Unwinding snail chirality by a massive subtractive linkage analysis (MSLA)
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Biology
Abstract
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.
Technical Summary
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.
Publications
Davison A
(2016)
Formin Is Associated with Left-Right Asymmetry in the Pond Snail and the Frog.
in Current biology : CB
Davison A
(2019)
Discrete or indiscrete? Redefining the colour polymorphism of the land snail Cepaea nemoralis.
in Heredity
Liu MM
(2013)
Fine mapping of the pond snail left-right asymmetry (chirality) locus using RAD-Seq and fibre-FISH.
in PloS one
Liu MM
(2014)
A conserved set of maternal genes? Insights from a molluscan transcriptome.
in The International journal of developmental biology
Richards PM
(2017)
Single-gene speciation: Mating and gene flow between mirror-image snails.
in Evolution letters
Richards PM
(2013)
RAD-Seq derived markers flank the shell colour and banding loci of the Cepaea nemoralis supergene.
in Molecular ecology
Description | The main objective of the grant was to use next generation sequencing technologies to identify the gene that determines shell coiling or chirality in snails. This was always a very ambitious aim, given that the genome of the snail was completely uncharacterised, and also, repeat rich. Yet, we were successful - see paper in Current Biology. http://www.cell.com/current-biology/fulltext/S0960-9822(16)00056-7 |
Exploitation Route | The example of variable snail chirality, or left -right symmetry, is useful in communicating ideas to the general public, specifically the issue of our own external bilateral symmetry and internal asymmetry, as well as the asymmetry of molecules - some of the snails that we bred were used as props in filming for Brian Cox's Wonder of the Universe. Davison developed a highly inbred line of snails (99.9% inbred). From this, ~ 6000 snails were scored for chirality. This inbred line is being used to generate a better genome assembly (e.g. funded by French Genoscope, Davison is a consortium member) and to fine map the chirality gene. PDRA Davey (Edinburgh) developed a suite of tools for RAD-Seq analysis, RADtools, which have been released openly through a dedicated web site. They are the toolset of choice for many RAD-Seq applications. |
Sectors | Education,Environment,Other |
URL | http://www.angusdavison.org |
Description | Yes, widely covered in media, including press and radio. Search "snail asymmetry" on Google News for results. Research widely featured on mainstream media including interviews on BBC R4, television etc |
First Year Of Impact | 2016 |
Impact Types | Cultural,Societal |
Description | BBRSC funded PhD studentships |
Amount | £150,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2009 |
End | 09/2012 |
Description | The Genome Analysis Centre (TGAC) Capacity and Capability Challenge (CCC), Round 6, |
Amount | £20,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2012 |
End | 05/2012 |
Description | Collaboration with Professor Mark Blaxter |
Organisation | University of Edinburgh |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Edinburgh collaborators have had two main functions, first to generate next generation sequencing data, second to develop bioinformatic methods for the bespoke analysis of the data. One of these methods, a programme called RADtools (https://www.wiki.ed.ac.uk/display/RADSequencing/Home), has been widely adopted by the community. We have worked closely with the Co-I over the existing term, especially through named PDRAs on the grant. |
Start Year | 2009 |
Description | New collaboration with staff at Wellcome Trust Sanger Institute |
Organisation | The Wellcome Trust Sanger Institute |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | In 2011 we initiated a collaboration with the Wellcome Trust Sanger Institute. Together, we developed a method for the fluorescent labelling of pond snail chromosomes. More specifically, we used a FIBRE FISH method to finely pinpoint the gene of interest to a defined region. |
Start Year | 2011 |
Description | Outreach event - communicating science to school children |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | local |
Primary Audience | Schools |
Results and Impact | Two BBSRC funded PhD students were trainined in science communication. They visited two separate local schools, on several occasions, to communicate the research that we are carrying out in the lab. One of them also communicated the science at a British Science Association "Science in the Park" event last year, and also to GCSE students visiting the university as part of a STEM activity day. All of the communication activities above were direcyly related to the existing grant. Training for PhD students in science communication. Science communication to general public and school students. no actual impacts realised to date |
Year(s) Of Engagement Activity | 2011 |
Description | Training of students and PDRAs from other UK universities |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | local |
Primary Audience | Participants in your research or patient groups |
Results and Impact | We have run several training days for RAD sequencing, with guests from University of Nottingham University of Sheffield and University of Manchester. Training event for researchers/postgraduates in RAD Seq technology no actual impacts realised to date |
Year(s) Of Engagement Activity | 2011 |
Description | University Open days - science communication |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Communicating BBSRC funded research to the general public during University Open Days Science communication / outreach Several potential students that I have talked to became actual students at this university |
Year(s) Of Engagement Activity | 2009,2010,2011,2012,2013,2014 |