Dynamic expression of ancient and novel molluscan shell genes during ecological transitions
© Jackson et al; licensee BioMed Central Ltd. 2007
Received: 22 December 2006
Accepted: 10 September 2007
Published: 10 September 2007
The Mollusca constitute one of the most morphologically and ecologically diverse metazoan phyla, occupying a wide range of marine, terrestrial and freshwater habitats. The evolutionary success of the molluscs can in part be attributed to the evolvability of the external shell. Typically, the shell first forms during embryonic and larval development, changing dramatically in shape, colour and mineralogical composition as development and maturation proceeds. Major developmental transitions in shell morphology often correlate with ecological transitions (e.g. from a planktonic to benthic existence at metamorphosis). While the genes involved in molluscan biomineralisation are beginning to be identified, there is little understanding of how these are developmentally regulated, or if the same genes are operational at different stages of the mollusc's life.
Here we relate the developmental expression of nine genes in the tissue responsible for shell production – the mantle – to ecological transitions that occur during the lifetime of the tropical abalone Haliotis asinina (Vetigastropoda). Four of these genes encode evolutionarily ancient proteins, while four others encode secreted proteins with little or no identity to known proteins. Another gene has been previously described from the mantle of another haliotid vetigastropod. All nine genes display dynamic spatial and temporal expression profiles within the larval shell field and juvenile mantle.
These expression data reflect the regulatory complexity that underlies molluscan shell construction from larval stages to adulthood, and serves to highlight the different ecological demands placed on each stage. The use of both ancient and novel genes in all stages of shell construction also suggest that a core set of shell-making genes was provided by a shared metazoan ancestor, which has been elaborated upon to produce the range of molluscan shell types we see today.
The evolutionary success of certain major metazoan groups, such as the Mollusca, Bryozoa, Scleractinia, Echinodermata and Crustacea partly can be attributed to an ability to assemble a wide diversity of mineralised structures [1, 2]. This capacity, in combination with environmental changes at the end of the Proterozoic [3, 4], has been proposed as one of the biological characters that aided the Cambrian radiation . However, it is unknown if the genetic programming directing the biofabrication of calcified and other mineralised structures in disparate animals is homologous . This is because the molecular and cellular mechanisms underlying metazoan biomineralisation remain largely unknown (however see  for an example of a highly conserved biomineralisation gene). Further complicating this analysis is the fact that many animals with calcified skeletons produce different types of skeletons at different times in their lives, with marked changes in skeletal form, mineralogy and function often accompanying ecological transitions . For example, most marine invertebrates have pelagobenthic life cycles, where metamorphosis of the microscopic larva into a benthic juvenile dramatically changes the ecology and body plan of the animal. This major ecological and morphological transition often includes a dramatic change in the form and function of the molluscan shell. It is currently unknown if ontogenetic changes in skeletal construction are the result of the expression of different batteries of biomineralisation genes, i.e. are discrete genetic networks required for larval shell formation versus adult shell formation?
All shell bearing molluscs employ a homologous organ (the mantle) to construct their shells in a way that permits an amazing phenotypic diversity . The mantle consists of a variety of cell types that are directly responsible for synthesis of the shell via the secretion of an organic matrix that is able to initiate and regulate the cell-autonomous assembly of CaCO3 crystals . Once initiated upon synthetic substrates in vitro, ordered CaCO3 crystal growth can reflect that observed in vivo [10, 11]. A number of proteins implicated in the calcification process have been identified from the shells of mature animals including oysters [12–14], mussels [15–17] and abalone [11, 18–20]. Despite the formulation of detailed hypotheses regarding the molecular basis of molluscan shell formation [21–24], these do not account for the initiation of biomineralisation or changes in shell construction during a mollusc's life [see  for a review of the mineralogical transitions that occur in larval forms]. Currently, only a handful of genes are known to play a role in the larval stages of biomineralisation [26–30], with all of these encoding molecules that either define biomineralising cells, the boundaries between biomineralising and non-biomineralising fields, or act to regulate the expression of downstream actuators of the biomineralisation process.
Here, we investigate the ontogenetic expression of a suite of genes expressed in shell forming cells and tissues in the tropical abalone Haliotis asinina. The juvenile mantle of H. asinina expresses a diverse set of genes, a large proportion of which are evolutionarily novel, are predicted to be secreted and are likely to be directly involved in shell synthesis . The regionalised structure of the juvenile mantle and shell also allows inferences to be made regarding gene function . Here we describe the developmental expression of nine mantle genes during the life of H. asinina, specifically testing if ontogenetic changes in gene expression correlate with ecological and morphological transitions. These genes are all expressed in dynamic patterns in shell forming cells and tissues, revealing the complexity of the genetic network underlying molluscan skeletogenesis. These results serve to highlight the interplay between ecology, evolution and development that has shaped the diversity of molluscan shells we see today.
Temporal expression of biomineralising genes
Gene characterisation and spatial expression
We recently have shown that the juvenile Haliotis asinina mantle transcriptome is rapidly evolving and extremely complex . It is evident that hundreds of proteins are secreted from the gastropod mantle into the vicinity where biomineralisation occurs. These are likely to be involved in shell synthesis, presumably contributing directly to the patterning and construction of the calcified shell. The regulation and production of the abalone shell is at least an order more complex than has been inferred from a compilation of previous studies on shell matrix proteins in numerous other molluscs as acknowledged by Marin and Luquet . This 'secretome' complexity, in combination with the modular organisation of the mantle into distinct territories responsible for the biofabrication of discrete shell layers [31, 40], provides a foundation for the generation of diverse shell types. While we cannot yet provide functional data for any of the genes we have studied here, based on their expression profiles we can infer another level of complexity in the regulation of these shell genes at different life cycle stages. The temporal regulation of biomineralisation genes is likely to be an important factor in the production of ontogenetically discrete shell types. For H. asinina, ontogenetic changes in the expression of genes likely to be directly involved in the process of biomineralisation correlate with habitat and ecological transitions.
Changes in shell structure and pattern track with H. asinina's ecology
H. asinina has a pelagobenthic life cycle that includes a minimal period of three to four days in the plankton [33, 41]. The first biomineralisation events occur shortly after hatching, with the fabrication of the larval shell (protoconch) over about a 10 h period. These structures allow the veliger larva to completely retract into a protective environment and rapidly fall out of the water column. The next phase of biomineralisation does not commence until the competent veliger larva contacts an environmental cue that induces metamorphosis . Postlarval shell (teleoconch) is laid down rapidly following metamorphosis with marked variation in the rate of its production between individuals. While the initial teloconch is not pigmented (Fig. 1D), it is textured and opaque such that postlarval shell growth is easily discerned from the larval shell (Fig. 1D inset). Subsequently, the teloconch rapidly develops a uniform maroon colouration similar to the crustose coralline algae (CCA) that the larva has settled upon (Fig. 1E). At about 1 mm in size further changes in the morphogenetic program of the mantle are reflected in the shell. Structurally, a pronounced series of ridges and valleys and a line of respiratory pores (tremata) have appeared (Fig. 1F). Furthermore, it is at this stage of development that the first recognisable tablets of nacre can be detected (Fig. 1J). Colourmetrically, the uniform maroon background is now interrupted by oscillations of a pale cream colour, and is punctuated by a pattern of dots (that only occur on ridges) which are blue when overlying a maroon field and orange when overlying a cream field. This shell pattern may enhance the juvenile's ability to camouflage on the heterogeneous background of the CCA they inhabit at this stage of development.
At 10 to 15 mm, this ornate colouration pattern begins to fade, with maroon and cream fields apparently blending to give a brown background. Blue and orange dots however persist on the ridges (Fig. 1G). With further growth, the ridge-valley structure fades to give rise to a smooth adult shell, with irregular brown-green triangles on a light brown background (Fig. 1H). These larger animals are nocturnal, graze amongst turf algae  and inhabit the undersides of boulders and coral bommies . Overall, ontogenetic changes in H. asinina shell pigmentation and structure match changes in the habitats occupied during development.
Differential expression of mantle genes reflect changes in shell structure
The spatial and temporal expression patterns of the nine genes investigated here reveal a complexity to the genetic networks that coordinate the deposition of larval, juvenile and adult shell. Many of the genes analysed in this study are expressed in the mantle during the production of larval, juvenile and adult shells (Has-Ubfm, Has-ferrt, Has-calmbp1), while others are restricted to one or two shell phases (Has-tsfgr1, Has-cam1, Has-vm1, Has-vm2, Has-lustA, Has-Som). While the lack of a detailed cell fate map through metamorphosis prevents conclusions from being drawn regarding cellular developmental homologies, the continuous expression of Has-tsfgr1 and Has-vm1 in cells no other than shell forming cells in both larval and postlarval stages suggests that a proportion of the postlarval mantle is derived from cells of the larval shell field.
Analyses of the expression profiles of the genes included in this study provide insight into the morphogenetic activity of shell production at different stages in the life of H. asinina. Genes that are continuously expressed in the mantle – Has-ferrt, Has-ubfm and Has-calmbp1 – are likely to play fundamental roles in biomineralisation. Has-ubfm encodes a highly conserved ubiquitin fold-like modifying protein  and is expressed in the expanding shell field suggests that specific intracellular processing of gene products is required to generate functional extracellular components of the biomineralising secretome. Two other evolutionarily conserved proteins, Has-ferrt and Has-calmbp1, are also expressed within the trochophore shell field and later in the mantle, and also are likely to be involved in intracellular events necessary for shell deposition. Iron is known to affect calcification processes in mammals [44, 45], algae  and molluscs . The high expression level of Has-ferrt in a range of cell types in the juvenile mantle is compatible with iron being essential for shell construction or pigmentation. Has-calmbp1 is similar to calcium dependent protein kinases, however its role in shell production remains unknown.
In contrast to these constitutively expressed genes, Has-tsfgr1 displays a dynamic expression profile in the shell forming tissue during development. The putative protein is composed of a set of glycine-rich repeats (over 52% Gly in the mature protein), suggesting it may possess elastomeric properties known to be important in various calcification processes . The highly repetitive nature of Has-tsfgr1 also suggests that this protein may be involved in forming the organic template upon which initial CaCO3 nucleation occurs . Recently Yano et al.  isolated a family of glycine rich, repetitive motif proteins (Shematrins) from a mantle cDNA library of the pearl oyster Pinctada fucata. The Shematrin family is currently known to encode 7 proteins with similar C-terminal motifs which terminate in a tyrosine residue and are expressed in the mantle edge, apparently localised to the prismatic layer of the mature oyster shell . Interestingly, Has-tsfgr1 also possesses a C-terminal motif of 5 residues terminating in a tyrosine residue, suggesting that this feature may be of functional importance to this class of protein. Although sequence alignments of the Shematrins and Has-tsfgr1 do not reveal any close sequence homology, the high glycine content, repetitive nature and shared spatial expression suggest these proteins may play similar functional roles. Unlike Has-ubfm, Has-calmbp1, and Has-ferrt, which all maintain expression within juvenile and adult mantle tissue, Has-tsfgr1 is significantly down-regulated in the mantle tissue of >20 mm animals. This observation is compatible with different stages of shell development requiring the secretion of discrete sets of structural proteins, which act to alter the physical and mineralogical characteristics of the shell.
Two other novel genes – Has-vm1 and -vm2 – also display a dynamic temporal expression during the development of the shell. As expression of Has-vm1 is activated in the larval mantle after completion of the construction of the larval shell, this gene is likely to be involved in the construction of the postlarval shell following metamorphosis. This pattern of expression reveals a linkage between larval and postlarval mantles and is similar to that observed for the developmental regulator Has-Hox4 . It also demonstrates that although larval shell synthesis has ceased, transcriptional activity in the larval mantle continues in anticipation for the next life cycle phase .
Has-vm2 is also differentially regulated during shell growth, and encodes a protein with the hallmarks of being involved in biomineralisation including a signal sequence, repetitive proline rich motifs and two putative O-linked glycosylation sites. Has-vm2 is also down-regulated in larger individuals, with a concomitant reduction in transcript size suggesting that alternative splicing of this gene product takes place in animals larger than 40 mm, again highlighting the different requirements for shell construction at different life cycle stages.
Three genes – Has-cam, Has-lustA and Has-Som – are expressed in patterns that are indicative of roles in biomineralisation during post-larval shell growth. Reflective of the various roles it plays within well studied mammalian systems including signal transduction and regulation of the cell cycle [50–53], Has-cam1 appears to play diverse roles during development. Highly expressed in the prototroch of trochophores, it is not until the veliger larva attains competence to metamorphose that Has-cam1 is detected within the mantle. This suggests that Has-cam1 is not directly involved in larval shell synthesis. In agreement with studies on bivalves [12, 54]Has-cam1 is expressed within the gills and the outer fold of the mantle of juvenile animals. The expression pattern of Has-lustA supports its proposed role of binding aragonitic tablets of nacre together [18, 55], and coincides with the appearance of ordered aragonitic tablets. Interestingly, Has-lustA is down-regulated in the mantle tissue of mature abalone of 100 mm (Fig. 2) possibly reflecting a cessation of shell growth as this is close to the maximum size of 11 cm reported for this species . Has-Som has previously been shown to play a role in pigmentation of the juvenile shell  and is expressed in the mantle tissue of juvenile animals at the time complex colour patterning commences.
Many planktonic molluscan larvae face similar challenges during larval life and the evolution of a larval shell has clearly been a successful response to these challenges . On the molecular level, it is currently unknown the degree to which construction of the molluscan protoconch is conserved. Previous studies have revealed that the expression of the engrailed transcription factor in polyplacophoran , gastropod [27, 29, 57] and scaphopod  representatives is restricted to cells that form boundaries between shell forming and non-shell forming ectoderm, suggesting that the regulatory mechanisms that establish shell forming structures in these clades were inherited from a common ancestor. Following metamorphosis, planktonic molluscan larvae inhabit a broad diversity of benthic ecological niches from sediments, coral reefs, deep sea hydrothermal vents and temperate rocky reefs. The shell of the tropical abalone undergoes several major transitions in morphology, mineralogy and pigmentation during its construction, each of which is adapted to suit the different habitats that larval, juvenile and adult forms occupy. These varied morphologies are the result of differential gene expression of both evolutionarily ancient and novel genes within the mantle tissue.
Given the number of reports of molluscan biomineralising genes that do not share homology with any other phyla (see  for a review) and the data reported here, we suggest that the rapid evolution of the mantle secretome has greatly contributed to the radiation and evolutionary success of the Mollusca . This study demonstrates that the regulation of these genes can be complex, with different batteries of structural genes activated in different parts of the mantle at different phases of the life cycle. We show that changes in expression correlate with changes in shell structure, colour and pattern, and that these changes map closely with ecological transitions. We propose that the regulation of this rapidly evolving mantle secretome is achieved through the action of highly conserved transcription factors and signalling molecules. Dissection of the gene regulatory networks controlling the construction of both larval and postlarval shells promises to shed light on the interplay between ecology and development on evolution of the molluscan body plan.
Analysis of mantle genes
The genes investigated in this study were originally identified either through (1) an expressed sequence tag (EST) screen of genes expressed in the mantle of juvenile abalone , (2) an EST survey of developmentally expressed genes  or (3) a differential display analysis of developmentally regulated genes . Full length cDNA sequences for the genes used in this study were obtained using a RACE approach as described in Jackson et al. . cDNA sequences were initially characterised as described in Jackson et al.  and classified as either having conceptually derived amino acid sequence similarity with proteins in public databases, or encoding a novel secreted protein. The presence of signal peptides was inferred using the SignalP 3.0 server  and glycosylation predictions were made using the NetOGlyc server . Putative open reading frames (ORFs) for the evolutionarily novel and divergent genes Has-vm1 (H. as inina – v eliger m antle 1), Has-vm2 (veliger mantle 2), Has-tsfgr1 (trochophore shell field glycine rich 1) and Has-Som (Sometsuke) were identified using ORF Finder .
For genes encoding conserved proteins (Has-ubfm, ubiquitin fold modifier; Has-cam1, calmodulin; Has-ferrt, ferritin; Has-calmbp1, calcium binding protein; Has-lustA, Lustrin A), tBLASTx and BLASTp searches were conducted against the GenBank database using default settings (Has-Som, Sometsuke, has been previously characterised ). Publicly available protein sequences that displayed significant similarity to H. asinina sequences and representing a broad taxonomic range were downloaded and aligned in ClustalX. Alignments were manually edited in MacClade. Percent identity and biochemical similarity for each sequence relative to the respective H. asinina sequence were calculated using the NCBI bl2seq algorithm .
Animals and whole mount in situ hybridisation
Animals were procured from natural spawnings conducted at the Bribie Island Aquaculture Research Centre, Queensland, Australia as described in Jackson et al. . Larvae and juvenile abalone were relaxed in approximately 0.3 M MgCl2 in FSW prior to fixation in 4% paraformaldehyde in 0.1 M 3-(N-morpholino) propane sulfonic acid pH 7.5 (MOPS), 2 mM MgSO4, 1 mM ethyleneglycoltetraacetic acid (EGTA) and 0.5 M sodium chloride (NaCl) for 30 min at room temperature. Fixed samples were then rinsed several times with PBS buffer plus 0.1% Tween 20 and stepped into 75% ethanol and stored at -20°C. Decalcification of the juvenile shell was achieved by incubation in a solution of 4% paraformaldehyde, 1× PBS buffer and 350 mM ethylenediaminetetraacetic acid (EDTA) for 1 – 3 h depending on shell size. The remaining periostracum and proteinaceous components of the shell were manually dissected away from the animal with fine dissecting forceps. Whole mount in situ hybridisation using digoxigenin-labeled riboprobes synthesised from PCR templates was performed following Giusti et al.  and Jackson et al. .
Reverse transcriptase PCR
Total RNA was extracted from eggs, 10 h old newly hatched trochophores, 134 h old competent veligers, whole 4 mm (shell length) juveniles, 20 mm juvenile mantle tissue, 40 mm juvenile mantle tissue and 100 mm adult mantle tissue using TriReagent following the manufactures instructions. cDNA was synthesised from 1 μg of intact total RNA following Jackson et al. . Relative levels of gene expression across the 7 cDNA samples were assessed by empirically determining the linear phase of PCR amplification for each gene using gene specific primers (available upon request). Briefly, each PCR was run for 20 cycles after which time 4 μl was removed. Reactions were then allowed to continue for a further 3 cycles and the process repeated until aliquots had been obtained from cycles 20 – 34. Samples were then separated on 2% agarose gels . Each PCR reaction was run in duplicate with cDNA synthesised in the absence of MMLV-RT as a control for genomic DNA contamination. Histone H1 was used as a constitutively expressed housekeeping gene and as an indicator of equivalent cDNA synthesis efficiency and PCR template quality [33, 65].
Scanning electron microscopy
Nine, 10 and 11 h old trochophores were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer for 30 min then washed in the same buffer prior to postfixing in 1% osmium tetroxide (OsO4) in 0.1 M sodium cacodylate buffer. Samples were dehydrated through a graded series of ethanol before being infiltrated and dried overnight in hexamethyldisilasane (HMDS). The soft tissue of competent veligers and newly metamorphosed post-larvae was dissolved using 2.8% v/v sodium hypochlorite for approximately 5 min. The remaining shells were then washed extensively with de-ionised water and dehydrated with 100% ethanol before mounting. All samples were mounted either on double-sided tape or Leit-C conductive carbon cement on aluminium stubs and sputter-coated with gold. Samples were viewed with an S-2300 Hitachi scanning electron microscope at 10 kV.
- Has-ubfm :
Haliotis asinina Ubiquitin fold modifier 1
- Hasferrt :
Haliotis asinina ferritin
- Has-calmbp1 :
Haliotis asinina calcium binding protein 1
- Has-tsfgr1 :
Haliotis asinina trochophore shell field glycine rich 1
- Has-Cam1 :
Haliotis asinina calmodulin 1
- Has-vm1 :
Haliotis asinina veliger mantle 1
- Has-vm2 :
Haliotis asinina veliger mantle 2
- HaslustA :
Haliotis asinina lustrinA
- Has-Som :
Haliotis asinina sometsuke
We are grateful to Kathryn Green for the trochophore SEM and Andreas Reimer for SEM advice. Alina Craigie and Elizabeth Williams provided the in situ image shown in Fig. 7E. Andreas Wanninger provided valuable discussions, which greatly improved this manuscript. Two anonymous reviewers provided valuable comments. This work was supported by Australian Research Council funds to BMD and German Research Foundation funds (DFG, Project Wo896/4-1 COSMAP) to GW.
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