Do you remember seeing a land snail lately? Was the shell coiling clockwise or counter-clockwise? Most of the snail that are seen in USA is a species called Cornu aspersa and they usually have dextral (clockwise) shell. Chirality is a characteristic of snail shell. Usually the direction of coiling is the same for all members of a given species. Occasionally, there are mutant that coils in different direction but they rarely reproduce due to the difficulties in mating. A pair of gene that’s responsible for chirality was identified but we still don’t know exactly how that gene results in different direction of coiling.
A paper was published on Nature (2009) that suggested a signaling pathway called Nodal as a solution. In fact, this Nodal pathway is also involved in animal chirality and the paper proved that they might be an ancestral feature of all the Bilateria. In order to understand this hypothesis, we need to understand how snail goes through embryonic development.
Spiral cleavage of snail embryo
Snail embryo goes through spiral holoblastic cleavage. Unlike normal (non-spiral) cleavage, the cleavage planes are not parallel or perpendicular to the animal-vegetal axis of the egg. Rather, they are in oblique angles, forming a spiral arrangement of daughter blastomeres. Cells touch one another at more places than do those of radially cleaving embryos hence resulting in most thermodynamically stable packing. Also, spirally cleaving embryos usually undergo fewer divisions before gastrulation, making it possible to follow the fate of each cell of the blastula.
Figure 1 Spiral cleavage of the mollusc Trochus viewed (A) from the animal pole and (B) from one side. In (B), the cells derived from the A blastomere are shown in color. The mitotic spindles, sketched in the early stages, divide the cells unequally and at an and at an angle to the vertical and horizontal axes. From: The Early Development of Snails. Developmental Biology 6th edition. Gilbert SF. Sounderland (MA): Sinauer Associates;2000
Unequal division of cell produces big daughter cell called macromere, and small daughter cell called micromere. The first two cleavages produce four large macromeres (A,B,C and D) with different size (D being the largest)(see fig.1). Each macromere buds off a small micromere at its animal pole. Each successive quartet of micromere is displaced to the right or to the left of its sister macromere, creating the characteristic spiral pattern.
Figure 2 spiral cleavage of the snail Ilyanassa. Cleavage is dextral A)8-cell stage B)12-cell stage C) 32-cell stage From: The Early Development of Snails. Developmental Biology 6th edition. Gilbert SF. Sounderland (MA): Sinauer Associates;2000
Polar lobe store morphogenetic determinant
Morphogentic determinants are placed in a specific region of the oocyte. These factors are actively moved to one pole of the cell so that a blastomere containing these factors can pass on to only one of its two daughter cells. To make sure, these factors are stored in particular extrusion called polar lobe. This polar lobe is extruded and absorbed twice during the cleavage.(see fig.3)
Figure 3 Cleavage in the mollusk Dentalium. From: The Early Development of Snails. Developmental Biology 6th edition. Gilbert SF. Sounderland (MA): Sinauer Associates;2000
The resulting four cell blastomeres are in different size, D blastomere being the largest with polar lobe. Clement(1962) did an experiment to observe the further appropriation of these determinants. By removing daughter cells one at a time (1D,2D,3D and 4D in fig.1), he could tell how the determinant move during cleavage.
- Removal of first or second derivatives of D (1D or 2D): the larva lacks heart, intestine, velum, shell gland, eyes and foot
- Removal of third derivative (3D): almost normal embryo with eyes, foot,velum and some shell gland, but no heart or intestine -> some determinants should have apportioned to the 3d cell
- Removal of fourth derivative (4D): no difference, all essential determinants for heart and intestine formation are in 4d
- 4d is responsible for forming the two mesentoblasts which gives rise to both mesodermal(heart) and endodermal(intestine) organs
Nodal signaling is a candidate signal pathway that cause left-right asymmetry
In vertabrates, the molecular pathway that leads to this asymmetry uses the signaling molecule Nodal, a member of the transforming growth factor-beta (TGF-β) superfamily. Nodal gene is expressed in the left lateral plate mesoderm, and loss of nodal function produces a randomization of the left-right asymmetry of visceral organs.
One paper published in nature (2009) tested if this signaling pathway is also involved in snail. Scientists expected genes that are previously described to be critical for left–right determination in other organisms will also play role in snail. They isolated nodal and pitx gene in two species of snails and found that the side of the embryo that expresses nodal and pitx is related to body chirality : both genes are expressed on the right side of the embryo in the dextral species and on the left in the sinistral species. The inhibition of the gene caused loss of shell chirality.
Figure 4 Anterior is up, L and R indicate left and right sides. The blue arrowhead in c–h indicates non-specific staining of the shell. a, b, nodal is expressed in the right cephalic region (upper green arrowhead) and in the right lateral ectoderm (lower green arrowhead) in L. gigantea, as seen from dorsal (a) and right lateral (b) views. c, Expression is maintained in the right lateral ectoderm (green arrowhead); the right lateral view (d) shows that nodal expression (green arrowhead) is near the right side of the developing shell (blue arrowhead). e–h, nodal is expressed in the left lateral ectoderm (green arrowhead) in B. glabrata, as seen from dorsal (e) and posterior (f) views; g, h, Expression is maintained in the left lateral ectoderm (green arrowhead); the posterior view (h) shows that nodal expression (green arrowhead) is near the left side of the developing shell (blue arrowhead). i–l, hedgehog (black arrowheads in i and k) is expressed along the ventral midline, and nodal (red arrowheads in j and l) is expressed on the right side of L. gigantea (j) and on the left side of B. glabrata (l). m, n, Pitx is expressed in the visceral mass (orange arrow) and right lateral ectoderm (orange arrowhead) in L. gigantea, as seen from dorsal (m) and right lateral (n) views. o, p, Pitx is expressed in the stomodeum (orange arrow), visceral mass (orange triangle) and the left lateral ectoderm (orange arrowhead) in B. glabrata, as seen from dorsal (o) and posterior (p) views. Scale bars: 50 m. Nodal signalling is involved in left-right asymmetry in snails Cristina Grande &Nipam H. Patel Nature 457, 1007-1011(19 February 2009) doi:10.1038/nature07603
In order to specify the function of Nodal pathway, experiment was done with drug SB-431542. This chemical inhibitor specifically interferes with type І receptors such as Nodal, Activin and TGF-β. Among the genes identified in L.gigantea, only Nodal and Activin signaling can be affected by SB-431542. By varying the time of drug application, they figured out that only by applying the drug before the blastula stage could they obtain coiling defect. This result suggests that left-right asymmetry have a role in the earliest function of nodal.
Figure 5 Control animals (a, e) display the normal sinistral shell morphology. Drug-treated animals (b–d, f, g, exposed to SB-431542 from the 2-cell stage onwards) have straight shells. b–d show three different living individuals; f and g are a fourth individual, ethanol-fixed, and shown from the side (f) and slightly rotated (g). h–k, Pitx expression in embryos exposed to SB-431542. Dorsal (h) and posterior (i) views of an embryo showing reduced levels of expression. Pitx expression is maintained in the stomodeum (orange arrow in h) and the visceral domain (orange triangle in i), but asymmetric expression in left ectoderm is greatly reduced (orange arrowhead). Dorsal (j) and posterior (k) views of an embryo in which the asymmetric ectodermal expression of Pitx is undetectable (orange arrowhead in j and k show where expression would be expected), although the stomodeal (orange arrow in j) and visceral (orange triangle in k) domain expression of Pitx is normal. Pitx expression levels shown in h–k should be compared to levels in wild-type embryos in Fig. 2o and p, which are the same levels seen in DMSO-treated animals. L and R indicate the left and right sides of the embryo, respectively. Scale bars: a–d, 1.0 mm; e–g, 0.5 mm; h–k, 50 m.
Shells and human hearts and its application
In recent paper, Oliverio M et al. they addressed interesting question ; are human laterality and chirality of snails controlled by the same maternal genes? This paper propose that an evolutionarily conserved genetic basis of chirality is a major synapomorphy of the Bilateria. Identification of the maternal genes involved will allow for the identification of homozygous females of having affected children and spontaneous abortion. Also it will provide a general medical framework for understanding the genetics of most alterations of chirality.