Microarray analysis identifies candidate genes for key roles in coral development
© Grasso et al; licensee BioMed Central Ltd. 2008
Received: 15 August 2008
Accepted: 14 November 2008
Published: 14 November 2008
Anthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development – when skeletogenesis is initiated, and symbionts are first acquired.
Of 5081 unique peptide coding genes, 1084 were differentially expressed (P ≤ 0.05) in comparisons between four different stages of coral development, spanning key developmental transitions. Genes of likely relevance to the processes of settlement, metamorphosis, calcification and interaction with symbionts were characterised further and their spatial expression patterns investigated using whole-mount in situ hybridization.
This study is the first large-scale investigation of developmental gene expression for any cnidarian, and has provided candidate genes for key roles in many aspects of coral biology, including calcification, metamorphosis and symbiont uptake. One surprising finding is that some of these genes have clear counterparts in higher animals but are not present in the closely-related sea anemone Nematostella. Secondly, coral-specific processes (i.e. traits which distinguish corals from their close relatives) may be analogous to similar processes in distantly related organisms. This first large-scale application of microarray analysis demonstrates the potential of this approach for investigating many aspects of coral biology, including the effects of stress and disease.
Cnidarians are the simplest animals at the tissue level of organization, and are of particular importance in terms of understanding the evolution of metazoan genomes and developmental mechanisms. Members of the basal cnidarian Class Anthozoa, which includes the sea anemone Nematostella and the coral Acropora, have proved to be surprisingly complex and vertebrate-like in terms of gene repertoire [1–3], and are therefore of particular interest. Scleractinian corals are also of fundamental ecological significance in tropical and sub-tropical shallow marine environments as the most important components of coral reefs. Surprisingly, both the general molecular principles of cnidarian development and many aspects of the functional biology of corals are only poorly understood. Whole genome sequences are now available for both the textbook cnidarian Hydra magnipapillata and the sea anemone Nematostella vectensis. However, corals are distinguished from Nematostella and other cnidarians by the presence of an extensive skeleton composed of calcium carbonate in the form of aragonite. The ability to carry out calcification on a reef-building scale is enabled by the obligate symbiosis between scleractinians and photosynthetic dinoflagellates in the genus Symbiodinium.
Expressed Sequence Tag (EST) projects carried out on Acropora millepora and Nematostella vectensis have provided insights into the evolution of animal genomes [2, 3]. The latter publication, based on ca 5800 unigenes from the coral Acropora and 10,500 unigenes from the sea anemone Nematostella, revealed the surprisingly rich genetic repertoire of these morphologically simple animals. The genomes of anthozoan cnidarians encode not only homologs of numerous genes known from higher animals (including many that had been assumed to be 'vertebrate-specific'), but also a significant number of genes not known from any other animals ('non-metazoan' genes; ). This picture of genetic complexity has been augmented by the recently completed whole genome sequence (WGS) of Nematostella vectensis , for which approximately 165,000 ESTs are now available. Similar resources exist for Hydra magnipapillata [4, 5] although the much larger genome size of this organism has consequences for the completeness of the assembly. Both of these other cnidarians not only lack a calcified skeleton, but also do not enter symbioses. Entry into a symbiosis can have profound effects on gene expression patterns, with changes to immune function, and to many metabolic functions including CO2 cycling, nutrient cycling, metabolite transfer and reactive oxygen quenching [6, 7]. The phylogenetic position of Nematostella makes this a particularly useful comparator because both Nematostella and Acropora are classified into the anthozoan subclass Hexacorallia (Zoantharia).
The identification and composition of synexpression clusters
Of the 5081 unigenes giving rise to predicted peptides that are represented on the arrays, a total of 1084 unigenes (2462 spots) were found to be up- or down-regulated (P = < 0.05) between any two consecutive stages. The microarray results were validated by virtual northern blots. The results for eight arbitrarily chosen clones are shown in Additional File 1; in each case the observed expression pattern corresponds with the microarray results.
Percentage of unigenes within expression clusters in each functional category.
Functions that many kinds of cells use
Transportation and binding proteins for ions and other small molecules
RNA processing, polymerising, splicing, and binding proteins and enzymes
Cell replication; histones, cyclins and allied kinases DNA polymerases, topoisomerases, DNA modification
Cytoskeleton and membrane proteins
Protein synthesis cofactors, tRNA synthetase, ribosomal proteins
Intermediary synthesis and catabolism enzymes
Stress response, detoxification and cell defence proteins (GFPs in here)
Protein degradation and processing, proteases, apoptosis related
Transportation and binding proteins for proteins and other macromolecules
Signalling receptors, including cytokine and hormone receptors and signalling ligands
Intracellular signal transduction pathway molecules including kinases and signal intermediates
Extracellular matrix proteins and cell adhesion
Transcription factors and other gene regulatory proteins
Sequence-specific DNA binding proteins
Non-DNA-binding proteins with positive or negative regulatory roles
Chromatin proteins other than AIII with regulatory function
Not enough information to classify
Approximately 10% of cluster I (genes down-regulated after embryogenesis) consists of genes in functional category AIII, genes involved in cell replication , probably reflecting the extent to which cell proliferation dominates early embryogenesis. 29.1% of cluster VI (genes up-regulated after embryogenesis) were classified into functional category AV: protein synthesis cofactors, tRNA synthetase, and ribosomal proteins, whereas all other clusters contained very few genes in this category. 27.2% of cluster III (genes up-regulated in planula and primary polyp) were classified into AVI: Intermediary synthesis and catabolism enzymes; this is significantly more than in any other cluster.
Planula larvae are primarily dependent upon stored lipid, whereas the energy requirements of adult corals are often largely met by photosynthetic products exported from their dinoflagellate symbionts. These physiological changes are reflected by shifts in the coral transcriptome. For example, lipases are highly represented amongst the planula ESTs, but strongly down-regulated thereafter. Also of note are dramatic differences in representation of genes in category BII (intracellular signalling) between cluster I (10.5%) and cluster II (0.9%), and in genes in category BIII (extracellular matrix and cell adhesion) between cluster I (0.9%) and cluster II (14.4%). These shifts, and the sharp spike in expression of ECM and cell adhesion genes, are associated with the transition from an undifferentiated proliferative stage and the emergence of differentiated cell types.
Lectins related to sea cucumber CEL-III are strongly expressed during metamorphosis in Acropora
Whilst our understanding of metamorphoses in marine invertebrates is very incomplete, in several cases key molecules implicated in the underlying processes have been identified, and these include lectins [11, 12]. Studies of coral settlement and metamorphosis have indicated that the inductive morphogenetic cue is exogenous/environmental and, whilst the exact structure of the metamorphosis inducing morphogen remains elusive, lipopolysaccharides are prime candidates  suggesting that cell surface recognition by coral larvae may be mediated by lectins. Lectins are therefore of particular interest as candidates for roles in settlement and metamorphosis as well as in other developmental processes including the uptake of Symbiodinium (see below). Indeed, a mannose-binding lectin has recently been described from A. millepora which binds both bacteria and Symbiodinium and may therefore have roles in both immunity and symbiosis 
Other lectins in nematocyst differentiation
Whereas the five proteins discussed above all contain galactose-binding lectin domains, the last of these six differentially expressed proteins (A043-D8) contains a C type lectin domain. Moreover, whilst a signal peptide is present, A043-D8 does not contain a mini-collagen domain. As in the case of A044-C2 and A032-H1, expression of A043-D8 appears in scattered ectodermal cells as the planula is developing (Figure 4C), although the distribution of these cells appears to differ somewhat from those shown in Figures 4A and 4B. Histological sections fail to reveal any evidence of expression in obvious cnidoblasts.
A potential mediator of symbiont uptake
Acropora species acquire symbionts directly from the environment and although uptake in the wild has only been observed a few days after settlement , larvae of Acropora [22, 23] and a number of other coral species [24, 25] are competent to take up symbionts. However, the exact time and mode of uptake remain to be established. Lectin/polysaccharide signalling is used in many systems as a mechanism for symbiotic recognition , and has been implicated in the establishment of symbiosis in various marine invertebrates (e.g). In the octocoral Sinularia lochmodes a lectin is involved in the conversion of Symbiodinium from a motile to the non-motile form required for symbiosis [28, 29]. Also, masking cell surface glycoproteins with lectins decreases the rate of Symbiodinium infection of the sea anemone Aiptasia pulchella  and enzymatic digestion of cell surface glycans prevents Symbiodinium recognition and the establishment of symbiosis in the coral Fungia scutaria . Although Smith  has argued otherwise, these more recent experiments point to a possible role for lectins in symbiont recognition/uptake in corals.
The one differentially regulated coral protein containing a lectin domain and with an expression pattern consistent with a role in symbiont uptake is A043-H7, introduced in the previous section as a mini-collagen-like protein. Unlike those in the proteins with similar domain architecture (A044-C2 and A032-H1), the mini-collagen domain of A043-H7 is interrupted, (which may have structural consequences) and the gene's expression pattern is completely different. The expression pattern of A043-H7 immediately prior to settlement (Figure 4D) is consistent with a role in symbiont uptake since, in contrast to many other cnidarians, the endoderm of the Acropora planula is tightly packed with yolk cells and frequently is hollow only immediately adjacent to the oral pore. As the endoderm is the most common route of cnidarian infection (see Discussion), the endodermal region immediately adjacent to the oral pore (i.e. the zone of A043-H7 expression) is a probable site of symbiont infection in the case of Acropora larvae. Confocal microscopy was recently used to demonstrate the binding of an A millepora mannose-binding lectin, which was not among our ESTs, to Symbiodinium, but its localization within the coral remains unknown .
Conserved and novel genes with roles in calcification
The molecular basis of calcification in corals is not well understood; the process involves the deposition of calcium carbonate in an area defined by an organic matrix  and is initiated immediately after settlement and prior to metamorphosis . Initially a flattened plate is laid down, upon which are deposited radiating vertical walls corresponding to the septa which give the polyp its six-fold symmetry. Initial calcification can, and in the case of Acropora millepora does, happen in the absence of Symbiodinium, but the massive calcification of larger colonies is dependent on the photosynthetic symbiont through interacting cycles of respiration, photosynthesis and calcification. Although many animal phyla include calcifying representatives, few components of the calcification machinery appear to be conserved between different lineages. For example, in the scleractinian Galaxea fascicularis, one of the most prevalent protein components of the calcifying organic matrix is galaxin , which appears to be unique to corals. One exception to this heterogeneity is the alpha type carbonic anhydrase family, which has been implicated in CaCO3 deposition from sponges to vertebrates . Most animals have multiple carbonic anhydrases; distinct subfamilies are recognised [37, 38] each of which is widely distributed phylogenetically, but in addition some calcifying animals have atypical carbonic anhydrases that may represent lineage specific adaptations to facilitate CaCO3 deposition. For example, nacrein – a soluble organic matrix protein in the nacreous layer of pearl oysters – contains a carbonic anhydrase domain that is split by a Gly-X-Asn repeat domain  which may have a regulatory role . In a directly relevant example, Tambutte et al.  have recently demonstrated that active carbonic anhydrase is present in the organic matrix of Tubastrea aurea and plays a direct role in the calcification process. In another recent paper Moya et al  have cloned, sequenced and immunolocalized a previously undescribed CA from the coral Stylophora pistillata. It is localized in the calicoblast ectoderm, from which it is secreted, and has a CA catalytic function. In terms of understanding the bases of skeleton deposition, carbonic anhydrases are therefore of particular interest.
The second carbonic anhydrase, A030-E11, was expressed in the oral half of the metamorphosing larva (Figure 5B1) and the entire ectoderm of the primary polyp, except the aboral disc (Figure 5B2) and the oral pore (data not shown). In older polyps this carbonic anhydrase is expressed in the septa, where calcification is occurring to form adult structures (Figure 5B4).
Expression analysis reveals that some "unique" coral genes have spatial expression patterns strikingly like that of carbonic anhydrase C007-E7, i.e. consistent with roles in the initiation of calcification. Figure 5C1 and 5D1 show genes with expression at the aboral end of the metamorphosing larva and in the basal plate of the metamorphosing larva, respectively. However, differences are apparent slightly later – C012-D9 expression becomes restricted to an aboral ring, and then appears to be switched off (Figure 5C3, C4). Whilst B036-D5 expression also appears to be down-regulated in the basal plate, transcripts can be visualised in the mesenteries (Figure 5D4) at a stage when C012-D9 transcripts are undetectable. Neither of these genes encodes known domains or could be functionally classified (using BlastP, Phi-Blast and InterPro Scan). However, their expression patterns are consistent with roles in early calcification.
A synexpression cluster of coral-specific genes
Discussion and conclusion
Validation of the approach and methodology
Virtual northern blots for eight genes were consistent with the microarray results, thus confirming them. In addition, and consistent with the microarray results being accurate, several mini-collagen-like proteins were upregulated in the planula. Mini-collagens have thus far only been described from nematocysts, cnidarian-specific structures which first appear at the planula stage in A. millepora (Additional Files 2, 3).
Taxonomic and functional breakdown of the genes
The composition of the EST set used in these microarray experiments has previously been considered specifically with respect to the complement of developmental signalling pathway components [2, 3], but this paper is the first to examine broad scale changes in gene expression during development for any cnidarian. The use of different criteria and thresholds, and the ever-changing baseline provided by the databases, complicates making direct comparisons with other developmental studies. For example, although a recent paper on developmental gene expression in the ascidian, Molgula  addressed many of the same questions, it focussed specifically on highly expressed genes (i.e. only those accounting for more than 0.2% of the total number of ESTs) so it is not possible to interpret apparent differences, such as in the percentage of unique genes. In terms of developmental changes, it is particularly noteworthy that the percentage of "core" genes (59%; i.e. those genes shared with members of other kingdoms as well as other animals) is highest in cluster VI and that the percentage of unique genes (12%) is lowest in cluster I. Presumably these figures reflect shifts from common cellular pathways during very early development to greater cellular and molecular diversification later. As in many other animals, the early development of Acropora appears to involve many stored maternal mRNAs. The composition of the maternal mRNA pool is complex, consisting principally of low abundance transcripts including those involved with cell division, RNA metabolism, and regulation of gene transcription (L McFarlane, unpublished). Among genes of particular interest, H2A.Z and H1, histones with roles in priming chromatin for developmental gene expression  in a variety of other systems, are highly represented in the prawn chip ESTs and strongly down regulated thereafter, as are cyclins A and B3. In Drosophila and Xenopus, maternal cyclin transcript levels are initially very high and then decrease dramatically after the onset of gastrulation [50–52]. Acropora may therefore follow this pattern of abundant maternal cyclin transcripts that drive very rapid cell proliferation early in embryogenesis, followed by lower transcript levels with the onset of slower developmentally regulated cell cycles. Cell cycle transcripts such as cyclin A and B were also abundant among the cleaving embryo ESTs of Molgula tectiformis  and in pre-gastrulation stages of Xenopus  and Drosophila .
Lectin domain proteins are potentially involved in diverse processes
There are a number of precedents for the involvement of lectin-containing proteins in metamorphosis. Lectins are differentially expressed at metamorphosis in two ascidians, Herdmania curvata  and Boltenia villosa [11, 12]. In Boltenia, four lectins and two key lectin pathway genes are up-regulated in the larva or the newly settled adult . The lectin induced complement pathway, which is initiated by a mannose-binding lectin, is important in Boltenia for the recognition of those bacteria which induce metamorphosis and tissue remodeling . It is possible that the lectins up-regulated at metamorphosis in Acropora have an analogous role in activating tissue remodelling. Consistent with this idea, a possible complement effector, the perforin domain protein apextrin, is expressed in a strikingly similar pattern to those of the CELIII lectins during metamorphosis in Acropora .
Lectin domain-containing proteins also potentially function in the recognition of symbionts by corals. Lectin/polysaccharide signalling is used in many systems as a mechanism for symbiont recognition, the most widely known example being the recognition of sugars on the surface of nitrogen-fixing bacteria by the lectins of their host legume during the establishment of their symbiosis. Symbiodinium in scleractinian corals reside in the endoderm, and two mechanisms of entry have been described in those corals that acquire them from the environment. The first is directly into the endoderm via the oral pore after it is formed 3–5 days post fertilization in association with feeding, as was demonstrated in the coral Fungia scutaria  and the anemone Anthopleura elegantissima . The second, also demonstrated in Fungia , is that they can enter via the epithelium pre- or post-gastrulation. Those which have entered by the ectoderm are then shunted to the endoderm where they are retained . Elegant studies in the latter half of the last century described the cell biology of symbiont uptake and retention, for example , and it has recently been established that members of the Rab family of proteins are involved in determining whether symbionts are digested or retained [59–61]. Symbiodinium are not transmitted through the eggs of A. millepora, and while planulae can be infected  this may only occur after the oral pore has opened shortly before settlement ( and AH Baird, pers comm.) although the timing and mode of symbiont uptake remain to be firmly established. The limited available field observations indicate that infection normally does not occur until a few days after settlement in A. millepora . These observations point to the endoderm as the likeliest point of Symbiodinium uptake, but do not rule out a possible role for the ectoderm. There is clear evidence from a number of cnidarian species of selective maintenance of the most "appropriate" clade of symbiont, while conclusions on specificity of uptake and its possible mechanisms are equivocal, perhaps due to interspecific variabity. Nevertheless, there is evidence that lectins function in symbiont recognition, as previously summarised, and these molecules therefore remain obvious candidates for roles in symbiont uptake and maintenance by Acropora.
Genes involved in calcification
Two alpha type carbonic anhydrases are expressed in patterns that are consistent with roles in calcification. However, these genes are not restricted to heavily calcifying cnidarians, as both have probable orthologs in sea anemones and other cnidarians. This is perhaps not surprising, as carbonic anhydrases are involved in pH and CO2/bicarbonate homeostasis in all organisms, and the ability to deposit some form of calcified exoskeleton is taxonomically widespread among cnidarians. For example, polyps of the hydrozoan Hydractinia symbiolongicarpus secrete a mat of calcium carbonate, in the form of aragonite, on their substrate . Two membrane-associated carbonic anhydrases have been described from planulae of the coral Fungia scutaria, but they are short and missing amino acids thought to be necessary for CA activity, although the authors hypothesize that they could play a role in the onset of calcification at the time of settlement . The first Acropora carbonic anhydrase, C007-E7, matches most strongly to vertebrate IV/XV-type carbonic anhydrases, and consistent with this, is predicted to be GPI anchored. C007-E7 has likely orthologs in both Nematostella and Hydra. The second carbonic anhydrase, A30-E11, is a I/II-type carbonic anhydrase and is likely to be the Acropora ortholog of a protein identified in the sea anemone, Anthopleura elegantissima (29.8% identity and 43.1% similarity) as a "symbiosis gene" – it is strongly up-regulated when this facultatively symbiotic anemone takes up endosymbionts . However, clear counterparts of this soluble cytosolic type carbonic anhydrase are present in both Nematostella and Hydra magnipapillata, neither of which harbours symbionts. Whereas the two carbonic anhydrase genes are not restricted to calcifying cnidarians, a number of other coral genes with similar expression patterns have no apparent sea anemone or Hydra homologs. One possible scenario is that many of the genes involved in calcium processing will have a widespread distribution while some of those involved in secreting the organic matrix may be more specific, as in the case of galaxin. It will be particularly interesting to see whether different gene repertoires play a significant part in the determining the dramatic differences in colony morphology that are characteristic of the various corals or whether this is due mainly to deploying the same genes in different ways.
"Coral-specific" processes as variations on known themes
One conclusion that follows from the work presented above is that many of the molecules involved in "coral-specific" processes such as metamorphosis and calcification are not coral specific – genes whose expression patterns imply key roles in implementing metamorphosis, such as the lectins A036-E7 and A049-E7 and apextrin  have homologs in other animals even though they are not present in Nematostella. Both of the carbonic anhydrases implicated in calcification also have clear counterparts in non-calcifying cnidarians. A second conclusion is that processes central to coral biology, such as symbiont recognition, may have analogous biochemical bases in phylogenetically distant systems. Lectins function in symbiont recognition in the legume-Rhizobium system; this analogy may be useful in understanding how specificity might be achieved in the coral/dinoflagellate symbiosis and in exploring the roles of the candidate molecules identified here. As in ascidians, metamorphosis in Acropora involves activation of an innate immune response, as both lectins and the perforin domain protein apextrin are strongly and specifically expressed at this time. Inevitably, other genes implicated in coral-specific processes appear at this stage to be taxon-restricted, but it is unclear to what extent this simply reflects the limited number and range of animals for which whole genome data are yet available. Genes that are today considered "coral-specific" may actually be more widely distributed; the number of genes considered vertebrate-specific shrinks with the publication of each additional animal whole genome sequence. Moreover, genes with no clear homologs may simply be old genes that have evolved beyond recognition.
One promising approach arises from the prediction that genes involved in "coral-specific" processes such as symbiont recognition are under positive selection. With the imminent availability of large EST datasets for several corals, a combination of in silico and in situ approaches should identify these genes and build on the pioneering study reported here.
The microarrays used in this experiment consisted of 13,392 spots derived from 12,240 cDNA clones (1,152 clones are represented more than once) and 432 spots representing positive and negative controls. The cDNA clones spotted onto the array were randomly selected from cDNA libraries that had been constructed in Lambda ZAP (Stratagene), and include 3456 clones from the prawnchip developmental stage, 4608 clones from the planula larva stage , and 4128 clones from the primary polyp. All of the material used for making the libraries came from Nelly Bay, Magnetic Island, Queensland, Australia (19°08'S 146°50'E).
All cDNAs spotted onto the slides were derived from cDNA libraries of the appropriate developmental stages. They were isolated by TempliPhi (GE Life Sciences) on excised clones except for 2,000 postsettlement polyp clones which were PCR amplified directly from individual phage suspensions and 3,012 planula larva cDNAs which were isolated previously 
Generation and spotting of cDNAs
PCR (1× HotMaster Taq Reaction buffer, 0.25 mM each dNTP, 25 pmoles of each of M13 Forward and M13 Reverse primer, 1.25 units of HotMaster Taq Polymerase (Eppendorf) spiked with Pfu (Promega) in a 25 ul reaction) was used to generate DNA for spotting onto microarray slides. Phage suspension was used as template by adding 4 ul to the PCR mix. TempliPhi was used as a template by dipping a pin into the TempliPhi reaction and then into the PCR mix. PCR was carried out in 96-well plates (ABGene) under the following conditions: 94°C for 30 s, 50°C for 30 s and 72°C for 1 min, for 30–35 cycles. PCR products were purified using 96 well Multiscreen plates (Millipore).
Microarrays were generated by spotting the amplified cDNA onto GAPSII slides using a Biorad Chipwriter Pro, and then fixed by UV light exposure (150 mJ) followed by baking at 80°C for 3 hours. All cDNA clones represented on the arrays were sequenced from the 5' direction using standard Sanger (ABI Big Dye) sequencing technology.
After data filtering, ESTs were clustered using CAP3 . The coding potential of the resulting unigenes was analysed using ESTScan . 5081 were predicted to give rise to bonafide proteins, using the criterion of a coding potential of 25 or greater. The EST contigs which had predicted peptides were used to search the Uniprot database using BlastX  with a threshold of e = 1 × 10-5 in order to functionally classify the predicted proteins according to the scheme in .
cDNA for probing arrays was produced from unamplified total RNA which was extracted using TRI Reagent (Ambion) according to the manufacturer's instructions. The quality was assessed using denaturing gel electrophoresis using standard methods . For each hybridized sample, total RNA (80 ug) was reverse transcribed, labelled and hybridised using standard protocols .
Data analysis and verification
Slides were scanned using a GenePix 4200A scanner, and data extracted using Spot . All further analyses were carried out using the limma package  for the R system . Print-tip loess normalisation  was performed on each slide. Quantile normalisation was applied to mean log-intensities in order to make the distributions essentially the same across arrays.
The methodology used for statistical analysis is described in Smyth . The prior probability of differential expression, for each pair of comparisons between stages, was taken as 0.1. The Benjamini and Hochberg method  was used to adjust the sequence-wise p-values, so that a choice of sequences for which the adjusted p-value is at most 0.05 identifies a set of differentially expressed genes in which 5% may be falsely identified as differentially expressed (see Additional File 4 for more detail). Array data have been deposited in the Gene Expression Omnibus (GEO) database (accession number GSE11251).
Results were also verified using M vs A plots, where M = the log ratio of the spot fluorescence intensity values and A = the log of the average spot fluorescence intensity. An example is given in Additional File 5. Spots for which no fluorescence was expected, including salmon sperm DNA, empty vector and primers, plotted near the origin of the MA plot, as expected. Negative controls for differential expression (i.e. spots expected to show hybridization but no differential expression), had an M value of or near to zero, but ranged in fluorescence intensity, also in accordance with expectations. Differentially expressed positive controls (i.e, spots expected to show both hybridization and differential expression between presettlement and postsettlement on the basis of virtual northern results) were positioned on either side of an M value of zero with a range of fluorescence intensities.
Cluster analysis was used to search for clusters of expression profiles in the data. K-means clustering was used to split the genes into 6 groups of differential expression profiles. Clustering was carried out using Cluster 3.0  and the results viewed with Java TreeView . Unigenes with protein coding potential > 25 and p-value < 0.05 in the test for differential expression between temporally sequential developmental stages were removed prior to cluster analysis.
Results for the microarray experiments were verified using "virtual northern blots" which were made using the Clontech SMART cDNA Synthesis Kit, according to the manufacturer's instructions using RNA from the same stages used in the microarray experiment. DNA used to probe the blots was generated by PCR (see section 2.5.4 PCR and spotting of cDNAs), purified using the Qiagen PCR Purification kit according to the manufacturer's instructions, and radiolabelled with 32P-dATP using the Prime-A-Gene Labeling System (Promega) according to the manufacturer's instructions. Hybridization was conducted according to standard protocols  and visualized by exposure to a Phosphorimager (Molecular Dynamics) cassette overnight. Digital images were viewed with Quantity One software.
In order to obtain the entire open reading frame, some unigenes selected for in situ hybridization required further sequencing. This was done either as described for EST sequencing or using 300 ng of plasmid as template. Raw data were viewed and edited with Chromas Lite and sequences were aligned with LaserGene (DNASTAR). cDNA sequences for genes characterized by in situ hybridization have been deposited in GenBank under accession numbers EU863776–EU863788.
In situ hybridization
Templates for riboprobe production were generated by PCR. Riboprobe synthesis and in situ hybridization were performed as reported by . In order to view further histological detail embryos stained in whole mount were embedded in LR White Resin sectioned at various thicknesses and counterstained with Saffranin O.
The authors would like to thank Dr William Leggat for critically reading the manuscript. This work was supported by the Australian Research Council through the Centre for the Molecular Genetics of Development.
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