BACKGROUND
for over  <dig> million years, mollusks have successfully used a variety shells to populate the world over  <cit> .
due in part to the simple sheer prevalence of mollusks in past and present environments and their variety of shell formation strategies, these organisms represent the current model from which biomineralization is studied.
facilitating the shell formation in molluscs is the mantle organ.
phylum mollusca is typically classified by an invertebrate unsegmented body, a mantle and a calcareous shell.
the latter two are the subject of this investigation.
the shell is internally lined by the mantle, composed of a thin sheath of tissue radiating out to the shell margins.
in the case of a bivalve this organ is zootomically divided into two regions: the mantle pallial located proximal to the shell hinge, and the mantle edge situated distal to the hinge  <cit> .
the distal mantle is further characterized by enlargement of the sheath at the shell margin into three terminal folds: the outer fold , middle fold , and the inner fold .
they are arranged such that of is closest to the shell and the if furthest.
the main function of the mantle is recognized as the secretion of organic components necessary for shell biomineralization but it also has other purposes  <cit> .
the mantle has a sensory function and can initiate closure of the valves in response to unfavourable environmental conditions  <cit> .
in addition, the mantle also controls inflow of water into the shell's internal chamber responsible for respiratory and filter feeding purposes.
these functions are said to be zone-specific in the greater mantle organ, referencing the if as muscular, mf as sensory, and the of as secretory in task  <cit> .
likewise the mantle edge and mantle pallial are considered principally secretory tissues.
to date, the secretory function of the mantle has been the focus of significant research with regard to biomineralization of the shell  <cit> .
this is especially the case within pearl oyster species, considering pearl cultivation's reliance on mantle tissue.
the pearl oyster shell typically consists of an outermost organic layer termed the periostracum, and calcium carbonate oriented in two distinct microlaminates, the outer calcite prismatic layer and the inner aragonite nacreous layer  <cit> .
evidence in the microstructure of both prismatic and nacreous layers has credited an organic framework as being central to the ordered mineralization  <cit> .
as such, the organic component has been the subject of much research devoted to its extraction and characterization  <cit> .
primarily these investigations have identified a number of matrix proteins, a subset of which have had their corresponding gene sequence determined.
some of those identified include: nacrein  <cit> , msi60/msi <dig>  <cit> , n66/n <dig>  <cit>  prismalin- <dig>  <cit> , and caspartin/calprismin  <cit> .
however, many of the proteins remain to be identified, due in part to insolubility, self-aggregation of the molecules or an unusual resistance to temperature, chemicals and enzymes  <cit> .
more recently, alternative techniques to identify organic matrix proteins have been employed, including the use of expression cdna libraries generated from mantle tissue screened with antibodies elicited from unfractionated organic matrix  <cit> .
although this technique has yielded positive identification of matrix proteins, it is largely inefficient and has meant the expense is inhibitory for most laboratories.
moreover, mantle tissue cdna libraries have been screened with degenerate primers based on the signal peptide sequences of known proteins  <cit> .
while this approach has successfully identified a number of organic matrix proteins, this technique is restricted to related proteins, providing little latitude for novel matrix protein detection.
also noteworthy are subtractive cdna libraries enriched with hundreds of putative organic matrix gene sequences  <cit> .
although the most encompassing method used thus far, subtractive cdna libraries inherently report only presence or absence of putative organic matrix gene sequences and are incapable of detecting more subtle expression differences.
overall, all the techniques outlined have diverse advantages and limitations however they are still largely inadequate to address the likely complexity of shell biomineralization.
a need remains for developing technology by which clusters of genes can be identified and analyzed simultaneously.
transcriptomics is a recently developing field now readily available for gene discovery and is rapidly being put to use in many novel applications  <cit> .
high-throughput sequencing and est microarrays facilitate a comprehensive and inclusive experimental approach in which alterations in the state of entire transcriptomes can be simultaneously assayed.
this technology has begun to be applied allowing the large scale investigation of gene products expressed in the mantle tissue with reference to biomineralization and other mantle-associated processes  <cit> .
although gene products identified may not necessarily be incorporated in the shell, this technique would circumvent the aforementioned technology limitations.
additionally it should be noted that a transcriptomic approach would not prejudice against gene products potentially involved in biomineralization but not integrated into the shell.
in order to expedite the elucidation of biological processes associated with the mantle organ this investigation has spatially mapped the differential expression of numerous expressed sequence tags  derived from the mantle of p. maxima using the custom microarray chip pmaxarray  <dig>  developed for this investigation.
RESULTS
a kruskal-wallis test of the data generated from pmaxarray  <dig>  was performed against the five experimental conditions.
outer fold , middle fold , inner fold , ventral mantle  and dorsal mantle  comparisons identified  <dig> ests of the total  <dig> ests present on the microarray as statistically differentially expressed in reference to the experimental control .
hierarchical cluster analysis of these  <dig> ests grouped them according to similar expression profiles across the conditions.
this analysis assisted the selection of four major expression profiles designated clusters a, b, d and e. a sub-cluster of b, termed cluster c was also selected.
clusters of interest were primarily selected based on the likelihood they would be informative in relation to biomineralization characterization.
cluster c was additionally selected due to its extreme difference in expression between the conditions from cluster b as indicated by the colour intensity .
a subset of the  <dig> ests, representing approximately 33% of the corresponding clones, were sequenced and batch blasted against blastx  and blastn  databases .
this subset of the total ests identified was deemed sufficient sequence coverage due to redundancy measurements.
many of the smaller cluster's ests were sequenced almost in entirety.
sequence alignment software resolved these microarray ests to  <dig> unique sequences.
a number of ests were selected from each of the five clusters to determine specific local expression in the mantle .
these selections were founded on several factors including whether they were either: novel and highly differentially expressed, or share significant homology with annotated genes of interest.
a list of p. maxima mantle ests with significant sequence similarity to annotated genes and proteins.
these ests are significantly differentially expressed  among specific regions of the mantle and have been clustered according to similar expression profiles: cluster a, b, d and e. description of best sequence hit = highest sequence comparison match of ests with blastx or blastn search tools, including accession number and brief identification of matching sequence.
e-valve = likelihood of random occurrence of sequence match, values approaching zero indicate increasing sequence match significance.
cluster a
cluster a consisted of  <dig> microarray ests typical of the highest relative expression in dm, slightly less expression in vm, and low expression among of, mf and if .
<dig> of the total  <dig> ests were sequenced from which  <dig> unique sequences were resolved,  <dig> were contigs and  <dig> singletons.
putative sequence homologies could only be found for  <dig> of these ests including known shell matrix proteins n <dig> matrix protein and msi <dig> protein.
other noteworthy matches identified are papilin, trypsin inhibitor protein, mantle gene  <dig> and calconectin .
a functional domain search of the ests significantly similar in sequence with papilin and the trypsin inhibitor protein both revealed tandem kuntiz trypsin inhibitor domains.
the majority of sequences identified from cluster a bear no significant similarity to sequences in public databases, furthermore many of the sequences aligned with poorly described genes and translated proteins.
in situ hybridization was able to further resolve the localized expression for three ests, including; pm <dig>  pm <dig> and pm <dig>  these ests were chosen because they were among the most highly differentially expressed ests in cluster a and sequence similarity searches indicated they were novel.
the three ests were all detected as expressed in the outer epithelium of the dorsal mantle region .
of particular note is that expression of these ests is conspicuously absent at what appears the border of the ventral mantle zone and throughout this region.
cluster b
cluster b contained  <dig> ests detailing a relative expression profile as highly expressed in dm, mf, if, no differential expression in vm, while lowly expressed in the of in comparison to the control condition .
<dig> ests were randomly selected and sequenced.
<dig> unique sequences were detected of which  <dig> resolved as contigs and the remaining  <dig> singletons.
this cluster is principally unannotated; however  <dig> ests are noted for significant sequence similarity to cellular maintenance proteins including: ferritin-like protein, ribosomal proteins, cytochrome oxidase subunits, glutathione peroxidise and radixin .
in situ hybridization was unable to precisely locate any of these sequences in the mantle tissue potentially due to diffuse expression of the target mrnas impeding in situ resolution and/or transcript concentrations being outside the range of detection for the in situ hybridization protocol employed in this investigation.
cluster c
cluster c is a small sub-cluster of  <dig> ests within cluster b, characterized by relative high expression present in if, mf compared to low expression in dm, vm, of .
all  <dig> ests were sequenced, condensing into three contigs and two singletons.
sequence analysis revealed no significant sequence similarity to sequences in the public databases.
in situ hybridization revealed localized regions of expression of three of the ests.
pm <dig> was localised to outer epithelial cells of the mf as well as the inner epithelial cells of the entire mantle organ .
pm <dig> was predominantly expressed in the outer and inner epithelial cells of the ventral sections of folds of and mf respectively .
pm <dig> was found to be expressed sub-cutaneously in the if and mf, specifically appearing interspersed among these regions .
cluster d
cluster d is represented by  <dig> ests almost exclusively expressed in the of mantle region .
<dig> ests were sequenced resolving as  <dig> unique sequences including  <dig> contigs and nine singletons.
approximately half of these ests show significant sequence homologies, the majority of which align with the family of lysine-rich matrix proteins  .
additional matches include tyrosinase and thermoacin .
alignment of krmp deduced amino acid sequences with existing protein family members showed these ests were significantly divergent from p. fucata, p. margaritifera and p. maxima  krmp's particularly by a general absence of the c-terminal gly/tyr region.
of the cluster d homologs only pm <dig> did not align with all of the  <dig> cysteine residues present in the basic region .
local spatial expression of six cluster d ests was mapped to the mantle.
pm <dig>  pm <dig> and pm <dig> had similar patterns of expression, detected on the inner epithelium of the outer fold extending the length of the fold .
conversely, pm <dig> is expressed only in the proximal most inner epithelial cells of the outer fold , notably absent in expression of the three preceding ests .
pm <dig> and pm <dig> indicate a further difference in local expression, observed midway along the inner epithelium of the outer fold .
cluster e
cluster e consists of  <dig> ests showing high levels of expression in of, no difference to low expression in vm and very low expression in all other conditions in comparison to the control condition .
<dig> ests were sequenced revealing  <dig> unique sequences,  <dig> of which are contigs and  <dig> singletons.
sequence analysis shows  <dig> of these ests have significant similarities to shematrin and krmp isoforms.
other sequence similarities include a coat protein, mantle protein  <dig> and articulin .
the deduced amino acid sequence for the latter two was analyzed for signal peptides and both indicated likely signal peptide sequences.
alignment of krmp deduced amino acid sequences from cluster e with existing protein family members showed these ests all conformed to the typical protein primary structure, particularly with the signal peptide region, basic region and the gly/tyr region.
additionally the positions of all six cysteine residues were conserved .
in situ hybridization indicates spatial expression for five of the cluster's ests.
pm <dig>  pm <dig>  pm <dig> and pm <dig> were detected in the mantle outer epithelium extending from the distil region of the of into the vm zone after which expression is abruptly absent towards the dm region .
notably, the directly adjacent epithelium is likewise marked by expression of three cluster a ests showing a precise border of expression between the ests .
pm <dig> has a similar pattern of expression to the four other cluster e ests, however, it is additionally expressed in the inner epithelium of the outer fold .
discussion
the molluscan mantle is a thin tissue from which proteins are secreted into the extrapallial fluid; these proteins dictate the animals shell construction and microstructure.
as a conserved organ involved in shell formation throughout mollusks, the mantle is an excellent foundation from which to study biomineralization  <cit> .
in this study a p. maxima mantle tissue-specific cdna microarray has been generated termed pmaxarray  <dig> , comprising  <dig> cdna clones derived from the mantle tissue of several p. maxima individuals.
this tool has provided significant power to interrogate the role of proteins in shell formation.
microarray analysis has spatially mapped the expression of a number of known and unknown ests with reference to specific mantle zones.
<dig> ests present on pmaxarray  <dig>  were expressed as significantly different to the control condition and approximately one third of those were sequenced and aligned resolving a total of  <dig> unique ests.
the majority of those sequences could not be annotated via the genbank database as no molluscan genome has yet been sequenced, let alone functionally annotated.
other non-model organisms also report a high proportion of unannotated genes .
as such, where sequence homologies are absent, functional significance of ests identified in this study are interpreted with reference to their pattern of expression  and the relevance this bears to mantle associated responsibilities.
five major expression profiles were observed among the mantle zones indicative of specialized molecular functions and ests clustering in each of these profiles will be discussed within these groupings.
cluster a
the spatial expression profile in cluster a suggests a role associated with the nacreous shell formation of p. maxima.
sudo et al.
<cit>  along with others  <cit>  support this supposition noting a close spatial link between transcript expression in mantle zones and shell microstructure inclusion.
of particular interest within this cluster are pm <dig> and pm <dig>  as both ests possess two tandem kuntiz/bovine pancreatic trypsin domains .
pm <dig> is a significant match to papilin; an extracellular matrix glycoprotein occurring widely from nematodes to humans and known to contain several kuntiz domains  <cit> .
likewise the presence of kuntiz domains is expected for pm <dig> which shares sequence similarity with a pancreatic trypsin inhibitor domain protein.
kuntiz bpt <dig> domains are generally regarded as serine protease inhibitors involved in clotting and tissue remodeling  <cit> .
similarly shell formation is known to involve a number of inhibitory components limiting mineralization.
proteoglycans are one such component, essential to shell formation yet intrinsically inhibit biomineralization  <cit> .
the protease inhibiting domains of pm <dig> and pm <dig> may act to maintain the viscous silk gel detailed by adaddi et al.
<cit>  as necessary for nacre formation.
pm <dig> is expressed in the dm epithelial cells overlying the nacre microstructure in conjunction with the immediate cessation of expression toward the vm zone and prismatic microstructure.
taken together, tissue localization and sequence homologies suggest that pm <dig> and possibly pm <dig> are glycoproteins with inhibitory protease activity specific for nacre formation.
ests pm <dig> and pm <dig> are unannotated however in situ hybridization demonstrated a very specific localization to the epithelium of the dm zone, as already described for pm <dig>  this same distribution of expression is also demonstrated for n <dig> gene  <cit>  and msi <dig> gene  <cit>  both of which code for nacre matrix proteins.
the exclusive expression of these two novel ests, pm <dig> and pm <dig>  suggest a role in nacre formation which along with pm <dig>  are the only reported cases of in situ hybridization localizing ests to the dm zone since sudo et al.
<cit>  reported msi <dig> gene expression.
cluster b
this cluster is the largest and most ubiquitous of all the expression profiles identified in this study.
ests in cluster b display similar expression values across a number of seemingly unrelated mantle tissues.
the anatomy and function of the mantle organ is generally considered as follows: of is secretory , mf is sensory, if is muscular, vm and dm are secretory   <cit> .
therefore in the perceived absence of a specialized function uniting these tissues, cluster b most likely represents ests involved in general cellular maintenance and regulation rather than shell formation.
this proposition is supported by the identification of a number of 'housekeeping' genes  not seen in any of the other clusters including cytochrome c oxidase, glutathione peroxidase, ezrin/radixin/moesin binding proteins and ribosomal proteins.
cluster c
this cluster is the smallest and contains ests which showed no significant similarities with any reported protein or nucleotide sequences.
the in situ hybridization results for ests pm <dig> and pm <dig> showed association with the periostracal groove in which the outer epithelium of the mf is included.
the main function of the periostracal groove is to secrete a glycocalyx coating forming the periostracum.
a glycocalyx is a network of polysaccharides that project from cellular surfaces usually secreted by epithelial cells for a range of adhesion functions.
the distil expression of pm <dig> in the mf outer epithelium indicates this est may code for a glycoprotein incorporated in the mature stages of the outer glycocalyx coating  <cit> .
similarly, expression of pm <dig> in the proximal epithelial cells of the periostracal groove may also code a protein involved in glycocalytic coatings and the stepwise construction of the periostracum.
est pm <dig> has a peculiar in situ expression pattern in that the transcript is found below the epidermal layer, interspersed throughout the inner region of the mf and the outer region of the if.
bivalves expose these mantle folds to the external environment  <cit> .
chemoreceptors, photoreceptors and mechanoreceptors are all usually present in the epidermal layer of these folds in order to elicit closure of the shell valves in response to negative stimulus  <cit> .
considering pm <dig> is expressed sub-dermally, it is less likely that this est has a direct sensory role but rather associated with what appears to be nerve fibres  <cit> , possibly involved in a signal transduction cascade  <cit> .
cluster d
the expression profile of cluster d ests suggests an exclusive role of these genes in the of tissue, specifically concerned with the inner epithelia.
this epithelium forms the bottom half of the periostracal groove, which is a highly dynamic tissue responsible for formation, maturation and extrusion of the complex periostracum layer.
the proteinaceous layer functions to seal the extrapallial space and protect the shell from dissolution as well as serve as an initial matrix for mineralization  <cit> .
in situ localization to the inner epithelium of the of tissue signify a periostracum-related function.
neuromacin  <cit>  and theromacin  <cit>  are a family of antimicrobial peptides known to occur in a number of invertebrates.
these peptides are part of a immediate immune response characterized predominantly by cationic and hydrophobic amino acids  <cit> .
est pm <dig> shows a significant sequence similarity to the gene encoding these antimicrobial peptides and its in situ expression profile maps it to where the internal periostracum is formed.
cationic and hydrophobic properties of these peptides  <cit>  are synonymous with the characteristics of the periostracum and water insoluble matrix  of shells  <cit> .
specifically, a scenario for pm <dig> may be that poly-anionic glycoproteins  bind to cationic peptides in the periostracum, effectively anchoring the hydrophilic macromolecules to the hydrophobic wism.
this in turn facilitates active nucleation sites by which microstructure mineralization occurs.
lysine-rich matrix protein  is a family of proteins seemingly specific to mollusks and shell formation of the prismatic design.
cluster d includes seven ests significantly similar to the krmp gene class.
zhang et al.
<cit>  first described these proteins noting predominate expression in the inner epithelial cells of the of and outer epithelium of the mantle edge region.
the deduced amino acid sequence includes an n-terminal signal peptide, a lysine-rich basic region potentially interacting with acidic proteins or co32-, and a glycine/tyrosine-rich region considered involved in protein cross-linking via the quinone-tanning process.
the expression in the mantle edge region and similarities among the signal peptide of other prismatic shell matrix proteins lead zhang et al.
to assign a putative prismatic microstructural function to the krmp family in p. fucata.
however unlike zhang's et al.
<cit>  observations of dual expression in the periostracal groove and the prismatic mantle region, these p. maxima ests are exclusively expressed in the of, a number of which are localized by in situ hybridization to the inner epithelium of the fold , representing the lower half of the periostracal groove.
this deviation from zhang's et al.
<cit>  original characterization is potentially explained by sequence analysis.
the newly identified krmp members appear to be concatenated versions of p. fucata krmp possessing only the signal peptide and lysine rich region typical of the class.
in many of the ests the c-terminal region is significantly reduced and/or replaced with serine and aspartic acid residues.
the absence of the glycine/tyrosine-rich region suggests that the predicted proteins coded by these ests are not quinone-tanned.
pm <dig> is the most divergent of the krmp members and displays a different local expression being present along the middle region of the of inner epithelium, suggesting a different function, specific to periostracum formation.
unclear however, is whether these ests are a novel sub-family of krmp or a species specific evolutionary adaption of krmp in p. maxima.
the conservation of the lysine-rich region confers the positive charge required to attract and bind acidic glycoproteins necessary for nucleation  <cit>  while expression in the periostracal groove suggests they are incorporated in the periostracum.
in summary, the seven krmp homologs in cluster d are considered specifically adapted for periostracal formation in p. maxima.
pm <dig> is a novel transcript expressed in the inner epithelia cells of the of at the base of the periostracal groove.
periostracum development begins with the formation of the pellicle providing a framework on which coatings of the glycocalyx thicken and develop the periostracum  <cit> .
in bivalves, the pellicle typically originates from a row of basal cells at the bottom of the periostracal groove  <cit> .
the spatial expression of pm <dig> closely matches the area described for pellicle formation and its deduced sequence is dominated by tyrosine and glycine, typical of a quinone-tanned protein  <cit> .
as the pellicle provides the structural backbone on which ensuing glycocalyx coatings mature the periostracum, its formation would be largely concerned with the hardening of the structure.
cluster e
ests in this cluster are characterised by expression primarily in the of and vm tissues.
the outer epithelia of both these tissues are considered homogenous in function, attributed to prismatic shell formation  <cit> .
in situ hybridization of several cluster e ests confirms dual expression in the outer epithelia of the of and vm, consistent with involvement in prismatic shell formation.
ests pm <dig>  pm <dig>  pm <dig>  pm <dig>  pm <dig>  pm <dig> and pm <dig> represent the shematrin protein family.
while p. maxima isoforms for shematrin have already been reported  all the ests presented here, except pm <dig>  are novel isoforms.
shematrin is a family of glycine-rich shell matrix proteins known to be present in the prismatic microstructure of several pearl oyster species.
yano et al.
<cit>  suggests shematrins are framework proteins facilitating calcification of the prismatic microstructure.
this investigation maps shematrin isoform pm <dig> via in situ hybridization to the outer epithelium from the tip of the of to the vm/dm mantle border, parallel with the prismatic/nacreous shell border, adding to the characterization of the shematrin family in relation to the prismatic microstructure.
ests pm <dig> and pm <dig> show significant sequence homologies with mantle protein  <dig> and alveolin <dig> respectively, and both appear to be related to cytoskeletal protein family articulin.
articulins are part of the membrane skeleton of eukaryotic cells stabilizing plasma membranes  <cit> .
it is suggested that ests pm <dig> and pm <dig>  function as plateins, a new family of articulins described by kloetzel et al.
<cit> .
plateins contain modified articulin core domains typical of secreted structural proteins as well as a novel predicted signal peptides detected in intra-alveolar sacs, an extracellular environment.
likewise, pm <dig> and pm <dig> also contain predicted signal peptides indicating a secretory pathway and est pm <dig> has been detected by in situ hybridization specifically to the epithelial cells of both the lower periostracal groove and mantle outer epithelium, in contact with the prismatic shell.
these tissues are noted for their secretions reinforcing the secretory pathway of pm <dig> and pm <dig>  in summary, gene sequence homology of pm <dig> and pm <dig> with membrane skeleton proteins, coupled with their differential expression to secretory tissues and detection of signal peptides suggest these ests are putative members of a new articulin family, differentiated by extracellular function.
these ests may encode framework proteins involved in the formation of the prismatic microstructure in p. maxima shell.
a functional link between the periostracal groove secretions and prismatic shell formation has previously been suspected based on a structural continuity between the outer periostracum and interprismatic matrices  <cit> .
zhang et al.
<cit>  demonstrated shell matrix protein krmp as expressed in both secretory tissues.
however, the presence of ten krmp related ests found to be expressed specifically in the outer epithelia of the ventral mantle zone three of which were confirmed with in situ hybridisation  represent a break from that observed by zhang et al.
<cit> .
the krmp family has already been discussed in reference to seven est homologs found to be specific to the periostracal groove of p. maxima.
the observation of these two separate expression patterns for krmp related ests in the periostrcum and prismatic shell formation mantle regions differs from reports in the related pearl oyster p. fucata  <cit> .
in contrast, where krmp homologues appear to perform dual periostracum/prismatic microstructure roles; p. maxima appear to use additional krmp homologs to accomplish the periostracum related task.
this corroborates jackson's et al.
<cit>  supposition that the 'secretome' is a rapidly evolving collection of proteins capable of significant molecular differences in building molluscan shells.
in summary, cluster e contains specific krmp isoforms potentially involved in the prismatic microstructure formation of the p. maxima shell.
a functional linkage between the periostracum and prismatic shell formation is probable, however the mode by which this occurs is highly adaptable, and unlikely to be conserved among species.
CONCLUSIONS
this investigation has mapped the spatial distribution for over  <dig> ests present on pmaxarray  <dig>  with reference to specific locations of the mantle.
five major expression profiles were distinguishable from these differentially expressed ests  relating to the examined mantle divisions: dorsal mantle , ventral mantle , inner fold , middle fold  and outer fold .
these expression profile clusters have indicated at least five unique functioning zones in the mantle.
three of these zones are considered involved in shell related activities including cluster a's role in nacre formation, cluster d's link to periostracum formation and cluster e's implication in calcitic prismatic microstructure formation.
a number of known and novel ests have been identified from these clusters.
microarray differential expression, in situ expression localization and comparative sequence analysis have begun the task of characterizing novel ests identified herein, in addition to further elucidating the functions of previously reported biomineralization related genes.
the microarray approach utilized here has alleviated many of the past difficulties plaguing the molluscan biomineralization discipline, however, this method and its' outcomes is in no way seen as a standalone conclusion.
rather, microarray analysis is intended to spearhead preliminary investigations of shell formation targeting ests for subsequent in-depth characterization including protein isolation and activity studies.

 for over million years, mollusks have successfully used a variety shells to populate the world over. due in part to the simple sheer prevalence of mollusks in past and present environments and their variety of shell formation strategies, these organisms represent the current model from which biomineralization is studied. facilitating the shell formation in molluscs is the mantle organ. phylum mollusca is typically classified by an invertebrate unsegmented body, a mantle and a calcareous shell. the latter two are the subject of this investigation. the shell is internally lined by the mantle, composed of a thin sheath of tissue radiating out to the shell margins. in the case of a bivalve this organ is zootomically divided into two regions: the mantle pallial located proximal to the shell hinge, and the mantle edge situated distal to the hinge. the distal mantle is further characterized by enlargement of the sheath at the shell margin into three terminal folds: the outer fold, middle fold, and the inner fold. they are arranged such that of is closest to the shell and the if furthest. the main function of the mantle is recognized as the secretion of organic components necessary for shell biomineralization but it also has other purposes. the mantle has a sensory function and can initiate closure of the valves in response to unfavourable environmental conditions. in addition, the mantle also controls inflow of water into the shell's internal chamber responsible for respiratory and filter feeding purposes. these functions are said to be zone-specific in the greater mantle organ, referencing the if as muscular, mf as sensory, and the of as secretory in task. likewise the mantle edge and mantle pallial are considered principally secretory tissues. to date, the secretory function of the mantle has been the focus of significant research with regard to biomineralization of the shell. this is especially the case within pearl oyster species, considering pearl cultivation's reliance on mantle tissue. the pearl oyster shell typically consists of an outermost organic layer termed the periostracum, and calcium carbonate oriented in two distinct microlaminates, the outer calcite prismatic layer and the inner aragonite nacreous layer. evidence in the microstructure of both prismatic and nacreous layers has credited an organic framework as being central to the ordered mineralization. as such, the organic component has been the subject of much research devoted to its extraction and characterization. primarily these investigations have identified a number of matrix proteins, a subset of which have had their corresponding gene sequence determined. some of those identified include: nacrein, msi60/msi, n66/n prismalin-, and caspartin/calprismin. however, many of the proteins remain to be identified, due in part to insolubility, self-aggregation of the molecules or an unusual resistance to temperature, chemicals and enzymes. more recently, alternative techniques to identify organic matrix proteins have been employed, including the use of expression cdna libraries generated from mantle tissue screened with antibodies elicited from unfractionated organic matrix. although this technique has yielded positive identification of matrix proteins, it is largely inefficient and has meant the expense is inhibitory for most laboratories. moreover, mantle tissue cdna libraries have been screened with degenerate primers based on the signal peptide sequences of known proteins. while this approach has successfully identified a number of organic matrix proteins, this technique is restricted to related proteins, providing little latitude for novel matrix protein detection. also noteworthy are subtractive cdna libraries enriched with hundreds of putative organic matrix gene sequences. although the most encompassing method used thus far, subtractive cdna libraries inherently report only presence or absence of putative organic matrix gene sequences and are incapable of detecting more subtle expression differences. overall, all the techniques outlined have diverse advantages and limitations however they are still largely inadequate to address the likely complexity of shell biomineralization. a need remains for developing technology by which clusters of genes can be identified and analyzed simultaneously. transcriptomics is a recently developing field now readily available for gene discovery and is rapidly being put to use in many novel applications. high-throughput sequencing and est microarrays facilitate a comprehensive and inclusive experimental approach in which alterations in the state of entire transcriptomes can be simultaneously assayed. this technology has begun to be applied allowing the large scale investigation of gene products expressed in the mantle tissue with reference to biomineralization and other mantle-associated processes. although gene products identified may not necessarily be incorporated in the shell, this technique would circumvent the aforementioned technology limitations. additionally it should be noted that a transcriptomic approach would not prejudice against gene products potentially involved in biomineralization but not integrated into the shell. in order to expedite the elucidation of biological processes associated with the mantle organ this investigation has spatially mapped the differential expression of numerous expressed sequence tags derived from the mantle of p. maxima using the custom microarray chip pmaxarray developed for this investigation.  a kruskal-wallis test of the data generated from pmaxarray was performed against the five experimental conditions. outer fold, middle fold, inner fold, ventral mantle and dorsal mantle comparisons identified ests of the total ests present on the microarray as statistically differentially expressed in reference to the experimental control. hierarchical cluster analysis of these ests grouped them according to similar expression profiles across the conditions. this analysis assisted the selection of four major expression profiles designated clusters a, b, d and e. a sub-cluster of b, termed cluster c was also selected. clusters of interest were primarily selected based on the likelihood they would be informative in relation to biomineralization characterization. cluster c was additionally selected due to its extreme difference in expression between the conditions from cluster b as indicated by the colour intensity. a subset of the ests, representing approximately 33% of the corresponding clones, were sequenced and batch blasted against blastx and blastn databases. this subset of the total ests identified was deemed sufficient sequence coverage due to redundancy measurements. many of the smaller cluster's ests were sequenced almost in entirety. sequence alignment software resolved these microarray ests to unique sequences. a number of ests were selected from each of the five clusters to determine specific local expression in the mantle. these selections were founded on several factors including whether they were either: novel and highly differentially expressed, or share significant homology with annotated genes of interest. a list of p. maxima mantle ests with significant sequence similarity to annotated genes and proteins. these ests are significantly differentially expressed among specific regions of the mantle and have been clustered according to similar expression profiles: cluster a, b, d and e. description of best sequence hit = highest sequence comparison match of ests with blastx or blastn search tools, including accession number and brief identification of matching sequence. e-valve = likelihood of random occurrence of sequence match, values approaching zero indicate increasing sequence match significance. cluster a cluster a consisted of microarray ests typical of the highest relative expression in dm, slightly less expression in vm, and low expression among of, mf and if. of the total ests were sequenced from which unique sequences were resolved, were contigs and singletons. putative sequence homologies could only be found for of these ests including known shell matrix proteins n matrix protein and msi protein. other noteworthy matches identified are papilin, trypsin inhibitor protein, mantle gene and calconectin. a functional domain search of the ests significantly similar in sequence with papilin and the trypsin inhibitor protein both revealed tandem kuntiz trypsin inhibitor domains. the majority of sequences identified from cluster a bear no significant similarity to sequences in public databases, furthermore many of the sequences aligned with poorly described genes and translated proteins. in situ hybridization was able to further resolve the localized expression for three ests, including; pm pm and pm these ests were chosen because they were among the most highly differentially expressed ests in cluster a and sequence similarity searches indicated they were novel. the three ests were all detected as expressed in the outer epithelium of the dorsal mantle region. of particular note is that expression of these ests is conspicuously absent at what appears the border of the ventral mantle zone and throughout this region. cluster b cluster b contained ests detailing a relative expression profile as highly expressed in dm, mf, if, no differential expression in vm, while lowly expressed in the of in comparison to the control condition. ests were randomly selected and sequenced. unique sequences were detected of which resolved as contigs and the remaining singletons. this cluster is principally unannotated; however ests are noted for significant sequence similarity to cellular maintenance proteins including: ferritin-like protein, ribosomal proteins, cytochrome oxidase subunits, glutathione peroxidise and radixin. in situ hybridization was unable to precisely locate any of these sequences in the mantle tissue potentially due to diffuse expression of the target mrnas impeding in situ resolution and/or transcript concentrations being outside the range of detection for the in situ hybridization protocol employed in this investigation. cluster c cluster c is a small sub-cluster of ests within cluster b, characterized by relative high expression present in if, mf compared to low expression in dm, vm, of. all ests were sequenced, condensing into three contigs and two singletons. sequence analysis revealed no significant sequence similarity to sequences in the public databases. in situ hybridization revealed localized regions of expression of three of the ests. pm was localised to outer epithelial cells of the mf as well as the inner epithelial cells of the entire mantle organ. pm was predominantly expressed in the outer and inner epithelial cells of the ventral sections of folds of and mf respectively. pm was found to be expressed sub-cutaneously in the if and mf, specifically appearing interspersed among these regions. cluster d cluster d is represented by ests almost exclusively expressed in the of mantle region. ests were sequenced resolving as unique sequences including contigs and nine singletons. approximately half of these ests show significant sequence homologies, the majority of which align with the family of lysine-rich matrix proteins. additional matches include tyrosinase and thermoacin. alignment of krmp deduced amino acid sequences with existing protein family members showed these ests were significantly divergent from p. fucata, p. margaritifera and p. maxima krmp's particularly by a general absence of the c-terminal gly/tyr region. of the cluster d homologs only pm did not align with all of the cysteine residues present in the basic region. local spatial expression of six cluster d ests was mapped to the mantle. pm pm and pm had similar patterns of expression, detected on the inner epithelium of the outer fold extending the length of the fold. conversely, pm is expressed only in the proximal most inner epithelial cells of the outer fold, notably absent in expression of the three preceding ests. pm and pm indicate a further difference in local expression, observed midway along the inner epithelium of the outer fold. cluster e cluster e consists of ests showing high levels of expression in of, no difference to low expression in vm and very low expression in all other conditions in comparison to the control condition. ests were sequenced revealing unique sequences, of which are contigs and singletons. sequence analysis shows of these ests have significant similarities to shematrin and krmp isoforms. other sequence similarities include a coat protein, mantle protein and articulin. the deduced amino acid sequence for the latter two was analyzed for signal peptides and both indicated likely signal peptide sequences. alignment of krmp deduced amino acid sequences from cluster e with existing protein family members showed these ests all conformed to the typical protein primary structure, particularly with the signal peptide region, basic region and the gly/tyr region. additionally the positions of all six cysteine residues were conserved. in situ hybridization indicates spatial expression for five of the cluster's ests. pm pm pm and pm were detected in the mantle outer epithelium extending from the distil region of the of into the vm zone after which expression is abruptly absent towards the dm region. notably, the directly adjacent epithelium is likewise marked by expression of three cluster a ests showing a precise border of expression between the ests. pm has a similar pattern of expression to the four other cluster e ests, however, it is additionally expressed in the inner epithelium of the outer fold. discussion the molluscan mantle is a thin tissue from which proteins are secreted into the extrapallial fluid; these proteins dictate the animals shell construction and microstructure. as a conserved organ involved in shell formation throughout mollusks, the mantle is an excellent foundation from which to study biomineralization. in this study a p. maxima mantle tissue-specific cdna microarray has been generated termed pmaxarray, comprising cdna clones derived from the mantle tissue of several p. maxima individuals. this tool has provided significant power to interrogate the role of proteins in shell formation. microarray analysis has spatially mapped the expression of a number of known and unknown ests with reference to specific mantle zones. ests present on pmaxarray were expressed as significantly different to the control condition and approximately one third of those were sequenced and aligned resolving a total of unique ests. the majority of those sequences could not be annotated via the genbank database as no molluscan genome has yet been sequenced, let alone functionally annotated. other non-model organisms also report a high proportion of unannotated genes. as such, where sequence homologies are absent, functional significance of ests identified in this study are interpreted with reference to their pattern of expression and the relevance this bears to mantle associated responsibilities. five major expression profiles were observed among the mantle zones indicative of specialized molecular functions and ests clustering in each of these profiles will be discussed within these groupings. cluster a the spatial expression profile in cluster a suggests a role associated with the nacreous shell formation of p. maxima. sudo et al. along with others support this supposition noting a close spatial link between transcript expression in mantle zones and shell microstructure inclusion. of particular interest within this cluster are pm and pm as both ests possess two tandem kuntiz/bovine pancreatic trypsin domains. pm is a significant match to papilin; an extracellular matrix glycoprotein occurring widely from nematodes to humans and known to contain several kuntiz domains. likewise the presence of kuntiz domains is expected for pm which shares sequence similarity with a pancreatic trypsin inhibitor domain protein. kuntiz bpt domains are generally regarded as serine protease inhibitors involved in clotting and tissue remodeling. similarly shell formation is known to involve a number of inhibitory components limiting mineralization. proteoglycans are one such component, essential to shell formation yet intrinsically inhibit biomineralization. the protease inhibiting domains of pm and pm may act to maintain the viscous silk gel detailed by adaddi et al. as necessary for nacre formation. pm is expressed in the dm epithelial cells overlying the nacre microstructure in conjunction with the immediate cessation of expression toward the vm zone and prismatic microstructure. taken together, tissue localization and sequence homologies suggest that pm and possibly pm are glycoproteins with inhibitory protease activity specific for nacre formation. ests pm and pm are unannotated however in situ hybridization demonstrated a very specific localization to the epithelium of the dm zone, as already described for pm this same distribution of expression is also demonstrated for n gene and msi gene both of which code for nacre matrix proteins. the exclusive expression of these two novel ests, pm and pm suggest a role in nacre formation which along with pm are the only reported cases of in situ hybridization localizing ests to the dm zone since sudo et al. reported msi gene expression. cluster b this cluster is the largest and most ubiquitous of all the expression profiles identified in this study. ests in cluster b display similar expression values across a number of seemingly unrelated mantle tissues. the anatomy and function of the mantle organ is generally considered as follows: of is secretory, mf is sensory, if is muscular, vm and dm are secretory. therefore in the perceived absence of a specialized function uniting these tissues, cluster b most likely represents ests involved in general cellular maintenance and regulation rather than shell formation. this proposition is supported by the identification of a number of'housekeeping' genes not seen in any of the other clusters including cytochrome c oxidase, glutathione peroxidase, ezrin/radixin/moesin binding proteins and ribosomal proteins. cluster c this cluster is the smallest and contains ests which showed no significant similarities with any reported protein or nucleotide sequences. the in situ hybridization results for ests pm and pm showed association with the periostracal groove in which the outer epithelium of the mf is included. the main function of the periostracal groove is to secrete a glycocalyx coating forming the periostracum. a glycocalyx is a network of polysaccharides that project from cellular surfaces usually secreted by epithelial cells for a range of adhesion functions. the distil expression of pm in the mf outer epithelium indicates this est may code for a glycoprotein incorporated in the mature stages of the outer glycocalyx coating. similarly, expression of pm in the proximal epithelial cells of the periostracal groove may also code a protein involved in glycocalytic coatings and the stepwise construction of the periostracum. est pm has a peculiar in situ expression pattern in that the transcript is found below the epidermal layer, interspersed throughout the inner region of the mf and the outer region of the if. bivalves expose these mantle folds to the external environment. chemoreceptors, photoreceptors and mechanoreceptors are all usually present in the epidermal layer of these folds in order to elicit closure of the shell valves in response to negative stimulus. considering pm is expressed sub-dermally, it is less likely that this est has a direct sensory role but rather associated with what appears to be nerve fibres, possibly involved in a signal transduction cascade. cluster d the expression profile of cluster d ests suggests an exclusive role of these genes in the of tissue, specifically concerned with the inner epithelia. this epithelium forms the bottom half of the periostracal groove, which is a highly dynamic tissue responsible for formation, maturation and extrusion of the complex periostracum layer. the proteinaceous layer functions to seal the extrapallial space and protect the shell from dissolution as well as serve as an initial matrix for mineralization. in situ localization to the inner epithelium of the of tissue signify a periostracum-related function. neuromacin and theromacin are a family of antimicrobial peptides known to occur in a number of invertebrates. these peptides are part of a immediate immune response characterized predominantly by cationic and hydrophobic amino acids. est pm shows a significant sequence similarity to the gene encoding these antimicrobial peptides and its in situ expression profile maps it to where the internal periostracum is formed. cationic and hydrophobic properties of these peptides are synonymous with the characteristics of the periostracum and water insoluble matrix of shells. specifically, a scenario for pm may be that poly-anionic glycoproteins bind to cationic peptides in the periostracum, effectively anchoring the hydrophilic macromolecules to the hydrophobic wism. this in turn facilitates active nucleation sites by which microstructure mineralization occurs. lysine-rich matrix protein is a family of proteins seemingly specific to mollusks and shell formation of the prismatic design. cluster d includes seven ests significantly similar to the krmp gene class. zhang et al. first described these proteins noting predominate expression in the inner epithelial cells of the of and outer epithelium of the mantle edge region. the deduced amino acid sequence includes an n-terminal signal peptide, a lysine-rich basic region potentially interacting with acidic proteins or co32-, and a glycine/tyrosine-rich region considered involved in protein cross-linking via the quinone-tanning process. the expression in the mantle edge region and similarities among the signal peptide of other prismatic shell matrix proteins lead zhang et al. to assign a putative prismatic microstructural function to the krmp family in p. fucata. however unlike zhang's et al. observations of dual expression in the periostracal groove and the prismatic mantle region, these p. maxima ests are exclusively expressed in the of, a number of which are localized by in situ hybridization to the inner epithelium of the fold, representing the lower half of the periostracal groove. this deviation from zhang's et al. original characterization is potentially explained by sequence analysis. the newly identified krmp members appear to be concatenated versions of p. fucata krmp possessing only the signal peptide and lysine rich region typical of the class. in many of the ests the c-terminal region is significantly reduced and/or replaced with serine and aspartic acid residues. the absence of the glycine/tyrosine-rich region suggests that the predicted proteins coded by these ests are not quinone-tanned. pm is the most divergent of the krmp members and displays a different local expression being present along the middle region of the of inner epithelium, suggesting a different function, specific to periostracum formation. unclear however, is whether these ests are a novel sub-family of krmp or a species specific evolutionary adaption of krmp in p. maxima. the conservation of the lysine-rich region confers the positive charge required to attract and bind acidic glycoproteins necessary for nucleation while expression in the periostracal groove suggests they are incorporated in the periostracum. in summary, the seven krmp homologs in cluster d are considered specifically adapted for periostracal formation in p. maxima. pm is a novel transcript expressed in the inner epithelia cells of the of at the base of the periostracal groove. periostracum development begins with the formation of the pellicle providing a framework on which coatings of the glycocalyx thicken and develop the periostracum. in bivalves, the pellicle typically originates from a row of basal cells at the bottom of the periostracal groove. the spatial expression of pm closely matches the area described for pellicle formation and its deduced sequence is dominated by tyrosine and glycine, typical of a quinone-tanned protein. as the pellicle provides the structural backbone on which ensuing glycocalyx coatings mature the periostracum, its formation would be largely concerned with the hardening of the structure. cluster e ests in this cluster are characterised by expression primarily in the of and vm tissues. the outer epithelia of both these tissues are considered homogenous in function, attributed to prismatic shell formation. in situ hybridization of several cluster e ests confirms dual expression in the outer epithelia of the of and vm, consistent with involvement in prismatic shell formation. ests pm pm pm pm pm pm and pm represent the shematrin protein family. while p. maxima isoforms for shematrin have already been reported all the ests presented here, except pm are novel isoforms. shematrin is a family of glycine-rich shell matrix proteins known to be present in the prismatic microstructure of several pearl oyster species. yano et al. suggests shematrins are framework proteins facilitating calcification of the prismatic microstructure. this investigation maps shematrin isoform pm via in situ hybridization to the outer epithelium from the tip of the of to the vm/dm mantle border, parallel with the prismatic/nacreous shell border, adding to the characterization of the shematrin family in relation to the prismatic microstructure. ests pm and pm show significant sequence homologies with mantle protein and alveolin respectively, and both appear to be related to cytoskeletal protein family articulin. articulins are part of the membrane skeleton of eukaryotic cells stabilizing plasma membranes. it is suggested that ests pm and pm function as plateins, a new family of articulins described by kloetzel et al.. plateins contain modified articulin core domains typical of secreted structural proteins as well as a novel predicted signal peptides detected in intra-alveolar sacs, an extracellular environment. likewise, pm and pm also contain predicted signal peptides indicating a secretory pathway and est pm has been detected by in situ hybridization specifically to the epithelial cells of both the lower periostracal groove and mantle outer epithelium, in contact with the prismatic shell. these tissues are noted for their secretions reinforcing the secretory pathway of pm and pm in summary, gene sequence homology of pm and pm with membrane skeleton proteins, coupled with their differential expression to secretory tissues and detection of signal peptides suggest these ests are putative members of a new articulin family, differentiated by extracellular function. these ests may encode framework proteins involved in the formation of the prismatic microstructure in p. maxima shell. a functional link between the periostracal groove secretions and prismatic shell formation has previously been suspected based on a structural continuity between the outer periostracum and interprismatic matrices. zhang et al. demonstrated shell matrix protein krmp as expressed in both secretory tissues. however, the presence of ten krmp related ests found to be expressed specifically in the outer epithelia of the ventral mantle zone three of which were confirmed with in situ hybridisation represent a break from that observed by zhang et al.. the krmp family has already been discussed in reference to seven est homologs found to be specific to the periostracal groove of p. maxima. the observation of these two separate expression patterns for krmp related ests in the periostrcum and prismatic shell formation mantle regions differs from reports in the related pearl oyster p. fucata. in contrast, where krmp homologues appear to perform dual periostracum/prismatic microstructure roles; p. maxima appear to use additional krmp homologs to accomplish the periostracum related task. this corroborates jackson's et al. supposition that the'secretome' is a rapidly evolving collection of proteins capable of significant molecular differences in building molluscan shells. in summary, cluster e contains specific krmp isoforms potentially involved in the prismatic microstructure formation of the p. maxima shell. a functional linkage between the periostracum and prismatic shell formation is probable, however the mode by which this occurs is highly adaptable, and unlikely to be conserved among species.  this investigation has mapped the spatial distribution for over ests present on pmaxarray with reference to specific locations of the mantle. five major expression profiles were distinguishable from these differentially expressed ests relating to the examined mantle divisions: dorsal mantle, ventral mantle, inner fold, middle fold and outer fold. these expression profile clusters have indicated at least five unique functioning zones in the mantle. three of these zones are considered involved in shell related activities including cluster a's role in nacre formation, cluster d's link to periostracum formation and cluster e's implication in calcitic prismatic microstructure formation. a number of known and novel ests have been identified from these clusters. microarray differential expression, in situ expression localization and comparative sequence analysis have begun the task of characterizing novel ests identified herein, in addition to further elucidating the functions of previously reported biomineralization related genes. the microarray approach utilized here has alleviated many of the past difficulties plaguing the molluscan biomineralization discipline, however, this method and its' outcomes is in no way seen as a standalone conclusion. rather, microarray analysis is intended to spearhead preliminary investigations of shell formation targeting ests for subsequent in-depth characterization including protein isolation and activity studies. 
the mantle is a conserved organ involved in shell formation throughout molluscs. in situ hybridization validated and further localized the expression for a subset of these ests. this investigation has mapped the spatial distribution for over ests present on pmaxarray with reference to specific locations of the mantle. expression profile clusters have indicated at least five unique functioning zones in the mantle. this study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization. pmaxarray consists of expressed sequence tags originating from mantle tissue. the organic component incorporated within the shell is said to dictate this architecture. this microarray was used to analyze the spatial expression of ests throughout the mantle organ.
 biomineralization is a process encompassing all mineral containing tissues produced within an organism. one of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. the organic component incorporated within the shell is said to dictate this architecture. however general understanding of how this process is achieved remains ambiguous. the mantle is a conserved organ involved in shell formation throughout molluscs. specifically the mantle is thought to be responsible for secreting the protein component of the shell. this study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization.  a microarray platform was custom generated from the pearl oyster pinctada maxima. pmaxarray consists of expressed sequence tags originating from mantle tissue. this microarray was used to analyze the spatial expression of ests throughout the mantle organ. the mantle was dissected into five discrete regions and analyzed for differential gene expression with pmaxarray. over ests were determined to be differentially expressed among the tissue sections, identifying five major expression regions. in situ hybridization validated and further localized the expression for a subset of these ests. comparative sequence similarity analysis of these ests revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes.  this investigation has mapped the spatial distribution for over ests present on pmaxarray with reference to specific locations of the mantle. expression profile clusters have indicated at least five unique functioning zones in the mantle. three of these zones are likely involved in shell related activities including formation of nacre, periostracum and calcitic prismatic microstructure. a number of novel and known transcripts have been identified from these clusters. the development of pmaxarray, and the spatial map of its ests expression in the mantle has begun characterizing the molecular mechanisms linking the organics and inorganics of the molluscan shell. 