Unwinding snail chirality by a massive subtractive linkage analysis (MSLA)

尽管许多动物的外表是对称的，但它们中的很多都是内部不对称的，或者说是手性的：脊椎动物（包括我们自己）有一个或几个器官移向一侧，线虫有一个不对称的神经系统，甚至长期以来被认为具有完美“镜像”对称的果蝇也有一个不对称的肠道。对于不对称的动物来说，对称在发育过程中一定会被打破。这就提出了一个有趣的问题：考虑到我们称之为“正确”的一方本质上是任意的，如何始终如一地将一方与另一方区分开来。解决方案是，在开发的早期阶段，假设一个不对称的“f分子”与前/后和上/下平面排列在一起，从而产生左右不对称。后来的器官不对称是由于f分子将一些物质向一侧或另一侧（即向右或向左）输送而产生的。为了理解不对称是如何形成的，科学家们把研究重点放在了老鼠、小鸡和斑马鱼等模式生物上。在这些动物中，研究人员发现，在早期胚胎中，小毛（纤毛）的旋转跳动产生了不对称的流体运动，这导致了一种建议，即这是关键的对称性打破步骤：纤毛周围运动蛋白的不对称导致定向流体运动，最终决定了分子和生物体的不对称。虽然这些发现是优雅的，但一些最近和更早的研究表明，对称性破坏事件发生的时间要早得多，这使上述研究的最终相关性受到质疑。例如，在线虫中，左右不对称是在六细胞阶段建立起来的，可能更早。在池塘蜗牛手性是由一种物质，母亲沉积在未受精卵。在青蛙中，分子的不对称是在四细胞阶段建立起来的，甚至在斑马鱼和小鸡中，在纤毛阶段之前就有差异的迹象。总之，结果表明，在许多动物中，包括我们的近亲，分子不对称在胚胎发育的早期就建立起来了，形态不对称只是在后来才变得明显。我们相信蜗牛可能是理解对称性打破步骤的关键模式生物，因为它们的不对称性很早就建立起来了，但近年来几乎完全被忽视了。因此，该项目的目标是利用新的DNA测序技术的力量，直接识别决定蜗牛卵手性的基因序列。有一种观点认为，这个基因也是f分子，那么这项工作将在未来导致对蜗牛体内对称性破坏事件的理解。研究结果将为其他生物（包括脊椎动物）中相同或相关分子的分析提供动力。最后，由于我们将使用的方法是超高通量DNA测序的全新应用，那么这个项目的成功将是一个跳板，使用相同的方法来识别其他基因。

Although many animals are symmetric on the outside, very many of them are inwardly asymmetric, or chiral: vertebrates (including ourselves) have one or several organs displaced to one side, nematode worms have an asymmetric nervous system, and even fruitflies, long-supposed to have perfect 'mirror image' symmetry, have an asymmetric intestine. For an animal to become asymmetric, symmetry must somehow be broken during development. This raises the interesting problem of how one side is consistently distinguished from the other, given that the side that we call 'right' is essentially arbitrary. The solution is that in the early stages of development a hypothetical, asymmetric 'F-molecule' lines up with the front/back and top/bottom planes, so creating a left-right asymmetry. Later organ asymmetry comes about because the F-molecule sends some substance(s) toward one side or the other (i.e. to the right, or, to the left). To attempt to understand how asymmetry is established, scientists have focussed their research on model organisms such as the mouse, chick and zebrafish. In these animals, it has been found that rotational beating of small hairs ('cilia') in the early embryo create a fluid movement that is asymmetric, leading to a suggestion that this is the critical symmetry-breaking step: the asymmetry of motor proteins around the cilia leads to directional fluid movement, ultimately determining the molecular and organismal asymmetry. While these findings are elegant, some recent and also much older research indicates that the symmetry-breaking event is much earlier, putting the ultimate relevance of the above research into doubt. For instance, in the nematode worm, left-right asymmetry is established by the six-cell stage, and probably earlier. In the pond snail chirality is determined by a substance that the mother deposits in the unfertilised egg. In the frog, molecular asymmetry is established by the four-cell stage, and even in zebrafish and chick, there are indications of differences prior to the cilial stage. Together, the results suggest that in many animals, including our close relatives, molecular asymmetry is established early in the development of embryos, with morphological asymmetry only becoming apparent later. We believe that snails may be a crucial model organism in coming to understand the symmetry-breaking step, because their asymmetry is established very early, yet they have been almost completely neglected in recent years. The objective of this project, therefore, is to utilise the power of new DNA sequencing technologies to directly identify the gene sequence that determines chirality in snail eggs. As one idea is that this gene is also the F-molecule, then this work will lead in the future to an understanding of the symmetry-breaking event in snails. The results will then invigorate analyses of the same or related molecules in other organisms, including vertebrates. Finally, as the methodology that we will use is an entirely new application of ultrahigh-throughput DNA sequencing, then success in this project would be a springboard towards using the same method to identify other genes with the same methodology.