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

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.

Although multiple lines of enquiry remain, a deep-seated theoretical problem has stoked a burning interest in understanding the symmetry-breaking event during development / how is one side of an organism consistently distinguished from the other, given that the side that is called 'right' is essentially arbitrary? In the hypothetical view of Brown and Wolpert, the solution is provided by a pre-existing asymmetric molecular reference: an asymmetric distribution is created if an 'F-molecule' aligns with anterior-posterior and dorsal-ventral axes, so transporting an effector molecule towards the left or right. 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. The objective of the project, therefore, is to use the almost exhaustive power of massively parallel DNA sequencing to directly clone the maternal determinant of chirality in snails, by a method that we term 'massive subtractive linkage analysis' (MSLA). The basic principle is that if the dextral gene product is only present in snails that are genetically dextral, and the sinistral gene product is only present in snails that are genetically sinistral, then a comparative bioinformatic analysis of DNA sequence reads can be used to rapidly discover candidate genes, finally identifying the gene with a functional assay. As one hypothesis is that the maternal determinant is the F-molecule in snails, or else a molecule that interacts with it, then this work will not only lead in the future to a precise understanding of the symmetry-breaking event, but will also likely stimulate comparative / investigative analyses of the same or related molecules in other organisms, including vertebrates. As the methodology is a new application of ultrahigh-throughput DNA sequencing, then success would be a springboard towards identifying other genes via the same methodology.