Snailed it, knowing our left from right

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Externally, most organisms including us humans look bilateral. That being, if we cut a human being directly in half (theoretically, murder is not endorsed by the author) down the middle each half would have one eye, one arm, one leg and so on, a mirror image of itself. However, the internal composition is very different. If we were to inspect the inside of our half human, we would find only the left side would contain a heart. The asymmetry of the internal organs of most bilateral organisms has previously not been extensively studied, the genetic basis largely unknown.

A team of researchers lead by Angus Davison investigated the origins of this asymmetry using Lymnaea stagnalis, a snail which shows naturally inherited difference in the coiling of their shell, known as chirality. Unlike humans and other bilateral organisms, these snails wear their asymmetry on their backs, making it very easy to observe the phenotype. The coil of the shell is controlled by a single maternally inherited gene. Naturally, most have a shell that coils clockwise (two dominant alleles DD), those with the recessive alleles (dd) exhibit an anti-clockwise shell.

Using genetic mapping (finding a genes location) and genomic approaches they were able to determine the chirality locus. This method narrowed the candidate genes down to six, of which only one, Ldia2, showed significant difference in expression associated with genotype. Ldia2 is one copy of tandemly-duplicated diaphanous-related formin genes (aka formin), the other being Ldia1. Whilst that sounds complicated, tandem duplication only means that a single gene has been duplicated beside itself and diversified in function to give Ldia1 and Ldia 2. The Ldia2 gene in dd individuals was found to have a single base pair deletion resulting in a frameshift, and a non-functional protein. Formin are a group of proteins involved in the formation of actin filaments (also known as microfilaments) the support structure or scaffold of the cell.

In DD individuals (clockwise shells) there was high levels of Ldia2 transcripts compared to the dd (anticlockwise) where its expression was negligible. The frameshift mutation is not lethal to the dd snails, owing the to paralog Ldia1 which likely has similar roles in embryonic development allowing the embryo to continue developing.

Asymmetrical inheritance of Ldia2 during cell division. At the two cell stage it has localised to one cell, and at the four cell stage is again only localised in one of the cells

Figure 1

When a fertilized egg begins to divide, it initially divides into two, then four, then eight and so forth. Interestingly, when investigating the expression of Ldia2 mRNA during these divisions, they found its expression was already asymmetrical by the two cell stage only being expressed in one of the cells (Figure 1), and further confined to only one cell by the four-cell stage. This demonstrates that asymmetry is established very early in development, and occurs before any morphological features appear.

Taking this finding one step further, they decided to look at how inhibition of the Ldia2 formin gene would affect phenotype of DD individuals. They found that they could partially convert those with clockwise shells to being anti-clockwise using formin inhibitor drugs, those snails, however, unfortunately did not survive. That aside, it showed that formin was acting in the way they suspected in chirality, and disruption of its function resulted (as found in those individuals with the mutant Ldia2) an anti-clockwise shell.

The question then arises? Is this solely the case in L. stagnalis or is this asymmetry of the cytoskeleton which provides the blue-print for development a conserved mechanism across other groups

Using the the model organism Xenopus laevis (a frog, not some kind of warrior princess) they conducted two experiments to investigate the left-right patterning of this vertebrate. Firstly, they applied the same formin inhibiting drugs as used on the snails to embryos of various stages of development. They found that exposure to these drugs caused heterotaxia, where organs develop on the incorrect side of the body. To further this, they injected into the embryo the mouse formin gene dia1 into Xenopus embryos to stimulate a gain of formin function. Again, they observed heterotaxia, with disorganization of organs occurring which can be seen in Figure 2. From this figure we can see that over expression of formin leads to a pretty messed up tadpole. And so it appears that formin is not restricted to the asymmetrical patterning of snails but also of vertebrate.

Figure 2: Embryos were injected into the animal pole with mRNA encoding mouse dia1 formin and scored for visceral organ situs at stage 45. Images: Examples of organ situs for experimental microinjection with wild-type mouse dia1 mRNA. The control shows a wild-type (situs solitus) tadpole, ventral view, demonstrating the normal arrangement of the stomach (yellow arrowhead), heart apex (red arrowhead), and gall bladder (green arrowhead). Heterotaxic tadpoles (ventral view) resulting from formin overexpression show reversal of all three organs, i.e., situs inversus; the gut position and looping and gall bladder; or the heart.

Figure 2: Embryos were injected  with mRNA encoding mouse dia1 formin Images:  The control shows a wild-type (situs solitus) tadpole, ventral view, demonstrating the normal arrangement of the stomach (yellow arrowhead), heart apex (red arrowhead), and gall bladder (green arrowhead). Heterotaxic tadpoles (ventral view) resulting from formin overexpression show reversal of all three organs, i.e., situs inversus; the gut position and looping and gall bladder; or the heart.

But what does it all mean? And why should you care?

While which way a snail shell coils may seem insignificant to some, the same mechanism likely underpins why we are the way we are. This research shows the asymmetry is likely an ancient basal trait of animals, showing that formin is the earliest “symmetry-breaking” determinant in snails and frogs, and possibly even all bilateral animals. Now knowing the role formin plays in asymmetry means new research could explore formins function in other organisms. This may be of particular interest for human disease and those individuals who suffer from Heterotaxy Syndrome, where organs are not where they are suppose to be (for example, the heart on the right instead of the left). It’s hard to believe that those slow often overlooked critters in our gardens could hold the key to understanding the evolution of bilateral asymmetry.

“You have to know the past to understand the present” – Carl Sagan

Check out the video below to see Angus talk about his research

 


References:

Davison, A., McDowell, G. S., Holden, J. M., Johnson, H. F., Koutsovoulos, G. D., Liu, M. M., … & Yang, F. (2016). Formin is associated with left-right asymmetry in the pond snail and the frog. Current Biology, 26(5), 654-660.

Figures 1 & 2 adapted from Davison et al., (2016)

Snail and tapole picture © Esther de Roij and Gary McDowel

University of Nottingham (25 February 2016). Snail shells offer clue in unravelling origins of body asymmetry [File video]. Retrieved from https://www.youtube.com/watch?v=Mh8-QyJsDVo

 

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15 thoughts on “Snailed it, knowing our left from right

    • Well of course, understanding developmental processes in humans is very complicated involving both genetic and nongenetic influences. For example, situs ambiguus is a disorder where organs are not where they are suppose to be (ie. liver in the middle, no spleen etc), and this disorder is often found to run in families. Researchers have found this is a complicated disease where there is a cascade of genes involved, one of these being Pitx2. Pitx2 is known to be directly involved in forming and maintaining asymmetry, in the chick, it is expressed on the left side of the animal in tissue that will become the heart and internal organs. Numerous other genes have been implicated in left-right asymmetry in human disease including nodal, lefty1/2, PKD2 to name a few. You can check out the following paper if you want to know more http://journals.cambridge.org/download.php?file=%2FERM%2FERM16%2FS1462399414000222a.pdf&code=48353be258956be6388169c5ae3c5095. The research carried out by Davison shows how using other non-model organisms can aid in understand the very earliest symmetry breaking molecules and further research will show how this might influence human development. 

      • Hi Katie! Wow, that sounds so cool. Thanks for that info, having a read through the article now!
        Kathy 

  1. Hey Katie,
    So i figure most of the brunt work was done on the snails and i particularly liked how they looked at Ldia2 mRNA expression very early on. Have they looked at analogous pathways in other vertebrates to determine when asymmetry begins to take place or is it just in L stagnalis for now? 
    Thanks!

    • Interesting question. Ldia2 encodes for formin, which has found to be in all (studied) eukaryotes. The form and function of formin is well known in other species, playing an important role in polymerization of actin. When the researchers undercovered the the Ldia2 gene was those responsible for the anti-clockwise shell it was interesting that this gene are already well described across other groups. 

      Research into left-right symmetry has been done, especially with relation to human disease, and they’ve found that genes such as NODAL, PCSK6, Pitx6, are important in formation/maintenance of left-right asymmetry. But this research shows formin is the first molecule to break symmetry in both snails and frogs, doing so at only the second cleavage. Further research would need to determine if this was the case in other bilateral organisms. After formin, there are other genes that play a role in asymmetry such those mentioned above. PCSK6 is an enzyme that cleaves NODAL to an active form which establishes left-right asymmetry, knockouts of these result in heterotaxia (abnormal organ positioning). Considering the important role of formin as the cellular scaffold, and its conserved nature across phyla, it is worth investigating if this is the case. These researchers believe that the formin scaffold of the cell determines if the shell is clockwise or anticlockwise and this mechanism will likely to conserved in determining that our hearts are on the left instead of the right

      I hope that makes sense! 

  2. Hi Katie, do you know what the distribution of phenotypes is within the population? Is it possible that there could be some sort of evolutionary or reproductive advantage of having one over the other? I know that sometimes the less common phenotype can be favourable, is there any evidence of this being the case here?

    • In the species here, L stagnalis, or pond snails, there is a difference in productive fitness. In the rare coiling type (anti clockwise) their eggs do not hatch as well, and as such this phenotype is not widespread in the population. So no, the less favourable phenotype is not favourable. In another snail species, however, the same mutation causing the difference in coiling has no effect on the reproductive fitness of either type, leading the researches to believe there may be another gene involved 

  3. Hi Katie, great blog! 
    This may be a dumb question, but I just wanted to clarify if the heterozygote (Dd) had the same exact same phenotype as the the homozygote dominant (DD) in the snail? Interesting if so as the results in Xenopus suggest that it may be dose dependent as with the gain of function they exhibit heterotaxia also! Hope this makes sense!

    • Hi Chi, not a dumb question, I probably should’ve made that clear in the blog! Yes the heterozygotes exhibit the same phenotype as the dominant homozygotes! The anti-clockwise phenotype in the recessive (dd) individuals is due to a mutation which causes a frameshift in Lidx2 

  4. Hi Katie, 

    Great blog – seems like a super neat experiment! How similar and relevant are studies conducted on snails to humans or other vertebrates as per se? Has much research been done on any other animals that may be more closely related to humans? 

    Thanks 🙂

    • The experiment also used the vertebrate model organisms Xenopus to demonstrate that what is happening in snails is also happening in vertebrates (like us). Xenopus are very easy to use as they develop outside their mother and can be exposed to drugs and have mRNA injected. The difficulty comes with using other model organisms that are closer to humans, such as the mouse, that their development is internal which makes it hard to manipulate and observe their phenotype. These pond snails present an interesting and useful study organism, whilst not a model organism their asymmetry is naturally occurring, and considering the formin gene associated with this phenotype is found across all eukaryotes the authors think it is likely a conserved mechanisms. 

      Some work has been done in humans, in a reverse genetics fashion, to understand the genetics of human disorders. I’ve talked about those previously in the replies to Jeremy and Kathy, or check out the following article http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=9491902. So other experiments have been done in asymmetry but these have focussed on other genes, this study represents likely the first symmetry breaking molecule during development 

  5. Hi Katie, Excellent blog post. Did they have a reason for injecting mouse formin gene into Xenopus? Rather than ectopic expression of the Xenopus formin gene (assuming that it does have one). How early in development did they inject the Xenopus embryos? Thanks,
    Megan

    • Thanks Megan! The authors do not give any justification for injecting the mouse formin as opposed to ectopic expression, less work maybe? But from what I’ve found Xenopus does have a formin gene (Inverted-formin 2 Inf2). The embryos were injected in the animal pole at 30 and 60 min post-fertilization, and in one of two or four cells and then phenotype scored at stage 45. Overexpression of dia1 resulted in significant proportion of heterotaxia regardless of what stage they were injected.  

  6. Hi Katie!
    Loved the Blog, super interesting! I was just wondering where you think the research should go in terms of disease and possible treatment of those people unfortunately born with Heterotaxy Syndrome?
    Thanks Bex

    • Well it’s pretty hard to say. This research here probably doesn’t offer any medical interventions, only understanding how the initial asymmetry might be occurring in the cytoskeleton. Heterotaxy Syndrome, along with other similar disorders have been studied in understanding the genetics of these disorders. However, I’m not sure what kind of medical interventions might come from these, obviously the organs cannot be reversed, and often there are defects of the heart, liver, and other organs associated with these disorders. The understanding and correct diagnosis of these disorders may lend to better treatment, such as the best surgery options (most patients will require heart surgery). But at this stage, I think the research is unable to provide a medical treatment.