The identification of zebrafish mutants showing alterations in senescence-associated biomarkers.
The identification of zebrafish mutants showing alterations in senescence-associated biomarkers.
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DOI：
10.1371/journal.pgen.1000152
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发表时间：
2008-08-15
期刊：
影响因子：
4.5
通讯作者：
Roberts TM
中科院分区：
文献类型：
--
作者：
Kishi S;Bayliss PE;Uchiyama J;Koshimizu E;Qi J;Nanjappa P;Imamura S;Islam A;Neuberg D;Amsterdam A;Roberts TM
在许多生物体中，调节应激反应的基因和调节衰老表型（在某些情况下，还包括寿命）的基因在功能上存在有趣的重叠。因此，在我们目前的研究中，我们筛选了诱变斑马鱼胚胎中应激生物标志物衰老相关β-半乳糖苷酶（SA-β-gal）表达的改变。我们通过分析逆转录病毒插入突变体的集合来验证胚胎SA-β-gal生产作为筛选工具的用途。从306个这样的突变体库中，我们鉴定出11个表现出更高胚胎SA-β-gal活性的候选者，其中两个被选择用于进一步研究。这些突变体之一是空的同源果蝇spinster，已知调节寿命的基因在苍蝇，而另一个窝藏在同源的人类端粒重复序列结合因子2（terf 2）基因，它在端粒保护和端粒长度调节中发挥作用的突变。虽然纯合的spinster和terf 2突变体是胚胎致死的，但杂合的成鱼是可行的，并显示出加速出现的衰老症状，包括脂褐质积累，这是另一种生物标志物，和较短的寿命。接下来，我们使用相同的SA-β-gal测定来筛选化学诱变的斑马鱼，在氧化应激的致敏条件下，每种斑马鱼在多个基因中的病变都是杂合的。我们从该筛选中获得了8个额外的突变体，当繁殖至纯合性时，即使在没有应激的情况下也显示出增强的SA-β-gal活性，并进一步显示出胚胎神经和肌肉退行性表型。这些突变杂合的成鱼也显示出衰老生物标志物的过早表达和衰老表型的加速发作。因此，我们目前的策略突变体筛选衰老相关的生物标志物在斑马鱼胚胎可能被证明是一个有用的新工具，脊椎动物的应激反应和衰老机制的遗传解剖。通过使用衰老相关生物标志物进行遗传突变筛选，我们表明斑马鱼是衰老研究的一个易处理的模型系统。在脊椎动物生物体中，以前不可能以无偏见的方式进行对应激反应和衰老重要的基因的系统筛选。然而，这样的脊椎动物模型是相当重要的，考虑到挑衅性的证据，共同的生化和功能途径调节应激反应和寿命，以及在广泛的生物老化。我们目前的研究成功地采用了一种比色高通量方法，使用衰老相关的β-半乳糖苷酶为基础的测定来筛选改变斑马鱼胚胎应激反应的突变，希望这些突变可能代表潜在的衰老突变体。随后，通过胚胎衰老鉴定的突变确实在斑马鱼中显示了与成年衰老相关的表型。因此，我们的突变斑马鱼的鉴定方法具有直接的潜力，以加速发现新的基因和新的功能，我们的理解在脊椎动物的衰老过程。这些知识将是必不可少的药理学，营养和行为干预的最终发展，以改善氧化应激和年龄相关的疾病和残疾的人。
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. 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.
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DOI：
10.1073/pnas.92.20.9363
发表时间：
1995-09-26
影响因子：
11.1
作者：
DIMRI, GP;LEE, XH;CAMPISI, J
通讯作者：
CAMPISI, J
影响因子：
6.1
作者：
Ken, CF;Lin, CT;Shaw, JF
通讯作者：
Shaw, JF
影响因子：
5.3
作者：
Cheng, Aiwu;Shin-ya, Kazuo;Mattson, Mark P.
通讯作者：
Mattson, Mark P.
DOI：
10.1016/s0305-0491(00)00285-6
发表时间：
2000-12-01
影响因子：
2.2
作者：
Gerhard, GS;Kauffman, EJ;Grundy, MA
通讯作者：
Grundy, MA
影响因子：
10.5
作者：
Keyes, WM;Wu, Y;Mills, AA
通讯作者：
Mills, AA