Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development
Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development
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DOI：
10.1101/gad.10.24.3141
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发表时间：
1996-12-15
影响因子：
10.5
通讯作者：
Hopkins, N
中科院分区：
文献类型：
--
作者：
Allende, ML;Amsterdam, A;Hopkins, N
最近，我们的实验室描述了一种在斑马鱼生殖系中产生逆转录病毒前病毒插入的有效方法，我们发现前病毒插入以大约每70个插入一个突变体的频率诱导胚胎突变。到目前为止，我们已经分离出四个插入突变体，并使用前病毒作为分子标记，克隆了其中三个被破坏的基因。所有三种突变体中的前病毒位于第一编码外显子内或仅位于第一编码外显子的5 '端，指向与基因相反的转录方向，并破坏转录。在这里，我们提出了一个分子表征的两个基因，通过这种方法确定，并描述了相关的突变表型。pescadillo（pes）基因被预测编码一个582个氨基酸的蛋白质，没有可识别的功能基序，这是高度保守的酵母到人类。PES mRNA在胚胎发生的前3天广泛动态表达。突出的网站的表达是眼睛和视顶盖的第1天，鳍芽，肝原基，和肠道的第2天，和鳃弓的第3天。从胚胎发育的第三天开始，pes突变胚胎表现出小眼睛，大脑和内脏骨骼减少，鳍缩短，肝脏和肠道缺乏扩张，然后在发育的第六天死亡。死眼（染料）基因编码820个氨基酸的蛋白质，其与人类、小鼠和非洲爪蟾中的未知功能的基因同源，并且与酵母NIC 96（核孔蛋白相互作用组分）基因具有弱同源性。在胚胎发生的第2天，可以通过顶盖和眼睛中坏死细胞的存在来识别染料突变体。染料突变体在发育的第5天死亡。这些结果表明，插入突变的权力在斑马鱼快速发现和表征新的基因胚胎发育所必需的。使用我们目前的方法，我们估计我们的实验室可以在2 - 3年内筛选出大约25，000个插入，识别出大约250 - 350个胚胎致死基因。假设所有的基因都可以被前病毒插入，这种方法的广泛应用可能会导致快速鉴定这种脊椎动物胚胎发育所需的大多数基因。
Recently our laboratory described an efficient method for generating retroviral provirus insertions in the zebrafish germ line, and we showed that provirus insertions induce embryonic mutations at a frequency of roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all three mutants lie within or just 5' of the first coding exon, point in the opposite transcriptional orientation from the gene, and disrupt transcription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotypes. The pescadillo (pes) gene is predicted to encode a protein of 582 amino acids with no recognizable functional motifs, which is highly conserved from yeast to humans. pes mRNA is expressed widely and dynamically during the first 3 days of embryogenesis. Prominent sites of expression are the eyes and optic tectum on day 1, the fin buds, liver primordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a reduced brain and visceral skeleton, shortened fins, and a lack of expansion of the liver and gut, and then die on the sixth day of development. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopus, and that has weak homology with the yeast NIC96 (nucleoporin-interacting component) gene. dye: mutants can be recognized on day 2 of embryogenesis by the presence of necrotic cells in the tectum and eyes. dye mutants die on day 5 of development. These results demonstrate the power of insertional mutagenesis in zebrafish for rapidly finding and characterizing novel genes essential for embryonic development. Using our current methodology, we estimate that our laboratory could screen similar to 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to proviral insertion, the wider application of this approach could lead to the rapid identification of the majority of genes that are required for embryonic development of this vertebrate.