Left-right asymmetric expression of dpp in the mantle of gastropods correlates with asymmetric shell coiling
© Shimizu et al.; licensee BioMed Central Ltd. 2012
Received: 18 December 2012
Accepted: 21 March 2013
Published: 28 May 2013
Various shapes of gastropod shells have evolved ever since the Cambrian. Although theoretical analyses of morphogenesis exist, the molecular basis of shell development remains unclear. We compared expression patterns of the decapentaplegic (dpp) gene in the shell gland and mantle tissues at various developmental stages between coiled-shell and non-coiled-shell gastropods.
We analyzed the expression patterns of dpp for the two limpets Patella vulgata and Nipponacmea fuscoviridis, and for the dextral wild-type and sinistral mutant lineage of the pond snail Lymnaea stagnalis. The limpets had symmetric expression patterns of dpp throughout ontogeny, whereas in the pond snail, the results indicated asymmetric and mirror image patterns between the dextral and sinistral lineages.
We hypothesize that Dpp induces mantle expansion, and the presence of a left/right asymmetric gradient of the Dpp protein causes the formation of a coiled shell. Our results provide a molecular explanation for shell, coiling including new insights into expression patterns in post-embryonic development, which should aid in understanding how various shell shapes are formed and have evolved in the gastropods.
KeywordsLeft-right asymmetry Decapentaplegic Shell coiling Gastropods
Elongation factor 1 alpha
To understand the origin of such morphological diversity, we need to look at the developmental mechanisms of the shells. The developmental process of gastropod shells has already been described [4, 5]. The shell gland is formed by the invagination of ectodermal cells at the early trochophore stage . In the trochophore, shell-secreting cells in the shell gland start to form the initial shell. The mantle tissue begins to develop at the veliger stage, and takes over the role of shell secretion for most of the organism’s life . Thus, the shell gland is important in early shell formation, when the initial trigger and early processes of shell formation occur. Meanwhile, the mantle is involved in shell growth during and after the veliger stage. Accordingly, some previous studies of shell development have focused on these two ‘tissues’.
Despite existence of some studies on gastropod shell formation, molecular embryological insight into shell development remains meager. Nederbragt et al. and Iijima et al. [6, 7] reported that the decapentaplegic (dpp) gene is expressed around the shell gland, suggesting involvement of dpp in shell formation. These studies were not conclusive, however, because they studied dpp only in the early stages of embryonic development (late gastrula and trochophore stages). To remedy such lack of information, and to conclusively show if dpp is involved in shell development in gastropods, we checked the expression patterns of dpp in the later developmental stages in three gastropod species: two limpets with a non-coiling shell (Patella vulgata and Nipponacmea fuscoviridis) and a pond snail with a coiled shell (Lymnaea stagnalis). Because in previous studies, dpp expression patterns in early developmental stages up to the trochophore were reported in these three species [6–8], we confirmed the expression patterns in the veligers and adults. To understand the involvement of dpp expression in shell coiling, we confirmed the dpp expression pattern in the trochophore, veliger, and adults of the sinistral mutant of L. stagnalis, which have a left-wise coiled shell, and compared the expression patterns with the wild-type (dextral, right-wise coiled shell) strain of the same species .
Animal handling followed the guidelines for animal experiments of the University of Tokyo.
Individuals of P. vulgata were collected in Shaldon, Devon, UK, and N. fuscoviridis in Tateyama, Chiba, Japan. The strains of L. stagnalis were reared in tap water in the laboratory. We cultured the dextral strain and sinistral mutant strain of L. stagnalis (derived from Shinshu University). Throughout the year, these organisms lay eggs in capsules coated with jelly. Methods of egg collection and culturing followed those in the previous studies on N. fuscoviridis and L. stagnalis[10, 11].
RNA extraction, cDNA synthesis, and gene cloning
We used the mantle tissues of P. vulgata, N. fuscoviridis, and L. stagnalis for RNA extraction. The mantle tissues were cut off into two parts, left and right. The total RNA was extracted (ISOGEN; Nippon Gene Co. Ltd, Tokyo, Japan), and cDNA synthesis was performed (ReverTra Ace; Toyobo, Osaka, Japan) in accordance with the product protocols. We isolated elongation factor 1 alpha (EF-1α) sequences from P. vulgata and N. fuscoviridis using degenerate primers designed for Mollusca  (see Additional file 1: Figure S1). We used EF-1α-specific primers for L. stagnalis as reported previously . After purification of PCR products using a commercial kit (Gel Extraction Kit; Qiagen Science Inc., Valencia, CA, USA), amplicons were ligated into a vector (pGEM-T Easy Vector; Promega Corp., Madison, WI, USA) using a DNA ligation kit (Promega Corp.), and then transformed to DH5α competent cells (Toyobo).
Quantitative reverse transcriptase PCR
Because it is difficult to analyze gene expression patterns in adult specimens using whole-mount in situ hybridization, we performed quantitative reverse transcription (qRT)-PCR instead. We designed qRT-PCR primers using the software Primer 3 (see Additional file 2: Table S1). Relative quantification of total RNA was performed using a commercial solution (SsoFast EvaGreen supermix with low ROX; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a real-time PCR system (Step One; Applied Biosystems, Foster City, CA, USA). The production of gene-specific products was confirmed by checking their melting curves at the end of qRT-PCR reactions. Data acquisition and analysis were performed (ABI Step One™ software version 2.0; Applied Biosystems). Baselines and thresholds for Ct were set automatically. Quantifications of the target genes were performed by the relative standard curve method. To normalize the quantification of the target gene (dpp) expression, we used the housekeeping gene, EF-1α.
Whole-mount in situ hybridization
We performed in situ hybridization as described previously for amphioxus , except for the following changes in the conditions to make it suitable for molluscan embryos. We fixed the L. stagnalis embryos with 4% paraformaldehyde in MTSTr (50 mmol/l PIPES-KOH pH 6.9, 25 mmol/l EGTA, 150 mmol/l KCl, 25 mmol/l MgCl2, and 0.1% Triton X-100) . For the other limpet, P. vulgata, embryos were fixed with MEMPFA-T (0.1 mol/l MOPS pH 7.4, 2 mmol/l EGTA, 1 mmol/l MgSO4, 4% paraformaldehyde, and 0.1% Tween 20)  overnight at 4°C.
Proteins in the mantle tissues were extracted (ISOGEN; Nippon Gene, Tokyo, Japan) in accordance with the manufacturer’s protocol, and were dissolved afterwards in buffer (NuPAGE LDS Sample Buffer; Life Technologies, Corp., Carlsbad, CA, USA). We carried out electrophoresis using 20 μg protein samples on pre-cast polyacrylamide gels with a linear gradient of 4 to 20% (Bio-Rad, Laboratories, Inc., Hercules, CA, USA), and transferred the separated proteins to nitrocellulose membranes. Blocking was performed overnight using 3% BSA in Tris-buffered saline with Tween (TBS-T: 25 mmol/l Tris HCl pH 7.4, 137 mmol/l NaCl, 2.7 mmol/l KCl, and 0.1% Tween-20) at 4°C. Immunodetection was performed using phosphorylated SMAD1/5/8 polyclonal antibody (#9516; Cell Signaling Technology, Danvers, MA, USA) and SMAD1/5/8 polyclonal antibody (sc-6031-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:1000 dilution in a commercial solution (Can Get Signal solution 1; Toyobo Co. Ltd, Osaka, Japan). After overnight incubation with the primary antibody at 4°C, the membrane was washed three times in TBS-T, and incubated overnight at 4°C with horseradish peroxidase (HRP)-labeled anti-rabbit antibodies (Thermo Fisher Scientific Inc., Rockford, IL, USA) that were diluted 1:2000 in a commercial solution (Can Get Signal solution 2; Toyobo,). After washing the membrane three times in TBS-T, it was incubated with a western blotting detection reagent (ECL Prime; GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire UK). The enhanced chemiluminescence signals were detected with a lumino image analyzer (LAS-1000 Plus; Fuji Film, Japan). We measured these signals using ImageJ software (version 1.46.)
The Wilcoxon–Mann–Whitney test was performed using the statistical software R (version 2.7.1) to evaluate the significant differences in expression levels between the left and right parts of the mantle tissue. P<0.05 was considered significant.
In the field of theoretical morphology of biological shapes, coiling shells have drawn considerable interest for many years. Rice  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.. Urdy et al. . 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].
A recent report  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 . 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].
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).
We found crucial differences in dpp expression patterns between non-coiled-shell limpets and coiled-shell gastropods with a dextral or a sinistral shell, not only in the early developmental stages but also in the late stages. By cross-referencing with previous functional analyses of dpp in gastropods and other animals [8, 11, 19, 20] and previous mathematical models ([16–18], we suggest a hypothesis of shell coiling based on the presence of a Dpp gradient. We hypothesize that Dpp induces mantle expansion, corresponding to the pattern of the concentration gradient of the Dpp morphogen (Figure 5). This hypothesis provides plausible biological grounds for previously published mathematical models of shell formation [16–18]. Our results also suggest a molecular explanation for he shell-coiling mechanism in gastropods, and thus provide robust preliminary information to answer the question about how the diverse gastropod shell shapes evolved.
We thank Hiroshi Wada and Naoki Hashimoto (University of Tsukuba) for the gift of adult specimens of N. fuscoviridis. We thank Takenori Sasaki (University of Tokyo) for useful discussion and for providing us with some shell photographs. We thank Koji Noshita (University of Kyushu) for useful discussion about mathematical models. We thank Michinari Sunamura and Katsunori Yanagawa (The University of Tokyo) for help with qRT-PCR analysis. We thank Michio Suzuki, Makiko Ishikawa (The University of Tokyo), Aya Takesono, Sulayman Mourabit and Yujirou Higuchi (University of Exeter) for help with protein analysis. This study was supported by the JSPS Grants-in-Aid for Scientific Research 15104009.
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