In the field of theoretical morphology of biological shapes, coiling shells have drawn considerable interest for many years. Rice [16] provided a theoretical model based on the idea that the animal must keep a constant gradient of shell growth rate between the outer and inner edge (the gradient) to produce a coiling shell. This idea has been incorporated in many recent models for shell growth (for example, Hammer et al.[17]. Urdy et al. [18]. By contrast, the molecular basis of shell coiling is poorly understood to date. Probably a morphogen-like gradient substance exists, but no candidate for such a concentration gradient has yet been identified. Our results suggest that the left–right gradient of the Dpp protein (caused by a left–right asymmetric expression of the dpp gene) could be the most likely candidate for the gradient in shell coiling, as discussed for some previous mathematical models [16–18].

In this study, we found that in the coiled-shell snail L. stagnalis, dpp is expressed in the local spot of the left or right side mantle edge that corresponds with the shell-coiling direction at the veliger stage, and continues being expressed asymmetrically until the adult stage (Figure 2A-H; Figure 3). By contrast, in the limpets, dpp continues to be expressed symmetrically from the late trochophore stage to the adult stage (Figure 2I,K,L; Figure 3). Furthermore, we found by western blotting using anti-phosphorylated SMAD1/5/8 antibodies that Dpp signals are indeed distributed asymmetrically in the mantle edge in the coiled-shell snail and symmetrically in the non-coiled-shell limpet (Figure 4). In the fruit fly, Dpp works as a morphogen during wing development, spreading through the target point and forming a concentration gradient that provides positional information [19]. Rogulja et al. [20] further showed that Dpp triggers cell division, and the division activity correlates positively with the concentration of Dpp gradient. Hashimoto et al. [8] suggested that in gastropods, Dpp might function by triggering the regulation of cell division in the mantle during shell formation. The cells of the mantle edge secrete shell-matrix proteins, and these proteins are transferred to the outer edge of the shell and mineralized with CaCO 3 . Therefore, if cells rapidly proliferate, more cells can secrete shell-matrix proteins in any one unit of time. We thus propose that during coiled-shell development, Dpp acts as a trigger for an asymmetric cell proliferation, by producing a concentration gradient in the mantle from one spot of expression, and diffuses to the other side of the mantle (Figure 5A). The Dpp gradient might then cause several different reaction thresholds, which in turn induce different levels of cell proliferation along the aperture (Figure 5B). These different levels of cell division might then cause an asymmetric aperture expansion, causing a non-uniform shell growth (Figure 5C) and resulting in a coiled shell (Figure 5D). Constant asymmetric expression of dpp, and thus a constant presence of the gradient until the veliger and adult stage of the snail, ensures the constant coiling during shell growth. Meanwhile, in the non-coiled-shelled limpets, symmetric aperture expansion and shell growth occurs because dpp is expressed symmetrically in the shell gland and the mantle edge, causing uniform cell division (Figure 2, Figure 3, Figure 4, Figure 5).

Figure 5 A molecular hypothesis of shell coiling in Gastropoda. (A) In a snail with a coiled shell, dpp (red) is expressed asymmetrically in the mantle, and Dpp diffusion causes an asymmetric concentration gradient in the mantle. (B) Asymmetric mantle expansion is induced by asymmetric Dpp localization, because Dpp controls cell proliferation in the mantle [8]. (C, D) As a result of the asymmetric mantle expansion, non-uniform shell growth occurs, and produces a coiled shell. By contrast, in the limpets, a non-coiled shell is formed because the lack of expression of dpp in the mantle results in symmetric mantle expansion and shell growth. L, left; R, right. Full size image

A recent report [11] of functional analysis of Dpp in L. stagnalis supports this hypothetical mechanism of shell coiling. When the embryos were treated with a Dpp signal inhibitor (dorsomorphin) at the trochophore and veliger stages, the juvenile shells showed a cone-like form rather than a normal coiled form [11]. These results indicated that Dpp signals induce differences in shell growth rates around the aperture by their gradient. The molecular results presented here support this mathematical models for shell growth [16–18].

The molecular developmental insights into shell coiling reported here also explain how shell coiling was lost several times during the evolution of gastropods. Although it is difficult to infer the ancestral shell shape (coiled or non-coiled shell), previous phylogenetic studies showed that the non-coiled-shelled gastropod Patellogastropoda is placed as the sister group to the rest of extant gastropods (Figure 1; Figure 6). However, considering the fossil record, Paragastropoda that have coiled shells are possibly the most recent common ancestor of gastropods [1], hence suggesting that the coiled-shell feature is probably synplesiomorphy and the non-coiled shell shape has evolved independently several times in gastropods (Figure 1; Figure 6) [1, 2]. Our current results suggest that the loss of coiling might have happened relatively easily, by losing the asymmetric expression of dpp (or its upstream regulators) in the shell gland at the trochophore stage, and leading to symmetric dpp expression n the veliger and adult stages. Further investigations are needed to understand the molecular mechanisms of shell formation and evolution, because the process of shell development is very complex. However, the new insight provided by the current study into dpp expression patterns in the mantle edge, not only in the early developmental stages but also in later stages, is the key basis for understanding how various shell shapes evolved and are formed in gastropods.

Figure 6 Evolutionary hypothesis of the shell-coiling mechanism in Gastropoda. The most recent common ancestor of Gastropoda acquired the asymmetric dpp expression pathway in the mantle at one stage (orange line). Later, the Patellogastropoda lost this pathway and the non-coiled shell shape evolved in this group (blue line). Moreover, other species with non-coiled shells in Vetigastropoda, Caenogastropoda or Heterobranchia most likely evolved like Patellogastropoda (broken blue lines). Full size image

In this study, we found that continuous expression of dpp in the mantle edge until the adult stage might explain the mechanism of these two variations in gastropod shell shapes, that is, the coiled and the non-coiled shapes. However, because in this study we used only patellogastropod species (P. vulgata and N. fuscoviridis), further molecular studies of the species other than those of the Patellogastropoda, such as those from other non-coiled-shell snails are needed in order to be able to infer a decisive conclusion about the evolution of shell-coiling loss in gastropods (Figure 1).