Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte.
Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte.
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DOI：
10.1371/journal.pgen.1001073
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发表时间：
2010-08-19
期刊：
影响因子：
4.5
通讯作者：
Mullins MC
中科院分区：
文献类型：
--
作者：
Gupta T;Marlow FL;Ferriola D;Mackiewicz K;Dapprich J;Monos D;Mullins MC
尽管在发育生物学中具有基本的重要性，但脊椎动物卵母细胞和卵子极化对称性破坏事件的遗传基础在很大程度上是未知的。在脊椎动物中，卵母细胞的第一个形态不对称是巴比亚尼小体，这是一种高度保守的瞬时结构，存在于脊椎动物和无脊椎动物中，包括果蝇、非洲爪哇、人类和老鼠。我们报告了斑马鱼麦哲伦(MGN)突变体的鉴定，该突变体表现出一种新的扩大的Balbiani小体表型和卵母细胞极性的破坏。为了确定MGN基因的分子同源性，我们对该基因进行了定位克隆，采用了一种新的DNA捕获方法来针对600kb的区域特异性基因组DNA进行大规模平行测序。使用这项技术，我们能够在一周内丰富与我们的突变相关的基因组区域，然后使用大规模平行测序鉴定MGN中的突变。这是基因组DNA浓缩和大规模并行测序相结合的第一次成功应用之一，以确定与突变表型相关的基因的分子身份。我们预计，这些技术的结合将在有效识别所有生物体中的突变基因方面具有广泛的适用性。我们鉴定了MGN中的突变是斑马鱼微管肌动蛋白交联素1基因编码序列的缺失。Macf1是高度保守的细胞骨架连接蛋白spectraplakin家族的成员，在神经元、肌肉细胞和上皮细胞等极化细胞中发挥着不同的作用。在MGN突变体中，卵母细胞核定位错误，卵母细胞外围缺乏Balbiani小体、定位的mRNAs和细胞器，这与macf1在核锚定和皮质定位中的功能一致。这些数据首次证明了镜板蛋白在脊椎动物卵母细胞和卵子极化过程中所起的作用。胚轴是如何建立的，是发育生物学中的一个重要问题。在许多生物体中，胚轴是在卵子发生过程中通过产生极化的卵子而建立的。脊椎动物卵母细胞和卵子中极性的建立和维持机制知之甚少。我们已经鉴定出一种名为麦哲伦的斑马鱼突变体，它在卵子极性上显示出缺陷。在麦哲伦突变体中中断的基因编码细胞骨架连接蛋白微管肌动蛋白交联因子1(Macf1)。在脊椎动物中，可能需要数年时间才能确定突变的分子性质。我们使用了一种新技术来鉴定麦哲伦突变，这使我们能够快速分离与突变相关的基因组DNA并对其进行测序。我们的结果描述了Macf1在卵母细胞和卵子极化中的一个重要的新功能，并证明了这一新技术用于有效识别突变的可行性。
Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg. How the axes of the embryo are established is an important question in developmental biology. In many organisms, the axes of the embryo are established during oogenesis through the generation of a polarized egg. Very little is known regarding the mechanisms of polarity establishment and maintenance in vertebrate oocytes and eggs. We have identified a zebrafish mutant called magellan, which displays a defect in egg polarity. The gene disrupted in the magellan mutant encodes the cytoskeletal linker protein microtubule actin crosslinking factor 1 (macf1). In vertebrates, it can take years to identify the molecular nature of a mutation. We used a new technique to identify the magellan mutation, which allowed us to rapidly isolate genomic DNA linked to the mutation and sequence it. Our results describe an important new function for macf1 in polarizing the oocyte and egg and demonstrate the feasibility of this new technique for the efficient identification of mutations.
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