Formin, an opinion

In previous work (Davison et al., 2016), we used both genetic mapping and a chemical knockdown to show that a frameshift mutation in one copy of a duplicated formin gene is most likely the mutation that causes changes in left-right (LR) asymmetry, or chirality, in the pond snail Lymnaea stagnalis. We also showed that the asymmetric morphology is preceded by asymmetric formin expression in snails, and that overexpressing the same gene in frogs reverses LR asymmetry.

We are therefore pleased that research by Abe and Kuroda (2019) not only corroborates our own findings, but gains definitive proof for causation. The new work puts beyond any doubt that LR asymmetry in snails originates in the cellular architecture. However, we are troubled by several errors or omissions, and also that the authors dismiss previous experimental work, by ourselves and others. These points need making because they detract from what is otherwise an important step forward, causing confusion amongst colleagues in an important area of developmental biology.

• (1)

  The new work confuses the gene names, to the extent that several colleagues mistakenly believed that we misidentified the locus in 2016. In being the first to identify the causative gene (Davison et al., 2016; submitted in August 2015, published in February 2016), we named it Ldia2 because it is evidently the derived version, compared with Ldia1. Ldia2 is located on a long branch (evidence of rapid evolution), and Ldia2 transcripts are enriched in the embryo relative to Ldia1 (indicating specialized function). Despite submitting their work after ours was published (Kuroda et al., 2016; received in July 2016, published in October 2016), the Kuroda group reversed the naming of the same genes, Lsdia1 for the mutated version and Lsdia2 for the other copy. This fact is not mentioned at all in their new work (Abe and Kuroda, 2019). It is therefore important that these differences are made clear, and that, as in other fields, precedence should be used for describing the genes in future publications.

• (2)

  The authors' title is that the work ‘establishes the formin Lsdia1 as the long-sought gene for snail dextral/sinistral coiling’. Notwithstanding the fact that we established the formin as the causative gene (Davison et al., 2016), and they gained definitive proof (Abe and Kuroda, 2019), all experiments prior to ∼2003 were carried out in another species, L. peregra, for which the causative gene remains unidentified. In this latter species, we agree that it is reasonable to suspect that formin may also be involved. This is because sinistral development in two separate isolates of L. peregra is pathological (Boycott et al., 1930; Freeman and Lundelius, 1982), just as in L. stagnalis (Davison et al., 2009; Utsuno et al., 2011). Although not cited, prior to the new work, a formin was also shown to be duplicated and associated with chiral variation and pathology in another land snail (Noda et al., 2019).

  However, the genes that determine natural chiral variation in snails, without pathological effect, remain unknown. We would argue that formin is not a good candidate, mainly because of the associated pathology, but also because we ruled out one formin as causative in two snail genera, Euhadra and Partula (Davison et al., 2016).

  In our opinion, there is likely no single ‘long-sought gene’ that flips chirality, although it is possible that there is a universal pathway that sets up an asymmetric cellular architecture in all animals and plants (e.g. via microtubules; Lobikin et al., 2012). The important question from a developmental point of view is to understand how the asymmetry is set up and amplified. The important question from an evolutionary standpoint is to understand how other species of snail can flip their chirality without apparent pathology, unlike any other animal.

• (3)

  Abe and Kuroda state that this is ‘the first application of CRISPR/Cas9 to a mollusc’. This is repeated in a press release put out by The Company of Biologists. This is incorrect. Perry and Henry (2015) used gene CRISPR/Cas9-mediated genome modification in the mollusc Crepidula fornicata, albeit for transient transgenesis.

• (4)

  The new work shows that there are also morphological asymmetries in the first cleavage, stating that this is ‘the earliest observed symmetry-breaking event linked directly to body handedness in the animal kingdom’. We agree that this is a fascinating finding but it is not unexpected. Preceding the current vogue for cellular chirality by more than a century, Conklin (1903) and later Meshcheryakov and Beloussov (1975) reported that the spiral character of cleavage begins with the first division of the molluscan egg. Abe and Kuroda (2019) extend the findings to show that the morphological asymmetry of the single-cell embryo varies within chirally variable species. Moreover, although it is rarely so evident morphologically, it is not correct that this is the only animal for which it has been shown to have such an early symmetry-breaking event. For example, the frog has a defined left and right as early as the first cell cleavage (Vandenberg et al., 2013), as do ascidians (Albrieux and Villaz, 2000).

• (5)

  Abe and Kuroda conclude that experiments using a chemical knockdown to corroborate the genetic results ‘do not provide any meaningful insights’, and imply that our findings of asymmetric gene expression are a technical artefact. We disagree. In their previous work, they applied the inhibitor drug very early, resulting in complete developmental arrest, and among other differences, they used an in situ hybridization protocol that may be less sensitive than ours (Herlitze et al., 2018), included fixing overnight and using ten times more antibody. In our view, it is not surprising that different methods in different laboratories may produce different outcomes. To resolve the debate regarding asymmetric gene expression, an independent method, such as single cell transcriptomics and/or single molecule RNA FISH may be necessary.

• (6)

  It is stated in the new work (Abe and Kuroda, 2019) and in the press release that changing chirality by gene editing is likely to lead to the generation of a new species. So-called ‘single-gene speciation’ is a persistent idea, but not likely the case for these snails. It has long been known that chiral reversal per se is not sufficient to create new species (Richards et al., 2017 and references therein). This is especially the case in pond snails, in which sinistrals and dextrals are able to mate, and also because the mutation in the formin causes a ∼50% reduction in egg hatch rate (supplementary figures S3 and S4 in Abe and Kuroda, 2019; see also Davison et al., 2009; Utsuno et al., 2011).