Transcriptome profiling of the demosponge Amphimedon queenslandica reveals genome-wide events that accompany major life cycle transitions
© Conaco et al.; licensee BioMed Central Ltd. 2012
Received: 9 August 2011
Accepted: 2 April 2012
Published: 30 May 2012
The biphasic life cycle with pelagic larva and benthic adult stages is widely observed in the animal kingdom, including the Porifera (sponges), which are the earliest branching metazoans. The demosponge, Amphimedon queenslandica, undergoes metamorphosis from a free-swimming larva into a sessile adult that bears no morphological resemblance to other animals. While the genome of A. queenslandica contains an extensive repertoire of genes very similar to that of complex bilaterians, it is as yet unclear how this is drawn upon to coordinate changing morphological features and ecological demands throughout the sponge life cycle.
To identify genome-wide events that accompany the pelagobenthic transition in A. queenslandica, we compared global gene expression profiles at four key developmental stages by sequencing the poly(A) transcriptome using SOLiD technology. Large-scale changes in transcription were observed as sponge larvae settled on the benthos and began metamorphosis. Although previous systematics suggest that the only clear homology between Porifera and other animals is in the embryonic and larval stages, we observed extensive use of genes involved in metazoan-associated cellular processes throughout the sponge life cycle. Sponge-specific transcripts are not over-represented in the morphologically distinct adult; rather, many genes that encode typical metazoan features, such as cell adhesion and immunity, are upregulated. Our analysis further revealed gene families with candidate roles in competence, settlement, and metamorphosis in the sponge, including transcription factors, G-protein coupled receptors and other signaling molecules.
This first genome-wide study of the developmental transcriptome in an early branching metazoan highlights major transcriptional events that accompany the pelagobenthic transition and point to a network of regulatory mechanisms that coordinate changes in morphology with shifting environmental demands. Metazoan developmental and structural gene orthologs are well-integrated into the expression profiles at every stage of sponge development, including the adult. The utilization of genes involved in metazoan-associated processes throughout sponge development emphasizes the potential of the genome of the last common ancestor of animals to generate phenotypic complexity.
For many animals, the genome controls the construction and function of two or more discrete life forms, each with its own morphological, physiological, behavioral and ecological characteristics. These life cycles, which typically include larval and adult phases, require the successive unfolding of morphogenetic programs at embryogenesis and metamorphosis. The pelagobenthic life cycle, in which a pelagic, microscopic larva metamorphoses into a large, benthic adult, is one common variation. An inherent feature of the pelagobenthic life cycle is the settlement of the planktonic larva, usually in response to an environmental cue, and its subsequent metamorphosis into the benthic form. This major ecological transition dictates the likelihood of future reproductive success because adults usually are sessile or sedentary, and therefore, larvae must settle at a distance from conspecifics to allow subsequent effective matings. Before settling and metamorphosing, the larvae must first develop the capacity, or competence, to detect and respond to benthic environmental cues typically associated with post-settlement survival and reproduction (e.g. signals from food items and conspecific adults) .
During development, a single gene inventory is drawn upon to implement the vast morphological and ecological changes associated with metamorphosis from one body form into another. Gene expression studies at developmental transitions, therefore, offer a window into the genetic framework that corresponds to distinct morphological features and ecological demands. Genome-wide activity as the animal prepares and moves from the pelagic to the benthic phase has been studied in just a few animals, including ascidians, gastropods and corals [2–8]. These gene expression studies of metamorphosis in animals from diverse phyla have uncovered few conserved features of this essential life cycle transition.
A strong theme has emerged among the growing number of complete genomes from extant representatives of the earliest diverging metazoans, including the sea anemone, Nematostella vectensis, and the hydra, Hydra magnipapillata, the placozoan, Trichoplax adhaerens, and as studied here, the haplosclerid demosponge, A. queenslandica, that despite their relatively simple body plans they possess a gene repertoire that is comparable to that of the bilateria [23–26]. For example, sequencing of the A. queenslandica genome has revealed a surprisingly complex complement of genes important for metazoan multicellularity, including those with known roles in cell adhesion, self and non-self distinction, immunity, controlled proliferation, cell death, and differentiation . However, it is yet unknown which sets of genes are expressed at each developmental stage to coordinate the changing morphological features and ecological demands throughout the sponge life cycle. Whether the adult sponge, which lacks discernable features that readily link it to other animals, utilizes the bilaterian gene set or exploits uniquely Poriferan genes to implement its distinct morphology remains undetermined.
With the rudiments of the animal gene inventory in place at the time the earliest metazoans diverged, a large portion of evolutionary innovation thus has likely arisen by modification of gene regulatory networks . Given the early-branching position of Porifera and their success in widely speciating through diverse eco-niches while maintaining a simple body plan for the past 580 million years , these organisms offer a unique opportunity to explore the origin and evolution of metazoan gene networks and the biphasic lifestyle. Our global study of the A. queenslandica transcriptome highlights the genomic events that accompany the transition from pelagic larvae to the benthic adult stage and point to an intricate network of regulatory mechanisms that coordinate the changes experienced by the sponge during the pelagobenthic transition. This study also reveals gene families with potential roles in competence, settlement, and metamorphosis in the sponge, including transcription factors, G-protein coupled receptors and other signaling molecules.
Deep sequencing of the sponge poly(A) RNA transcriptome
Transcriptional complexity across the A. queenslandica life cycle
Expression of annotated and non-annotated sponge genes
A. queenslandica genes without identifiable homologs in other organisms are also likely to contribute to environmental adaptations, as was reported in the microcrustacean Daphnia pulex, a genome in which more than a third of genes lack detectable homologs in any other available proteome, and are the most responsive genes to ecological challenges . About 36% (10,645 out of 29,915 genes) of A. queenslandica protein coding genes have no identifiable homologs in other species, as determined by alignment to proteins in the UniProt database . 1,536 of these genes without identifiable homologs have recognizable PFAM domains. Expressed sequence tag analysis in two other demosponges, Suberites domuncula and Lubomirskia baicalensis, revealed a similar percentage of genes that could not be assigned to any known function based on homology to other species . The set of non-annotated sequences is likely to include novel ‘sponge-specific’ genes, although some erroneous gene models may also be present . This is supported by the finding that only 28% of non-annotated genes (2,968 out of 10,644 genes) were robustly expressed above the cutoff threshold of 64 reads compared to 55% (10,535 out of 19,271 genes) of annotated genes (Figure 3C). Sponge-specific genes also tend to have lower expression levels than annotated genes, however, they exhibit the same overall patterns of variation across development (Figure 3D, Additional file 3).
Expression trends across development
A limitation of the previous method is that it only detects stage-specific changes that are robust across all life cycle stages and overlooks genes with variable expression patterns. Thus, to identify genes that are differentially expressed at stage transitions, we followed the expression trajectory of every gene by performing pairwise comparisons of their expression between successive stages. Genes were categorized as upregulated, downregulated, or unchanged. From this analysis, we identified 8,219 genes (37.8%) that exhibited greater than four-fold change in expression between any two successive stages, of which 2,178 (26.5%) are sponge-specific (Additional file 6). If the cutoff threshold is lowered to two-fold, the number of differentially expressed genes increases to 11,888 with 2,759 (23%) sponge-specific transcripts ( Additional file 7). At the transition from the precompetent to the competent stage, a total of 491 genes were upregulated at least four-fold while 804 were downregulated. As settled postlarvae began metamorphosis into the juvenile body plan, 1,582 genes were upregulated and 2,204 were downregulated. Maturation from postlarval juvenile to adult was accompanied by the upregulation of 3,848 genes and downregulation of 1,980 genes. It should be noted, however, that because the competent and postlarval samples were sequenced at lesser depth than the other stages, genes expressed at low abundance in these samples may not be detected above the cutoff threshold. Mapped reads in the range of 20 million, as is the case for the postlarval sample, allow a close estimate of expression level for approximately 70% of low abundance genes . Thus, the number of genes that appear to be downregulated at the precompetent-competent and at the competent-postlarva transition, as well as genes that are upregulated at the postlarva-adult transition may be overestimated.
Larval stage expression profiles and determinants of competence
Functionally related A. queenslandica genes were identified by Gene Ontology (GO)  annotation based on their best sequence similarity match to proteins in the UniProt database . GO annotation enrichment analysis was performed on groups of genes that are differentially expressed by greater than four-fold at specific developmental transitions to determine the processes that are up- or downregulated at particular ontogenetic stages (Figure 4C, Additional file 8). Although the set of genes that change in expression between each stage encodes diverse cellular functions, we were able to observe enrichment of specific functional categories within gene groups with similar expression trends. GO analysis on the set of genes that are differentially expressed by greater than two-fold between successive stages revealed enrichment of a comparable set of GO categories (Additional file 8). Further analysis of available PANTHER  annotations for the same gene groups yielded enrichment of specific domains or enzymatic activities that fall under the more general categories seen by GO analysis (Additional file 9).
As larvae attain metamorphic competence (the capacity to initiate metamorphosis by detecting and responding to inductive environmental cues), genes encoding dyneins, kinesins, and other proteins involved in microtubule cytoskeleton assembly and microtubule dynamics become downregulated (Figure 4C). Cilia and flagellar components are further downregulated at the transition to postlarva when cilia appear to be resorbed and major morphogenic changes begin. Ubiquitin thiolesterase genes, which protect proteins from proteasome degradation, are also downregulated in the competent stage, suggesting that protein turnover may be an important mechanism regulating this transition.
In competent larvae, transcripts that encode genes with potential protective functions, such as negative regulators of apoptosis (Bcl2, Bax), antioxidants (glutathione metabolism enzymes, superoxide dismutase, peroxiredoxin, catalase), and stress response proteins (Hsp70, Sap16, pirin, sequestosome) are upregulated (Figure 4C, Additional file 8 and Additional file 9). Genes that regulate transcription, including CCAAT-box binding proteins, CRE-binding proteins, bHLH factors, and homeobox proteins are also enriched. As expected for lecithotrophic larvae that rely on metabolism of yolk stores for energy generation, many genes involved in cellular respiration are found at their highest levels in pelagic larvae (Additional file 4). Furthermore, there is an enrichment of transcripts regulating oxidation and reduction, including mitochondrial genes such as glycerol-3-phosphate dehydrogenase, electron transfer flavoprotein-ubiquinone oxidoreductase, and glycine dehydrogenase, in the set of genes that are upregulated in competent larvae. A similar upregulation of lipases and mitochondrial genes has been observed in lecithotrophic coral planula [3, 6].
Genome-wide expression profiles from the pelagobenthic transition
The transition from the planktonic competent larval stage to the benthic postlarval stage results in extensive cellular transdifferentiation, proliferation and rearrangement, resulting in the appearance of most of the cell types associated with the adult body plan arranged into functional morphological units such as pinacoderm and choanocyte chambers [9, 19]. We observed transcriptional changes consistent with these processes and the extensive intercellular signaling pathways that may regulate morphogenetic events during metamorphosis. For example, many transcripts encoding transmembrane receptors, kinases, and signal transduction factors are upregulated in the postlarva (Figure 4C, Additional file 8 and Additional file 9). We also observed an increase in the expression of genes involved in DNA replication, DNA recombination, and methyltransferase activity that occur in preparation for cell division. Extracellular matrix components that make up the sponge mesohyl are also upregulated at this stage. Furthermore, there appears to be an overall decrease in the requirement for energy production as evidenced by downregulation of many mitochondrial enzymes with oxidation-reduction activity.
The adult transcriptome
Although previous systematics suggest that the only clear homology between Porifera and other animals was in the embryonic and larval stages , analysis of the A. queenslandica genome revealed the presence of many of the genes involved in metazoan-associated cellular processes . Transcriptome analysis showed extensive use of these genes throughout the sponge life cycle, particularly at the adult stage, where the differentially upregulated gene set is enriched for functions that are characteristically metazoan, such as cell adhesion, immune response, tissue morphogenesis, cell proliferation, and apoptosis. Transcripts encoding transmembrane receptors, adhesion molecules, and extracellular matrix components (Figure 4C, Additional file 8 and Additional file 9) are widely expressed in sponge with marked enrichment in the adult (Additional file 4).
With transition to the adult stage, upregulated genes include extracellular matrix components required to form the mesohyl or gelatinous matrix that fills the space between the external pinacoderm and the internal choanoderm. Multiple sulfatases and metallopeptidase-like proteases that are upregulated in the adult may also have a role in extracellular matrix remodeling (Figure 4C). The reliance of the adult on phagocytosis of food particles from surrounding seawater is reflected in the transcriptome by an increase in the expression of scavenger receptors and polysaccharide binding molecules (lectins) that enable the sponge to differentiate between food bacteria and symbiotic bacteria [38–42]. These same molecules may mediate cell-to-cell adhesion and allorecognition in the sponge [39, 43, 44]. The adult sponge upregulates the expression of various catabolic enzymes, including lysosomal enzymes such as sulfatases and fucosidases that degrade glycoproteins, glycolipids, and sulfated proteoglycans, as well as cysteine-type endopeptidases that degrade proteins (Additional file 8 and Additional file 9). Other enzymes upregulated in the adult, including multi-copper oxidases and aromatic compound metabolizing enzymes, may be involved in the synthesis of secondary metabolites that allow sponges to select for or against specific types of microorganisms [29, 45].
Notably, about 70% (2,568 out of 3,661 genes) of the genes that are upregulated at the transition from postlarva to adult exhibit an expression change on the order of ten-fold or greater, with many of the transcripts displaying either larval/postlarval- or adult-specific expression. Overall, we identified 703 transcripts exhibiting a greater than 100-fold expression differential between one stage and the other three stages (Additional file 10). The majority of these genes are enriched in the adult (640 genes). These include the PDZ domain-containing protein Nherf2, which regulates targeting and trafficking of receptors, ion channels, and other membrane proteins [46–48], and syntrophin gamma, a cytoplasmic peripheral membrane protein that binds to components of mechanosensitive sodium channels . Ion channels, including amiloride-sensitive sodium channels are also upregulated in the adult, suggesting a possible role in water homeostasis or coordination of cellular contraction .
Functional gene families associated with competence and metamorphosis
Developmental signaling pathways
Members of developmental signaling pathways were expressed at variable levels in all the stages that were sequenced, indicating that the same pathways involved in early embryonic patterning [10, 11] may also have a role in morphogenetic changes occurring after larval settlement (Additional file 5). Several Wnt receptors, such as Frizzled and Low-density lipoprotein receptor-related protein (Lrp5/6), Wnt ligands, and inhibitory molecules (Apc, Sfrp, Invs), are upregulated after settlement in postlarva. The expression of Wnt pathway genes in the adult sponge suggests roles for these genes beyond setting up embryonic polarity and may extend to the formation of water canal openings in the exopinacoderm [51–53]. Molecules in the Notch and TGF-β signaling pathways were also detected in all stages, although the expression of ligands and receptors were not always coordinated. While genes in the Wnt pathway appear to be generally downregulated at the postlarval stage (p < 0.05, Fisher’s exact test), there is no stage-specific enrichment or depletion observed for the Notch and TGF-β pathways.
Genes that are selectively expressed in the same developmental stage may be regulated by the same set of transcription factors. For example, short-chain collagens and members of the cadherin domain and scavenger receptor domain-containing gene families (Figure 5B), show coordinated upregulation at the adult stage (p < 0.0005, Fisher’s exact test). In the adult, transcriptionally active genomic loci with three or more scavenger receptor domain-containing genes are organized in tandem arrays and are potentially regulated through a shared cis-regulatory element (Figure 5C).
Upon emerging from the brood chamber A. queenslandica larvae are light-sensitive but are initially indifferent to environmental signals that can induce metamorphosis. They need to swim in the water column for at least four hours before they are able to respond behaviorally to settlement cues associated with benthic coralline algae . Physiological differences between younger, unreactive larvae and competent older larvae are likely to include the deployment of additional functional sensory systems that allow them to sense and react to the benthic environment. Several mechanisms, including photosensory molecules, ion channels, G-protein coupled receptors (GPCR) and kinases may be key to this response.
Studies in A. queenslandica and S. domuncula have shown that cryptochromes and bioluminescence proteins, whose transcripts are regulated by light exposure, play a role in the sponge photosensory system [58–60] and may mediate the negative phototactic response exhibited by larvae . Transcripts for these genes are expressed in all stages of A. queenslandica, with an increase in the expression level of cryptochromes, luciferase, and luciferin binding protein in the competent stage (Additional file 4). Upregulation of genes involved in calcium-mediated signaling, including the luciferin regenerating enzyme, occurred in the same stage. Some ion channels were expressed highly in the pre-settlement stages, suggesting that ionic concentration may be a contributing factor for larval settlement, as has been observed in the sponge Aplysilla.
G-protein coupled receptors and kinases
The A. queenslandica genome encodes a wide array of kinases , a majority of which were detectable in all stages sampled (Additional file 4, Additional file 5). While we observed an increase in transcript expression for members of most kinase classes as the larvae mature into adults, the AGC/RGC and CAMK classes have higher average read counts in pelagic larvae (Figure 6C).
Expression of genes found in bilaterian cell types
Porifera does not possess neurons or recognizable synapses but homologs of neuronal post-synaptic genes have been identified in the sponge genome (Figure 7C) and have been shown to be expressed in the globular cells of the outer epithelial layer of sponge larvae [16, 23]. In the absence of true synapses, the role of these genes must be distinct from the function of bilaterian homologs. Indeed, while we observed a general downregulation of synaptic gene expression at the postlarval stage (p < 0.005, Fisher’s exact test), the overall expression pattern reveals a lack of coordinated regulation between the different elements that might be expected to generate a functional synapse (Figure 7D). Members of complexes involved in regulating vesicle dynamics, such as vesicular ATPases, synaptic vesicle proteins, and presynaptic signaling molecules show relatively higher expression in precompetent and competent stages, where the processes of intracellular transport, membrane trafficking, and signaling may be needed for the maintenance of ciliated epithelia and for the rapid response to the environment required by a pelagic lifestyle .
Deep sequencing of the transcriptome of the demosponge, A. queenslandica, as it develops from a pelagic larva to a benthic adult, revealed genome-wide transcriptional events accompanying this major life history transition. While metamorphosis is widespread throughout the animal kingdom, it is thought to have evolved several times in metazoans, thus there are likely to be different regulatory mechanisms controlling metamorphosis in various taxa . For example, the metamorphic transition in marine invertebrates occurs rapidly compared to insects and other arthropods, making the period immediately after settlement, when the larva is most vulnerable, as brief as possible [73, 74]. In this study, we discovered that competent A. queenslandica larvae retain a similar transcriptome profile to the precompetent larvae, but differentially express many transcription factors and regulators. This suggests that swimming sponge larvae already possess most of the gene products required at the pelagobenthic transition and are poised to enact rapid and widespread changes in gene expression upon settlement. One of the key mechanisms in this regulation may be the utilization of bivalent chromatin marks, as occurs in pluripotent cells . Although the acquisition of metamorphic competence itself is not accompanied by much global change in transcription, the expression of select functional gene groups (e.g. transcription factors, rhodopsin family GPCRs, cryptochromes, calcium signaling proteins) does occur. The timely expression of competence genes allows the larva to disperse widely before settlement, navigate towards a suitable habitat, detect appropriate inductive cues, and adhere to its chosen substrate. GPCRs and membrane trafficking mechanisms that are expressed in the larval stages are candidate regulators of competence and metamorphosis, and indeed, exposure of precompetent larvae to an inductive cue results in habituation or a decrease in responsiveness that delays settlement .
For some species, the metamorphic transition can occur even with the inhibition of transcription and translation [1, 73]. In contrast, we found that transformation of A. queenslandica from larva to the juvenile/adult is accompanied by a large change in transcription consistent with gross changes in morphology and the different environmental demands faced by free-swimming lecithotrophic larva and the sessile, filter-feeding adult. Pelagic larvae express genes required for metabolism of yolk stores and the maintenance of cilia and flagellar structures that allow them to remain in the water column. The decreased expression of genes involved in assembly of cilia/flagellar structures may be responsible, in part, for the decrease in swimming behavior that accompanies settlement on a substrate at the beginning of metamorphosis. The adult sponge, on the other hand, shows the hallmarks of a filter-feeding lifestyle, expressing genes that allow it to recognize food bacteria and to protect itself from potentially harmful microorganisms. Interestingly, metazoan developmental and structural gene orthologs are well-integrated into the expression profiles at every stage of sponge development. Rather than expressing mainly sponge-specific genes, the adult sponge expresses many genes that encode typical metazoan features, such as cell adhesion, cell differentiation, and immunity. No stage-selective expression was observed for the sponge-specific gene set (Figure 3, Additional file 3).
The genetic controls for metamorphosis are linked to evolutionarily ancient ‘toolkits’ that generate each staged body plan. In fact, the pelagobenthic transition is coordinated by a host of conserved developmental regulators (i.e. signaling pathway components and transcription factors) that are differentially expressed in larval and adult stages. For example, canonical Wnt signaling functions in the establishment of the anterior-posterior (AP) axis; the use of posterior Wnt signaling and anterior Wnt inhibition appears to be a unifying principle of the metazoan body plan development . In A. queenslandica, Wnt expression is the earliest marker of asymmetry in the embryo; it becomes localized to the posterior pole as embryos begin to display AP polarity and this polar expression continues until the swimming larva stage [10, 11]. Conservation is also apparent among those genes that have a likely role as downstream effectors of metamorphosis. Apoptosis, stress response, immunity, and calcium signaling are common functional groups upregulated during metamorphosis in various phyla [4, 6, 7, 77]. Similar gene groups are expressed in the developing sponge, however, there is no evidence of extensive autolysis or apoptosis during sponge metamorphosis . Like Acropora millepora coral larvae, A. queenslandica upregulates Bcl2, pirin, and catalase upon induction of metamorphosis . Pirin and pirin-related genes are transcription cofactors that interact with nuclear factor I/CCAAT box transcription factor and forms a complex with Bcl-3 and NFκB to promote the transcription of anti-apoptotic genes in response to stress [78, 79]. A decrease in the requirement for energy production, reflected in the downregulation of many mitochondrial enzymes with oxidation-reduction activity and of structural genes, is observed in the larvae of A. queenslandica, abalone (Haliotis asinina), and mollusc (Aplysia californica) as they undergo the metamorphic transition, as well as in fly larvae entering the pupal stage [5, 7, 80]. Thus, life cycle transitions may have deep homologies in their effector genes despite the widely different environmental settings in which these transitions unfold among organisms.
The current study was limited to the sequencing of single pooled samples from four well-defined developmental stages. Although each sample represents profiles from hundreds of larvae, this is a potential source of sampling bias since ensemble measurements may not account for greater variation in gene expression between individuals. The lack of biological replicates also limited our ability to detect genes with subtle changes over time. To minimize the effect of these biases, we estimated sampling error from technical replicates and used this as a benchmark to gauge the gene detection threshold and to set conservative criteria for the identification of differentially expressed genes. Similar gene ontology results for transcripts detected at two thresholds of differential expression, as well as validation by quantitative RT-PCR for a subset of genes and for biological replicates, provide some assurance that the changes we observed are robust. Inclusion of biological replicates in future studies, as well as increased sequencing depth, will allow more sensitive detection of differentially expressed genes. Further analysis of sponge gene expression at greater spatial and temporal resolution will allow the detection of genes with transient, cell type-specific, or stimulus-induced expression patterns. It is also important to note that some key regulatory events may be occurring at the post-transcriptional and post-translational levels that will not be observed by transcriptome sequencing. Expression studies, along with functional interrogation of genes identified here as potentially important for sponge development and metamorphosis, will ultimately provide a more complete picture of the pelagobenthic transition.
Our findings point to a network of regulatory mechanisms that coordinate morphological changes with the ecological demands of the pelagobenthic transition. While differentially expressed groups of transcriptional regulators may mediate the widespread changes in gene expression that accompany the metamorphic transition, the identification of specific subsets of receptors that are upregulated during the period of competence suggests a potential link between morphogenesis and the environment. The vastly different transcriptome of the adult sponge, which expresses recently evolved metazoan genes involved in secondary metabolism, immune system, and stress response, most likely contribute to its ability to readily adapt to changing ecological conditions. The utilization of a conserved metazoan gene set throughout sponge development emphasizes the potential of the genome of the last common ancestor of animals to generate phenotypic complexity. This study provides a rich resource for the identification of mechanisms regulating major life cycle transitions, and will contribute to our understanding of sponge biology.
Materials and methods
A. queenslandica were collected from Heron Island Reef, southern Great Barrier Reef, Queensland, Australia using a standardized protocol . Precompetent larvae were collected not more than 3 hours after emergence from the brood chambers of the adult. Competent larvae were collected 6 hours after emergence. Postlarvae exhibiting a flattened juvenile body plan were collected after 2 days of settlement on glass coverslips. Due to the limited quantity of sponge material collected, we pooled about 1000 precompetent or competent larvae and 100 postlarvae to obtain sufficient RNA for library construction. Adult tissues were collected as a 5 mm core from apical to basal surface of individuals without brood chambers to avoid inclusion of early embryonic stages. All tissues were stored in RNAlater [Ambion] before processing.
RNA extraction and poly(A) RNA purification
RNA from early larvae and postlarvae was extracted directly with Trizol [Invitrogen] following the manufacturer’s protocol. Adult samples were cleaned of macroscopic debris then ground in liquid nitrogen before RNA extraction with Trizol. Contaminating DNA was removed using the DNAfree kit [Ambion]. The poly(A) RNA fraction was enriched using the MicroPoly(A)Purist kit [Ambion] and ribosomal RNA was depleted using the RiboMinus Eukaryote kit [Invitrogen]. RNA quality was monitored using the Agilent Bioanalyzer RNA 6000 Pico Assay.
Poly(A) RNA fragment library preparation and sequencing
Fragment libraries were prepared as previously described . Briefly, approximately 250 ng of purified poly(A) RNA was subjected to 95°C until most of the RNA formed 50–200 nt fragments. First-strand cDNA synthesis was primed with a 3’ adapter-tagged random hexamer primer (sFDVhex) using SuperScript II reverse transcriptase [Invitrogen]. Second strand cDNA was then synthesized using a 5’ template switching adapter-tagged oligonucleotide (RDV-GGG). cDNA libraries were amplified using limited PCR cycles and fragments averaging 150 bp in length were purified. Libraries were quantified by PCR and fragment size was verified using the Agilent Bioanalyzer DNA 1000 Assay. 280 pg of each library was loaded onto beads by emulsion PCR. Approximately 40–80 million library beads were loaded on to separate slide sections and sequenced to 50 bases using the Applied Biosystems SOLiD V3 chemistry.
Sequencing data analysis
Assembled sponge contigs and gene models (Aqu1) are publicly available . Redundant transcripts for predicted gene models were filtered out retaining only the longest of overlapping transcripts originating from the same locus (29,915 unique genes). All 50 nucleotide reads were analyzed in color space using the SOLiD RNA Analysis Pipeline Tool . Reads were aligned to sponge contigs using the ‘anchor-extend’ method as previously described . Estimates of transcript expression were obtained by counting reads aligning to exons of predicted gene models. Sequencing data has been submitted to the NCBI Gene Expression Omnibus (GEO) as series GSE29978.
Read normalization and identification of differentially expressed transcripts
Comparison of sequencing results from different libraries prepared from the same sample or libraries prepared from different biological samples showed that variable sequencing depths did not induce any detectable read count-dependent bias but rather affected read counts by a single scaling factor which is of the order of the difference in the total number of mapped reads. Read counts from different libraries were normalized by multiplying with a proportionality constant reflecting different sequencing depths to obtain read count distributions similar to the precompetent library, which was used as the reference sample. The distribution of reads in each sample after normalization is shown in Additional file 1. The transcript detection cutoff was determined to be the minimal read count above which all transcripts are detected in two independently prepared libraries from the same starting RNA sample (Additional file 1). Sampling statistics accounts for most of the differences between libraries in that read count differences between technical replicates of the same library, or between libraries derived from the same biological sample, scaled as √N irrespective of the read count N. The variation in read counts between two libraries was always smaller than 5√N for a given read count. The read count error equals 5√N/N, which decreases as the read count increases and is negligible for large read counts. Thus, reads with raw counts below 32 are likely to be detected in just one library out of two. To account for different sequencing depths, we set a normalized read count of 64 as the detection threshold (this number is equivalent to a threshold of 32 for the non-normalized postlarval sample, which had the lowest depth of sequencing).
In technical replicates of libraries derived from the same biological sample, the variation in expression for genes detected above 64 reads did not exceed three-fold. Fold change cutoffs as calculated based on read count error within technical replicates are 1.36, 1.68, and 2.25-fold for false calling rates of 5%, 1%, and 0.1%, respectively. Thus, we selected a four-fold change as a conservative criterion for differential expression. To identify genes with stage-specific expression across the pelagobenthic transition, we compared gene expression at each stage to the mean expression of the other three stages by performing a two-tailed t-test on the log of read counts and filtered out transcripts for which expression varied by less than four-fold. This method would filter out any gene that does not exhibit a strong stage-specific expression, particularly genes with dynamic expression across development. Therefore, to identify genes that exhibit differential expression between stages, we tracked the expression trajectory for each expressed gene in pair-wise comparisons between successive stages. Individual genes were categorized as either up or downregulated if their expression changed more than four-fold and were greater than the sampling error on the read count between stages. We also identified genes differentially expressed by two-fold between stages.
Sequence alignment, annotation, and GO analysis
Sponge gene models were aligned to proteins in the UniProt database  using BLASTp  with an e-value cutoff ≤ 1x10-4. Genes were assigned the names and Gene Ontology (GO)  annotations of their best match. GO term enrichment p-values were estimated by comparing the enrichment of a particular GO term within a gene set to the enrichment distribution determined by re-sampling the set of all detected transcripts. GO enrichments are presented relative to the number of annotated genes and not of the entire genome, which includes about 30% genes without functional annotation. The statistical significance threshold was set to P < 0.05 (corrected for multiple hypothesis testing using the Benjamini-Hochberg method). Stage enrichment or depletion for genes in a specific functional group was determined using Fisher’s exact test on the number of genes at each stage that are detected above or below the upper 25% of its expression range across development.
Quantitative real-time PCR
Primers for quantitative RT-PCR were designed to span exon junctions of predicted transcripts whenever possible to ensure specificity for target mRNAs (Additional file 11). All primers were synthesized by Integrated DNA Technologies (IDT). Total RNA for quantitative RT-PCR analysis was extracted from three separate pools of sponge material at each developmental stage. cDNA was synthesized using random primers and SuperScriptIII reverse transcriptase [Invitrogen] following the manufacturer’s protocol. Quantitative RT-PCR was performed using Power SYBR Green PCR master mix [Applied Biosystems] on an ABI 7500 Fast Real-Time PCR System. RNA expression was normalized to β-actin and quantified using the ΔΔCt method . Variance of the pooled sample replicates was assessed using one-way ANOVA with Bonferroni’s post-test.
G-protein coupled receptor
We thank Todd Oakley, Mansi Srivastava, Mark Martindale, Robert Steele, Christine Henzler, Pantelis Tsoulfas and members of the Kosik lab for insightful comments and conversation. Support for this project came from generous gifts by Harvey Karp and Gus Gurley. PN is supported in part by the National Science Foundation under Grant No. PHY05-51164.
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