Snails have nodal!

Blogging on Peer-Reviewed Research

My first column in the [Guardian science blog](http://www.guardian.co.uk/science) will be coming out soon, and it's about a recent discovery that I found very exciting…but that some people may find strange and uninteresting. It's all about the identification of _nodal_ in snails.

nodal_guts.jpg

Why should we care? Well, _nodal_ is a rather important — it's a gene involved in the specification of left/right asymmetry in us chordates. You're internally asymmetric in some important ways, with, for instance, a heart that is larger on the left than on the right. This is essential for robust physiological function — you'd be dead if you were internally symmetrical. It's also consistent, with a few [rare exceptions](http://en.wikipedia.org/wiki/Situs_inversus), that _everyone_ has a stronger left ventricle than right. The way this is set up is by the activation of the cell signaling gene _nodal_ on one side, the left. _Nodal_ then activates other genes (like _Pitx2_) farther downstream, that leads to a bias in how development proceeds on the left vs. the right.

In us mammals, the way this asymmetry in gene expression seems to hinge on the way [cilia rotate to set up a net leftward flow of extraembryonic fluids](http://scienceblogs.com/pharyngula/2006/08/generating_rightleft_asymmetri.php). This flow activates sensors on the left rather than the right, that upregulate _nodal_ expression. So _nodal_ is central to differential gene expression on left vs. right sides.

nodal_cilia.jpg

What about snails? Snails are cool because their asymmetries are just hanging out there visibly, easy to see without taking a scalpel to their torsos (there are also internal asymmetries that we'd need to do a dissection to see, but the external markers are easier). The assymetries also appear very early in the embryo, in a process called [spiral cleavage](http://scienceblogs.com/pharyngula/2006/04/spiral_cleavage.php), and in the adult, they are obvious in the handedness of shell coiling. We can see shells with either a left-handed or right-handed spiral.

nodal_spiral.jpeg
Chirality in snails. a, Species with different chirality: sinistral Busycon pulleyi (left) and dextral Fusinus salisbury (right). b, Sinistral (left) and dextral (right) shells of Amphidromus perversus, a species with chiral dimorphism. c, Early cleavage in dextral and sinistral species (based on ref. 27). In sinistral species, the third cleavage is in a counterclockwise direction, but is clockwise in dextral species. In the next divisions the four quadrants (A, B, C and D) are oriented as indicated. Cells coloured in yellow have an endodermal fate and those in red have an endomesodermal fate in P. vulgata (dextral)15 and B. glabrata (sinistral)28. L and R indicate left and right sides, respectively. d, B. glabrata possesses a sinistral shell and sinistral cleavage and internal organ organization. e, L. gigantea displays a dextral cleavage pattern and internal organ organization, and a relatively flat shell characteristic of limpets. Scale bars: a, 2.0 cm; b, 1.0 cm; d, 0.5 cm; e, 1.0 cm.

Until now, the only organisms thought to use _nodal_ in setting up left/right asymmetries were us deuterostomes — chordates and echinoderms. In the other big (all right, _bigger_) branch of the animals, the protostomes, _nodal_ seemed to be lacking. Little jellies, the cnidaria, didn't have it, and one could argue that with radial symmetry it isn't useful. The ecdysozoans, animals like insects and crustaceans and nematodes, which do show asymmetries, don't use _nodal_ for that function. This suggests that maybe _nodal_ was a deuterostome innovation, something that was not used in setting up left and right in the last common ancestor of us animals.

That's why this is interesting news. If a major protostome group, the lophotrochozoa (which includes the snails) use _nodal_ to set up left and right, that implies that the ecdysozoans are the odd group — they secondarily _lost_ _nodal_ function. That would suggest then that our last common ancestor, a distant pre-Cambrian worm, used this molecule in the same way.

Look in the very early mollusc embryo, and there's _nodal_ (in red, below) switched on in one or a few cells on one side of the embryo, the right. It's asymmetrical gene expression!

nodal_early_exp.jpeg
Early expression of nodal and Pitx in snails. a, 32-cell stage L. gigantea expressing nodal in a single cell. b, Group of cells expressing Pitx in L. gigantea. c, Onset of nodal expression in B. glabrata. d, A group of cells expressing Pitx in B. glabrata. e, 32-cell L. gigantea expressing nodal (red) in a single cell (2c) and brachyury (black) in two cells (3D and 3c). f-h, brachyury (black) is expressed in a symmetrical manner in progeny of 3c and 3d blastomeres (blue triangles in g), thus marking the bilateral axis, and nodal (red) is expressed on the right side of L. gigantea in the progeny of 2c and 1c blastomeres, as seen from the lateral (f) and posterior (g, h) views of the same embryo. i, A group of cells expressing nodal (red) in the C quadrant and Pitx (black) in the D quadrant of the 120-cell-stage embryo of L. gigantea. j, nodal (red) and Pitx (black) expression in adjacent areas of the right lateral ectoderm in L. gigantea. L and R indicate the left and right sides of the embryo, respectively. The black triangle in b and i, the green, yellow and pink arrows in f and i, and the black and pink arrows in f and h point to the equivalent cells. Scale bars: 50┬Ám.

Seeing it expressed is tantalizing, but the next question is whether it actually does anything in these embryos. The test is to interfere with the _nodal-Pitx2_ pathway and see if the asymmetry goes away…and it does, in a dramatic way. There is a chemical inhibitor called SB-431542 that disrupts this pathway, and exposing embryos to it does interesting things to the formation of the shell. In the photos below, the animal on the left is a control, and what you're seeing is a coiled shell (opening to the right). The other two views are of an animal treated with SB-431542…and look! Its shell doesn't have _either_ a left- or right-handed twist, and instead extends as a straight tube.

nodal_shell.jpeg
Wild-type coiled and drug-treated non-coiled shells of B. glabrata. Control animals (e) display the normal sinistral shell morphology. Drug-treated animals (f, g, exposed to SB-431542 from the 2-cell stage onwards) have straight shells. f and g show an
individual, ethanol-fixed, and shown from the side (f) and slightly rotated (g).

What this all means is that we've got a slightly better picture of what genes were present in the ancestral bilaterian animal. It probably had both _nodal_ and _Pitx2_, and used them to build up handedness specializations. Grande and Patel spell this out:

Although Pitx orthologues have also been identified in non-deuterostomes such as _Drosophila melanogaster_ and _Caenorhabditis elegans_, in these species Pitx has not been reported in asymmetrical expression patterns. Our results suggest that asymmetrical expression of _Pitx_ might be an ancestral feature of the bilaterians. Furthermore, our data suggest that _nodal_ was present in the common ancestor of all bilaterians and that it too may have been expressed asymmetrically. Various lines of evidence indicate that the last common ancestor of all snails had a dextral body. If this is true, then our data would suggest that this animal expressed both _nodal_ and _Pitx_ on the right side. Combined with the fact that _nodal_ and _Pitx_ are also expressed on the right side in sea urchins, this raises the possibility that the bilaterian ancestor had left-right asymmetry controlled by _nodal_ and _Pitx_ expressed on the right side of the body. Although independent co-option is always a possibility, the hypotheses we present can be tested by examining nodal and _Pitx_ expression and function in a variety of additional invertebrates.

It's also, of course, more evidence for the unity of life. We are related to molluscs, and share key genes between us.


Grande C, Patel NH (2009) Nodal signalling is involved in left-right asymmetry in snails. Nature 457(7232):1007-11.