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Cis-regulatory DNA sequences causally mediate patterns of gene expression, but efficient experimental analysis of these control systems has remained challenging. Here we develop a new version of “barcoded" DNA-tag reporters, “Nanotags" that permit simultaneous quantitative analysis of up to 130 distinct cis-regulatory modules (CRMs). The activities of these reporters are measured in single experiments by the NanoString RNA counting method and other quantitative procedures. We demonstrate the efficiency of the Nanotag method by simultaneously measuring hourly temporal activities of 126 CRMs from 46 genes in the developing sea urchin embryo, otherwise a virtually impossible task. Nanotags are also used in gene perturbation experiments to reveal cis-regulatory responses of many CRMs at once. Nanotag methodology can be applied to many research areas, ranging from gene regulatory networks to functional and evolutionary genomics.

The current gene regulatory network (GRN) for the sea urchin embryo pertains to pregastrular specification functions in the endomesodermal territories. Here we extend gene regulatory network analysis to the adjacent oral and aboral ectoderm territories over the same period. A large fraction of the regulatory genes predicted by the sea urchin genome project and shown in ancillary studies to be expressed in either oral or aboral ectoderm by 24h are included, though universally expressed and pan-ectodermal regulatory genes are in general not. The loci of expression of these genes have been determined by whole mount in situ hybridization. We have carried out a global perturbation analysis in which expression of each gene was interrupted by introduction of morpholino antisense oligonucleotide, and the effects on all other genes were measured quantitatively, both by QPCR and by a new instrumental technology (NanoString Technologies nCounter Analysis System). At its current stage the network model, built in BioTapestry, includes 22 genes encoding transcription factors, 4 genes encoding known signaling ligands, and 3 genes that are yet unknown but are predicted to perform specific roles. Evidence emerged from the analysis pointing to distinctive subcircuit features observed earlier in other parts of the GRN, including a double negative transcriptional regulatory gate, and dynamic state lockdowns by feedback interactions. While much of the regulatory apparatus is downstream of Nodal signaling, as expected from previous observations, there are also cohorts of independently activated oral and aboral ectoderm regulatory genes, and we predict yet unidentified signaling interactions between oral and aboral territories.

The diagnosis of medulloblastoma likely encompasses several distinct entities, with recent evidence for the existence of at least four unique molecular subgroups that exhibit distinct genetic, transcriptional, demographic, and clinical features. Assignment of molecular subgroup through routine profiling of high-quality RNA on expression microarrays is likely impractical in the clinical setting. The planning and execution of medulloblastoma clinical trials that stratify by subgroup, or which are targeted to a specific subgroup requires technologies that can be economically, rapidly, reliably, and reproducibly applied to formalin-fixed paraffin embedded (FFPE) specimens. In the current study, we have developed an assay that accurately measures the expression level of 22 medulloblastoma subgroup-specific signature genes (CodeSet) using nanoString nCounter Technology. Comparison of the nanoString assay with Affymetrix expression array data on a training series of 101 medulloblastomas of known subgroup demonstrated a high concordance (Pearson correlation r = 0.86). The assay was validated on a second set of 130 non-overlapping medulloblastomas of known subgroup, correctly assigning 98% (127/130) of tumors to the appropriate subgroup. Reproducibility was demonstrated by repeating the assay in three independent laboratories in Canada, the United States, and Switzerland. Finally, the nanoString assay could confidently predict subgroup in 88% of recent FFPE cases, of which 100% had accurate subgroup assignment. We present an assay based on nanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials.

Electronic supplementary material

The online version of this article (doi:10.1007/s00401-011-0899-7) contains supplementary material, which is available to authorized users.

The serum prostate-specific antigen (PSA) test has a high false positive rate. As a single marker, PSA provides limited diagnostic information. A multi-marker test capable of detecting not only tumors but also the potentially lethal ones provides an unmet clinical need. Using the nanoString nCounter gene expression system, a 20-gene multiplex test was developed based on digital gene counting of RNA transcripts in urine as a means to detect prostate cancer. In this test, voided urine is centrifuged to pellet cells and the purified RNA is amplified for hybridization to preselected probesets. Amplification of test cell line RNA appeared not to introduce significant bias, and the counts matched well with gene abundance levels as measured by DNA microarrays. For data analysis, the individual counts were compared to that of β2 microglobulin, a housekeeping gene. Urine samples of 5 pre-operative cases and 2 non-cancer were analyzed. Pathology information was then retrieved. Signals for a majority of the genes were low for non-cancer and low Gleason scores, and 6/6 known prostate cancer markers were positive in the cases. One case of Gleason 4+5 showed, in contrast, strong signals for all cancer-associated markers, including CD24. One non-cancer also showed signals for all 6 cancer markers, and this man might harbor an undiagnosed cancer. This multiplex test assaying a natural waste product can potentially be used for screening, early cancer detection and patient stratification. Diagnostic information is gained from the RNA signatures that are associated with cell types of prostate tumors.

Background

A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples.

Results

We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53).

Conclusions

Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

Background

The nCounter analysis system (NanoString Technologies, Seattle, WA) is a technology that enables the digital quantification of multiplexed target RNA molecules using color-coded molecular barcodes and single-molecule imaging. This system gives discrete counts of RNA transcripts and is capable of providing a high level of precision and sensitivity at less than one transcript copy per cell.

Results

We have designed a web application compatible with any modern web browser that accepts the raw count data produced by the NanoString nCounter analysis system, normalizes it according to guidelines provided by NanoString Technologies, performs differential expression analysis on the normalized data, and provides a heatmap of the results from the differential expression analysis.

Conclusion

NanoStriDE allows biologists to take raw data produced by a NanoString nCounter analysis system and easily interpret differential expression analysis of this data represented through a heatmap. NanoStriDE is freely accessible to use on the NanoStriDE website and is available to use under the GPL v2 license.

Pax-6 is known to be a key regulator of vertebrate eye development. We have now isolated cDNA for an invertebrate Pax-6 protein from sea urchin embryos. Transcripts of this gene first appear during development at the gastrula stage and are later expressed at high levels in the tube foot of the adult sea urchin. The sea urchin Pax-6 protein is highly homologous throughout the whole protein to its vertebrate counterpart with the paired domain and homeodomain being virtually identical. Consequently, we found that the DNA-binding and transactivation properties of the sea urchin and mouse Pax-6 proteins are very similar, if not identical. A potent activation domain capable of stimulating transcription from proximal promoter and distal enhancer positions was localized within the C-terminal sequences of both the sea urchin and mouse Pax-6 proteins. The homeodomain of Pax-6 was shown to cooperatively dimerize on DNA sequences consisting of an inverted repeat of the TAAT motif with a preferred spacing of 3 nucleotides. The consensus recognition sequence of the Pax-6 paired domain deviates primarily only at one position from that of BSAP (Pax-5), and yet the two proteins exhibit largely different binding specificities for individual, naturally occurring sites. By creating Pax-6-BSAP fusion proteins, we were able to identify a short amino acid stretch in the N-terminal part of the paired domain which is responsible for these differences in DNA-binding specificity. Mutation of three Pax-6-specific residues in this region (at positions 42, 44, and 47 of the paired domain) to the corresponding amino acids of BSAP resulted in a complete switch of the DNA-binding specificity from Pax-6 to BSAP. These three amino acids were furthermore shown to discriminate between the Pax-6- and BSAP-specific nucleotide at the divergent position of the two consensus recognition sequences.

Echinoderms, which are phylogenetically related to vertebrates and produce large numbers of transparent embryos that can be experimentally manipulated, offer many advantages for the analysis of the gene regulatory networks (GRN) regulating germ layer formation. During development of the sea urchin embryo, the ectoderm is the source of signals that pattern all three germ layers along the dorsal-ventral axis. How this signaling center controls patterning and morphogenesis of the embryo is not understood. Here, we report a large-scale analysis of the GRN deployed in response to the activity of this signaling center in the embryos of the Mediterranean sea urchin Paracentrotus lividus, in which studies with high spatial resolution are possible. By using a combination of in situ hybridization screening, overexpression of mRNA, recombinant ligand treatments, and morpholino-based loss-of-function studies, we identified a cohort of transcription factors and signaling molecules expressed in the ventral ectoderm, dorsal ectoderm, and interposed neurogenic (“ciliary band”) region in response to the known key signaling molecules Nodal and BMP2/4 and defined the epistatic relationships between the most important genes. The resultant GRN showed a number of striking features. First, Nodal was found to be essential for the expression of all ventral and dorsal marker genes, and BMP2/4 for all dorsal genes. Second, goosecoid was identified as a central player in a regulatory sub-circuit controlling mouth formation, while tbx2/3 emerged as a critical factor for differentiation of the dorsal ectoderm. Finally, and unexpectedly, a neurogenic ectoderm regulatory circuit characterized by expression of “ciliary band” genes was triggered in the absence of TGF beta signaling. We propose a novel model for ectoderm regionalization, in which neural ectoderm is the default fate in the absence of TGF beta signaling, and suggest that the stomodeal and neural subcircuits that we uncovered may represent ancient regulatory pathways controlling embryonic patterning.

Author Summary

Echinoderms (sea urchins, starfish, etc.) are marine invertebrates that share a close ancestry with vertebrates. Their embryos offer many advantages for the analysis of transcriptional circuits that control developmental programs. During early development of the common sea urchin Paracentrotus lividus, a signaling center located within the ventral ectoderm sends two key signals, Nodal and BMP2/4, that control patterning of the embryo along the whole dorsal-ventral axis. How this signaling center works is not understood. We have conducted a large-scale functional analysis of the genes responsible for patterning of the ectoderm along the dorsal-ventral axis. We identified direct targets of Nodal and BMP2/4 and identified several key regulators that mediate the effects of these factors and drive essential and probably ancient regulatory circuits that together constitute a transcriptional program controlling morphogenesis of the embryo. In addition, we uncovered a striking parallel between the mouse embryo and the sea urchin embryo by showing that in both models a neurogenic ectoderm is the default state of ectoderm differentiation in the absence of Nodal and BMP signaling. Our results support the idea that inhibition of Nodal and BMP signaling was probably an ancient mechanism to specify neural cells in the ancestor of vertebrates.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor α (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples.

Background

MicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level. While they have been implicated in various diseases, the profile changes in allergen inhalation challenge are not clarified in human. We aimed to evaluate changes in the microRNA profiles in the peripheral blood of asthmatic subjects undergoing allergen inhalation challenge.

Results

Seven mild asthmatic subjects participated in the allergen inhalation challenge. In addition, four healthy control subjects (HCs) were recruited. MicroRNA profiles in peripheral blood samples (pre-challenge and 2 hours post-challenge) were measured by the NanoString nCounter assay to determine changes in miRNA levels as these asthmatic subjects underwent an allergen inhalation challenge. One common miRNA, miR-192, was significantly expressed in both comparisons; HCs vs. pre-challenge and pre- vs. post-challenge, showing that miR-192 was significantly under-expressed in asthmatics compared to HCs and decreased in post-challenge at an FDR of 1%. Cell-specific statistical deconvolution attributed miR-192 expression in whole blood to PBMCs. MiR-192 was technically validated using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showing that the level in asthmatics (pre-challenge) was significantly lower than HCs and that post-challenge was significantly lower than pre-challenge. The normalized relative miR-192 expression quantified using RT-qPCR specific to PBMCs was also validated. Ontology enrichment and canonical pathway analyses for target genes suggested several functions and pathways involved in immune response and cell cycle.

Conclusions

The miRNA profile in peripheral blood was altered after allergen inhalation challenge. Change in miR-192 levels may be implicated in asthma mechanisms. These results suggest that allergen inhalation challenge is a suitable method to characterize peripheral miRNA profiles and potentially elucidate the mechanism of human asthma.

Gene regulatory networks for development underlie cell fate specification and differentiation. Network topology, logic and dynamics can be obtained by thorough experimental analysis. Our understanding of the gene regulatory network controlling endomesoderm specification in the sea urchin embryo has attained an advanced level such that it explains developmental phenomenology. Here we review how the network explains the mechanisms utilized in development to control the formation of dynamic expression patterns of transcription factors and signaling molecules. The network represents the genomic program controlling timely activation of specification and differentiation genes in the correct embryonic lineages. It can also be used to study evolution of body plans. We demonstrate how comparing the sea urchin gene regulatory network to that of the sea star and to that of later developmental stages in the sea urchin, reveals mechanisms underlying the origin of evolutionary novelty. The experimentally based gene regulatory network for endomesoderm specification in the sea urchin embryo provides unique insights into the system level properties of cell fate specification and its evolution.

Motivation: The NanoString nCounter Platform is a new and promising technology for measuring nucleic acid abundances. It has several advantages over PCR-based techniques, including avoidance of amplification, direct sequence interrogation and digital detection for absolute quantification. These features minimize aspects of experimental error and hold promise for dealing with challenging experimental conditions such as archival formalin-fixed paraffin-embedded samples. However, systematic inter-sample technical artifacts caused by variability in sample preservation, bio-molecular extraction and platform fluctuations must be removed to ensure robust data.

Results: To facilitate this process and to address these issues for NanoString datasets, we have written a pre-processing package called NanoStringNorm in the R statistical language. Key features include an extensible environment for method comparison and new algorithm development, integrated gene and sample diagnostics, and facilitated downstream statistical analysis. The package is open-source, is available through the CRAN package repository, includes unit-tests to ensure numerical accuracy, and provides visual and numeric diagnostics.

Availability: http://cran.r-project.org/web/packages/NanoStringNorm

Contact: paul.boutros@oicr.on.ca

Supplementary information: Supplementary data are available at Bioinformatics online.

A global scan of transcription factor usage in the sea urchin embryo was carried out in the context of the S.purpuratus genome sequencing project, and results from six individual studies are here considered. Transcript prevalence data were obtained for over 280 regulatory genes encoding sequence-specific transcription factors of every known family, but excluding genes encoding zinc finger proteins. This is a statistically inclusive proxy for the total “regulome” of the sea urchin genome. Close to 80% of the regulome is expressed at significant levels by the late gastrula stage. Most regulatory genes must be used repeatedly for different functions as development progresses. An evolutionary implication is that animal complexity at the stage when the regulome first evolved was far simpler than even the last common bilaterian ancestor, and is thus of deep antiquity.

One of the polyketide compounds, the naphthoquinone pigment echinochrome, is synthesized in sea urchin pigment cells. We analyzed polyketide synthase (pks) and sulfotransferase (sult) gene expression in embryos and larvae of the sea urchin Strongylocentrotus intermedius from various stages of development and in specific tissues of the adults. We observed the highest level of expression of the pks and sult genes at the gastrula stage. In unfertilized eggs, only trace amounts of the pks and sult transcripts were detected, whereas no transcripts of these genes were observed in spermatozoids. The addition of shikimic acid, a precursor of naphthoquinone pigments, to zygotes and embryos increased the expression of the pks and sult genes. Our findings, including the development of specific conditions to promote pigment cell differentiation of embryonic sea urchin cells in culture, represent a definitive study on the molecular signaling pathways that are involved in the biosynthesis of pigments during sea urchin development.

We have isolated and sequenced a gene encoding a late H1 histone subtype from the sea urchin species L. pictus. The primary structure of the late H1 subtype encoded by this gene is 209 amino acids in length, and has a net positive charge of 67. This gene is present in a single copy per haploid genome and encodes an mRNA of 752 nucleotides. Late H1 transcripts are detected in the unfertilized egg and are most prevalent in gastrulating embryos. Comparison of 375 bp of 5' flanking sequences of the L. pictus late H1 gene and the H1-gamma gene of a distantly related sea urchin species, S. purpuratus, reveals large blocks of sequences that are identical between the two genes. To determine if these conserved 5' sequences are present in other members of the sea urchin H1 gene family, the analogous region of S. purpuratus H1-alpha, an early H1 gene, was sequenced. The homology between the flanking sequences of the early and late families was limited to consensus sequences which are found upstream of all H1 genes. The possible regulatory implications of these findings are discussed.

Images

Three different Wnt signaling pathways function to restrict the anterior neuroectoderm state to the anterior end of the sea urchin embryo, a mechanism of anterior fate restriction that could be conserved among deuterostomes.

Patterning the neuroectoderm along the anterior–posterior (AP) axis is a critical event in the early development of deuterostome embryos. However, the mechanisms that regulate the specification and patterning of the neuroectoderm are incompletely understood. Remarkably, the anterior neuroectoderm (ANE) of the deuterostome sea urchin embryo expresses many of the same transcription factors and secreted modulators of Wnt signaling, as does the early vertebrate ANE (forebrain/eye field). Moreover, as is the case in vertebrate embryos, confining the ANE to the anterior end of the embryo requires a Wnt/β-catenin-dependent signaling mechanism. Here we use morpholino- or dominant negative-mediated interference to demonstrate that the early sea urchin embryo integrates information not only from Wnt/β-catenin but also from Wnt/Fzl5/8-JNK and Fzl1/2/7-PKC pathways to provide precise spatiotemporal control of neuroectoderm patterning along its AP axis. Together, through the Wnt1 and Wnt8 ligands, they orchestrate a progressive posterior-to-anterior wave of re-specification that restricts the initial, ubiquitous, maternally specified, ANE regulatory state to the most anterior blastomeres. There, the Wnt receptor antagonist, Dkk1, protects this state through a negative feedback mechanism. Because these different Wnt pathways converge on the same cell fate specification process, our data suggest they may function as integrated components of an interactive Wnt signaling network. Our findings provide strong support for the idea that the sea urchin ANE regulatory state and the mechanisms that position and define its borders represent an ancient regulatory patterning system that was present in the common echinoderm/vertebrate ancestor.

Author Summary

The initial regulatory state of most cells in many deuterostome embryos, including those of vertebrates and sea urchins, supports anterior neural fate specification. It is important to restrict this neurogenic potential to the anterior end of the embryo during early embryogenesis, but the molecular mechanisms by which this re-specification of posterior fate occurs are incompletely understood in any embryo. The sea urchin embryo is ideally suited to study this process because, in contrast to vertebrates, anterior–posterior neuroectoderm patterning occurs independently of dorsal-ventral axis patterning and takes place before the complex cell movements of gastrulation. In this study, we show that a linked, three-step process involving at least three different Wnt signaling pathways provides precise spatiotemporal restriction of the anterior neuroectoderm regulatory state to the anterior end of the sea urchin embryo. Because these three pathways impinge on the same developmental process, they could be functioning as an integrated Wnt signaling network. Moreover, striking parallels among gene expression patterns and functional studies suggest that this mechanism of anterior fate restriction could be highly conserved among deuterostomes.

Background

We assessed NanoString's nCounter™ Analysis System for its ability to quantify gene expression of forty-eight genes in a single reaction with 100 ng of total RNA or an equivalent amount of tissue lysate. In the nCounter™ System, multiplexed gene expression target levels are directly detected, without enzymatic reactions, via two sequence-specific probes. The individual mRNA is captured with one mRNA target sequence-specific capture probe that is used in a post-hybridization affinity purification procedure. The second mRNA target specific-sequence and fluorescent-labeled colored coded probe is then used in the detection with the 3-component complex separated on a surface via an applied electric field followed by imaging. We evaluated reproducibility, accuracy, concordance with quantitative RT-PCR, linearity, dynamic range, and the ability of the system to assay different inputs (matched samples of total RNA from Flash Frozen (FF) and Formalin Fixed Paraffin Embedded Tissues (FFPET), and crude tissue lysates (CTL)).

Findings

The nCounter™ Analysis System provided data equivalent to that produced by Taqman®-based assays for genes expressed within the ranges of the calibration curves (above ~0.5 mRNA copies per human cell based on an assumption of 10 pg of total RNA per cell). System response was linear over more than two orders of magnitude with typical CVs of ~6% for concentrations above 1 fM (105 molecules per mL). Profiling the industry-standard MAQC data set yielded correlation coefficients of >0.83 for intensity values and >0.99 for measured ratios. Ninety percent of nCounter™ ratio measurements were within 1.27–1.33 fold changes of the Taqman® data (0.34–0.41 in log2 scale) for FF total RNA samples.

Conclusion

The nCounter™ Analysis System generated robust data for multi-gene expression signatures across three different sample preparation conditions.

The Hedgehog (Hh) signaling pathway is essential for patterning many structures in vertebrates including the nervous system, chordamesoderm, limb and endodermal organs. In the sea urchin, a basal deuterostome, Hh signaling is shown to participate in organizing the mesoderm. At gastrulation the Hh ligand is expressed by the endoderm downstream of the Brachyury and FoxA transcription factors in the endomesoderm gene regulatory network. The co-receptors Patched (Ptc) and Smoothened (Smo) are expressed by the neighboring skeletogenic and non-skeletogenic mesoderm. Perturbations of Hh, Ptc and Smo cause embryos to develop with skeletal defects and inappropriate non-skeletogenic mesoderm patterning, although initial specification of mesoderm occurs without detectable abnormalities. Perturbations of the pathway caused late defects in skeletogenesis and in the non-skeletogenic mesoderm, including altered numbers of pigment and blastocoelar cells, randomized left-right asymmetry of coelomic pouches, and disorganized circumesophageal muscle causing an inability to swallow. Together the data support the requirement of Hh signaling in patterning each of the mesoderm subtypes in the sea urchin embryo.

A major goal of contemporary studies of embryonic development is to understand large sets of regulatory changes that accompany the phenomenon of embryonic induction. The highly resolved sea urchin pregastrular endomesoderm–gene regulatory network (EM-GRN) provides a unique framework to study the global regulatory interactions underlying endomesoderm induction. Vegetal micromeres of the sea urchin embryo constitute a classic endomesoderm signaling center, whose potential to induce archenteron formation from presumptive ectoderm was demonstrated almost a century ago. In this work, we ectopically activate the primary mesenchyme cell–GRN (PMC-GRN) that operates in micromere progeny by misexpressing the micromere determinant Pmar1 and identify the responding EM-GRN that is induced in animal blastomeres. Using localized loss-of -function analyses in conjunction with expression of endo16, the molecular definition of micromere-dependent endomesoderm specification, we show that the TGFβ cytokine, ActivinB, is an essential component of this induction in blastomeres that emit this signal, as well as in cells that respond to it. We report that normal pregastrular endomesoderm specification requires activation of the Pmar1-inducible subset of the EM-GRN by the same cytokine, strongly suggesting that early micromere-mediated endomesoderm specification, which regulates timely gastrulation in the sea urchin embryo, is also ActivinB dependent. This study unexpectedly uncovers the existence of an additional uncharacterized micromere signal to endomesoderm progenitors, significantly revising existing models. In one of the first network-level characterizations of an intercellular inductive phenomenon, we describe an important in vivo model of the requirement of ActivinB signaling in the earliest steps of embryonic endomesoderm progenitor specification.

Author Summary

In recent years, “gene regulatory networks” (GRNs) have provided integrated views of gene interactions that control biological processes. One of the earliest networks to be activated in the developing zygotes is the one controlling endomesoderm development. In the sea urchin, this network includes several subnetworks that function in adjacent tiers of cells that form the endoderm and mesoderm of the developing embryo. Although classic embryological manipulations have shown that the precursors of the embryonic skeleton induce endomesoderm fate in adjacent cells, the GRNs regulating this interaction are not understood. To investigate these networks, we ectopically activated a GRN that operates in skeletogenic precursors and characterized the responding GRN in neighboring cells, which adopt an endomesoderm fate. By testing the responsiveness of every core factor in the responding GRN, which allowed us to identify a subset that executes the response to the induction, we demonstrated that the signaling molecule, ActivinB, is an essential component of this induction and that its function is physiologically relevant: it is required during normal embryonic development to activate the same GRN that responds to signals from skeletogenic precursors. Furthermore, the network response to ActivinB signaling reveals greater complexity in an additional uncharacterized inductive signal emitted by skeletogenic precursors. Our results thus highlight how interacting GRNs can be used to understand a fundamental signaling process.

A classic embryonic induction in sea urchins is described in terms of interacting gene regulatory networks and a signal transduction system that connects them.

Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. This gene encodes a TGFβ ligand, and in Strongylocentrotus purpuratus its transcription is activated in the presumptive oral ectoderm at about the 30-cell stage. Thereafter Nodal signaling occurs among all cells of the oral ectoderm territory, and nodal expression is required for expression of oral ectoderm regulatory genes. The cis-regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. Functional cis-regulatory control modules of the nodal gene were identified by interspecific sequence conservation. A 5′ cis-regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression.

Background

The mechanisms by which the conserved genetic “toolkit” for development generates phenotypic disparity across metazoans is poorly understood. Echinoderm larvae provide a great resource for understanding how developmental novelty arises. The sea urchin pluteus larva is dramatically different from basal echinoderm larval types, which include the auricularia-type larva of its sister taxon, the sea cucumbers, and the sea star bipinnaria larva. In particular, the pluteus has a mesodermally-derived larval skeleton that is not present in sea star larvae or any outgroup taxa. To understand the evolutionary origin of this structure, we examined the molecular development of mesoderm in the sea cucumber, Parastichopus parvimensis.

Results

By comparing gene expression in sea urchins, sea cucumbers and sea stars, we partially reconstructed the mesodermal regulatory state of the echinoderm ancestor. Surprisingly, we also identified expression of the transcription factor alx1 in a cryptic skeletogenic mesenchyme lineage in P. parvimensis. Orthologs of alx1 are expressed exclusively within the sea urchin skeletogenic mesenchyme, but are not expressed in the mesenchyme of the sea star, which suggests that alx1+ mesenchyme is a synapomorphy of at least sea urchins and sea cucumbers. Perturbation of Alx1 demonstrates that this protein is necessary for the formation of the sea cucumber spicule. Overexpression of the sea star alx1 ortholog in sea urchins is sufficient to induce additional skeleton, indicating that the Alx1 protein has not evolved a new function during the evolution of the larval skeleton.

Conclusions

The proposed echinoderm ancestral mesoderm state is highly conserved between the morphologically similar, but evolutionarily distant, auricularia and bipinnaria larvae. However, the auricularia, but not bipinnaria, also develops a simple skelotogenic cell lineage. Our data indicate that the first step in acquiring these novel cell fates was to re-specify the ancestral mesoderm into molecularly distinct territories. These new territories likely consisted of only a few cells with few regulatory differences from the ancestral state, thereby leaving the remaining mesoderm to retain its original function. The new territories were then free to take on a new fate. Partitioning of existing gene networks was a necessary pre-requisite to establish novelty in this system.

Recent work is reviewed on several molecular aspects of gene expression during oocyte maturation and early development. Several analyses have been carried out on protein synthesis patterns using the high resolution two-dimensional polyacrylamide gel electrophoresis technique. The protein pattern of some 400–500 spots in sea urchin oocytes before and just after fertilization are quite similar. By gastrula, major stage specific protein changes have been noted. In a similar study major protein changes are noted during the meiotic maturation process in mammals. With the use of the single-copy DNA hybridization technique, the quantitative levels of rare mRNA sequence expression have been determined during oogenesis and early development in the sea urchin model. The mature oocyte sequence set of some 37 × 106 nucleotides of information (or potentially 18,500 different proteins) is synthesized during oogenesis and slowly utilized during development. The sea urchin gastrula mRNA is predominantly synthesized by the embryo genome; however, essentially all those gastrula mRNA sequences can also be found in the mature oocyte (maternal) set. It is proposed by Hough-Evans et al. that this maternal sequence set, which is made during oogenesis and both utilized (translated in 10,000–20,000 proteins), and in part, continually synthesized by all stages of the embryo, plays a critical role in the morphogenic formation of a sea urchin embryo.

Dishevelled (Dsh) is a phosphoprotein key to beta-catenin dependent (canonical) and beta-catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta-catenin independent pathways in embryogenesis, we used Dsh-DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca2+ signaling. Embryos overexpressing Dsh-DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta-catenin failed to rescue Dsh-DEP embryos, indicating that Dsh-DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh-DEP-like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh-DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis. Developmental Dynamics 238:1649–1665, 2009. © 2009 Wiley-Liss, Inc.

Summary

microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and would serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of the major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. All miRNAs have dynamic accumulation profiles through early development as do a large population of putative piRNAs (piwi-interacting RNAs). Defects in morphogenesis caused by loss of Drosha can be rescued with four miRNAs which permits a strong miRNA functional assay. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.

The expression of three different actin genes in the sea urchin, Strongylocentrotus purpuratus, was monitored in embryos and adult tissues by using untranslated mRNA sequences as specific hybridization probes. Three distinct patterns of expression were found: muscle specific, embryo specific, and constitutive (i.e., present in all tissues examined). The actin genes encoding the muscle-specific and constitutively expressed genes were each found to be present once in the haploid genome. The embryo-specific probe could derive from either a single gene or a small subset of actin genes. These data demonstrate that at least three members of the sea urchin actin gene family are expressed in distinct ways and thus are probably associated with different regulatory programs of gene expression necessary for development of this metazoan.

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