The juvenile shell of H. asinina is an ideal model system with which to study the molecular events of shell construction and patterning. A complex but regular chromatic pattern adorns the outermost layer of the shell, the periostracum. Here, a series of dots (either blue or orange) are laid down on top of the ridges of the shell. The color of the dot will be blue if it overlies a brown/red background, or orange if it overlies a cream background (Figure 1a–c). This model system is such that it allows for the spatial mapping of gene expression profiles within the mantle to patterning and structural events at the leading edge of the shell.

The mineralogical composition of the shell is partitioned dorso-ventrally into two major layers; a dorsal calcitic layer and a ventral aragonitic layer (Figure 1d). The mantle epithelium, which secretes the proteins responsible for the construction of these structures, is convoluted at its anterior edge. Between the two main folds (the inner and outer folds) lies the periostracal groove (Figure 1e–g), into which the periostracum is secreted and then extruded onto the dorsal surface of the shell. We have identified a second minor fold within the mantle of H. asinina that we have termed the anterior crease of the outer fold (Figure 1e, f and 1h). Although we cannot yet assign a specific function to this structure, the fact that it possesses an abundance of microvilli (Figure 1h) suggests that it is actively secreting substances responsible for shell or periostracum construction.

We have sequenced 530 randomly selected clones from a cDNA library constructed from the juvenile mantle tissue of the tropical abalone, Haliotis asinina (Linnaeus). These sequences are available under GenBank accession numbers DW986183 to DQ298397. This tissue was taken from juveniles maintained in warm water (26–28°C) that were rapidly depositing shell material, approximately 50 μm per day 32. From this survey, we have identified 331 unique EST clusters (unigenes) that are expressed in the mantle (Figure 2; Additional file 1). Based on the presence of conserved signal sequences and similarity to secreted proteins in GenBank (Figure 2) we conservatively estimate that 26% of these (85 unigenes) encode secreted proteins. Amongst the 140 unigenes that lack both significant similarity to sequences lodged in public databases (Figure 2) and a signal sequence, some may be secreted via mechanisms alternative to the classical signal peptide pathway. Of the 106 intracellular unigenes that encode proteins with significant similarity to GenBank sequences, 15 encode trafficking and mineral binding proteins, and are likely to represent mechanisms essential for the supply of shell building components. For example, based upon in situ hybridization analyses (see below) and previous studies on bivalves 3334, ferritin and calmodulin are likely to be playing fundamental and evolutionarily ancient roles in shell construction within the Mollusca.

When compared with EST surveys in other metazoan tissues (e.g. various mouse 35, pufferfish 36 and human 3637 proteomes), a markedly higher proportion (~twofold) of the genes expressed in the abalone mantle encode secreted proteins. By extrapolation, we estimate that hundreds of proteins are released extracellularly from the mantle, and contribute to the fabrication of the shell. This estimate however needs to take into consideration non-biomineralizing secreted proteins that do not possess similarity with previously described GenBank sequences. This estimate is in marked contrast to the current number of biomineralizing proteins isolated from the shells of other gastropods and bivalves. With efforts chiefly focused on the characterization of proteins from the nacreous layer 38394041, fewer than 20 protein families have been shown to contribute to shell formation to date 78. Strikingly, of the 85 unigenes that encode putative secreted proteins in the Haliotis asinina mantle, 67 (80% of the secretome) do not share significant similarity to any sequences in GenBank (Figure 2). However, several novel secreted open reading frames (ORFs) possess motifs comparable to proteins in other organisms, such as GGYGLGL repeats – similar to the elasticity regions of Spidroin, an elastomeric spider silk protein 42 – and proline-rich repeats (LXPLSXIPVXXPXAX) as found in plant cell walls 43. In comparison, 140 of the 246 intracellular genes (57%) do not match sequences in GenBank.

Interestingly, BLAST alignments between H. asinina mantle ESTs and genomic trace sequences from the gastropod L. scutum, which is estimated to be sequenced to 8× coverage and is currently being assembled 44, reveals that only 13 of the 67 (19%) novel secreted proteins in Haliotis appear to have identifiable homologues in the Lottia genome (Additional file 2). These 13 genes may be involved in conserved aspects of shell construction in gastropods and possibly other mollusks. In contrast, the remaining 54 novel secreted proteins are likely to either represent genes that evolved after these gastropod lineages split, have been lost in the Lottia lineage or a combination of these two scenarios. Likewise, many previously discovered molluskan shell matrix genes do not have clear homologues in the Lottia genome (See Additional File 3 for results of these searches against Lottia genome traces). The few shell matrix proteins, such as Lustrin 11, Perlucin 45 and Mucoperlin 46, are likely to be conserved components of molluskan shells, although their general function in shell fabrication and evolutionary origins are currently unknown. Together, these data suggest that the complex secretome involved in mollusk shell construction is encoded primarily by rapidly evolving genes.

In order to infer the function of a subset of these ESTs, we selected 22 for spatial expression analysis on the basis of whether they were novel, predicted to be secreted, possessed repetitive domains and/or shared significant similarity with a gene likely to play a role in biomineralization (Figure 3). Of these, 11 are novel and one appears to have a homologue only in the Lottia genome (i.e. gastropod-specific; Figure 3M). The spatial expression patterns of these 22 mantle ESTs reveals a diversity of territories and cell types in the mantle (Figure 3), and highlights the modular nature of the mantle tissue 24. Some of these (9 of 22) are expressed homogenously in one or more zones responsible for the creation of different shell layers. For example, LustrinA, first isolated from Haliotis rufescens, the gene product of which contributes to the elastomeric properties of the nacreous layer 112747484950, is expressed, along with two other novel genes, in the mantle territory responsible for the production of the inner nacreous layer (Figure 3X–Z). Other evolutionarily conserved genes, including calmodulin and a calcium-binding protein, are expressed continuously along the length of the mantle within both the inner fold and the anterior crease of the outer fold (Figure 3I–L), while others, such as ferritin, are expressed only in the outer fold (Figure 3M, N). The shared spatial expression of ferritin and calmodulin genes between the Bivalvia and Gastropoda suggests they play a conserved role in the formation or modification of the periostracum 3451. The remaining 13 genes analyzed here are restricted to specific mantle territories and are expressed discontinuously along the length of the mantle; either in the inner fold alone (Figure 3E–G), the inner mantle fold and the anterior crease of the outer fold (Figure 3H), the anterior crease of outer fold alone (Figure 3O–V), or the anterior zone of the outer fold the mantle (Figure 3W). The dynamic spatial expression of these genes along the length of the mantle, regardless of precise zone, implies that they are contributing to the patterning of shell structure and/or coloration 5253.

Has-sometsuke (HasSom) is the only gene in this study to be expressed in the anterior zone of the outer fold of the mantle (Figure 3W and 4) and the only gene to map precisely to a shell pigmentation pattern (Figure 4). HasSom transcripts are only detected in territories of the mantle that underlie regions of the shell that are producing blue dots and fields of red, which become blue upon de-calcification (Figure 1). Blue dots (Figure 1a) correspond to zones of high HasSom expression relative to regions of red (Figure 4). The relative abundance (blue dot or red field) or absence (cream field) of this single gene product is consistent with a role in creating the juvenile shell pattern. Interestingly, BLAST alignments indicate the derived Sometsuke protein shares weak similarity to the ependymins, a family of rapidly evolving extracellular glycoproteins previously only found in deuterostomes ([54; Supplementary Figure 1] and Additional File 4). There is currently no evidence of this protein family encoded in ecdysozoan and basal metazoan genomes; we could not detect an ependymin gene within the Lottia, Nematostella, Amphimedon or Hydra genomes. Ependymin is highly expressed in vertebrate cerebrospinal fluid 55 and may be involved in echinoderm tissue regeneration 56. With disparate roles in these three phyla and apparent loss from many genomes, it is difficult to infer the ancestral role of this protein in the Bilateria. However, various functional features of this protein, including its ability to bind calcium 57 and to undergo polymerization into insoluble fibrils 55 support a role for Sometsuke in shell construction and patterning.

A directionally cloned cDNA library was constructed from total RNA extracted from the mantle tissue of ten 7–15 mm juvenile H. asinina using a BD Biosciences SMART library construction kit. Phages were converted into plasmid DNA following the manufacturer's instructions and sequenced using ABI chemistry v 3.1 59. EST sequences were clustered using ClustalW 6061 and inspected visually to yield consensus contiguous sequences and a non-redundant collection of ESTs. ESTs were first annotated based on SWISSPROT and NCBI nucleotide, protein and EST database searches at the National Center for Biotechnology Information (NCBI) using the BLAST 62 family of programs 63 and classified according to function 64. At the time of writing 900,172,457 L. scutum traces were available from the NCBI trace archive 65. These traces were downloaded and searched against our dataset using the standalone BLAST package (TBLASTx and TBLASTn algorithms with default gap costs and the BLOSUM62 matrix). Putative ORFs were identified in ESTs either through sequence similarity or ORF Finder 66 and manual inspection. Among the novel ESTs (those ESTs sharing no significant similarity with publicly available sequences) only ORFs larger than 50 codons, encoded by the positive strand, and beginning with a methionine residue were accepted. All conceptually derived protein sequences were assessed for the presence of a leader sequence using the SignalP 3.0 67 server 68 and were classified into extracellular or intracellular categories. An EST encoding a novel protein was only accepted as destined for secretion if both SignalP 3.0 algorithms (neural network and hidden Markov model) identified the presence of a signal peptide and a cleavage site, and if the Markov model probability was higher than 90% (in most cases this was >95%). Sequences are deposited at NCBI under accession numbers DW986183 to DW986511. Has-vm2 and Has-lustrin have accession numbers DQ298397 and DQ298402 respectively.

Juveniles (1–10 mm) of H. asinina were relaxed with 1 M MgCl₂ in seawater and then fixed for 1 h in 4% paraformaldehyde, 0.1 M 3-(N-morpholino) propane sulfonic acid pH 7.5, (MOPS), 2 mM MgSO₄, 1 mM ethyleneglycoltetra-acetic acid (EGTA) and 0.5 M NaCl. Fixed animals were then washed five times with 100% ethanol and stored in 75% ethanol at -20°C. All DIG-labeled riboprobes were produced using either SP6, T3 or T7 RNA polymerase and a PCR amplicon of the desired clone. Several sense controls were performed with probes of various GC contents and lengths to assess background patterns. Endogenous alkaline phosphatase activity of the mantle tissue was also assessed and found not to be present following the in situ procedure. Whole mount in situ hybridization (WMISH) was performed as previously described 69 usually at a hybridization temperature of 62°C. Sections (6 μm) were produced by mounting juveniles that had undergone WMISH in EPON 812 and sectioning using a Leica Ultracut T. Prior to WMISH shells were removed by incubation in 1× PBS, 4% paraformaldehyde and 350 mM EDTA. Gene expression within the mantle was correlated to shell patterning activity by photographing individual shells prior to decalcification and relating this to WMISH results.

For transmission electron microscopy, juveniles were relaxed as described above and mantle tissue was dissected and processed according to 70. Briefly, tissue was prefixed in low osmium (0.05% OsO₄ in 4% glutaraldehyde, 0.2 M Na cacodylate, 0.1 M NaCl and 0.35 M sucrose, pH 7.2) for 10 min. This was followed with a primary fixation in the same fixative, but lacking osmium, for 1 h. The tissue was then washed twice in buffer (0.3 M NaCl and 0.2 M Na cacodylate, pH 7.2) before a post fixation in osmium (1% OsO₄, 0.3 M NaCl and 0.2 M Na cacodylate, pH 7.2) for 1 h. Samples were then dehydrated through a graded series of ethanol, embedded in EPON 812 and 60 nm sections taken using a Leica Ultracut T. Sections were stained with uranyl acetate and lead citrate and viewed in a JEOL JEM 1010 transmission electron microscope at 80 kV.

For scanning electron microscopy, whole juveniles with the shell removed were fixed and dehydrated as described above, then infiltrated and dried overnight in hexamethyldisilisane. Juveniles and unfixed shells were mounted on stubs, sputter-coated with platinum and viewed in a JEOL JSM 6300 scanning electron microscope at 15 kV.