) in Drosophila or Spinster homolog 1 (Spns1) in vertebrates is a putative lysosomal H+-carbohydrate transporter, which functions at a late stage of autophagy. The Spin/Spns1 defect induces aberrant autolysosome formation that leads to embryonic senescence and accelerated aging symptoms, but little is known about the mechanisms leading to the pathogenesis in vivo. Beclin 1 and p53 are two pivotal tumor suppressors that are critically involved in the autophagic process and its regulation. Using zebrafish as a genetic model, we show that Beclin 1 suppression ameliorates Spns1 loss-mediated senescence as well as autophagic impairment, whereas unexpectedly p53 deficit exacerbates both of these characteristics. We demonstrate that ‘basal p53’ activity plays a certain protective role(s) against the Spns1 defect-induced senescence via suppressing autophagy, lysosomal biogenesis, and subsequent autolysosomal formation and maturation, and that p53 loss can counteract the effect of Beclin 1 suppression to rescue the Spns1 defect. By contrast, in response to DNA damage, ‘activated p53’ showed an apparent enhancement of the Spns1-deficient phenotype, by inducing both autophagy and apoptosis. Moreover, we found that a chemical and genetic blockage of lysosomal acidification and biogenesis mediated by the vacuolar-type H+-ATPase, as well as of subsequent autophagosome-lysosome fusion, prevents the appearance of the hallmarks caused by the Spns1 deficiency, irrespective of the basal p53 state. Thus, these results provide evidence that Spns1 operates during autophagy and senescence differentially with Beclin 1 and p53.